Il-2 procytokine antibody fusion proteins

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 63 / 393,150, filed on July 28, 2022, which is incorporated herein by reference in its entirety.

[0003] Statement regarding sequence listing

[0004] The sequence listing XML associated with this application is provided in XML file format and is hereby incorporated into this specification by reference. The name of the XML file containing the sequence listing XML is PRVA_015_01WO_ST26.xml. The XML file is approximately 286,496 bytes, created on July 27, 2023, and submitted electronically through the USPTO Patent Center.

[0005] background Technical Field

[0006] The present invention relates to an activatable proprotein homodimer, which comprises two independent but identical polypeptide chains, each chain comprising a fragment antigen binding (Fab) region specifically binding to human PD-1 or 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. It also includes related pharmaceutical compositions and methods of use thereof.

[0007] Description of Related Technology

[0008] Interleukin-2 (IL-2) immunotherapy has been shown to be useful for treating cancers such as malignant melanoma and renal cell carcinoma. Programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) inhibitor therapy enhances anti-tumor T cell responses and mediates anti-tumor activity (Dermani et al., J Cell Physiol. 234: 1313-1325, 2019).

[0009] 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 primarily expand immunosuppressive regulatory T cells or T cells. reg(See, e.g., Arenas-Ramirez et al., Trends in Immunology. 36: 763-777, 2015). In addition, the effects of IL-2 therapy are mainly systemic, rather than confined to target tissues, leading to many serious side effects, such as respiratory problems, nausea, hypotension, loss of appetite, confusion, severe infections, seizures, allergic reactions, heart problems, renal failure, and vascular slit syndrome. Despite this, IL-2 therapy can be effective, and there are strategies for addressing these and other shortcomings (see, e.g., WO 2021 / 011353). However, there is still a need in the art to improve such strategies.

[0010] Embodiments of the present invention represent such an improvement by providing an anti-PD-1 / PD-L1 activatable proprotein comprising IL-2 (procytokine) that can be specifically targeted to the tumor microenvironment (TME) and activated therein.

[0011] Brief Overview

[0012] Embodiments of the present invention include an activatable preprotein homodimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprise, in the N-terminal to C-terminal direction: a fragment antigen binding (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, wherein the IL-2 protein of the first polypeptide binds to the IL-2Rα protein of the second polypeptide, and wherein the IL-2Rα of the first polypeptide binds to the IL-2 protein of the second polypeptide, wherein the binding masks a binding site of the one or more IL-2 proteins, which originally binds to IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of immune cells in vitro or in vivo; and wherein the second linker is a cleavable linker.

[0013] In some embodiments, the Fab region specifically binds to human PD-1, and optionally comprises a Fab region from an anti-PD-1 antibody selected from nivolumab, pembrolizumab, cemiplizumab, JTX-4014, spartalizumab, carrelizumab, sintilimab, tislelizumab, toripalizumab, dotalimumab, MGA012, AMP-22, and AMP-514. In some embodiments, the Fab region specifically binds to human PD-1, and comprises

[0014] A heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO: 1; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO: 2;

[0015] a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO:3; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO:4;

[0016] A heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions shown in SEQ ID NO:5; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions shown in SEQ ID NO:6; or

[0017] a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO:7; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO:8;

[0018] a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO:9; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO:10;

[0019] a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO: 11; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO: 12;

[0020] a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO: 13; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO: 14;

[0021] a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO: 15; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO: 16;

[0022] a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO: 17; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO: 18;

[0023] a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO: 19; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO: 20;

[0024] A heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions shown in SEQ ID NO: 21; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions shown in SEQ ID NO: 22; or

[0025] a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions shown in SEQ ID NO:23; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions shown in SEQ ID NO:24.

[0026] In some embodiments, the VH region comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical 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 that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24, respectively.

[0027] In some embodiments, the Fab region specifically binds to 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 to human PD-L1 and comprises

[0028] a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO: 25; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO: 26;

[0029] a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO: 27; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO: 28;

[0030] A heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions shown in SEQ ID NO: 29; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions shown in SEQ ID NO: 30; or

[0031] a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO:31; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO:32;

[0032] a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO:33; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO:34;

[0033] A heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions shown in SEQ ID NO:35; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions shown in SEQ ID NO:36; or

[0034] a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions shown in SEQ ID NO:37; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions shown in SEQ ID NO:38.

[0035] In some embodiments, the VH region comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical 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 that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 26, 28, 30, 32, 34, 36, and 38, respectively.

[0036] In some embodiments, the Fab region specifically binds to human B7H3, wherein the VH region comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:261, and the VL region comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:262, respectively.

[0037] 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 that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from Table F1, and wherein the Fc domain comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical 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 having L234A / L235A ("LALA") mutations and / or P329A or P329G mutations (EU numbering).

[0038] In some embodiments, the IL-2 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 an amino acid sequence selected from Table S1, optionally amino acids 21-153 of SEQ ID NO: 68 (full length wild-type human IL-2), optionally comprising a C145X (X is any amino acid) or C145S substitution as defined by SEQ ID NO: 68. In some embodiments, the IL-2 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 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 comprises 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 wherein the IL-2Rα protein of the first polypeptide forms a disulfide bond with the IL-2 protein of the second polypeptide, the disulfide bond is optionally formed through one or more of the aforementioned cysteines. 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.

[0039] 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 wherein the one or more amino acid substitutions are selected from T3A; 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; E62A and E62L; 8A and E68V; and L72A, L72G, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K, including combinations thereof, optionally 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 a combination of R38A, F42A, Y45A and E62A. In some embodiments, the IL-2 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 SEQ ID NO: 84 or 85 (human IL-2 mature sequence (26-153)), optionally wherein the IL-2 protein comprises or retains the R38D, K43E and C125S substitutions of SEQ ID NO: 85.

[0040] 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 an amino acid sequence selected from Table S2, optionally amino acids 22-187 of SEQ ID NO:86 (full-length wild-type human IL-2Rα), optionally wherein 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 SEQ ID NO:90, including wherein the IL-2Rα protein retains D6R and / or E29K substitutions. 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 as defined by SEQ ID NO: 88 (human IL-2Rα Sushi 1 to Sushi 2 domains), and / or a K38S substitution. 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 wherein the IL-2Rα protein of the second polypeptide forms a disulfide bond with the IL-2 protein of the first polypeptide, optionally through one or more of the aforementioned cysteines and one or more cysteines in the IL-2 protein, optionally through one or more cysteine ​​pairs 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 IL-2Rα protein and the second IL-2Rα protein comprise an alanine substitution at position 49 and / or 68 as defined by SEQ ID NO:88.

[0041] 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, and wherein the cleavable linker comprises a protease cleavage site, optionally wherein the cleavable linker is selected from Table S3. In a specific embodiment, the first linker is a non-cleavable or stable linker of 7 or less amino acids in length (or 1, 2, 3, 4, 5, 6, 7 amino acids in length), and the cleavable linker comprises a protease cleavage site, optionally wherein the cleavable linker is 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 stable linker of 4 amino acids, such as GGGS (SEQ ID NO: 188), and the cleavable linker comprises a protease cleavage site, optionally wherein the cleavable linker is selected from Table S3, e.g., SEQ ID NO: 93.

[0042] In some embodiments, the protease cleavage site can be cleaved by one or more proteases selected from metalloproteases, serine proteases, cysteine ​​proteases and aspartic proteases. In some embodiments, the protease cleavage site can be cleaved by one or more proteases selected from MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, TEV protease, matriptase, uPA, FAP, legumin, PSA, kallikrein, cathepsin A and cathepsin B. In some embodiments, the length of 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, 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.

[0043] 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 (κ); 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.

[0044] In certain activatable preprotein homodimers, cleavage of the second linker (optionally protease cleavage) exposes one or more binding sites of the IL-2 protein, which bind to the IL-2Rβ / γc chain present on the surface of an immune cell in vitro or in vivo. 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.

[0045] In some embodiments, the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to a sequence selected from Table S4 (chain 1 and chain 2), and a VL / CL region polypeptide that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to the corresponding sequence from Table S4 (chain 3 and chain 4), including wherein:

[0046] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 138 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 139;

[0047] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 140 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 141;

[0048] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 142 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 143;

[0049] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 146 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 147;

[0050] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 148 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 149;

[0051] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 152 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 153;

[0052] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 154 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 155;

[0053] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 156 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 157;

[0054] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 158 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 159;

[0055] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 209 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 210;

[0056] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 211 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 212;

[0057] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 213 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 214;

[0058] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 215 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 216;

[0059] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 217 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 218;

[0060] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 219 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 220;

[0061] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 221 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 222;

[0062] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 223 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 224;

[0063] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 225 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 226;

[0064] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 227 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 228;

[0065] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 229 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 230;

[0066] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 231 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 232;

[0067] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 233 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 234;

[0068] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 235 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 236;

[0069] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 237 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 238;

[0070] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 239 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 240;

[0071] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 241 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 242;

[0072] the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 243 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 244;

[0073] The first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence and a VL / CL region polypeptide that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 245 and a VL / CL region polypeptide that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 246; or

[0074] The first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence and a VL / CL region polypeptide that is at least 80%, 85%, 90%, 95%, 98% or 100% identical to SEQ ID NO:247 and a VL / CL region polypeptide that is at least 80%, 85%, 90%, 95%, 98% or 100% identical to SEQ ID NO:248.

[0075] Certain activatable proprotein homodimers described herein are substantially homodimers in physiological solution, or under physiological conditions, optionally under in vivo conditions.

[0076] Also included are one or more recombinant nucleic acid molecules encoding an activatable preprotein homodimer as described herein. In some embodiments, the first recombinant nucleic acid molecule encodes the VH / CH1 region, hinge / Fc domain, first linker, IL-2 protein, second linker, and IL-2Rα protein of the Fab region, and the 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 as described herein. Certain embodiments include a host cell comprising one or more recombinant nucleic acid molecules as described herein, or one or more vectors as described herein.

[0077] Certain embodiments include methods of producing an activatable proprotein, comprising culturing a host cell described herein under culture conditions suitable for expression of activatable proprotein homodimers, and isolating the activatable proprotein from the culture.

[0078] Also included are pharmaceutical compositions comprising the activatable proprotein homodimers described herein and a pharmaceutically acceptable carrier.

[0079] Certain embodiments relate to methods of treating a disease in a subject, and / or methods of enhancing an immune response in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.

[0080] In some embodiments, the disease is cancer, for example, a cancer that expresses or overexpresses PD-L1. In some embodiments, the cancer is a primary cancer or a metastatic cancer and is selected from melanoma (optionally metastatic melanoma), renal 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, liver cancer (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.

[0081] In some embodiments, after administration, the activatable preprotein homodimer is activated by protease cleavage in cancer cells or cancer tissues or tumor microenvironment (TME), which exposes one or more binding sites of the IL-2 protein that bind to the IL-2Rβ / γc chain present on the surface of immune cells in vitro or in vivo, thereby producing an activated protein. In some embodiments, the activated protein is bound to the IL-2Rβ / γc chain present on the surface of immune cells in vitro or in vivo by the IL-2 protein. In some embodiments, the immune cells are selected from one or more of T cells, B cells, natural killer cells, monocytes, and macrophages.

[0082] In some embodiments, administration and activation of the activatable proprotein enhances the anti-cancer immune response in the 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 relative to a control. In some embodiments, administration and activation of the activatable proprotein increases cancer cell killing in the 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 relative to a control.

[0083] In some embodiments, the pharmaceutical composition is administered to the subject by parenteral administration. In some embodiments, the parenteral administration is intravenous administration.

[0084] Certain embodiments include the use of a pharmaceutical composition as described herein for the preparation of a medicament for treating a disease in a subject, optionally a cancer (e.g., a cancer that expresses or overexpresses PD-L1), and / or for enhancing an immune response in a subject. Certain embodiments include a pharmaceutical composition as described herein for treating a disease in a subject, optionally a cancer (e.g., a cancer that expresses or overexpresses PD-L1), and / or for enhancing an immune response in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 An exemplary structure of a single polypeptide (IgG-proIL-2 motif) is shown, which forms a homodimer with a second polypeptide of the same structure (not shown). The IgG-proIL-2 motif consists of a high affinity neutralizing antibody (e.g., no FcγR binding and intact FcRn binding) and an IL-2 pro-cytokine module.

[0086] Figure 2 An exemplary structure of an activatable proprotein homodimer in its inactive (procytokine) state is shown. The dashed line indicates a cleavable linker. The complete IgG-proIL-2 construct shows no or little IL-2 activity because the IL-2Rβγ binding site is "masked" in this format.

[0087] Figure 3Illustration of homodimeric IL-2 "activation" by protease cleavage of a 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, releasing active IL-2 at the tumor site. This can reduce toxicity in peripheral blood and enhance antitumor activity.

[0088] Figure 4A-4C Shown are the ELISA binding activities 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.

[0089] Figure 5 Shown are the results of mixed lymphocyte reactions performed by co-culture of PBMC cells and allogeneic DCs with or without addition of parental PD-1 antibody or dose adjustment of PD-1-proIL-2 at the start of the assay. After 5 days, IFN-γ secretion in the culture supernatants was analyzed by ELISA.

[0090] Figure 6A-6B Shown are 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.

[0091] Figure 7A-7D Shown are the results of STAT5 assays of intact PD-1-proIL-2v and MMP2 protease-activated PD-1-proIL-2v in resting PBMCs of donor 1 (CD4 T cells (A), CD8 T cells (B), regulatory T cells (C), and NK cells (D); legend on 7B).

[0092] Figure 8A-8D Shown are the results of STAT5 assays of intact PD-L1-proIL-2v and MMP2 protease-activated PD-L1-proIL-2v in resting PBMCs of donor 1 (CD4 T cells (A), CD8 T cells (B), regulatory T cells (C), and NK cells (D); legend on 8B).

[0093] Figure 9A-9BIn vitro CD4 T cell activation and cytokine release are shown. Dose-dependent (A) GM-CSF and (B) IFN-γ secretion of human polyclonal CD4 T cells after 5 days of stimulation with increasing concentrations of PD-1 antibody or intact PD-1-proIL-2v or MMP2 protease-activated PD-1-proIL-2v is shown.

[0094] Fig. 10A Shown are the mean tumor volumes measured over time in an A375-PBMC xenograft model, demonstrating the in vivo antitumor activity of PD-L1-proIL-2v with different linkers as single agents in inhibiting tumor growth compared to anti-PD-L1 antibodies at the same molar concentration. Fig. 10B Shows Fig. 10A Mean tumor volume at day 24 of the same treatment in . Mice (n=5 or 6) were injected iv on days 0, 3, 7, 10 and 14; results are expressed as mean ± SEM.

[0095] Fig.11A Shown are the mean tumor volumes measured over time in the HT-29-PBMC xenograft model, demonstrating the in vivo antitumor activity of PD-L1-proIL-2v with different linkers as single agents in inhibiting tumor growth compared to the same molar concentration of anti-PD-L1 antibody. Fig. 11B Shows Fig.11A Mean tumor volume at day 23 after the same treatment in . Mice (n=6 per group) were injected iv on days 0, 3, 7 and 10; the results are expressed as mean ± SEM.

[0096] Fig. 12A Shown are the mean tumor volumes 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. Fig. 12B Shows Fig. 12A Mean tumor volume at day 29 after the same treatment in . Mice (n=5 or 6) were injected iv on days 0, 3, 7 and 10; the results are expressed as mean ± SEM.

[0097] Fig.13A Shown are the mean tumor volumes measured over time in a HT-29-PBMC xenograft model, demonstrating the in vivo antitumor activity of PD-1-proIL-2v with different linkers as single agents in inhibiting tumor growth compared to anti-PD-1 antibodies. Fig. 13B Shows Fig.13AMean tumor volume at day 27 after the same treatment in . Mice (n=5 or 6) were injected iv on days 0, 4, 7 and 11; results are expressed as mean ± SEM.

[0098] Fig.14A Shown are mean tumor volumes measured over time in an A375-PBMC xenograft model, demonstrating in vivo dose-dependent antitumor activity of PD-L1-proIL-2v and PD-1-proIL-2v as single agents in inhibiting tumor growth. Fig. 14B Shows Fig.14A Mean tumor volume at day 22 after the same treatment in . Mice (n=5 or 6) were injected iv on days 0, 3, 7, 10 and 14; results are expressed as mean ± SEM.

[0099] Fig.15A Shown are mean tumor volumes 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. Fig. 15B Shows Fig.15A Mean tumor volume at day 22 after the same treatment in . Mice (n=6) were injected iv on days 0 and 7; the results are expressed as mean ± SEM.

[0100] Fig.16A Shown are mean tumor volumes measured over time in an A375-PBMC xenograft model, demonstrating the in vivo antitumor activity of PD-L1-proIL-2v and PD-L1-proIL-2wt as single agents in inhibiting tumor growth. Fig. 16B Shows Fig.16A Mean tumor volume at day 19 after the same treatment in . Mice (n=5 or 6) were injected iv on days 0, 3, and 7; the results are expressed as mean±SEM.

[0101] Fig.17A Shown are mean tumor volumes 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 alone and in combination in inhibiting tumor growth. Fig. 17B Shows Fig.17A Mean tumor volume at day 29 after the same treatment in . Mice (n=5 or 6) were injected iv on days 0, 3, 7 and 10; the results are expressed as mean ± SEM.

[0102] Fig.18A Total drug (pro-cytokine + activated cytokine) and pro-cytokine concentrations of PD-1-proIL-2v in peripheral blood over time are shown. Fig.18B Total drug and pro-cytokine concentrations of PD-1-proIL-2v in tumors over time are shown. Fig.18C The concentration of activated cytokines of PD-1-proIL-2v in tumors over time is shown. Mice (n=3) were injected iv with P41222037 at 1 mg / kg on day 0; results are expressed as mean ± SEM.

[0103] Figure 19A-Figure 19C The results show that the expression of CD3 in peripheral blood was significantly increased after PD-1-proIL-2v treatment in the PBMC humanized A375 xenograft model. + T cells (A), CD4 + T cells (B) and CD8 + T cells (C) Cell number over time. Mice (n=5) were injected iv on days 0 and 7; results are expressed as mean ± SEM.

[0104] Figure 20A-20C Shows CD3 in tumors after PD-1-proIL-2v treatment in A375-PBMC xenograft model + T cells (A), CD4 + T cells (B) and CD8 + Cell number of T cells (C). Mice (n=3) were injected iv on days 0 and 8, and tumors were harvested on day 12; results are expressed as mean ± SEM.

[0105] Fig.21A Shown are the concentrations of masked or unmasked PD-1-proIL-2v in peripheral blood of cynomolgus monkeys over time. Fig. 21B Shown are albumin levels over time in 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.

[0106] Figure 22A-22C The protease activated P41222037 ( P41222037 ) compared to human recombinant IL-2 and the complete proprotein (no protease activation) P41222037, P45412037 or P45422037 constructs, respectively, are shown. Fig.22A )、P45412037( Fig. 22B ) or P45422037( Fig. 22C) construct-induced proliferation of the human acute megakaryoblastic leukemia cell line M-07e.

[0107] Figure 23A-23D Compared to human recombinant IL-2, intact and MMP2-cleaved P41222037 ( Fig.23A )、P45412037( Fig. 23B ) or P45422037( Fig.23C ) treatment of resting PBMCs. Analysis of STAT5 phosphorylation in CD4 T cells, CD8 T cells, and Tregs after treatment of resting PBMCs with the intact proprotein P41222037, P45412037, and P45422037 constructs were plotted together for comparison ( Fig.23D ).

[0108] Figure 24A-24C Compared to human recombinant IL-2, the expression of P78192037, P78202037, P78212037, P78222037, P78232037 and P78242037 ( Fig.24A ); P78252037, P78262037, P78272037, P78282037, P78292037 and P78302037( Fig. 24B ) or P78312037, P78322037, P78332037, P78342037, P78352037 and P78362037( Fig.24C ) treatment of resting PBMCs.

[0109] Fig.25 Shown is STAT5 phosphorylation in CD4 T cells, CD8 T cells and Tregs following treatment of pre-activated PBMCs with the intact precursor protein P41222037 construct as determined by flow cytometry.

[0110] Fig.26The amino acid sequences of the test constructs of the fusion junction between the antibody heavy chain and the IL-2 variant are shown. Exemplary junction sequences include various stable linker lengths, the deletion of the C-terminal Lys of the heavy chain and / or the 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).

[0111] Detailed Description

[0112] Embodiments of the present invention relate to activatable proprotein homodimers, which comprise two separate but identical polypeptides, each polypeptide comprising a fragment antigen binding (Fab) region that specifically binds to human PD-1 or human PD-L1 in the N-terminal to C-terminal direction, a hinge / Fc domain, a stable linker, an IL-2 protein, a protease-cleavable linker, and an IL-2Rα protein. In some cases, homodimers are formed by the following binding interactions: the binding of the hinge / Fc domain of one polypeptide to the hinge / Fc domain of another polypeptide, and the binding of each IL-2 protein of one polypeptide to each IL-2Rα protein of another polypeptide. These binding interactions form biologically inactive (proprotein) homodimers by masking the binding site of the IL-2 protein, which would originally bind to the IL-2Rβ / γc and / or IL-2Rα / β / γc chain present on the surface of immune cells. Proprotein homodimers remain inactive or substantially inactive in plasma.

[0113] In some cases, the homodimer is targeted to the tumor microenvironment (TME) by anti-PD-1 or anti-PD-L1 Fab and then activated within the TME by exposure to tumor site proteases, which cleave the protease-cleavable linker, thereby releasing the IL-2Rα protein and exposing the active site of the IL-2 protein. This results in enhanced IL-2 signaling activity on cells expressing PD-1 or PD-L1 due to a "cis-targeting" effect and / or a synergistic effect between the anti-tumor activity of the anti-PD-1 / anti-PD-L1 Fab and the immunostimulatory activity of the IL-2 protein.

[0114] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs. Although any methods, materials, compositions, reagents, cells similar or equivalent to those described herein can be used for the practice or testing of the subject matter of the present invention, preferred methods and materials are described. All publications and references (including but not limited to patents and patent applications) cited in this specification are incorporated herein by reference in their entirety, just as specifically and individually pointed out, as fully described, by reference to each separate publication or reference incorporated herein. Any patent application for which this application claims priority is also incorporated herein by reference in its entirety in the manner described above for publications and references.

[0115] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reaction and purification techniques can be carried out according to the manufacturer's instructions, or as usually completed in this area or as described herein. These and related techniques and procedures can usually be performed according to conventional methods described in various general and more specific references as well known in the art and as quoted and discussed in this specification in full. Unless specific definition is provided, the nomenclature used in combination with molecular biology, analytical chemistry, synthetic organic chemistry, medicine and medicinal chemistry as described herein and the laboratory procedures and techniques of the subject are well known in the art and commonly used. Standard techniques can be used for the treatment of recombinant technology, molecular biology, microbiology, chemosynthesis, chemical analysis, drug preparation, preparation, delivery and patients.

[0116] For purposes of the present invention, the following terms are defined below.

[0117] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. For example, "an element" includes "an element," "one or more elements," and / or "at least one element."

[0118] “About” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by up to 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0119] The terms "activatable proprotein", "proprotein", "activatable procytokine", "procytokine", "activatable prodrug" and "prodrug" are used interchangeably herein to refer to an activatable proprotein comprising at least one masking moiety and an active domain, or a derivative / variant thereof, as described herein. In one embodiment, the proprotein may further comprise one or more protein domains.

[0120] The term "antigen" refers to a molecule or part of a molecule that can be bound by a selective binding agent (such as an antibody) and can additionally be used in an animal to produce an antibody that can bind to the antigen and an epitope. An antigen may have one or more epitopes. As used herein, the term "antigen" includes substances that can induce an immune response to the substance under appropriate conditions and can react with the product of the immune response. More broadly, the term "antigen" includes any substance that an antibody binds to, or any substance that an antibody against it is required, whether or not the substance is immunogenic. For such antigens, antibodies can be identified by recombinant methods without relying on any immune response.

[0121] "Antagonist" refers to a biological structure or chemical agent that interferes with or otherwise weakens the physiological effect of another agent or molecule. In some cases, antagonists specifically bind to another agent or molecule. Both complete and partial antagonists are included.

[0122] "Agonist" refers to a biological structure or chemical agent that increases or enhances the physiological effect of another agent or molecule. In some cases, an agonist specifically binds to another agent or molecule. Both full and partial agonists are included.

[0123] As used herein, the term "amino acid" is intended to refer to naturally occurring and non-naturally occurring amino acids and amino acid analogs and mimetics. For example, naturally occurring amino acids include the 20 (L)-amino acids utilized in the protein biosynthesis process as well as other amino acids, such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine, and the like, known to those skilled in the art. Amino acid analogs include modified forms of natural and non-natural amino acids. Such modifications may include, for example, substitution or replacement of chemical groups and moieties on the amino acids, or by derivatization of amino acids. Amino acid mimetics include, for example, organic structures that exhibit functionally similar properties, such as charge and charge spacing characteristics of reference amino acids. For example, an organic structure that mimics arginine (Arg or R) will have a positively charged portion that is located in a similar molecular space and has the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of amino acids or amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0124] 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 have exhibited detectable disease or disease symptoms prior to the treatment methods described herein. "At risk" means that the subject has one or more risk factors, as described herein and known in the art, which are measurable parameters associated with the development of a disease. A subject with one or more of these risk factors has a higher likelihood of developing a disease or adverse reaction than a subject without one or more of these risk factors.

[0125] "Biocompatible" refers to materials or compounds that are generally not deleterious to the biological functions of cells or subjects and do not cause any degree of unacceptable toxicity, including allergies and disease states.

[0126] The term "binding" refers to the direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic and ionic and / or hydrogen bonding interactions (including interactions such as salt bridges and water bridges).

[0127] "Cis-targeting" refers to the preferential activity of one function from a multifunctional molecule (including bispecific or bifunctional molecules) on the same cell in which a second function (such as a binding activity) has an effect. This approach offers the potential to optimize the use of functional modulators (inhibitors or stimulators of the molecular target function) to treat cancer and other diseases by limiting or enhancing their effects on specific cell types.

[0128] "Coding sequence" refers to any nucleic acid sequence that contributes to encoding the polypeptide product of a gene. In contrast, the term "non-coding sequence" refers to any nucleic acid sequence that is not directly involved in encoding the polypeptide product of a gene.

[0129] Throughout the disclosure, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of stated steps or elements or groups of steps or elements but not the exclusion of any other steps or elements or groups of steps or elements.

[0130] “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 no other elements are present. “Consisting essentially of” means including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present, depending on whether they materially affect the activity or action of the listed elements.

[0131] The term "endotoxin-free" or "essentially endotoxin-free" generally refers to compositions, solvents and / or containers containing at most trace amounts of endotoxin (preferably undetectable amounts of endotoxin) (e.g., amounts that have no clinically adverse physiological effects on a subject). Endotoxins are toxins associated with certain microorganisms such as bacteria (usually gram-negative bacteria), although endotoxins may be present in gram-positive bacteria such as Listeria monocytogenes. The most common endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS) present in the outer membranes of various gram-negative bacteria, which represent the main pathogenic features of these bacteria's pathogenicity. A small amount of endotoxins in the human body may cause fever, hypotension, inflammation, and activation of coagulation, as well as other adverse physiological effects.

[0132] Therefore, in drug production, it is usually necessary to remove most or all of the trace endotoxins from drug products and / or drug containers, because even a small amount of endotoxins can also produce side effects on the human body. Since it is usually necessary to decompose most endotoxins at a temperature exceeding 300° C., a pyrogen removal oven can be used for this purpose. For example, based on primary packaging materials such as syringes or vials, a combination of a glass temperature of 250° C. and a holding time of 30 minutes is usually sufficient to achieve a 3-logarithmic reduction in endotoxin levels. It is contemplated that other methods of removing endotoxins include, for example, chromatography and filtration methods described herein and known in the art.

[0133] Endotoxin can be detected using conventional techniques known in the art. For example, the horseshoe crab amebocyte lysis assay using horseshoe crab blood is a very sensitive assay for detecting the presence of endotoxin. In this test, since a powerful enzymatic cascade amplifies this reaction, very low levels of LPS can cause detectable coagulation of horseshoe crab lysates. Endotoxin can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially free of endotoxin, the endotoxin level may be less than about 0.001 EU / mg, 0.005 EU / mg, 0.01 EU / mg, 0.02 EU / mg, 0.03 EU / mg, 0.04 EU / mg, 0.05 EU / mg, 0.06 EU / mg, 0.08 EU / mg, 0.09 EU / mg, 0.1 EU / mg, 0.5 EU / mg, 1.0 EU / mg, 1.5 EU / mg, 2 EU / mg, 2.5 EU / mg, 3 EU / mg, 4 EU / mg, 5 EU / mg, 6 EU / mg, 7 EU / mg, 8 EU / mg, 9 EU / mg, or 10 EU / mg of active compound. Typically, 1 ng of lipopolysaccharide (LPS) is equivalent to about 1-10 EU.

[0134] The term "half maximal effective concentration" or "EC 50 ” refers to the concentration of an agent described herein (e.g., an activatable proprotein) at which, after a specified exposure time, induces a response halfway between baseline and maximum; thus, the EC of a graded dose-response curve is 50 Represents the concentration of compound at which 50% of the maximal effect is observed. EC 50 It also represents the plasma concentration required to achieve 50% of the maximum effect in vivo. Similarly, “EC 90 ” refers to the concentration of an agent or composition at which 90% of the maximum effect of the agent or composition is observed. “EC 90 "Can be made by "EC 50 ” and the Hill slope, or it can be determined directly from the data using routine knowledge in the art. In some embodiments, the EC of an agent (e.g., an activatable proprotein) 50In some embodiments, the agent will have an EC of about 1 nM or less. 50 value.

[0135] "Immune response" refers to any immune response derived from the immune system, including responses from cells and body fluids, 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 subpopulations thereof. Cellular responses include, for example, effector functions, cytokine release, phagocytosis, efferocytosis, displacement, transport, proliferation, differentiation, activation, inhibition, cell-cell interactions, apoptosis, etc. Humeral responses include, for example, IgG, IgM, IgA, IgE responses and their corresponding effector functions.

[0136] " half-life " of agent such as activatable proprotein can refer to its pharmacology, physiology or other activity when being applied to the serum or tissue of organism, or the time point of any other restriction, the time that agent loses half of this activity. " half-life " can also refer to the amount or concentration of agent relative to this amount or concentration when being applied to the serum or tissue of organism, or relative to the time point of any other restriction, reduce the time that half of the initial amount in the serum or tissue of organism is spent. Half-life can be measured in serum and / or any one or more selected tissues.

[0137] The terms "modulate" and "alter" include "increase" or "enhance" and "decrease" or "reduce," typically by a statistically significant or physiologically significant amount or degree relative to a control. An "increased" or "enhanced" amount is typically a "statistically significant" amount and may include an amount that is about or at least about 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold or more relative to a control. An “increased” or “enhanced” amount may also include an amount of about 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 relative to a control. A "lowered" or "decreased" amount is typically a "statistically significant" amount and can include a reduction to an amount that is about or at least about 10 / 11, 10 / 12, 10 / 15, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 15, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 60, 1 / 70, 1 / 80, 1 / 90, 1 / 100, 1 / 200, 1 / 300, 1 / 400, 1 / 500, 1 / 600, 1 / 700, 1 / 800, 1 / 900, 1 / 1000, 1 / 2000, 1 / 3000, 1 / 4000, or 1 / 5000 of the amount of a control. "Reduced" or "decreased" amounts can also include 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 amounts relative to a control. Examples of comparisons and "statistically significant" amounts are described herein.

[0138] The terms "polypeptide", "protein" and "peptide" are used interchangeably and refer to polymers of amino acids that are not limited to any particular length. The term "enzyme" includes polypeptide or protein catalysts. The term includes modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of signal sequences. The term "polypeptide" or "protein" means one or more amino acid chains, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein the polypeptide or protein may comprise multiple chains non-covalently and / or covalently linked together by peptide bonds (which have the sequence of a natural protein (i.e., a protein produced by a naturally occurring, particularly non-recombinant cell or a genetically engineered cell or a recombinant cell)), and includes molecules having the amino acid sequence of a natural protein, or molecules having one or more amino acids of the natural sequence deleted, added and / or substituted. In certain embodiments, a polypeptide is a "recombinant" polypeptide produced by a recombinant cell comprising one or more recombinant DNA molecules, which are typically made from a combination of heterologous polynucleotide sequences or polynucleotide sequences that are not originally present in the cell.

[0139] The terms "polynucleotide" and "nucleic acid" include mRNA, RNA, cRNA, cDNA and DNA. The term generally refers to a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single-stranded and double-stranded forms of DNA. The terms "isolated DNA" and "isolated polynucleotides" and "isolated nucleic acids" refer to molecules isolated from the 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 is substantially isolated or purified from the total genomic DNA of the species from which the DNA segment was obtained. Non-coding polynucleotides (e.g., primers, probes, oligonucleotides) that do not encode polypeptides are also included. Recombinant vectors are also included, including, for example, expression vectors, viral vectors, plasmids, cosmids, phagemids, phages, viruses, etc.

[0140] Additional coding or non-coding sequences may, but need not be present in the polynucleotides described herein, and polynucleotides may, but need not be connected 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, such as expression control sequences.

[0141] As used herein, the term "isolated" polypeptide or protein means that the subject protein (1) is free of at least some other proteins with which it is normally found in nature, (2) is substantially free of other proteins from the same source, such as from the same species, (3) is expressed by cells from a different species, (4) has been separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated with protein moieties that are associated with the "isolated protein" in nature (by covalent or non-covalent interactions), (6) is operably associated with polypeptides that are not associated with it in nature (by covalent or non-covalent interactions), or (7) does not occur in nature. Such an isolated protein may be encoded by genomic DNA, cDNA, mRNA or other RNA, may be of synthetic origin, or any combination thereof. In certain embodiments, an isolated protein is substantially free of proteins or polypeptides or other contaminants that are present in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or other uses).

[0142] In certain embodiments, the "purity" of any given agent (e.g., an activatable proprotein) in a composition can be defined. For example, certain compositions may include agents, such as polypeptide agents, having a purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (including all decimals and ranges therebetween) on a protein basis or on a weight-weight basis, including by, for example and in no way limited to, high performance liquid chromatography (HPLC), which is a well-known form of column chromatography frequently used in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

[0143] 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 sequences described by name and sequences described in tables and sequence listings.

[0144] Certain embodiments include biologically active "variants" and "fragments" of the proteins / polypeptides described herein, and polynucleotides encoding them. A "variant" comprises one or more substitutions, additions, deletions and / or insertions relative to a reference polypeptide or polynucleotide (see, e.g., Tables and Sequence Listings). A variant polypeptide or polynucleotide comprises an amino acid or nucleotide sequence having 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 or similarity or homology to a reference sequence described herein, and substantially retains the activity of the reference sequence. Also included are additions, deletions, insertions or substitutions of 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 acids or nucleotides, or differ from the reference sequence in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 110, 120, 130, 140, 150 or more amino acids or nucleotides. In some embodiments, the invention relates to a sequence that has an addition, deletion, insertion or substitution of 1, 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 acids or nucleotides and substantially retains at least one activity of the reference sequence. In certain embodiments, the addition or deletion comprises a C-terminal and / or N-terminal addition and / or deletion.

[0145] As used herein, the term "sequence identity" or, for example, "a sequence that is 50% identical to ..." refers to the degree to which a sequence is identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis in a comparison window. Therefore, the "percentage of sequence identity" can be calculated by the following process: comparing two optimally aligned sequences within a comparison window, determining the number of positions at which the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) appear in both sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. The best alignment for comparing the sequence of the comparison window can be realized by computerized algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by observation and the best alignment (that is, producing the highest percentage homology in the comparison window) produced by any of the various methods selected. Also reference can be made to, for example, the BLAST series of programs disclosed in Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[0146] The term "solubility" refers to the property that an agent (e.g., activatable preprotein) provided herein is dissolved in a liquid solvent and forms a uniform solution. Solubility is usually expressed as a concentration, or as the mass of a solute per unit volume of solvent (grams of solute per kilogram of solvent, grams per deciliter (100 mL)), mg / ml, etc.), molar concentration, weight molar concentration, molar fraction, or other similar concentration descriptions. The maximum equilibrium amount of a solute soluble per unit amount of solvent is the solubility of the solute in the solvent under specified conditions (including the properties of temperature, pressure, pH, and solvent). In certain embodiments, at physiological pH or other pH, for example, at 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) solubility is measured. In certain embodiments, solubility is measured in water or physiological buffers such as PBS or NaCl (with or without NaPO4). In specific embodiments, solubility is measured at relatively low pH (e.g., pH 6.0) and relatively high salt (e.g., 500 mM NaCl and 10 mM NaPO4). 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°C, 21°C, 22°C, 23°C, 24°C, 25°C) or about body temperature (37°C). In certain embodiments, the agent has a solubility of at least about 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 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 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml or 100 mg / ml at room temperature or 37°C.

[0147] A "subject" or "subject in need thereof" or "patient" or "patient in need thereof" includes a mammalian subject, such as a human subject.

[0148] "Substantially" or "substantially" means nearly all or completely, for example, 95%, 96%, 97%, 98%, 99% or more of a given amount.

[0149] "Statistically significant" means that the result could not have occurred by chance. Statistical significance can be determined by any method known in the art. Common measures of significance include p-values, which are the frequency or probability of an observed event occurring if the null hypothesis is true. If the p-value obtained is less than the significance level, the null hypothesis is rejected. In simple cases, the significance level is defined as a p-value of 0.05 or less.

[0150] A "therapeutic response" refers to an improvement in symptoms (whether sustained or not) upon administration of one or more therapeutic agents.

[0151] As used herein, the terms "therapeutically effective amount," "therapeutic dose," "prophylactically effective amount," or "diagnostically effective amount" are the amount of an agent (eg, activatable proprotein, activated protein) required to elicit a desired biological response upon administration.

[0152] As used herein, "treatment" of a subject (e.g., a mammal, such as a human) or a cell is any type of intervention used to attempt to alter the natural course of an individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition, and may be performed prophylactically or after the onset of a pathological event or after contact with a pathogen. Also included are "preventive" treatments that can be used to reduce the rate of progression of the disease or disorder being treated, delay the onset of the disease or disorder, or reduce the severity of its onset. "Treatment" or "prevention" does not necessarily mean the complete eradication, cure, or prevention of a disease or disorder or its associated symptoms.

[0153] The term "wild-type" refers to a gene or gene product (eg, a polypeptide) that is most commonly observed in a population and is thus arbitrarily designated as the "normal" or "wild-type" form of the gene.

[0154] Unless expressly stated otherwise, each embodiment in this specification is applicable to all other embodiments.

[0155] Activatable proprotein homodimer

[0156] Certain embodiments relate to an activatable proprotein homodimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprise a fragment antigen binding (Fab) region that specifically binds to 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 in the N-terminal to C-terminal direction, wherein the hinge / Fc domain of the first polypeptide binds to the hinge / Fc domain of the second polypeptide, wherein the IL-2 protein of the first polypeptide binds to the IL-2Rα protein of the second polypeptide, and wherein the IL-2Rα of the first polypeptide binds to the IL-2 protein of the second polypeptide, wherein the binding masks one or more binding sites of the IL-2 protein, which originally bind to the IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of immune cells in vitro or in vivo, and wherein the second linker is a cleavable linker. In some embodiments, the total length of the stable linker affects the binding and masking.

[0157] As described above, one or more IL-2 proteins interact or bind together with one or more IL-2Rα proteins, for example, through non-covalent interactions or certain covalent bonds (e.g., disulfide bonds). In some cases, the binding of one or more IL-2 proteins to one or more IL-2Rα proteins sterically blocks or hinders the binding of one or more IL-2 proteins to their cognate IL-2Rβ / γc and / or IL-2Rα / β / γc receptor chains expressed on immune cells. Exemplary IL-2 proteins and IL-2Rα proteins are described elsewhere herein.

[0158] In some cases, 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 preprotein 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 chain). However, typically, the hinge / Fc domains of the first and second polypeptides are not bound together or dimerized by peptide or amide bonds. In some embodiments, the hinge / Fc domains are bound together into a homodimer, i.e., a homodimer consisting of two identical or nearly identical hinge / Fc domains. Thus, the hinge / Fc domains of the first and second polypeptides can be identical (or substantially identical) or different (e.g., knob-in-hole). Exemplary hinge / Fc domains are described herein.

[0159] As mentioned above, the second joint comprises a cleavable joint, for example, a joint that can be cleaved by a protease. In some cases, the first joint is a stable (e.g., physiologically stable) joint. In some cases, the protease is expressed in a target tissue or cell (e.g., cancerous tissue or cancer cell). In this case, the cutting of the joint releases the masking part, eliminates the steric hindrance of the IL-2 protein, and allows selective activation of the IL-2 protein in diseased tissues or cells (e.g., TME) relative to normal or healthy tissues or cells. This selectivity and local activation not only reduce the unnecessary consumption of the IL-2 administered, thereby extending its half-life, but also enhances tissue permeability and reduces the unwanted systemic effects of IL-2, and other advantages. Exemplary joints are described herein.

[0160] In some embodiments, homodimeric binding between the first polypeptide and the second polypeptide allosterically inhibits the binding of the IL-2 protein to its target (e.g., the cognate IL-2Rβ / γc and / or IL-2Rα / β / γc receptor chain on the surface of an immune cell). In these and related embodiments, the IL-2 portion of the activatable proprotein exhibits no binding to its target or substantially no binding to its target, or no more than 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50 ... 0% or 50%, optionally for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer, optionally as measured in vivo or in an in vitro target displacement assay available in the art.

[0161] In some cases, anti-PD-1 or anti-PD-L1 Fab not only improves the targeting of the activatable IL-2 proprotein (or procytokine) module to the TME, but also provides enhanced anti-cancer / immunostimulatory activity in addition to the activity of the IL-2 protein once the IL-2 protein is activated due to protease cleavage of the second linker.

[0162] In a specific embodiment, the first and second polypeptides of the activatable proprotein homodimer comprise anti-PD-1 Fab (SEQ ID NOs: 3 and 4) or anti-PD-L1 Fab (SEQ ID NOs: 25 and 26), and human IgG1 CH1 domain and CL domain (κ), IgG1 hinge (SEQ ID NO: 42), a modified IgG1 Fc domain with LALA and P329A mutations (see CH2 domain of SEQ ID NO: 57), an IgG1 CH3 domain (see CH3 domain of SEQ ID NO: 58), a stable linker (e.g., a linker of four amino acids in length, such as 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: 104). NO:88 or 90, optionally with D6R and E29K mutations), including active or other functional fragments and variants of the aforementioned sequences, as described herein.

[0163] In a specific embodiment, the first and second polypeptides of the activatable proprotein homodimer comprise SEQ ID NO: 146 (anti-PD-1 heavy chain 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).

[0164] The individual components of exemplary activatable proproteins are described in more detail herein.

[0165] Anti-PD-1 and anti-PD-L1 and anti-B7H3 Fab regions. The activatable preprotein described herein comprises at least one fragment antigen binding (Fab) region that specifically binds to human PD-1 or human PD-L1 or human B7H3. The "Fab" region consists of a constant domain and a variable domain of each of the heavy and light chains of the immunoglobulin molecule, for example, a VL / CL region or domain bound to the VH / CH1 region, or a domain (hinge / Fc domain) optionally fused to the Fc domain by a linker or hinge at its C-terminus. The VL:VH and CL:CH1 regions of Fab are typically combined as covalent heterodimers. In certain embodiments, the CL domain is a κ chain. In some embodiments, the CL domain is a λ chain. In some embodiments, the CH1 domain is an IgA, IgD, IgE, IgG, IgM domain, such as an IgA1, IgA2, IgG1, IgG2, IgG2, IgG3 or IgG4 CH1 domain. In a specific embodiment, the CL domain is a κ chain and the CH1 domain is an IgG1 domain.

[0166] In certain embodiments, the antibody or Fab region described herein includes a heavy chain and a light chain CDR group inserted between a heavy chain and a light chain framework region (FR) group, respectively, and the heavy chain and light chain framework region groups provide support for CDR and define the spatial relationship of CDR relative to each other. As used herein, the term "CDR group" refers to three hypervariable regions in a heavy chain or light chain V region. Starting from the N-terminal of a heavy chain or light chain, these regions are referred to as "CDR1", "CDR2" and "CDR3", respectively. Therefore, an antigen binding site includes six CDRs, which include a CDR group (VH CDR1, VH CDR2, VH CDR3, VLCDR1, VLCDR2, VLCDR3) from each of the heavy chain 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 many antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contacts with the bound antigen, wherein the most extensive antigen contact is contact with the heavy chain CDR3. Therefore, the molecular recognition unit is mainly responsible for the specificity of the antigen binding site.

[0167] As used herein, the term "FR group" refers to the four flanking amino acid sequences of the CDRs surrounding the CDR group of the heavy or light chain V region. Some FR residues may contact the bound antigen; however, FR is primarily responsible for folding the V region into the antigen binding site, particularly the FR residues directly adjacent to the CDR. In FR, certain amino acid residues and certain structural features are very conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region is folded into a binding site, the CDR appears as a protruding loop motif, forming an antigen binding surface. It is generally believed that there are conserved structural regions of FR that affect the shape of the CDR loop folding into certain "canonical" structures - regardless of the precise CDR amino acid sequence. In addition, it is known that certain FR residues are involved in non-covalent domain contacts that stabilize the interaction between the heavy and light chains of antibodies.

[0168] In certain embodiments, the Fab region is humanized. These embodiments relate to chimeric molecules that are usually prepared using recombinant technology, which have an antigen binding site of an immunoglobulin derived from a non-human species and a remaining immunoglobulin structure of a molecule based on the structure and / or sequence of a human immunoglobulin. The antigen binding site may comprise a complete variable domain fused to a constant domain, or may comprise only a CDR transplanted to an appropriate framework region in a variable domain. The epitope binding site may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in a human individual, but the possibility of an immune response to an external variable region still exists (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). Illustrative methods for antibody humanization include methods described in U.S. Pat. No. 7,462,697.

[0169] Another approach focuses not only on providing constant regions of human origin, but also on modifying variable regions to make them as close to human form as possible. As is well known, the variable regions of both heavy and light chains contain three complementary determining regions (CDRs), which vary according to the epitope and determine the binding capacity, flanked by four framework regions (FRs), which are relatively conserved in a given species and are considered to be the scaffolds of the CDRs. When non-human antibodies are prepared for specific epitopes, the variable regions can be "reshaped" or "humanized" by transplanting CDRs derived from non-human antibodies to FRs present in the human antibody to be modified. Application of this approach to various antibodies has been described 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., JImmunol.148:1149-1154,1992 reports. In some embodiments, humanized antibodies or Fab regions retain all CDR sequences (e.g., humanized mouse antibodies or Fabs, which contain all six CDRs from mouse antibodies). In certain embodiments, humanized antibodies or Fab regions have one or more CDRs (one, two, three, four, five, six) that are changed relative to the original antibody, also referred to as one or more CDRs "derived from" the original antibody's one or more CDRs.

[0170] 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 specifically binds to a target molecule, such as a PD-1 or PD-L1 protein or an epitope or complex thereof, with an equilibrium dissociation constant of about ≤10 -7 M or about 10 -8 M or ≤10 -7 M to about 10 -8In some embodiments, the equilibrium dissociation constant is about ≤10 -9 M or about ≤10 - 10 M or about ≤10 -9 M to about ≤10 -10 In certain illustrative embodiments, the affinity (Kd or EC) of the antibody or antigen-binding fragment thereof for the PD-1 or PD-L1 protein to which it specifically binds is 50 ) is about, at least about, or less than about 0.01 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, 40 nM, or 50 nM.

[0171] If a molecule such as an antibody or Fab region reacts or associates with a specific cell, substance or specific epitope more frequently, more rapidly, for a longer duration and / or with greater affinity than its reaction or association with an alternative cell or substance or epitope, the molecule is said to exhibit "specific binding" or "preferential binding". If the affinity, avidity, ease and / or duration of the antibody binding to the target molecule or epitope exceeds the affinity, avidity, ease and / or duration of its binding to other substances or epitopes, for example, by a statistically significant amount, the antibody "specifically binds" or "preferentially binds" to the target molecule or epitope. Typically, one member of a pair of molecules that exhibit specific binding has an area or cavity on its surface that specifically binds to and is therefore complementary to a specific spatial and / or polar organization of another member of the pair of molecules. Therefore, the pair of members has the characteristic of specific binding to each other. For example, an antibody that specifically or preferentially binds to a specific epitope is an antibody that binds to the specific epitope with greater affinity, avidity, greater ease and / or longer duration than it binds to other epitopes. It is also understood by reading this definition that, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. The term also applies, for example, to situations where an antibody is specific for a particular epitope carried by many antigens, in which case a particular binding member carrying an antigen binding fragment or domain will be able to bind to a variety of antigens carrying that epitope; for example, it may cross-react with many different forms of a target antigen from multiple species that share a common epitope.

[0172] Immunobinding generally refers to, for example (as an illustration and not by way of limitation), the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific 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 intensity or affinity of the immunobinding interaction can be represented by the dissociation constant (Kd) of the interaction, wherein the smaller the Kd, the greater the affinity. The immunobinding properties of the selected polypeptide can be quantified using methods well known in the art. One such method requires measuring the rates of formation and dissociation of the antigen binding site / antigen complex, wherein these rates depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that equally affect the rate in both directions. Therefore, both the "opening rate constant" (Kon) and the "closing rate constant" (Koff) can be determined by calculating the concentration and the actual binding and dissociation rates. The ratio of Koff / Kon enables the elimination of all parameters that are 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 agents and is expressed as Kd or EC 50 The affinity of the antibody for the PD-1 or PD-L1 protein or epitope can be, for example, about 100 nanomolar (nM) to about 0.1 nM, about 100 nM to about 1 picomolar (pM), or about 100 nM to about 1 femtomolar (fM). As used herein, the term "affinity" refers to the resistance of a complex of two or more agents to dissociation upon dilution.

[0173] Programmed cell death protein 1 (PD-1; CD279 (cluster of differentiation 279)) refers to a protein expressed on the surface of cells that regulates immune responses to human cells, for example by downregulating the immune system and promoting self-tolerance by inhibiting 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 of regulatory T cells (anti-inflammatory, suppressive T cells). Therefore, in certain embodiments, the Fab region specifically binds to the human PD-1 protein sequence described in Uniprot: Q15116. Anti-PD-1 antibodies are known in the art (see, e.g., U.S. Pat. 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. App. Nos. 2012 / 0039906, 2015 / 0203579). For example, in a specific embodiment, the Fab region is from an anti-PD-1 antibody selected from nivolumab, pembrolizumab, cemiplizumab, JTX-4014, spartalizumab, carrelizumab, sintilimab, tislelizumab, toripalizumab, dotalimumab, MGA012, AMP-22 and AMP-514.

[0174] Programmed death ligand 1 (PD-L1) is a 40kDa type 1 transmembrane protein that binds to its receptor PD-1, which is expressed on activated T cells, B cells, and myeloid cells to regulate activation or inhibition (see Uniprot: Q9NZQ7). In certain embodiments, the Fab region specifically binds to the human PD-L1 protein sequence described in Uniprot: Q9NZQ7. Anti-PD-L1 antibodies are known in the art (see, e.g., U.S. Patents Nos. 9,102,725, 9,393,301, 9,402,899, 9,439,962). For example, in a specific embodiment, the Fab region is from an anti-PD-L1 antibody selected from atezolizumab, avelumab, and durvalumab.

[0175] In certain embodiments, the anti-PD-1 or anti-PD-L1 Fab region is characterized by or comprises a heavy chain variable (VH) region sequence and a light chain variable (VL) region sequence, wherein the heavy chain variable region sequence comprises a complementary determining region VHCDR1, VHCDR2 and VHCDR3 sequence, and the light chain variable region sequence comprises a complementary determining region VLCDR1, VLCDR2 and VLCDR3 sequence. Exemplary VH, VHCDR1, VHCDR2, VHCDR3, VL, VLCDR1, VLCDR2 and VLCDR3 sequences are provided in Tables P1 and P2 below.

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183] Therefore, in certain embodiments, its anti-PD-1Fab region comprises a VH region containing VHCDR1, VHCDR2 and VHCDR3 regions (underlined) from Table P1 and a corresponding VL region containing VLCDR1, VLCDR2 and VLCDR3 regions (underlined) from Table P1. Also included are variants thereof that bind to human PD-1, for example, variants having a total of 1, 2, 3, 4, 5 or 6 changes in the combined CDR regions (e.g., VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and / or VLCDR3 sequences described herein). Exemplary "changes" include amino acid substitutions, additions and deletions. In certain embodiments, the anti-PD-1Fab 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 that is 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 that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a corresponding sequence selected from Table P1. Variants that bind to human PD-1 are also included, for example, variants with 1, 2, 3, 4, 5, 6 changes in one or more framework regions. Exemplary "changes" include amino acid substitutions, additions and deletions.

[0184] In certain embodiments, its anti-PD-L1 Fab region comprises a VH region containing VHCDR1, VHCDR2 and VHCDR3 regions (underlined) from Table P2 and a VL region containing VLCDR1, VLCDR2 and VLCDR3 regions (underlined) from Table P2. Also included are variants thereof that bind to human PD-L1, for example, variants having a total of 1, 2, 3, 4, 5 or 6 changes in the combined CDR regions (e.g., VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and / or VLCDR3 sequences described herein). Exemplary "changes" include amino acid substitutions, additions and deletions. In certain embodiments, the anti-PD-L1 Fab region comprises a VH region from Table P2 and a corresponding VL region from Table P2. In certain embodiments, the VH region comprises an amino acid sequence that is 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 that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a corresponding sequence selected from Table P2. Also included are variants thereof that bind to human PD-L1, for example, variants having 1, 2, 3, 4, 5, 6 changes in one or more framework regions. Exemplary "changes" include amino acid substitutions, additions and deletions.

[0185] 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 cell-mediated cytolysis and potential other anti-tumor immune responses. In specific embodiments, B7H3 is human B7H3 or a domain thereof. In certain embodiments, the anti-B7H3 Fab region specifically binds to a human B7H3 protein, for example, a human B7H3 domain selected from one or more of an Ig-like V-1 type domain, an Ig-like C2-1 type domain, an Ig-like V-2 type domain, and an Ig-like C2-2 type domain. In specific embodiments, the Fab region has a K of about 0.4nM or 0.5nM (400 or 500pM) or less. D Specifically binds human BH73.

[0186] In certain embodiments, the anti-B7H3 Fab region is characterized by or comprises a VH sequence and a VL sequence, wherein the VH sequence contains a complementary determining region VHCDR1, VHCDR2 and VHCDR3 sequence, and the VL sequence contains a complementary determining region VLCDR1, VLCDR2 and VLCDR3 sequence. Exemplary VH, VHCDR1, VHCDR2, VHCDR3, VL, VLCDR1, VLCDR2 and VLCDR3 sequences are provided in Table P3 below.

[0187]

[0188]

[0189] Therefore, in certain embodiments, the anti-B7H3 Fab region thereof comprises a VH region containing VHCDR1, VHCDR2 and VHCDR3 regions (underlined) from Table P3 and a corresponding VL region containing VLCDR1, VLCDR2 and VLCDR3 regions (underlined) from Table P3. Also included are variants thereof that bind to human B7H3, for example, variants having a total of 1, 2, 3, 4, 5 or 6 changes in the combined CDR regions (e.g., VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and / or VLCDR3 sequences described herein). Exemplary "changes" 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 that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from Table P3, and the VL region comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a corresponding sequence selected from Table P3. Variants that bind to human B7H3 are also included, for example, variants with 1, 2, 3, 4, 5, 6 changes in one or more framework regions. Exemplary "changes" include amino acid substitutions, additions and deletions.

[0190] Antibodies or Fab regions can be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Monoclonal antibodies specific for a polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and modifications thereof. Also included are methods of expressing human antibodies or Fab regions using transgenic animals such as mice. See, e.g., 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 regions can be determined by reference to Kabat, EA et al., Sequences of Proteins of Immunological Interest. 4th ed., US Department of Health and Human Services. 1987 and updates thereof.

[0191] It should be understood that any one or more of the aforementioned anti-PD-1 Fab or anti-PD-L1 Fab or anti-B7H3 Fab can be combined with any additional components described herein (e.g., CH1 domain, hinge / Fc domain, IL-2 protein, IL-2Rα protein, and linkers described herein) to produce one or more activatable proproteins.

[0192] Hinge / Fc domain.Some activatable preprotein homodimers include hinge / Fc domains. The hinge region (present in IgG, IgA and IgD) acts as a flexible spacer, allowing the Fab portion to move freely in space relative to the Fc domain. In contrast to the constant region, the hinge region is structurally diverse, and the sequence and length are variable between immunoglobulin classes and subclasses. The hinge region may also include one or more glycosylation sites, which include many structurally different types of sites for carbohydrate attachment. For example, IgA1 contains 5 glycosylation sites within the 17 amino acid segments of the hinge region, giving the hinge region polypeptide significant resistance to intestinal proteases. The residues in the hinge proximal region of the CH2 domain may also affect the specificity of the interaction between an immunoglobulin and one or more corresponding Fc receptors thereof (see, for example, Shin et al., Intern. Rev. Immunol. 10: 177-186, 1993).

[0193] The term "Fc domain" or "Fc fragment" or "Fc" refers to a protein containing one or more of the CH2 domain, CH3 domain and / or CH4 domain (including fragments and variants and combinations thereof) from one or more selected immunoglobulins. An "Fc domain" may also include one or more hinge regions of an immunoglobulin heavy chain constant region. In certain embodiments, the Fc domain does not include one or more of the CH1, CL, VL and / or VH regions of an immunoglobulin.

[0194] The Fc domain may be derived from any one or more immunoglobulin classes (including but not limited to IgA, IgD, IgE, IgG, IgM, including subclasses and combinations thereof) CH2 domain, CH3 domain, CH4 domain and / or one or more hinge regions. In some embodiments, the Fc domain is derived from IgA immunoglobulin, including subclasses IgA1 and / or IgA2. In certain embodiments, the Fc domain is derived from IgD immunoglobulin. In specific embodiments, the Fc domain is derived from IgE immunoglobulin. In some embodiments, the Fc domain is derived from IgG immunoglobulin, including subclasses IgG1, IgG2, IgG2, IgG3 and / or IgG4. In certain embodiments, the Fc domain is derived from IgM immunoglobulin. Exemplary hinge and Fc domain sequences are provided in Table F1 below.

[0195]

[0196]

[0197]

[0198] Thus, in some embodiments, the hinge comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical 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 an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from Table F1 (e.g., an IgA1 CH2, CH3, or a combined CH2CH3 sequence, an IgA2 CH2, CH3, or a combined CH2CH3 sequence, an IgD CH2, CH3, or a combined CH2CH3 sequence, an IgE CH2, CH3, CH4, or a combined CH2CH3 or CH2CH3CH4 sequence, an IgG1 CH2, CH3, or a combined CH2CH3 sequence, an IgG2 CH2, CH3, or a combined CH2CH3 sequence, an IgG3 CH2, CH3, or a combined CH2CH3 sequence, an IgG4 CH2, CH3, or a combined CH2CH3 sequence, or an IgM CH2, CH3, CH4, or a combined CH2CH3 or CH2CH3CH4 sequence from Table F1). In certain embodiments, the hinge is of the same Ig class as the Fc domain.

[0199] In certain embodiments, the Fc domain is a modified Fc domain. Such modifications can be used to change (e.g., increase, decrease) the binding properties of the Fc region to one or more specific FcRs (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, FcRn) relative to the corresponding wild-type Fc sequence, its pharmacokinetic properties (e.g., stability or half-life, bioavailability, tissue distribution, distribution volume, concentration, elimination rate constant, elimination rate, area under the curve (AUC), clearance, C max ,t max , C min , fluctuations), its immunogenicity, its complement fixation or activation and / or CDC / ADCC / ADCP-related activities of the Fc region, and other properties described herein.

[0200] In some embodiments, the modified Fc domain does not bind or substantially does not 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. The expression of FcγRI is upregulated by GM-CSF and γ-interferon (γ-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. The expression of FcγRIIa is upregulated by GM-CSF and γ-IFN and downregulated by IL-4. FcγIIb is expressed on B cells, PMNs, macrophages, and mast cells. FcγIIb inhibits responses mediated by immunoreceptor tyrosine-based activation motifs (ITAMs) and is therefore an inhibitory receptor. The expression of FcγRIIc is upregulated by intravenous immunoglobulin (IVIG) and IL-4 and downregulated by γ-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. The expression of FcγRIII is upregulated by C5a, TGF-β, and γ-IFN and downregulated by IL-4. FcγRIIIb is a GPI-linked receptor expressed on PMNs.

[0201] In a specific embodiment, the modified Fc domain comprises L234A / L235A ("LALA") mutations and / or P329A or P329G mutations (EU numbering) (see, e.g., CH2 domain 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.

[0202] It should be understood that any one or more of the aforementioned hinge and Fc domains can be combined with any additional components described herein (e.g., anti-PD-1 Fab, anti-PD-L1 Fab, IL-2 protein, IL-2Rα protein, and linkers described herein) to produce one or more activatable proproteins.

[0203] IL-2 protein. The activatable proproteins described herein comprise at least one "IL-2 protein" (or interleukin-2 protein), including human IL-2 protein. IL-2 is a cytokine signal transmitted through the IL-2 receptor (IL-2R), a complex consisting of three chains called α (CD25), β (CD122), and γc (CD132) chains. IL-2 is produced by T cells in response to antigens or mitogenic stimuli and is required for T cell proliferation and other activities that are critical for the regulation of immune responses. IL-2 can stimulate B cells, monocytes, lymphokine-activated killer cells, natural killer cells, and glioma cells, as well as other immune cells.

[0204] IL-2 is a 15-16 kDA protein consisting of a signal peptide (residues 1-20) and an active mature protein (residues 21-153). Table S1 below provides an exemplary human IL-2 amino acid sequence.

[0205]

[0206]

[0207]

[0208]

[0209] Thus, in certain embodiments, the IL-2 protein comprises, consists of, or consists essentially of the following amino acid sequence: an amino acid sequence selected from Table S1, or an active variant or fragment thereof having at least 80%, 85%, 90%, 95%, 98% or 100% identity with a sequence selected from Table S1. In some embodiments, an "active" IL-2 protein or fragment or variant is characterized by, for example, its ability to bind to IL-2Rβ / γc and / or IL-2Rα / β / γc receptor chains present on the surface of immune cells in vitro or in vivo and stimulate downstream signaling activity, without the steric hindrance of the masking moiety described herein. Examples of downstream signaling activity include IL-2-mediated signaling via one or more of the JAK-STAT, PI3K / Akt / mTOR, and MAPK / ERK pathways (including combinations thereof). In summary, IL-2 signals stimulate a series of downstream pathways, leading to responses that play an important role in the development, function, and survival of CD4 T cells, CD8 T cells, NK cells, NKT cells, macrophages, and intestinal intraepithelial lymphocytes, among others.

[0210] In certain embodiments, the IL-2 protein is a mature form of IL-2, or an active variant or fragment thereof, comprising, consisting of, or consisting essentially of an amino acid sequence that is 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), wherein X is any amino acid. In specific embodiments, the IL-2 protein comprises a C145S substitution as defined by SEQ ID NO: 68.

[0211] Certain IL-2 proteins comprise, consist of, or consist essentially of an amino acid sequence that is 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 defined therein.

[0212] Relative to the exemplary amino acid sequences in Table S1, certain IL-2 proteins comprise one or more defined amino acid substitutions. For example, some IL-2 proteins comprise 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 comprise one or more amino acid substitutions at positions 69, 74, and / or 28 as defined by SEQ ID NO: 69, including combinations thereof, and including, for example, wherein the one or more amino acid substitutions are selected from V69A, Q74P, and I128T as defined by SEQ ID NO: 69. Some IL-2 proteins at positions 69, 74, and / or 28 as defined by SEQ ID NO: 69, including combinations thereof, and including, for example, wherein the one or more amino acid substitutions are selected from V69A, Q74P, and I128T as defined by SEQ ID NO: 69. NO:69 defines positions R38, F42, K43, Y45, E62, E68 and / or L72 comprising one or more amino acid substitutions, including combinations thereof, and including, for example, wherein the one or more amino acid substitutions are selected from 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; E62A and E62L; E68A and E68V; and L72A, L72G, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K, including combinations thereof. Specific examples include where the IL-2 protein comprises an amino acid substitution 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 comprise an amino acid substitution or a combination of amino acid substitutions at T3 and / or E61 as defined by SEQ ID NO: 69, e.g., T3A and / or E61S. Certain IL-2 proteins (eg, mature IL-12 or residues 26-153) comprise a combination of R38D and K43E (see, e.g., SEQ ID NO: 85). Thus, the IL-2 protein may comprise any one or more of the aforementioned amino acid substitutions, including combinations thereof.

[0213] It should be understood that any one or more of the aforementioned IL-2 proteins can be combined with any additional components described herein (e.g., anti-PD-1 Fab, anti-PD-L1 Fab, hinge / Fc domain, IL-2Rα protein, and linkers described herein) to produce one or more activatable proproteins.

[0214] IL-2Rα proteins The activatable proprotein described herein comprises at least one "IL-2Rα protein" (or interleukin-2 receptor-α protein), including human IL-2Rα protein. The IL-2 receptor is composed of the following three subunits: IL-2Rα, CD122, and CD132. IL-2Rα specifically binds to IL-2 with very high affinity and is able to bind to IL-2 independently of other subunits. Exemplary IL-2Rα protein sequences are provided in Table S2.

[0215]

[0216]

[0217] 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 has at least 80%, 85%, 90%, 95%, 98% or 100% identity to a sequence selected from Table S2 and binds to an 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% of amino acids 22-187 or 22-240 of SEQ ID NO: 86 (full-length wild-type human IL-2Rα).

[0218] Relative to the exemplary amino acid sequences in Table S2, certain IL-2Rα proteins comprise one or more defined amino acid substitutions. For example, in some cases, the IL-2Rα protein comprises one or more amino acid substitutions at D6 and / or E29, for example, D6R and / or E29K as defined by SEQ ID NO:87 or 88. In some cases, the IL-2Rα protein comprises one or more cysteine ​​substitutions selected from D4C, D6C, N27C, K38C, S39C, L42C, Y43C, I118C, and H120C as defined by SEQ ID NO:88 (human IL-2RαSushi 1 to Sushi 2 domain). In some cases, the IL-2Rα protein comprises alanine substitutions at positions 49 and / or 68 as defined by SEQ ID NO:88. In some embodiments, the IL-2Rα protein comprises a K38S substitution as defined by SEQ ID NO:88. Thus, the IL-2Rα protein may comprise any one or more of the aforementioned amino acid substitutions (including combinations thereof). In a specific embodiment, 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% of the amino acid sequence of SEQ ID NO: 90, including wherein the IL-2Rα protein retains the D6R and / or E29K substitutions.

[0219] 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 aforementioned cysteines and one or more cysteines in the IL-2 protein. In a specific embodiment, IL-2Rα forms at least one disulfide bond with the IL-2 protein between one or more cysteine ​​pairs 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 a specific embodiment, as described above, the binding (e.g., disulfide bond binding) between the IL-2 protein and the IL-2Rα protein masks or sterically hinders the preferential binding of the IL-2 protein to the T42C. reg Binding sites for the IL-2Rα / β / γc chains expressed on the mitochondria.

[0220] It should be understood that any one or more of the aforementioned IL-2Rα proteins can be combined with any other components described herein (e.g., anti-PD-1 Fab, anti-PD-L1 Fab, hinge / Fc domain, IL-2 protein, and linkers described herein) to produce one or more activatable proproteins.

[0221] As described 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, e.g., a cleavable linker comprising a protease cleavage site.

[0222] In some embodiments, the first linker and / or the second linker is about 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 1-4, 1-3 amino acids in length, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 amino acids in length. In some 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. In some embodiments, both linkers are cleavable linkers.

[0223] In some embodiments, the cleavable linker comprises at least one protease cleavage site. Suitable protease cleavage sites and self-cleaving peptides are known to the skilled person (see, for example, Ryan et al., J. Gener. Virol. 78: 699-722, 1997; and Scymczak et al., Gener. Nature Biotech. 5: 589-594, 2004). In some embodiments, the protease cleavage site can be cleaved by one or more proteases selected from metalloproteases, serine proteases, cysteine ​​proteases, and aspartic proteases. In a specific embodiment, the protease cleavage site can be cleaved by one or more proteases selected from MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, TEV protease, matriptase, uPA, FAP, legumin, PSA, kallikrein, cathepsin A, and cathepsin B.

[0224] Exemplary cleavable linker sequences are provided in Table S3.

[0225]

[0226]

[0227]

[0228] Thus, in certain embodiments, the cleavable linker is selected from Table S3. Other 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).

[0229] Cleavable linkers also include amino acid sequences that can be cleaved by matrix metalloproteinases such as collagenase, stromelysin and gelatinase. Illustrative examples of amino acid sequences that can be cleaved by matrix metalloproteinases 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), wherein 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 an amino acid sequence cleavable by stromelysin is -Pro-Tyr-Ala-Tyr-Tyr-Met-Arg- (SEQ ID NO: 180); an example of an amino acid sequence cleavable by gelatinase is -Pro-Leu-Gly-Met-Tyr-Ser-Arg- (SEQ ID NO: 181).

[0230] 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.

[0231] In certain embodiments, the cleavable linker has a half-life of about 30 minutes 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 at pH 7.4, 25°C, e.g., at physiological pH, human body temperature (e.g., in vivo, in serum, in a given tissue), or any intervening half-life.

[0232] Typically, at least one of the first or second linkers is a non-cleavable linker. Exemplary non-cleavable linkers include Maratea et al., Gene 40:39-46, 1985; Murphy et al., PNAS USA.83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. Specific non-cleavable linker sequences contain Gly, Ser and / or Asn residues. If desired, other nearly neutral amino acids, such as Thr and Ala, may also be used in the peptide linker sequence.

[0233] Certain exemplary non-cleavable linkers include the following linkers containing Gly, Ser and / or Asn: [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.

[0234] Other examples of non-cleavable linkers include the following amino acid sequence: Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SE Q ID NO: 194); Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 195); Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 196);

[0235] Asp-Ala-Ala-Ala-Lys-Glu-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).

[0236] Other 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); KESGSVSSEQLAQFRSLD (SEQ ID NO: 204) (Bird et al., Science. 242: 423-426, 1988), GGRRGGGS (SEQ ID NO: 205); LRQRDGERP (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 comprising three glycine residues. In certain embodiments, flexible linkers can be rationally designed using computer programs capable of modeling DNA binding sites and the peptides themselves (Desjarlais & Berg, PNAS. 90:2256-2260, 1993; and PNAS. 91:11099-11103, 1994) or by phage display methods.

[0237] In some embodiments, the linker comprises a spacer element and a cleavable element to make the cleavage element more accessible to the enzyme responsible for cleavage.

[0238] It should be understood that any one or more of the aforementioned linkers can 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 pre-protein homodimer.

[0239] Exemplary activatable proprotein homodimers are provided in Table S4.

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273] Thus, in certain embodiments, the activatable proprotein comprises a first and a second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 100% identical to a sequence selected from Table S4 (i.e., chain 1 and chain 2), and a VL / C1 region polypeptide that is at least 80%, 85%, 90%, 95%, 98% or 100% identical to the corresponding sequence from Table S4 (i.e., chain 3 and chain 4).

[0274] In certain additional embodiments, the first polypeptide is different from the second polypeptide and forms a heterodimeric preprotein. For example, in some embodiments, the Fc domain from the first polypeptide and the Fc domain from the second polypeptide are modified, for example using a knob-hole structure mutation, 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 binding pair or a 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 binding pair or a preferential binding pair. In certain embodiments of these and related embodiments, the modification of the IL-2 and IL-2Rα proteins inhibits the formation of homodimers between (i) the IL-2 / IL-2Rα of the first polypeptide and (ii) the IL-2 / IL-2Rα protein of the second polypeptide.

[0275] Methods of use and pharmaceutical compositions

[0276] Certain embodiments include methods of treating, ameliorating symptoms of, and / or slowing progression of a disease or disorder in a subject in need thereof, comprising administering to the subject at least one activatable proprotein as described herein. Also included are methods of enhancing the immune response of a subject, comprising administering to the subject at least one activatable proprotein as described herein. In specific embodiments, the disease is cancer. In some embodiments, the cancer expresses or overexpresses PD-L1.

[0277] In some embodiments, after administration, the activatable proprotein is activated by protease cleavage in cells or tissues, which exposes a binding site for the IL-2 protein, which 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 producing an activated protein. In a specific embodiment, 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 immune cells. In a specific embodiment, the immune cells are selected from one or more of T cells, B cells, natural killer cells, monocytes, and macrophages.

[0278] In some embodiments, administration and activation of the activatable proprotein to produce an activated protein enhances the subject's anti-cancer immune response 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 relative to a control. In some embodiments, administration and activation of the activatable proprotein to produce the 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 relative to a control.

[0279] In certain embodiments, relative to the corresponding anti-PD-1Fab (or corresponding anti-PD-1 antibody) and / or the corresponding IL-2 protein alone, the activated anti-PD-1Fab / IL-2 protein stimulates an enhanced (e.g., synergistically enhanced) anti-cancer immune response, for example, relative to each individual component, the anti-cancer immune response is enhanced to 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times or more. In certain embodiments, relative to the corresponding anti-PD-1Fab (or corresponding anti-PD-1 antibody) and / or the corresponding IL-2 protein alone, the activated anti-PD-1Fab / IL-2 protein stimulates an enhanced (e.g., synergistically enhanced) cancer cell killing activity, for example, relative to each individual component, the cancer cell killing activity is enhanced to 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times or more.

[0280] In certain embodiments, the activated anti-PD-L1 Fab / IL-2 protein stimulates an enhanced (e.g., synergistically enhanced) anti-cancer immune response relative to the corresponding anti-PD-L1 Fab (or corresponding anti-PD-L1 antibody) alone and / or the corresponding IL-2 protein alone, for example, an enhancement of 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 more in terms of anti-cancer immune response relative to each individual component. In certain embodiments, the activated anti-PD-L1 Fab / IL-2 protein stimulates an enhanced (e.g., synergistically enhanced) cancer cell killing activity relative to the corresponding anti-PD-L1 Fab (or corresponding anti-PD-L1 antibody) alone and / or the corresponding IL-2 protein alone, for example, an enhancement of 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 more in terms of cancer cell killing activity relative to each individual component.

[0281] In some embodiments, the disease is cancer, that is, the subject in need has or is suspected of having cancer. Therefore, certain embodiments include methods of treating, ameliorating symptoms of cancer in a subject in need, or inhibiting its progression, comprising administering to the subject at least one activatable proprotein, as described herein. In specific embodiments, the cancer is a primary cancer or a metastatic cancer. In specific embodiments, the cancer is selected from one or more of the following cancers: 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, liver cancer (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.

[0282] In some embodiments, as described above, the cancer is a metastatic cancer. In addition to the above cancers, exemplary metastatic cancers include, but are not limited to, 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 glands, bones, brain, liver, and / or lungs; lung cancer that has metastasized to the adrenal glands, bones, 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 glands, bones, liver, and / or lungs; gastric cancer that has metastasized to the liver, lung, and / or peritoneum; thyroid cancer that has metastasized to the bone, liver, and / or lungs; and uterine cancer that has metastasized to the bone, liver, lung, peritoneum, and / or vagina; and the like.

[0283] In certain embodiments, as described herein, cancer expresses or overexpresses PD-L1. The PD-L1 expression level in a tissue (e.g., cancer tissue) sample can be determined by 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 of human AR protein or gene, and other determinations. PD-L1 mRNA levels can be measured, for example, by RT-PCR, such as quantitative competitive (QC) RT-PCR and other techniques known in the art. Therefore, some embodiments include a step of determining or detecting or measuring the PD-L1 level in a tissue sample from a subject in need. Also included is a step of comparing the PD-L1 level in a tissue sample with a control or reference level. Some embodiments include a step of determining the PD-L1 level in a cancer tissue sample (e.g., biopsy tissue) from a subject, and if the cancer tissue from the subject expresses or overexpresses PD-L1, an activatable preprotein homodimer is administered.

[0284] The method for treating cancer can be combined with other treatment modalities. For example, the combined therapy described herein can be applied to the subject before, during or after other therapeutic interventions, and the other therapeutic interventions include symptom care, radiotherapy, surgery, transplantation, hormone therapy, photodynamic therapy, antibiotic therapy or any combination thereof. Symptom care includes the use of corticosteroids to reduce brain edema, headache, cognitive dysfunction and vomiting, and the use of anticonvulsants to reduce epileptic seizures. Radiotherapy includes whole brain irradiation, fractionated radiotherapy and radiosurgery, such as stereotactic radiosurgery, which can be further combined with conventional surgery.

[0285] Therefore, certain embodiments include a combination therapy for treating cancer, including a method for treating a subject in need of a cancer that improves the symptoms of cancer or inhibits cancer progression, comprising administering to the subject at least one activatable proprotein described herein in combination with at least one additional agent (e.g., chemotherapeutic agent, hormone therapy agent, and / or kinase inhibitor). In some embodiments, administration of at least one activatable proprotein enhances the susceptibility of cancer to additional agents (e.g., chemotherapeutic agents, hormone therapy agents, and / or kinase inhibitors) 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.

[0286] Certain combination therapies use one or more chemotherapeutic agents, such as small molecule chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type 1 or type 2), anti-microtubule agents, and the like.

[0287] Examples of alkylating agents include nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, mechlorethamine, 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, mitomycin, and diazocone (AZQ)), cisplatin and its derivatives (e.g., carboplatin and oxaliplatin), and nonclassical alkylating agents (optionally, procarbazine and altretinoin).

[0288] 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);

[0289] Examples of cytotoxic antibiotics include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycin, mitomycin C, mitoxantrone, and dactinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mepalong, and aclarubicin.

[0290] Examples of anti-microtubule agents include taxanes (eg, paclitaxel and docetaxel) and vinca alkaloids (eg, vinblastine, vincristine, vindesine, vinorelbine).

[0291] The skilled artisan will appreciate 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.

[0292] Some combination therapies use at least one hormone therapy agent. General examples of hormone therapy agents include hormone agonists and hormone antagonists. Specific examples of hormone agonists include progesterone (progesterone), corticosteroids (e.g., prednisolone, methylprednisolone, dexamethasone), insulin-like growth factor, VEGF-derived angiogenic factors and lymphangiogenic factors (e.g., 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)-β, androgen, estrogen and somatostatin analogs. 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 analogs thereof. Also included are hormone receptor antagonists, such as selective estrogen receptor modulators (SERMs; eg, tamoxifen, raloxifene, toremifene) and anti-androgens (eg, flutamide, bicalutamide, nilutamide).

[0293] Also included are hormone pathway inhibitors, such as antibodies to hormone receptors. Examples include inhibitors of IGF receptors (e.g., IGF-IR1) such as citumumab, darotoxin, fentoxin, ganituzumab, estuximab, and lobatumumab; inhibitors of vascular endothelial growth factor receptors 1, 2, or 3 (VEGFR1, VEGFR2, or VEGFR3), such as pegol, bevacizumab, elucuumab, ramucirumab; inhibitors of TGF-β receptors R1, R2, and R3, such as fresolimumab and metitumumab; inhibitors of c-Met, such as nasituximab; inhibitors of EGF receptors, such as cetuximab, depatuxizumab, and sutuximab. mafodotin, fotuximab, imatinib, enstar-latuximab, matuzumab, zatuximab, nexitozumab, nimotuzumab, panitumumab, tomozutouximab, and zalutumumab; FGF receptor inhibitors such as aprutumab ixadotin and bematuzumab; and PDGF receptor inhibitors such as olaratumab and toviromab.

[0294] The skilled artisan will appreciate that the various hormone therapeutic 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.

[0295] Certain combination therapies utilize at least one kinase inhibitor, including a tyrosine kinase inhibitor. Examples of kinase inhibitors include, but are not limited to, adavosertib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mulitinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruzotinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemuafenib.

[0296] Those skilled in the art will appreciate that the various kinase inhibitors described herein can be combined with any one or more of the various activatable proproteins described herein and used in accordance with any one or more of the methods or compositions described herein.

[0297] In some embodiments, the methods and pharmaceutical compositions described herein extend the median survival time of the subject 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 longer. In certain embodiments, the methods and pharmaceutical compositions described herein extend the median survival time of the subject by 1 year, 2 years, 3 years or longer. In some embodiments, the methods and pharmaceutical compositions described herein extend the progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or longer. In certain embodiments, the methods and pharmaceutical compositions described herein extend the progression-free survival by 1 year, 2 years, 3 years or longer.

[0298] In certain embodiments, the methods and therapeutic compositions described herein are sufficient to cause tumor regression, as indicated by a statistically significant decrease in the amount of viable tumor (e.g., a decrease in tumor mass of at least 10%, 20%, 30%, 40%, 50% or more) or by an altered (e.g., a statistically significant decrease) scan size. In certain embodiments, the methods and therapeutic compositions described herein are sufficient to cause stable disease.

[0299] In certain embodiments, the methods and therapeutic compositions described herein are sufficient to result in a clinically relevant reduction in symptoms for a particular disease indication known to the skilled clinician.

[0300] For in vivo use, as described above, the agents described herein are typically incorporated into one or more therapeutic or pharmaceutical compositions (including veterinary therapeutic compositions) prior to administration for the treatment of disease or testing in humans or non-human mammals.

[0301] Therefore, as described herein, some embodiments relate to a medicine or therapeutic composition comprising at least one activatable proprotein. In some cases, the medicine or therapeutic composition comprises one or more activatable proproteins as described herein and a pharmaceutically or physiologically acceptable carrier or excipient. Some medicines or therapeutic compositions also comprise at least one additional agent, for example, a chemotherapeutic agent, a hormone therapy agent and / or a kinase inhibitor as described herein.

[0302] Some therapeutic compositions contain (and some methods utilize) only one activatable proprotein. Some therapeutic compositions contain (and some methods utilize) a mixture of at least two, three, four or five different activatable proproteins.

[0303] In certain embodiments, a pharmaceutical or therapeutic composition comprising at least one activatable proprotein is substantially pure on a protein basis or on a weight-to-weight basis, for example, the composition is at least about 80%, 85%, 90%, 95%, 98% or 99% pure on a protein basis or on a weight-to-weight basis.

[0304] In some embodiments, as known in the art, the activatable preprotein described herein does not form aggregates, has the desired solubility, and / or has an immunogenicity characteristic suitable for people. Therefore, in some embodiments, the therapeutic composition comprising an activatable preprotein is substantially free of aggregation. For example, some compositions include less than about 10% (based on protein) 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 include at least about 50%, about 60%, about 70%, about 80%, about 90% or about 95% of the activatable preprotein that is monodisperse according to its apparent molecular weight.

[0305] In some embodiments, the activatable proprotein is concentrated to about or at least 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 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml or 15 mg / ml and formulated for use as a biotherapeutic.

[0306] To prepare a treatment or pharmaceutical composition, an effective amount or a desired amount of one or more agents is mixed with one or more any pharmaceutical carriers or excipients known to those skilled in the art that are suitable for a particular agent and / or mode of administration. The pharmaceutical carrier can be liquid, semi-liquid or solid. Solutions or suspensions for parenteral, intradermal, subcutaneous or topical administration may include, for example, sterile diluents (such as water), saline solutions (such as phosphate buffered saline; PBS), fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antimicrobial agents (such as benzyl alcohol and methyl parabens); antioxidants (such as ascorbic acid and sodium bisulfite) and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as acetates, citrates and phosphates). If administered intravenously (e.g., by IV infusion), suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickeners and solubilizers (such as glucose, polyethylene glycol, polypropylene glycol and mixtures thereof).

[0307] The agents described herein can be administered in pure form or in the form of suitable treatment or pharmaceutical compositions by any acceptable mode of administration for agents of similar use. Treatment or pharmaceutical compositions can be prepared by combining compositions containing agents with suitable physiologically acceptable carriers, diluents or excipients, and can be formulated into solid, semisolid, 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, but not necessarily, present in the composition.

[0308] Administration can be achieved by a variety of different routes, including oral, parenteral, intranasal, intravenous, intradermal, intramuscular, subcutaneous or topical. The preferred mode of administration depends on the nature of the disease to be treated or prevented. Specific embodiments include administration by IV infusion.

[0309] The carrier may include, for example, a pharmaceutically or physiologically acceptable carrier, excipient or stabilizer that is nontoxic to the cell or mammal contacting them at the dosage and concentration used. Physiologically acceptable carriers are typically pH buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, 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 nonionic surfactants such as polysorbate 20 (TWEEN TM ), polyethylene glycol (PEG) and poloxamer (PLURONICS TM )wait.

[0310] In some embodiments, one or more agents may be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) 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 described in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., ed., (1980). One or more particles or liposomes may also contain other therapeutic or diagnostic agents.

[0311] The exact dosage and duration of treatment are a function of the disease being treated and can be determined empirically using known test protocols or by testing the composition in a model system known in the art and inferring therefrom. Controlled clinical trials can also be conducted. The dosage can also vary with the severity of the disease to be alleviated. Pharmaceutical compositions are usually formulated and administered to exert therapeutically useful effects while minimizing undesirable side effects. The composition can be administered once or divided into multiple smaller doses administered at different time intervals. For any particular subject, the specific dosage regimen can be adjusted over time according to individual needs.

[0312] Therefore, typical routes of administering these and related treatments or pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhaled, parenteral, sublingual, oral, rectal, vaginal and intranasal routes. The term "parenteral" as used herein includes subcutaneous injection, intravenous, intramuscular, intrasternal injection or infusion techniques. The treatment or pharmaceutical composition according to certain embodiments of the present invention is formulated to allow the active ingredients contained therein to be bioavailable when the composition is administered to a subject or patient. The composition to be administered to a subject or patient can take the form of one or more dosage units, wherein, for example, a tablet can be a single dosage unit, and a container of an agent in the form of an aerosol as described herein can accommodate multiple dosage units. The actual method of preparing such dosage forms is known or obvious to those skilled in the art; for example, see Remington: The Science and Practice of Pharmacy, 20th edition (Philadelphia College of Pharmacy and Science, 2000). In order to treat a target disease or illness, the composition to be administered generally comprises a therapeutically effective amount of an agent as described herein.

[0313] The treatment or pharmaceutical composition can be in solid or liquid form. In one embodiment, one or more carriers are particles, so the composition is, for example, in tablet or powder form. One or more carriers can be liquids, and the composition can be, for example, oral oils, injections or aerosols, which can be used, for example, for inhalation administration. When oral administration is intended, the pharmaceutical composition is preferably in solid or liquid form, wherein semi-solid, semi-liquid, suspension and gel forms are included in the forms considered as solid or liquid herein. Certain embodiments include sterile injectable solutions.

[0314] As a solid composition for oral administration, the pharmaceutical composition can be formulated into powders, granules, compressed tablets, pills, capsules, chewing gums, wafers, etc. Such solid compositions usually contain one or more inert diluents or edible carriers. In addition, one or more of the following substances may be present: a binder such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum or gelatin; an excipient such as starch, lactose or dextrin; a disintegrant such as alginic acid, sodium alginate, Primogel, corn starch, etc.; a lubricant such as magnesium stearate or Sterotex; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; a flavoring agent such as mint, methyl salicylate or orange flavoring; and a coloring agent. When the pharmaceutical composition is in the form of a capsule, such as a gelatin capsule, it may contain a liquid carrier such as polyethylene glycol or oil in addition to the above-mentioned types of substances.

[0315] Treatment or pharmaceutical composition can be in liquid form, such as elixir, syrup, solution, emulsion or suspension. As two examples, the liquid can be used for oral administration or by injection. When intended for oral administration, in addition to the compound of the present invention, the preferred composition also contains one or more of sweeteners, preservatives, dyes / colorants and spices. In the composition intended to be administered by injection, one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers and isotonic agents can be included.

[0316] Liquid therapeutic or pharmaceutical compositions, whether solutions, suspensions or other similar forms, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solutions, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic monoglycerides or diglycerides, polyethylene glycol, glycerol, propylene glycol or other solvents that may be used as solvents or suspension media; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or glucose. Parenteral formulations may be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0317] Liquid treatments or pharmaceutical compositions for parenteral administration or oral administration should contain a certain amount of agent so that a suitable dosage can be obtained. Typically, this amount is at least 0.01% of the target agent in the composition. When intended for oral administration, this amount can vary between 0.1% and about 70% of the composition weight. Some oral treatments or pharmaceutical compositions contain about 4% to about 75% of the target agent. In certain embodiments, preparation treatment or pharmaceutical compositions and preparations are made so that before dilution, the parenteral dosage unit contains 0.01% to 10% by weight of the target agent.

[0318] The treatment or pharmaceutical composition can be used for topical application, in which case the carrier can suitably comprise a solution, emulsion, ointment or gel matrix. For example, the matrix can comprise one or more of the following materials: vaseline, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickeners can be present in the treatment or pharmaceutical composition for topical application. If intended for transdermal administration, the composition can include a transdermal patch or an iontophoresis device.

[0319] The therapeutic or pharmaceutical composition can be used for rectal administration, for example, in the form of a suppository, which will 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.

[0320] Treatment or pharmaceutical compositions may include various materials that change the physical form of solid or liquid dosage units. For example, the composition may include a material that forms a coating shell around the active ingredient. The material that forms the coating shell is usually inert and can be selected from, for example, sugar, shellac and other enteric coating agents. Alternatively, the active ingredient can be loaded into a gelatin capsule. Treatment or pharmaceutical compositions in solid or liquid form may include components combined with an agent, thereby contributing to the delivery of the compound. Suitable components that can play this role include monoclonal or polyclonal antibodies, one or more proteins or liposomes.

[0321] Treatment or pharmaceutical composition can be basically composed of dosage units that can be used as aerosol.The term aerosol is used to represent various systems from those systems of colloidal nature to the system composed of pressurized packaging.Delivery can be carried out by liquefying or compressed gas or by a suitable pump system distributing active ingredient.Aerosol can be delivered in a single-phase, two-phase or three-phase system to deliver one or more active ingredients.The delivery of aerosol comprises necessary containers, activators, valves, sub-containers, etc., which can form a medicine box together.Those of ordinary skill in the art can determine preferred aerosols without excessive experiments.

[0322] The compositions described herein can be prepared with carriers such as sustained release formulations or coatings that protect the drug from rapid elimination from the body. Such carriers include controlled release formulations, such as but not limited to implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and other polymers known to those of ordinary skill in the art.

[0323] Therapeutic or pharmaceutical compositions can be prepared by methods well known in the pharmaceutical field. For example, a therapeutic or pharmaceutical composition intended to be administered by injection may contain one or more of a salt, a buffer and / or a stabilizer, together with sterile distilled water to form a solution. Surfactants may be added to promote the formation of a uniform solution or suspension. Surfactants are compounds that non-covalently interact with an agent to promote the dissolution or uniform suspension of an agent in an aqueous delivery system.

[0324] Treatment or pharmaceutical composition can be administered in a therapeutically effective amount, which will depend on a variety of factors, including the activity of the specific compound used; metabolic stability and duration of action of the compound; age, weight, general health, sex and diet of the subject; mode and time of administration; excretion rate; drug combination; severity of a particular condition or disorder; and the subject being treated. In some cases, the therapeutically effective daily dose is (for a 70 kg mammal) about 0.001 mg / kg (i.e., about 0.07 mg) to about 100 mg / kg (i.e., about 7.0 g); preferably, the therapeutically effective dose is (for a 70 kg mammal) about 0.01 mg / kg (i.e., about 0.7 mg) to about 50 mg / kg (i.e., about 3.5 g); more preferably, the therapeutically effective dose is (for a 70 kg mammal) about 1 mg / kg (i.e., about 70 mg) to about 25 mg / kg (i.e., about 1.75 g). In some embodiments, the therapeutically effective dose is administered once a week, every two weeks or every month. In specific embodiments, a therapeutically effective dose is administered, for example, once a week, once every two weeks, or once a month at a dose of about 1-10 mg / kg or 1-5 mg / kg, or about 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0325] The combination therapy described herein may include the administration of a single pharmaceutical dosage form containing an activatable proprotein and an additional therapeutic agent (e.g., a chemotherapeutic agent, a hormone therapeutic agent, a kinase inhibitor), as well as the administration of a composition comprising an activatable proprotein and an additional therapeutic agent in a separate pharmaceutical dosage form. For example, the activatable proprotein and the additional therapeutic agent may be administered to the subject together in a single oral dosage composition (such as a tablet or capsule), or each agent may be administered in a separate oral dosage formulation. Similarly, the activatable proprotein and the additional therapeutic agent may be administered to the subject together in a single parenteral dosage composition (such as in a saline solution or other physiologically acceptable solution), or each agent may be administered in a separate parenteral dosage formulation. As another example, for cell-based therapy, the activatable proprotein may be mixed with cells before administration, administered as part of a separate composition, or both. When a separate dosage formulation is used, the composition may be administered at substantially the same time, i.e., simultaneously, or at separate staggered times, i.e., sequentially and in any order; combination therapy is understood to include all of these therapies.

[0326] Also included are patient care kits comprising (a) at least one activatable proprotein, as described herein; and optionally (b) at least one additional therapeutic agent (e.g., a chemotherapeutic agent, a hormonal therapeutic agent, a kinase inhibitor). In some kits, (a) and (b) are in separate therapeutic compositions. In some kits, (a) and (b) are in the same therapeutic composition.

[0327] The kit herein may also include one or more additional therapeutic agents or other components suitable or desirable for the indication being treated or the diagnostic application desired. The kit herein may also include one or more syringes or other components (e.g., stents, implantable reservoirs, etc.) necessary or desirable to aid in the intended delivery mode.

[0328] In some embodiments, the patient care kit comprises separate containers, dividers or compartments for one or more compositions and one or more informational materials. For example, one or more compositions may be contained in a bottle, vial or syringe, and one or more informational materials may be contained in combination with the container. In some embodiments, the individual elements of the kit are contained in a single undivided container. For example, the composition is contained in a bottle, vial or syringe, and the informational material in the form of a label is attached thereto. In some embodiments, the kit comprises a plurality of (e.g., a pack) of separate containers, each container comprising one or more unit dosage forms (e.g., dosage forms described herein) of an activatable proprotein and optionally at least one additional therapeutic agent. For example, the kit comprises a plurality of syringes, ampoules, foil packets or blister packs, each comprising a single unit dose of an activatable proprotein and optionally at least one additional therapeutic agent. The container of the kit may be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation) and / or opaque.

[0329] The patient care kit optionally includes a device suitable for administration of the composition, such as a syringe, an inhaler, a dropper (e.g., an eye dropper), a swab (e.g., a cotton swab or a wooden stick), or any such delivery device. In some embodiments, the device is an implantable device that dispenses a metered dose of one or more agents. Also included are methods of providing a kit, such as by combining the components described herein.

[0330] Expression and purification systems

[0331] Certain embodiments include methods and related compositions for expressing and purifying activatable proproteins as described herein. Such recombinant activatable proproteins can be conveniently prepared using standard protocols, which are described in, for example, Sambrook et al. (1989, supra) (particularly Sections 16 and 17); Ausubel et al. (1994, supra) (particularly Chapters 10 and 16); and Coligan et al., Current Protocols in Protein Science (John Wiley & Sons, Inc. 1995-1997) (particularly Chapters 1, 5, and 6). As a general example, the activatable proprotein can be prepared by a procedure comprising one or more of the following steps: (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 region of the anti-PD-1 Fab region or the anti-PD-L1 Fab region described herein, wherein the polynucleotide sequences are 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 to express the first and second polypeptides and the VL / CL region, which are combined to form an activatable proprotein; and (d) isolating the activatable proprotein from the host cells.

[0332] In order to express the desired polypeptide, the nucleotide sequence encoding the first and / or second polypeptide chain of the activatable preprotein can be inserted into one or more suitable expression vectors, i.e., one or more vectors containing the necessary elements for transcribing and translating the inserted coding sequence. Methods well known to those skilled in the art can be used to construct expression vectors containing sequences encoding the target polypeptide and suitable transcription and translation control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. These techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., Current Protocols in Molecular Biology (1989).

[0333] A variety of expression vector / host systems are known and can be used 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 bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems, including mammalian cells, more specifically human cell systems.

[0334] "Control elements" or "regulatory sequences" present in expression vectors are those untranslated regions of the vector - enhancers, promoters, 5' and 3' untranslated regions - that interact with host cell proteins to transcribe and translate. The strength and specificity of such elements can be different. Depending on the vector system and host used, many suitable transcription and translation elements can be used, including constitutive and inducible promoters. For example, when cloning in a bacterial system, inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) can be used. In mammalian cell systems, promoters from mammalian genes or mammalian viruses are generally preferred. If it is necessary to produce a cell line containing multiple copies of a sequence encoding a polypeptide, a vector based on SV40 or EBV can be advantageously used together with a suitable selective marker.

[0335] In bacterial systems, depending on the use of the expressed polypeptide, many expression vectors can be selected. For example, when a large amount is required, a vector that directs high-level expression of fusion proteins that are easy to purify can be used. Such vectors include, but are not limited to, multifunctional Escherichia coli (E. coli) cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the target polypeptide can be connected to the vector in a frame-matched manner with the amino-terminal Met and subsequent 7 residues of β-galactosidase to produce a hybrid protein; pIN vector (Van Heeke & Schuster, J. Biol. Chem. 264: 5503 5509 (1989)); etc. pGEX vectors (Promega, Madison, Wis.) can also be used to express exogenous polypeptides in the form of fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption to glutathione-agarose beads and then eluted in the presence of free glutathione. Proteins produced in such systems can be designed to include heparin, thrombin, or Factor XA protease cleavage sites so that the cloned polypeptide of interest can be freely released from the GST moiety.

[0336] Certain embodiments employ an E. coli-based expression system (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 produce a suitable expression vector. In a specific embodiment, protein expression may be controlled by T7 RNA polymerase (e.g., pET vector series). These and related embodiments may utilize the expression host strain BL21 (DE3) (a λDE3 lysogenic bacterium of BL21 that supports T7-mediated expression and lacks lon and ompT proteases) to improve the stability of the target protein. Also included are expression host strains carrying plasmids encoding tRNAs that are rarely used in E. coli, such as ROSETTA TM (DE3) and Rosetta 2(DE3) strains. Cell lysis and sample processing can also be performed using the Nucleases and Protein extraction reagents are sold as an improvement over reagents sold as protein extraction reagents. For cell culture, auto-induction media can improve the efficiency of many expression systems (including high-throughput expression systems). This type of media (such as OVERNIGHT EXPRESS TMThe auto-induction system gradually triggers protein expression through metabolic shift without the addition of artificial inducers such as IPTG. Specific embodiments use a hexahistidine tag (such as the fusions) followed by immobilized metal affinity chromatography (IMAC) purification or related techniques. However, in some aspects, clinical-grade proteins can be isolated from E. coli inclusion bodies with or without affinity tags (see, e.g., Shimp et al., Protein Expr Purif. 50:58-67, 2006). As another example, certain embodiments can use a cold shock-induced high-yield production system in E. coli, because overexpression of proteins in E. coli at low temperatures improves their solubility and stability (see, e.g., Qing et al., Nature Biotechnology. 22:877-882, 2004).

[0337] High-density bacterial fermentation systems are also included. For example, high cell density culture of Ralstonia eutropha allows protein production at cell densities exceeding 150 g / L, and expression titers of recombinant proteins exceeding 10 g / L.

[0338] In Saccharomyces cerevisiae, many vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For review, see Ausubel et al. (supra) and Grant et al., Methods Enzymol. 153: 516-544 (1987). Also included are Pichia pastoris expression systems (see, e.g., 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 yeast with humanized N-glycosylation pathways, etc. (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. Pat. 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 15L, 50L, 100L, and 200L fermentors, etc.

[0339] In the case of using plant expression vectors, the expression of the sequence encoding the polypeptide can be driven by any of many promoters. For example, viral promoters such as 35S and 19S promoters of CaMV can be used alone or in combination with the ω leader sequence of TMV (Takamatsu, EMBO J.6:307-311 (1987)). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters (Coruzzi et al., EMBO J.3:1671-1680 (1984) can be used; 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 a number of generally available reviews (see, e.g., Hobbs in McGraw Hill, Yearbook of Science and Technology, pp. 191-196 (1992)).

[0340] Insect systems can also be used to express target polypeptides. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes in fall armyworm cells or cabbage looper cells. The sequence encoding the polypeptide can be cloned into a non-essential region of the virus, such as a polyhedrin gene, and placed under the control of a polyhedrin promoter. Successful insertion of the polypeptide coding sequence will inactivate the polyhedrin gene and produce a recombinant virus lacking coat protein. The recombinant virus can then be used to infect, for example, fall armyworm cells or cabbage looper cells (Engelhard et al., Proc. Natl. Acad. Sci. USA 91: 3224-3227 (1994)), wherein the target polypeptide can be expressed. Also included are baculovirus expression systems, including systems utilizing SF9, SF21, and T.ni cells (see, for example, Murphy and Piwnica-Worms, Curr Protoc Protein Sci. Chapter 5: Unit 5.4, 2001). Insect systems may provide post-translational modifications similar to those in mammalian systems.

[0341] In mammalian host cells, many virus-based expression systems are generally available. For example, when using adenovirus as an expression vector, the sequence encoding the target polypeptide can be connected to an adenoviral transcription / translation complex consisting of a late promoter and a tripartite leader sequence. Insertion of non-essential E1 or E3 regions of the viral genome can be used to obtain live viruses capable of expressing polypeptides in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. USA 81: 3655-3659 (1984)). In addition, transcription enhancers, such as Rous sarcoma virus (RSV) enhancers, can be used to increase expression in mammalian host cells.

[0342] Examples of useful mammalian host cell lines include monkey kidney CV1 cell line transformed by SV40 (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 CCL34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (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 cell line (HepG2). 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 lines 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 adherent cell lines, for example, in T-flasks, roller bottles, or cell factories or suspension cultures, for example, in 1L and 5L spinners, 5L, 14L, 40L, 100L, and 200L stirred tank bioreactors, or 20 / 50L and 100 / 200L WAVE bioreactors, and other reactors known in the art.

[0343] Cell-free expression of proteins is also contemplated. These and related embodiments typically utilize purified RNA polymerase, ribosomes, tRNA, and ribonucleotides; these reagents may be produced by extraction from cells or cell-based expression systems.

[0344] Specific start signals can also be used to achieve more efficient translation of the sequence encoding the target polypeptide. Such signals include ATG start codons and adjacent sequences. In the case where the sequence of the coded polypeptide, its start codon and upstream sequence are inserted into a suitable expression vector, additional transcription or translation control signals may not be required. However, in the case where only the coding sequence or its portion is inserted, an exogenous translation control signal including the ATG start codon should be provided. In addition, the start codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translation elements and start codons can have a variety of sources, including natural and synthetic. Expression efficiency can be improved by including enhancers suitable for the specific cell system used, such as those described in the document (Scharf. Results Probl. Cell Differ. 20: 125-162 (1994)).

[0345] In addition, host cell strains can be selected according to the ability of the host cell strain to regulate the expression of the insertion sequence or to process the protein expressed in a desired manner. This type of modification of polypeptide includes but is not limited to post-translational modification, such as acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. The post-translational processing of the "pre-form" form of cutting protein also can be used to promote correct insertion, folding and / or function. Except bacterial cells, different host cells can also be selected, such as yeast, CHO, HeLa, MDCK, HEK293 and W138, which have or even lack specific cell mechanisms and characteristic mechanisms for this type of post-translational activity, to ensure correct modification and processing of exogenous proteins.

[0346] For the long-term high-yield production of recombinant proteins, stable expression is generally preferred. For example, expression vectors can be used to transform cell lines that stably express the target polynucleotide, and the expression vectors can contain viral replication origins and / or endogenous expression elements and selective marker genes on the same vector or separate vectors. After the introduction of the vector, the cells can be allowed to grow in enriched medium for about 1 to 2 days, and then transferred to selective medium. The purpose of the selective marker is to confer resistance to selection, and its presence allows the cell growth and recovery of the sequence introduced by successful expression. The resistant clones of the stably transformed cells can be propagated using tissue culture techniques suitable for cell types. Transient production can also be used, such as by transient transfection or infection. Exemplary mammalian expression systems suitable for transient production include systems based on HEK293 and CHO.

[0347] Many selection systems can be used to reclaim transformed or transduced cell lines. These selection systems include but are not limited to 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 for tk-cells or aprt-cells, respectively. In addition, antimetabolite, antibiotic or herbicide resistance can also 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 aminoglycosides, neomycin and G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); and als or pat, which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, 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 widespread as 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 are widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a particular vector system (see, e.g., Rhodes et al., Methods Mol. Biol. 55:121-131 (1995)).

[0348] Also included are high-throughput protein production systems, or micro-production systems. In some aspects, protein expression and purification can be performed on a slide surface or MagneHis nickel particles modified with a six-histidine fusion tag (see, for example, Kwon et al., BMC Biotechnol. 9: 72, 2009; and Lin et al., Methods Mol Biol. 498: 129-41, 2009). Also included are high-throughput cell-free protein expression systems (see, for example, Sitaraman et al., Methods Mol Biol. 498: 229-44, 2009).

[0349] Various protocols for detecting and measuring the expression of products encoded by polynucleotides using binding agents or antibodies (such as polyclonal or monoclonal antibodies specific for the products) are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), protein immunoblotting, radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). These and other assays are described in, among others, Hampton et al., Serological Methods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med. 158: 1211-1216 (1983).

[0350] Many kinds of labels and conjugation techniques are known to those skilled in the art and can be used for various nucleic acid and amino acid determinations. The method of hybridization or PCR probes for detecting the labeling of sequences related to polynucleotides includes oligolabeling, nick translation, end labeling or PCR amplification using labeled nucleotides. Alternatively, the sequence or any part thereof can be cloned into a vector for producing mRNA probes. Such vectors are known in the art, are commercially available, and can be used for synthesizing RNA probes in vitro by adding suitable RNA polymerases 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, fluorescence, chemiluminescence or color developers and substrates, cofactors, inhibitors, magnetic particles, etc.

[0351] Host cells transformed with one or more polynucleotide sequences of interest can be cultured under conditions suitable for expressing the protein and recovering the protein from the cell culture. Certain embodiments utilize serum-free cell expression systems. Examples include HEK293 cells and CHO cells that can be grown in serum-free medium (see, e.g., Rosser et al., Protein Expr. Purif. 40:237-43, 2005; and U.S. Pat. No. 6,210,922).

[0352] The activatable preprotein produced by the recombinant cell can be secreted or contained in the cell, depending on the sequence and / or carrier used. As will be appreciated by those skilled in the art, the expression vector containing polynucleotides can be designed to contain a signal sequence, which instructs the encoded polypeptide to be secreted by a prokaryotic or eukaryotic cell membrane. Other recombinant constructs can be used to connect the sequence encoding the target polypeptide to the nucleotide sequence encoding the polypeptide domain, which will contribute to the purification and / or detection of soluble proteins. Examples of such domains include affinity purification and epitope tags that can be cut and cannot be cut, such as avidin, FLAG tags, polyhistidine tags (e.g., 6xHis), cMyc tags, V5 tags, glutathione S-transferase (GST) tags, etc.

[0353] Proteins produced by recombinant cells can be purified and characterized according to a variety of techniques known in the art. Exemplary systems for protein purification and analysis of 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 chemical methods 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 Ion exchange chromatography and hydrophobic interaction columns (HIC) and other methods known in the art. Also included are analytical methods such as SDS-PAGE (e.g., Coomassie Brilliant Blue staining, silver staining), immunoblotting, Bradford and ELISA, which can be used at any step of the production or purification process and are generally used to measure the purity of a protein composition.

[0354] Also include methods for concentrating activatable preproteins, and compositions comprising concentrated soluble activatable preproteins. In some aspects, the concentrated solution of such at least one activated preprotein comprises a concentration of about or at least about 5 mg / mL, 8 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or higher protein.

[0355] In some aspects, such compositions can be substantially monodisperse, meaning that the activatable proprotein exists predominantly (ie, at least about 90% or more) in one apparent molecular weight form when assessed, for example, by size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation.

[0356] In some aspects, such compositions are at least about 90% pure (on a protein basis), or in some aspects, at least about 95% pure, or in some embodiments, at least 98% pure. Purity can be determined by any conventional analytical method known in the art.

[0357] In some aspects, such compositions have a high molecular weight aggregate content of less than about 10%, 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%. The high molecular weight aggregate content can be determined by a variety of analytical techniques, including, for example, by size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation.

[0358] Examples of concentration methods contemplated herein include lyophilization, which is typically employed when the solution contains little soluble components other than the target protein. Lyophilization is typically performed after an HPLC run, and can remove most or all of the volatile components from the mixture. Ultrafiltration techniques are also included, which typically use one or more selectively permeable membranes to concentrate the protein solution. The membrane allows water and small molecules to pass through, while retaining the protein; the solution can be pressed against the membrane by techniques such as mechanical pumps, air pressure, or centrifugation.

[0359] In certain embodiments, as measured according to routine techniques in the art, activatable preprotein in the composition has a purity of at least about 90%. In certain embodiments, such as diagnostic compositions or some medicines or therapeutic compositions, activatable preprotein compositions have a purity of at least about 95% or at least about 97% or 98% or 99%. In some embodiments, such as when used as a reference or research reagent, activatable preprotein can have a lower purity, and can have a purity of at least about 50%, 60%, 70% or 80%. Can measure purity, for example, based on the purity of protein, from the whole or with respect to selected component (such as other proteins).

[0360] The purified activatable proprotein can also be characterized according to its biological properties. Binding affinity and binding kinetics can be measured according to various techniques known in the art, such as And related technologies utilizing surface plasmon resonance (SPR), an optical phenomenon that enables real-time detection of unlabeled interactors. SPR-based biosensors can be used for determination of active concentrations, screening, and characterization of affinity and kinetics. The presence or level of one or more biological activities can be measured according to cell-based assays, including those utilizing 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.

[0361] In certain embodiments, as described above, the activatable proprotein composition is substantially free of endotoxin, including, for example, about 95% free of endotoxin, preferably about 99% free of endotoxin, more preferably about 99.99% free of endotoxin. As described herein, the presence of endotoxin can be detected according to the routine techniques in the art. In a specific embodiment, the activatable proprotein composition is prepared in a substantially serum-free culture medium by eukaryotic cells such as mammals or human cells. In certain embodiments, as described herein, the endotoxin content of the activatable proprotein composition is less than about 10EU / mg activatable proprotein, or less than about 5EU / mg activatable proprotein, less than about 3EU / mg activatable proprotein, or less than about 1EU / mg activatable proprotein.

[0362] In certain embodiments, the activatable proprotein composition comprises less than about 10% wt / wt high molecular weight aggregates, or less than about 5% wt / wt high molecular weight aggregates, or less than about 2% wt / wt high molecular weight aggregates, or less than about 1% wt / wt high molecular weight aggregates.

[0363] Also included are protein-based analytical assays and methods that can be used to assess features such as protein purity, size, solubility, and aggregation. Protein purity can be assessed in a variety of ways. For example, purity can be assessed based on primary structure, higher structure, size, charge, hydrophobicity, and glycosylation. Examples of methods for assessing primary structure include N-terminal and C-terminal sequencing and peptide maps (see, e.g., Allen et al., Biologicals. 24: 255-275, 1996). Examples of methods for assessing higher structure 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 conformation-sensitive antibodies. Higher structure can also be evaluated as a function of various parameters such as pH, temperature, or added salt. Examples of 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 by, for example, reverse phase HPLC and hydrophobic interaction chromatography HPLC. Glycosylation can affect pharmacokinetics (e.g., clearance), conformation or stability, receptor binding, and protein function, and can be assessed by, for example, mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.

[0364] As mentioned above, some embodiments include using SEC-HPLC to assess protein characteristics, such as purity, size (e.g., size uniformity) or aggregation, and / or purifying proteins and other uses.SEC, also including gel filtration chromatography (GFC) and gel permeation chromatography (GPC), refers to a chromatographic method in which molecules in a solution are separated in a porous material according to their size, or more specifically according to their hydrodynamic volume, diffusion coefficient and / or surface properties. This method is commonly used to separate biomolecules, and to measure the molecular weight and molecular weight distribution of polymers. Typically, a biological or protein sample (such as a protein extract produced according to a protein expression method provided herein and known in the art) is loaded into a size exclusion column selected with a determined stationary phase (porous material) (preferably a phase that does not interact with the protein in the sample). In some aspects, the stationary phase is composed of inert particles in a dense three-dimensional matrix filled in a glass column or a steel column. The mobile phase can be pure water, an aqueous buffer, an organic solvent or a mixture thereof. The stationary phase particles typically have small pores and / or channels that only allow molecules less than a certain size to enter. Therefore, large particles are excluded from these pores and channels, and their limited interaction with the stationary phase causes them to elute in the form of a "complete exclusion" peak at the beginning of the experiment. Smaller molecules that can be suitable for entering the pores are removed from the mobile phase of the flow, and the time they spend fixing in the stationary phase pores depends in part on the depth they penetrate into the pores. Their removal from the mobile phase flow causes them to spend a longer time eluting from the post, and causes separation between particles based on their size differences. A given size exclusion chromatographic column has a separable molecular weight range. In general, molecules greater than the upper limit will not be captured by the stationary phase, and molecules less than the lower limit will fully enter the solid phase and elute as a single band, and molecules within this range will elute at different rates, depending on their properties, such as hydrodynamic volume. For examples of these methods in the practice of pharmaceutical proteins, see Bruner et al., Journal of Pharmaceutical and Biomedical Analysis.15:1929-1935,1997.

[0365] For example, Anicetti et al. (Trends in Biotechnology. 7: 342-349, 1989) also discussed protein purity for clinical applications. The technology recently used to analyze protein purity includes, but is not limited to, LabChip GXII (an automated platform for rapid analysis of proteins and nucleic acids), which provides high-throughput analysis of titer, sizing and purity analysis of proteins. In certain non-limiting embodiments, clinical-grade activatable preproteins can be obtained by utilizing a combination of chromatographic materials and other methods in at least two orthogonal steps (see, for example, Therapeutic Proteins: Methods and Protocols. Vol. 308, ed., Smales and James, Humana Press Inc., 2005). Typically, protein agents (e.g., activatable preproteins) are substantially free of endotoxins, as measured according to techniques known in the art and described herein.

[0366] Protein solubility determination is also included. Such determinations can be used, for example, to determine the optimal growth and purification conditions for recombinant production, optimize the selection of one or more buffers, and optimize the selection of activatable preproteins and variants thereof. Solubility or aggregation can be evaluated according to various parameters (including temperature, pH value, salt, and the presence or absence of other additives). Examples of solubility screening assays include, but are not limited to, microplate-based methods for measuring protein solubility using turbidity or other measurements as endpoints, high-throughput determinations for analyzing the solubility of purified recombinant proteins (see, for example, Stenvall et al., Biochim Biophys Acta. 1752: 6-10, 2005), structural complementation of genetic marker proteins is used to monitor and measure protein folding and solubility in vivo (see, for example, Wigley et al., Nature Biotechnology. 19: 131-136, 2001), and electrochemical screening of the solubility of recombinant proteins in Escherichia coli using scanning electrochemical microscopy (SECM) (see, for example, Nagamine et al., Biotechnology and Bioengineering. 96: 1008-1013, 2006), etc. Activatable proproteins with increased solubility (or reduced aggregation) can be identified or selected according to routine techniques in the art, including simple in vivo assays of protein solubility (see, e.g., Maxwell et al., Protein Sci. 8:1908-11, 1999).

[0367] Protein solubility and aggregation can also be measured by dynamic light scattering technology. Aggregation is a general term covering several types of interactions or features, including soluble / insoluble, covalent / non-covalent, reversible / irreversible and native / denatured interactions and features. For protein therapeutics, the presence of aggregates is generally considered undesirable because it is worried that aggregates may cause immunogenic reactions (such as small aggregates) or may cause adverse events when administered (such as microparticles). Dynamic light scattering refers to a technique that can be used to measure the size distribution characteristics of small particles in suspension or polymers such as proteins in solution. This technique is also called photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QELS), which uses scattered light to measure the diffusion rate of protein particles. Due to the Brownian motion of molecules and particles in solution, fluctuations in scattering intensity can be observed. The motion data can be routinely processed to derive the size distribution of the sample, where the size is given by the Stokes radius or hydrodynamic radius of the protein particles. The hydrodynamic size depends on mass and shape (conformation). Dynamic scattering can detect the presence of very small amounts of aggregated protein (<0.01 wt %), even in samples containing a wide range of masses. It can also be used to compare the stability of different formulations, including, for example, applications that rely on real-time monitoring of changes at high temperatures. Thus, certain embodiments include the use of dynamic light scattering to analyze the solubility and / or presence of aggregates in samples containing an activatable proprotein of the invention.

[0368] Although the foregoing embodiments have been described in considerable detail by way of illustration and example for the purpose of clear understanding, it will be readily apparent to those skilled in the art that certain changes and modifications may be made thereto in accordance with the teachings of the present invention without departing from the spirit or scope of the appended claims. The following examples are intended to illustrate and not to limit. Those skilled in the art will readily recognize various non-critical parameters that may be changed or modified to produce substantially similar results. Example

[0369] Example 1A

[0370] Preparation of PD-L1-proIL-2 fusion protein

[0371] Plasmids encoding PD-L1-proIL-2v (IL-2 variant) or PD-L1-proIL-2wt or anti-human PD-L1 with different protease-cleavable linkers were constructed by standard gene synthesis and subsequently subcloned into the pTT5 expression vector. Figure 1-Figure 2 Schematic representation of an illustrative IgG-proIL-2 fusion protein format is depicted.

[0372] Exemplary proteins of the PD-L1-proIL-2v ("v" = variant) form with different protease cleavage 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 of the PD-L1-proIL-2wt form include P47131942 (SEQ ID NOs: 154 and 155). Exemplary anti-human PD-L1 proteins include P40751942 (SEQ ID NOs: 144 and 145).

[0373] PD-L1-proIL-2 fusion protein or anti-human PD-L1 was produced by transient transfection in Expi293 cells and purified by one-step purification of MabSelect SuRe chromatography (GE Healthcare). The purified protein was characterized by SDS-PAGE and high-performance liquid chromatography (HPLC) for purity and homogeneity assessment. HPLC analysis was performed using a Nanofilm SEC-250 column (Sepax) and Agilent 1260 according to the manufacturer's instructions. The purified protein showed high purity on SDS-PAGE gel and good homogeneity based on the HPLC results.

[0374] Example 1B

[0375] Preparation of PD-1-proIL-2v fusion protein

[0376] Plasmids encoding PD-1-proIL-2v or 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. Figure 1-Figure 2 Schematic representation of an illustrative IgG-proIL-2 fusion protein format is depicted.

[0377] Exemplary proteins of the PD-1-proIL-2v form with different protease cleavage linkers include P41222037 (SEQ ID NOs: 146 and 147), P43492037 (SEQ ID NOs: 152 and 153). Exemplary proteins of the unmasked PD-1-proIL-2v form include P41252037 (SEQ ID NOs: 158 and 159). Exemplary proteins of the PD-1-null-proIL-2v form include P54475445 (SEQ ID NOs: 156 and 157). Exemplary proteins of anti-human PD-1 include P42412037 (SEQ ID NOs: 150 and 151).

[0378] 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 purification with MabSelect SuRe chromatography (GE Healthcare). The purified proteins were characterized by SDS-PAGE and high-performance liquid chromatography (HPLC) for purity and homogeneity assessment. HPLC analysis was performed using a Nanofilm SEC-250 column (Sepax) and Agilent 1260 according to the manufacturer's instructions. The purified proteins showed high purity on SDS-PAGE gels and good homogeneity based on HPLC results.

[0379] Example 1C

[0380] Preparation of B7H3-proIL-2 fusion protein

[0381] The plasmid encoding B7H3-proIL-2v was constructed by standard gene synthesis and then subcloned into the pTT5 expression vector. Figure 1-Figure 2 Schematic representation of an illustrative IgG-proIL-2 fusion protein format is depicted.

[0382] Exemplary proteins of the B7H3-proIL-2v form include P41713699 (SEQ ID NOs: 148 and 149).

[0383] B7H3-proIL-2v fusion protein was produced by transient transfection in Expi293 cells and purified by one-step purification of MabSelectSuRe chromatography (GE Healthcare). The purified protein was characterized by SDS-PAGE and high performance liquid chromatography (HPLC) for purity and homogeneity assessment. HPLC analysis was performed using Nanofilm SEC-250 columns (Sepax) and Agilent 1260 according to the manufacturer's instructions. The purified protein showed high purity on SDS-PAGE gels and showed good homogeneity based on the HPLC results.

[0384] Example 2A

[0385] Binding of PD-1-proIL-2 to human and cynomolgus monkey PD-1

[0386] The binding activity of P41222037 and P42412037 was determined by ELISA. Microtiter plates were coated with 100 μl of 2 ug / ml streptavidin overnight at 4°C. The next day, the plates were washed with PBS and blocked with 2% BSA (2% bovine serum albumin in PBS). 100 μl of 2 ug / ml biotinylated huPD-1 or CynoPD-1 was added to the corresponding wells and incubated for 1 hr at room temperature to capture the biotinylated proteins. The plates were washed with PBST (0.01% Tween in PBS) and PBS in sequence.

[0387] The designated samples (PD-1 IgG or PD-1-proIL-2v) used in the ELISA assay were prepared in 2% BSA at an initial concentration of 10ug / ml, followed by 1 / 3 serial dilutions. 100μl of the diluted protein was added to the corresponding wells and incubated at room temperature for 1h. The plate was washed with PBST and PBS in sequence.

[0388] The bound antibodies were detected with a peroxidase-conjugated anti-human IgG secondary antibody (Jackson Immunoresearch). The plates were washed with PBST and PBS in turn. 90ul of TMB substrate was added to each well and incubated for 5min in the dark at room temperature. The reaction was terminated with 45ul of 2M sulfuric acid and the absorbance was read at 450nm. Data were analyzed by Prism.

[0389] like Figure 4A-4B As shown, PD-1-proIL-2v and the corresponding PD-1 IgG bound similarly to human PD-1.

[0390] Example 2B

[0391] Binding of PD-L1-proIL-2 to human PD-L1

[0392] The binding activity of P40391942 and P40751942 was determined by ELISA. Microtiter plates were coated with 100 μl of 2 ug / ml streptavidin overnight at 4°C. The next day, the plates were washed with PBS and blocked with 2% BSA (2% bovine serum albumin in PBS). 100 μl of 2 ug / ml biotinylated huPD-L1 was added to the corresponding wells and incubated for 1 hr at room temperature to capture the biotinylated protein. The plates were washed with PBST (0.01% Tween in PBS) and PBS in sequence.

[0393] The designated samples (PD-L1 IgG or PD-L1-proIL-2v) used in ELISA assays were prepared in 2% BSA at an initial concentration of 10ug / ml, followed by 1 / 3 serial dilutions. 100μl of diluted protein was added to the corresponding wells and incubated at room temperature for 1h. The plates were washed with PBST and PBS in sequence.

[0394] The bound antibodies were detected with a peroxidase-conjugated anti-human IgG secondary antibody (Jackson Immunoresearch). The plates were washed with PBST and PBS in turn. 90ul of TMB substrate was added to each well and incubated for 5min in the dark at room temperature. The reaction was terminated with 45ul of 2M sulfuric acid and the absorbance was read at 450nm. Data were analyzed by Prism.

[0395] like Figure 4C As shown, PD-L1-proIL-2v and the corresponding PD-L1 IgG bound similarly to human PD-L1.

[0396] Example 2C

[0397] PD-1-proIL-2v restores T cell function inhibited by PD-1

[0398] Mature DC cells were recovered, treated with 50ug / ml of mitomycin C in the dark for 30 minutes, and washed twice with PBS. The recovered PBMCs (1e5 cells / well) and allogeneic DCs (5000 cells / well) were co-cultured with isotope control antibodies, parental PD-1 antibodies, or PD-1-proIL-2 fusion proteins titrated with an initial concentration of 100ug / ml. After incubation at 37°C for 5 days, IFNγ secretion in the culture supernatant was analyzed by human IFNγ ELISA Set (Biolegend Cat.430104). The pre-coated capture antibody plate was prepared as described in the manufacturer's instructions, and 100ul of diluted standards or samples were added to the corresponding wells and incubated at room temperature for 2h. The plate was washed 4 times with wash buffer, 100ul of diluted detection antibody solution was added to each well, and incubated with shaking at room temperature for 30min. The plate was then washed 5 times with wash buffer, 100ul of freshly mixed TMB substrate solution was added to each well and incubated for 20min at room temperature in the dark. The reaction was terminated with 100ul of stop solution and the absorbance was read at 450nm. Data were analyzed by Prism.

[0399] like Figure 5 As shown, mixed lymphocyte reaction assay data indicated that PD-1-proIL-2v restored the function of T cells suppressed by PD-1.

[0400] Example 2D

[0401] Proliferation of M07-e with PD-1-proIL-2v and PD-L1-proIL-2v

[0402] 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 during the logarithmic growth phase of M-07e cells and washed twice with PBS. 90 μl of cell suspension (2×10 4 Cells / well) were seeded into 96-well plates and incubated in assay medium (RPMI 1640 supplemented with 10% FBS) at 37° C. and 5% CO 2 for 4 h for cytokine starvation.

[0403] The IL-2, P40391942 or P41222037 protein samples used in the assay were prepared in the 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 diluted in 1 / 3 series. 10 μl of the diluted protein was added to the corresponding wells and incubated at 37 ° C and 5% CO2 for 72 hours. A colorimetric assay was performed using a cell counting kit-8 (CCK-8, Dojindo, CK04) to measure the amount of viable cells.

[0404] Fig. 6A It was shown that PD-1-proIL-2v could not induce proliferation of M07-e even at the highest concentration. MMP-2-cleaved PD-1-proIL-2v restored some IL-2 activity and exhibited an activity as low as about 1 / 30 of that of wild-type IL-2. Figure 6B The results showed that PD-L1-proIL-2v was almost inactive at a high concentration of 810 nM. MMP-2 protease-activated PD-L1-proIL-2v induced M07-e cell proliferation as efficiently as protease-activated PD-1-proIL-2v.

[0405] Example 3A

[0406] Cell activation of PBMCs with PD-1-proIL-2v (pSTAT5 assay)

[0407] Frozen human PBMC (SAILYBIO, donor 1) were recovered 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 for about 2 hours. PBMC were adjusted to 2.6×10 6 cells / ml and plated at 4×10 5 Cells were plated in 96-well plates. The recovered PBMCs were incubated with rhIL-2, P41222037, or P54475445 at 37°C for 15 min. After incubation, the cells were immediately fixed with Cytofix buffer (BD Bioscience) to maintain the phosphorylation state on ice for 15 min and stained with BD Pharmingen TM Wash once with staining buffer (FBS).

[0408] 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 (BD562442), CD56 BV510 (BD744218) for 30 min at 4°C. Cells were washed once with 1× PBS and centrifuged at 500 g for 5 min, and the supernatant was removed by aspiration. Cells were permeabilized with pre-chilled Phosflow Perm buffer III (BD Bioscience) for 30 min at 4°C. Before starting intracellular staining, cells were washed once with 1× PBS and centrifuged at 500 g for 5 min to collect the pellet. Cells were then incubated with Foxp3 PE (BD 560046) and anti-stat5 (pY694) Alexa Fluor (BD Bioscience) at RT. 647 (BD 562076) was used to stain the cells for 40 min. TM The cells were washed twice with staining buffer (FBS) and centrifuged at 500 g for 5 min to pellet the cells and remove the supernatant. The cell pellet was resuspended in 200 ul (per well) BD Pharmingen staining buffer (FBS) and analyzed by flow cytometry. The STAT5 phosphorylation status in PBMC subpopulations after prodrug treatment was obtained and processed by CytoFLEX (Beckman).

[0409] Figure 7A-7D Figure 2 shows that when resting PBMCs from donor 1 were treated with rhIL-2 and intact and protease-activated PD-1-proIL-2v, CD4 T cells ( Fig. 7A ), CD8 T cells ( Figure 7B ), regulatory T cells ( Figure 7C ) and NK cells ( Fig.7D ) STAT phosphorylation in . The masked or inactive pro-cytokine form of P41222037 cannot induce STAT5 phosphorylation in all tested cell subsets. The protease-activated form of P41222037 (matrix metalloproteinase-2 cleavage) is equally effective in activating STAT5 phosphorylation in CD8 and CD4 T cells, but is less effective in NK cells. In Treg cells, the effectiveness of activated P41222037 is as low as less than 1 / 30 of huIL-2. Therefore, compared with wild-type huIL-2, P41222037 no longer preferentially activates Treg cells. It is worth noting that the pre-blocking of the PD-1 receptor by the parental anti-PD-1 antibody reduces the effectiveness of P41222037 (activated form) to less than 1 / 20 for T cells expressing PD-1. Similarly, P54475445 (PD-1null-IL-2v fusion protein) shows an effectiveness as low as 1 / 20 for CD4+ T cells, CD8+ T cells, and Treg cells. These data confirm that preferential cis-targeting of PD-1-proIL-2v results in enhanced potency against PD-1-positive T cells.

[0410] Example 3B

[0411] Cell activation of PBMCs with PD-L1-proIL-2v (pSTAT5 assay)

[0412] Frozen human PBMC (SAILYBIO, donor 1) were recovered 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 for about 2 hours. PBMC were adjusted to 2.6×10 6 cells / ml, and 4×10 5 The cells were plated at a density of 10 cells / well in a 96-well plate. The recovered PBMCs were incubated with rhIL-2 and P40391942 (complete and activated forms) at 37°C for 15 min. After incubation, the cells were immediately fixed with Cytofix buffer (BD Bioscience) to maintain the phosphorylation state on ice for 15 min and stained with BD Pharmingen TM Wash once with staining buffer (FBS).

[0413] 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 (BD562442), CD56 BV510 (BD744218) for 30 min at 4°C. Cells were washed once with 1× PBS and centrifuged at 500 g for 5 min, and the supernatant was removed by aspiration. Cells were permeabilized with pre-chilled Phosflow Perm buffer III (BD Bioscience) for 30 min at 4°C. Before starting intracellular staining, cells were washed once with 1× PBS and centrifuged at 500 g for 5 min to collect the pellet. Cells were then incubated with Foxp3 PE (BD 560046) and anti-stat5 (pY694) Alexa Fluor (BD Bioscience) at RT. 647 (BD 562076) was used to stain the cells for 40 min. TM The cells were washed twice with staining buffer (FBS) and centrifuged at 500 g for 5 min to pellet the cells and remove the supernatant. The cell pellet was resuspended in 200 ul (per well) of BD Pharmingen staining buffer (FBS) and analyzed by flow cytometry. The STAT5 phosphorylation status in the PBMC subsets after pre-cytokine treatment was obtained and processed by CytoFLEX (Beckman).

[0414] Figure 8A-8D Figure 8 shows the expression of CD4 T cells (8A), CD8 T cells ( Figure 8B ), regulatory T cells ( Figure 8C ) and NK cells ( Fig.8D ). 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 effective in NK cells. In Treg cells, activated P40391942 was not as effective as huIL-2. It is worth noting that pre-blocking the PD-L1 receptor by the parental anti-PD-L1 antibody reduced the potency of P40391942 (activated form) to less than 1 / 100 on PD-L1-expressing CD4+ T cells, CD8+ T cells, and Treg cells. These data confirm that PD-L1 binding enhances the potency of IL-2v.

[0415] Example 3C

[0416] Activated PD-1-proIL-2v stimulates human CD4 T cells to secrete GM-CSF

[0417] Frozen PBMCs were recovered and treated with 10 μg / mL mitomycin C for 2 h in the dark. After washing 3 times, the 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 (complete and activated forms). Afterwards, frozen CD4 T cells from the same donor were suspended in RPMI1640 supplemented with 1 ug / ml soluble αCD3; 4e+5 cells / well of CD4 T cells were plated in PBMC 96-well plates.

[0418] After 5 days of incubation at 37°C, GM-CSF secretion in the culture supernatant was analyzed by Human GM-CSF ELISA Set (Biolegend Cat. 432004). The pre-coated capture antibody plate was prepared as described in the manufacturer's instructions, and 100ul of diluted standards or samples were added to the corresponding wells and incubated for 2h at room temperature. The plate was washed 4 times with wash buffer, 100ul of diluted detection antibody solution was added to each well, and incubated for 30min at room temperature with shaking. The plate was then washed 5 times with wash buffer, 100ul of freshly mixed TMB substrate solution was added to each well, and incubated in the dark at room temperature for 20min. The reaction was stopped with 100ul of stop solution, and the absorbance was read at 450nm. The data was analyzed by Prism.

[0419] like Fig. 9A As shown, protease-activated PD-1-proIL-2v, but not the intact pro-cytokine, stimulated CD4 T cells to produce GM-CSF. In contrast, PD-1 blockade alone did not induce any significant level of GM-CSF secretion.

[0420] Example 3D

[0421] Activated PD-1-proIL-2v stimulates human CD4 T cells to produce interferon gamma

[0422] Frozen PBMCs were recovered and treated with 10 μg / mL mitomycin C for 2 hr in the dark. After washing 3 times, the 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 (complete and activated forms). Afterwards, frozen CD4 T cells from the same donor were suspended in RPMI1640 supplemented with 1 ug / ml soluble αCD3; 4e5 cells / well of CD4 T cells were plated in PBMC 96-well plates.

[0423] After incubation at 37°C for 5 days, cells were collected for intracellular FACS staining. Cytokine accumulation in the Golgi complex was induced by stimulating cells again for 5 h with ionomycin (500 ng / ml) and PMA (50 ng / ml) and protein transport inhibitors (GolgiPlug and GolgiStop, BD). After incubation, cells were immediately fixed with Cytofix buffer (BD Bioscience) to maintain phosphorylation status on ice for 15 min and stained with BD Pharmingen TM Wash once with staining buffer (FBS). For surface staining, incubate the cells with CD3 Alexa Flour700 (BD 557943) and CD4 PerCP-Cytm5.5 (BD560650) at 4°C for 30min. Wash the cells once with 1×PBS and centrifuge at 500g for 5min, and remove the supernatant by aspiration. Permeabilize the cells with pre-cooled Phosflow Perm buffer III (BD Bioscience) for 30min at 4°C. Before starting intracellular staining, wash the cells once with 1×PBS and centrifuge at 500g for 5min to collect the precipitate. The cells were then stained with BV605 anti-human IFN-γ (BD 562974) at RT for 40min. BD Pharmingen TM The cells were washed twice with staining buffer (FBS) and centrifuged at 500 g for 5 min to pellet the cells and remove the supernatant. The cell pellet was resuspended in 200 ul (per well) of BD Pharmingen staining buffer (FBS) and analyzed using CytoFLEX (Beckman).

[0424] like Fig. 9B As shown, activated PD-1-proIL-2v stimulated CD4 T cells to produce IFNγ, but intact pro-cytokine or PD-1 antibody alone could not.

[0425] Example 4

[0426] In vivo efficacy of PD-L1-proIL-2v and PD-1-proIL-2v with different protease-cleavable linkers in a tumor-PBMC xenograft model

[0427] The antitumor efficacy of PD-L1-proIL-2v and PD-1-proIL-2v with different protease-cleavable linkers was tested alone and in comparison with the corresponding PD-L1 or PD-1 antibodies in a tumor-PBMC xenograft model.

[0428] 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 co-cultured with A375 tumor cells treated with mitomycin C for 6 days and maintained in vitro as a suspension 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.

[0429] 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 co-cultured with HT29 tumor cells treated with mitomycin C for 6 days and they were maintained in vitro as a suspension 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.

[0430] Female NCG mice (GemPharmatech Co., Ltd., Nanjing, China) aged 8 to 10 weeks were maintained at the beginning of the experiment under the condition of no specific pathogens with a daily cycle of 12h light / 12h dark. The mice were kept in independent ventilated cages at constant temperature (20 to 26°C) and constant humidity (40 to 70%), with ≤6 animals per cage. During the whole study, animals had free access to irradiated sterilized dry granular food and sterile drinking water. Before proceeding, all procedures related to animal handling, care and treatment in this study were carried out according to the guidelines approved by the Animal Care and Use Committee (IACUC) of Shanghai Viva. After arrival, the animals were maintained for at least 3 days to adapt to the new environment. During routine monitoring, animals were checked for any effects of tumor growth on normal behavior (such as activity, consumption of food and water (only by viewing), weight gain / reduction (weight measured twice a week), loss of gloss in eyes / hair) and any other abnormal effects.

[0431] Mice were inoculated subcutaneously in the right flank with 4×10 6 A375 cells and 4×10 5 hPBMCs (co-cultured with A375) or 5×10 6 HT29 cells and 1×10 6hPBMCs (co-cultured with HT29) were used for tumorigenesis. One week after tumor cell inoculation, mice were injected iv 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 a caliper and expressed in mm using the following formula: 3 Indicates volume: 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 the TGI and T / C values. Tumor growth inhibition (TGI%) and relative tumor growth inhibition (T / C%) were calculated according to the following equations.

[0432] TGI%=(1-(T n -T0) / (V n -V0))×100%

[0433] T / C%=(T n / T0) / (V n / V0)×100%

[0434] In the formula, T n and V n Respectively represent the tumor volume of the treatment group and the vehicle control group on the nth day after the start of treatment. T0 and V0 represent the tumor volume of the corresponding group on the day of grouping. The results were analyzed using Prism GraphPad.

[0435] Since the protease-cleavable linker in P40391942 was the easiest to cleave, and the linker in P40531942 was the most difficult to cleave, Figure 10A-10B As shown in Table 1, in the A375-PBMC xenograft model, P40391942 showed 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, showed complete remission.

[0436]

[0437]

[0438] Figure 11A-11B As shown in Table 2, in the HT-29-PBMC xenograft model, P40391942 showed the best antitumor activity, followed by P40621942>P40531942>P40751942. 2 / 6 mice treated with P40391942 showed complete remission.

[0439]

[0440] Since the protease-cleavable linker in P41222037 is more easily cleaved than the linker in P43492037, Figure 12A-12B As shown in Table 3 , P41222037 exhibited better antitumor activity than that of P43492037 in the A375-PBMC xenograft model.

[0441]

[0442]

[0443] Figure 13A-13B As shown in Table 4, in the HT-29-PBMC xenograft model, P41222037 showed the best anti-tumor activity, followed by P43492037 > P42412037 (anti-PD-1 antibody). 3 / 6 mice treated with P41222037 showed complete remission.

[0444]

[0445] Example 5

[0446] In vivo efficacy of IgG-proIL-2v with different targets in the A375-PBMC xenograft model

[0447] The anti-tumor efficacy of PD-L1-proIL-2v, PD-1-proIL-2v, and B7H3-proIL-2v were tested individually in the A375-PBMC xenograft model.

[0448] 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 co-cultured with mitomycin C-treated A375 tumor cells for 6 days and they were 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.

[0449] Female NCG mice (GemPharmatech Co., Ltd., Nanjing, China) aged 8 to 10 weeks were maintained at the beginning of the experiment under the condition of no specific pathogens with a daily cycle of 12h light / 12h dark. The mice were kept in independent ventilated cages at constant temperature (20 to 26°C) and constant humidity (40 to 70%), with ≤6 animals per cage. During the whole study, animals had free access to irradiated sterilized dry granular food and sterile drinking water. Before proceeding, all procedures related to animal handling, care and treatment in this study were carried out according to the guidelines approved by the Animal Care and Use Committee (IACUC) of Shanghai Viva. After arrival, the animals were maintained for at least 3 days to adapt to the new environment. During routine monitoring, animals were checked for any effects of tumor growth on normal behavior (such as activity, consumption of food and water (only by viewing), weight gain / reduction (weight measured twice a week), loss of gloss in eyes / hair) and any other abnormal effects.

[0450] Mice were inoculated subcutaneously in the right flank with 4 × 10 cells / mL of 0.2 ml of HBSS containing 0.1 ml of Matrigel (1:1). 6 A375 cells and 4×10 5 hPBMCs (co-cultured with A375) were used for tumorigenesis. One week after tumor cell inoculation, mice were injected iv 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 a caliper and expressed in mm using the following formula: 3 Indicates volume: 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 the TGI and T / C values. Tumor growth inhibition (TGI%) and relative tumor growth inhibition (T / C%) were calculated according to the following equations.

[0451] TGI%=(1-(T n -T0) / (V n -V0))×100%

[0452] T / C%=(T n / T0) / (V n / V0)×100%

[0453] In the formula, T n and V n Respectively represent the tumor volume of the treatment group and the vehicle control group on the nth day after the start of treatment. T0 and V0 represent the tumor volume of the corresponding group on the day of grouping. The results were analyzed using Prism GraphPad.

[0454] Figure 14A-14B and Table 5 show the dose-dependent anti-tumor efficacy mediated by activated PD-L1-proIL-2v and PD-1-proIL-2v in the A375-PBMC xenograft model, with PD-1-proIL-2v exhibiting superior efficacy to PD-L1-proIL-2v at doses of 0.1 mg / kg and 0.3 mg / kg.

[0455]

[0456] Figure 15A-15B Table 6 shows that PD-1-proIL-2v mediated better efficacy than B7H3-proIL-2v at a dose of 0.3 mg / kg in the A375-PBMC xenograft model. These data suggest that PD-1-proIL-2v is a more potent inhibitor of tumor growth than other IgG-proIL-2 targeting PD-L1 or B7H3.

[0457]

[0458]

[0459] Example 6

[0460] In vivo efficacy of PD-L1-proIL-2v and PD-L1-proIL-2wt in the A375-PBMC xenograft model

[0461] The antitumor efficacy of PD-L1-proIL-2v and PD-L1-proIL-2wt was tested individually in the A375-PBMC xenograft model.

[0462] A375 cells were maintained in vitro at 37°C in an atmosphere of 5% CO2 in air in DMEM supplemented with 10% FBS, 100 U / ml penicillin and 100 μg / ml streptomycin.

[0463] Female NCG mice (GemPharmatech Co., Ltd., Nanjing, China) aged 8 to 10 weeks were maintained at the beginning of the experiment under the condition of no specific pathogens with a daily cycle of 12h light / 12h dark. The mice were kept in independent ventilated cages at constant temperature (20 to 26°C) and constant humidity (40 to 70%), with ≤6 animals per cage. During the whole study, animals had free access to irradiated sterilized dry granular food and sterile drinking water. Before proceeding, all procedures related to animal handling, care and treatment in this study were carried out according to the guidelines approved by the Animal Care and Use Committee (IACUC) of Shanghai Viva. After arrival, the animals were maintained for at least 3 days to adapt to the new environment. During routine monitoring, the tumor growth of the animals was checked for any effects on normal behavior (such as activity, consumption of food and water (only by viewing), weight gain / reduction (weight measured twice a week), loss of gloss in eyes / hair) and any other abnormal effects.

[0464] Mice were inoculated subcutaneously in the right flank with 4 × 10 6 A375 cells and 4×10 5 hPBMCs were used for tumorigenesis. One week after tumor cell inoculation, mice were injected iv 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 a caliper and expressed in mm using the following formula: 3 Indicates volume: 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 the TGI and T / C values. Tumor growth inhibition (TGI%) and relative tumor growth inhibition (T / C%) were calculated according to the following equations.

[0465] TGI%=(1-(T n -T0) / (V n -V0))×100%

[0466] T / C%=(T n / T0) / (V n / V0)×100%

[0467] In the formula, T n and V n Respectively represent the tumor volume of the treatment group and the vehicle control group on the nth day after the start of treatment. T0 and V0 represent the tumor volume of the corresponding group on the day of grouping. The results were analyzed using Prism GraphPad.

[0468] Figure 16A-16BTable 7 and Table 7 show the anti-tumor efficacy mediated by PD-L1-proIL-2v and PD-L1-proIL-2wt in the A375-PBMC xenograft model, with PD-L1-proIL-2wt exhibiting superior efficacy to PD-L1-proIL-2v at a dose of 0.3 mg / kg.

[0469]

[0470] Example 7

[0471] In vivo efficacy of PD-1-proIL-2v and anti-PD-L1 antibody as single agents and in combination in an A375-PBMC xenograft model

[0472] In the A375-PBMC xenograft model, the anti-tumor efficacy of PD-1-proIL-2v and anti-PD-L1 antibody compared with their combination was tested.

[0473] 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 co-cultured with mitomycin C-treated A375 tumor cells for 6 days and they were 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.

[0474] Female NCG mice (GemPharmatech Co., Ltd., Nanjing, China) aged 8 to 10 weeks were maintained at the beginning of the experiment under the condition of no specific pathogens with a daily cycle of 12h light / 12h dark. The mice were kept in independent ventilated cages at constant temperature (20 to 26°C) and constant humidity (40 to 70%), with ≤6 animals per cage. During the whole study, animals had free access to irradiated sterilized dry granular food and sterile drinking water. Before proceeding, all procedures related to animal handling, care and treatment in this study were carried out according to the guidelines approved by the Animal Care and Use Committee (IACUC) of Shanghai Viva. After arrival, the animals were maintained for at least 3 days to adapt to the new environment. During routine monitoring, animals were checked for any effects of tumor growth on normal behavior (such as activity, consumption of food and water (only by viewing), weight gain / reduction (weight measured twice a week), loss of gloss in eyes / hair) and any other abnormal effects.

[0475] Mice were inoculated subcutaneously in the right flank with 4 × 10 6A375 cells and 4×10 5 hPBMCs (co-cultured with A375) were used for tumorigenesis. One week after tumor cell inoculation, mice were injected iv with PD-1-proIL-2v, anti-PD-L1 antibody, or the 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 a caliper and expressed in mm using the following formula: 3 Indicates volume: 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 the TGI and T / C values. Tumor growth inhibition (TGI%) and relative tumor growth inhibition (T / C%) were calculated according to the following equations.

[0476] TGI%=(1-(T n -T0) / (V n -V0))×100%

[0477] T / C%=(T n / T0) / (V n / V0)×100%

[0478] In the formula, T n and V n Respectively represent the tumor volume of the treatment group and the vehicle control group on the nth day after the start of treatment. T0 and V0 represent the tumor volume of the corresponding group on the day of grouping. The results were analyzed using Prism GraphPad.

[0479] Figure 17A-17B and Table 8 show that in the A375-PBMC xenograft model, the combination of PD-1-proIL-2v and PD-L1 mediated better efficacy than either monotherapy, and all tumors in the combination group were completely responded.

[0480]

[0481] Example 8

[0482] Pharmacokinetic and pharmacodynamic studies of PD-1-proIL-2v

[0483] The pharmacokinetics and pharmacodynamics of PD-1-proIL-2v in peripheral blood and tumors were tested.

[0484] 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 co-cultured with mitomycin C-treated A375 tumor cells for 6 days and they were 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.

[0485] Female NCG mice (GemPharmatech Co., Ltd., Nanjing, China) aged 8 to 10 weeks were maintained at the beginning of the experiment under the condition of no specific pathogens with a daily cycle of 12h light / 12h dark. The mice were kept in independent ventilated cages at constant temperature (20 to 26°C) and constant humidity (40 to 70%), with ≤6 animals per cage. During the whole study, animals had free access to irradiated sterilized dry granular food and sterile drinking water. Before proceeding, all procedures related to animal handling, care and treatment in this study were carried out according to the guidelines approved by the Animal Care and Use Committee (IACUC) of Shanghai Viva. After arrival, the animals were maintained for at least 3 days to adapt to the new environment. During routine monitoring, the tumor growth of the animals was checked for any effects on normal behavior (such as activity, consumption of food and water (only by viewing), weight gain / reduction (weight measured twice a week), loss of luster of eyes / hair) and any other abnormal effects.

[0486] For pharmacokinetic studies, mice were inoculated subcutaneously on the right flank with 4 × 10 6 A375 cells and 4×10 5 hPBMCs for tumorigenesis. 18 days after tumor cell inoculation, mice were injected iv with 1 mg / kg PD-1-proIL-2v. Blood and tumor samples were collected at 1hr, 6hr, 24hr, 48hr, 72hr, 96hr, 120hr and 168hr after administration. Blood samples will be centrifuged (6000rpm, 5min, 4°C) within 30 minutes after collection to obtain serum samples. Serum samples were stored at -80°C before use. The tumor was weighed, and then PBS with a volume of twice the tumor weight was added, and the tissue was homogenized on ice. The homogenate was then centrifuged at 6000rpm for 10min at 4°C, the supernatant was collected, and stored at -80°C before use.

[0487] Total drug (including intact pro-cytokines and cleaved cytokines) and pro-cytokines (intact pro-cytokines) were determined using ELISA using PD-1 as the capture antibody and various detection antibodies. Anti-human IgG or anti-IL-2Rα conjugated with HRP was used to detect total cytokine and pro-cytokine levels, respectively. The level of cleaved cytokines (activated cytokines) was calculated by deducting the pro-cytokines from the total drug concentration.

[0488] Fig.18A As shown in Table 9, the concentrations of total drug and procytokine of P41222037 in serum were almost the same, and there were no significant differences in the pharmacokinetic parameters of total drug and procytokine in serum, which meant that P41222037 was very stable in peripheral blood. Figure 18B-Figure 18C The results showed that the intratumoral concentrations of total drug and pro-cytokine of P41222037 reached the peak at 6 hours, and the intratumoral concentration of pro-cytokine was lower than the concentration of total drug after 48 hours ( Fig.18B ), which means that P41222037 is cleaved in the tumor and releases activated cytokines (18C).

[0489]

[0490] For pharmacodynamic studies in peripheral blood, mice were injected i.v. with 5 × 10 6 After 14 days, 4×10 hPBMC cells were cultured in 0.2 ml HBSS containing 0.1 ml Matrigel (1:1). 6 A375 cells were subcutaneously inoculated into the right flank of mice for tumorigenesis. One week after tumor cell inoculation, mice were injected iv with 1 mg / kg PD-1-proIL-2v. Mice in the vehicle group were injected with PBS.

[0491] Anticoagulated blood was collected at 0 hr, 72 hr, 168 hr, 240 hr and 336 hr, and lysed with red blood cell lysis buffer. The cells were stained with staining buffer containing CD45 (clone HI30, BioLegend), CD3 (clone UCHT1, BioLegend), CD4 (clone RPA-T4, BD Pharmingen), CD8 (clone HIT8a, BioLegend) at 4 ° C for 30 min. CD45 was gated using a flow cytometer (CytoFLEX S, Beckman coulter). + CD3 + Cells (CD3 T cells), CD45 + CD3 + CD4 + cells (CD4 T cells) and CD45 +CD3 + CD8 + Cells (CD8 T cells) were used to analyze the samples.

[0492] Figure 19A-Figure 19C The results showed that compared with the vehicle control, CD3 + 、CD4 + and CD8 + T cells in peripheral blood did not differ significantly or increase over time.

[0493] For pharmacodynamic studies in tumors, mice were inoculated subcutaneously in the right flank with 4×10 6 A375 cells and 4×10 4 hPBMCs (co-cultured with A375) were used for tumorigenesis. 18 days after tumor cell inoculation, mice were injected iv with 1 mg / kg PD-1-proIL-2v. Mice in the vehicle group were injected with PBS.

[0494] On day 12, tumors were collected, weighed, and dissociated using a validated tumor dissociation kit (MiltenyiBiotec) according to the protocol. The cell suspension was then filtered through a 70 μm cell strainer. Tumor cells were stained with live / dead (Live / Dead Horizon Fixable Viability Stain 700, BD Pharmingen, for 15 min) in PBS, and then the cells were stained with staining buffer containing CD45 (clone HI30, BioLegend), CD3 (clone UCHT1, BioLegend), CD4 (clone RPA-T4, BD Pharmingen), CD8 (clone HIT8a, BioLegend) at 4 ° C for 30 min. CD45 was gated using a flow cytometer (CytoFLEX S, Beckman coulter). + CD3 + Cells (CD3 T cells), CD45 + CD3 + CD4 + cells (CD4 T cells) and CD45 + CD3 + CD8 + Cells (CD8 T cells) were used to analyze the samples.

[0495] Figure 20A-20CThe results showed that compared with the vehicle control, CD3 + 、CD4 + and CD8 + T cells increased at the tumor site.

[0496] In summary, these data suggest that the PD-1-proIL-2v fusion protein P41222037 is very stable, releases very few activated cytokines in the peripheral blood (reducing toxicity), and can be cleaved in tumors to release activated cytokines to stimulate CD3 + 、CD4 + and CD8 + This stimulates the proliferation of T cells, which reduces toxicity in the peripheral blood and selectively enhances antitumor activity in the TME.

[0497] Example 9

[0498] Biological activity of PD-1-proIL-2v in cynomolgus monkeys

[0499] The in vivo biological activity of masked or unmasked PD-1-proIL-2v was evaluated in cynomolgus monkeys.

[0500] A dose-ranging study was conducted to test the safety profile and pharmacodynamics of PD-1-proIL-2v (P41222037) in cynomolgus monkeys. Each group included 1 male and 1 female cynomolgus monkey, receiving 5 repeated doses of 3 mg / kg, 10 mg / kg and 30 mg / kg of P41222037 (masked PD-1-proIL-2v) or 1 mg / kg of P41252037 (unmasked PD-1-proIL-2v).

[0501] Blood samples for pharmacodynamic studies were collected before dosing and 5min, 1h, 6hr, 24hr, 48hr, 72hr, 96hr, 120hr and 168hr after the first dose. The concentration of P41222037 was determined using ELISA using PD-1 as the capture antibody and anti-IL-2Rα as the detection antibody. The concentration of P41252037 was determined using ELISA using PD-1 as the capture antibody and anti-human IgG conjugated with HRP as the detection antibody.

[0502] Fig.21A The concentration of P41222037 in cynomolgus monkey serum was shown to be dose-dependent in the pro-cytokine exposure and the T of masked PD-1-proIL-2v (P41222037) was significantly higher than that of unmasked PD-1-proIL-2v (P41252037). 1 / 2 Longer.

[0503] Blood samples for serum chemistry were collected before dosing and on days 4, 11, 18, 25, and 30 after dosing. An automated blood analyzer was used to monitor changes in albumin. Fig. 21B It was shown that albumin levels in cynomolgus monkeys treated with 3 mg / kg, 10 mg / kg and 30 mg / kg of P41222037 were not significantly reduced compared to vehicle control. All three doses of P41222037 were well tolerated by all monkeys.

[0504] Example 10

[0505] Effects of linker length and alterations on immune cell stimulation

[0506] Experiments were conducted to test the effect of stable linker length (between Fc and IL-2) on the activity of pre-protein and activated protein on cell proliferation and specific immune cell subsets. Compared with human IL-2, the tested constructs included P41222037 (stable linker of four amino acids), P45412037 (stable linker of eight amino acids) and P45422037 (stable linker of 12 amino acids) with or without MMP-2 protease treatment. Fig.26 The heavy chain / IL-2 junction of the constructs tested is shown.

[0507] Proliferative effect of PD1-proIL-2v on M-07e. 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 the logarithmic growth phase and washed twice with PBS. 90 μl of cell suspension (2×10 4Cells / well) were seeded into 96-well plates and incubated in assay medium (RPMI 1640 supplemented with 10% FBS) for 4 hours at 37°C and 5% CO2 for cytokine starvation. IL-2, PD1-proIL-2v (P41222037, P45412037 and P45422037) or MMP-2-cleaved PD1-proIL-2v protein samples for assay were prepared in assay medium to an initial concentration (IL-2 was 300nM, PD1-proIL-2v was 8100nM, and MMP-2-activated PD1-pro IL-2v fusion protein was 2700nM), followed by 1 / 3 serial dilutions. 10 μl of the diluted protein was added to the corresponding wells and incubated at 37°C and 5% CO2 for 72 hours. A colorimetric assay was performed using a Cell Counting Kit-8 (CCK-8, Dojindo, CK04) to measure the amount of viable cells.

[0508] like Fig.22A As shown, the P41222037 proprotein was unable to induce M-07e proliferation even at the highest concentration tested; MMP-2 cleavage restored the biological activity of P41222037, but the activity was as low as about 1 / 30 relative to wild-type IL-2. Figure 22B-Figure 22C As shown, P45412037 and P45422037 proproteins were inactive at the highest concentration of 810 nM; MMP-2 cleavage restored the activity of P45412037 or P45422037, but the activity was as low as about 1 / 34 or 1 / 25, respectively, relative to wild-type IL-2.

[0509] Phosphorylation of STAT5 after treatment with PD-1-proIL2 on resting human PBMCs. Frozen human PBMCs (SAILYBIO, donor 1) were recovered 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 for approximately 2 hours. The PBMCs were then adjusted to 2.6×10 6 cells / ml, and 4×10 5 Cells were plated in 96-well plates. Recovered PBMCs were incubated with rhIL-2, P41222037, PD-1-proIL-2v linker length variants, or MMP-2 activating protein at 37°C for 15 min.

[0510] After incubation, cells were immediately fixed with Cytofix buffer (BD Bioscience) to maintain phosphorylation status for 15 min on ice and stained with BD Pharmingen TMWash once with 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 (BD557760), CD25 BV421 (BD562442), CD56 BV510 (BD 744218) at 4°C for 30 min, washed once with 1× PBS, centrifuged at 500 g for 5 min, and the supernatant was removed by aspiration. Cells were then permeabilized with pre-cooled Phosflow Perm buffer III (BD Bioscience) at 4°C for 30 min.

[0511] Before starting intracellular staining, cells were washed once with 1× PBS and centrifuged at 500 g for 5 min to collect the cell pellet. 647 (BD 562076) was used to stain the cells at room temperature for 40 min. TM The cells were washed twice with staining buffer (FBS) and centrifuged at 500 g for 5 min to pellet the cells. Each cell pellet was resuspended in 200 ul (per well) of BD Pharmingen staining buffer (FBS) and data were acquired using flow cytometry. The STAT5 phosphorylation status in PBMC subpopulations after prodrug treatment was obtained and processed by CytoFLEX (Beckman).

[0512] Fig.23A Shown with rhIL-2 and complete and activated P41222037 treatment donor 1 resting PBMC, STAT phosphorylation in CD4 T cells, CD8 T cells and regulatory T cells. The complete P41222037 preprotein at a concentration of up to 1000nM cannot induce STAT5 phosphorylation in all tested cell subsets. The protease activated form of P41222037 (MMP-2 cleavage) is equally effective in inducing STAT5 phosphorylation in CD8 and CD4 T cells. However, in Treg cells, the effectiveness of the activated form of P41222037 is as low as about 1 / 30 of huIL-2. Figure 23B-Figure 23C Shown is STAT5 phosphorylation in CD4 T cells, CD8 T cells and Tregs following treatment of resting PBMCs with intact and MMP2-cleaved P45412037 or P45422037 compared to human recombinant IL-2. Fig.23Dshowed that, unlike P41222037, both intact P45412037 and P45422037 induced STAT5 phosphorylation in CD4 T cells and regulatory T cells, but not in CD8 T cells.

[0513] Figure 24A-24C The activity of additional proprotein constructs in this assay is shown (see Fig.26 ). In contrast 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 cells and regulatory T cells, but not in CD8 T cells; the intact P78352037 proprotein had weak activity on STAT5 phosphorylation in CD4 T cells 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 for STAT5 phosphorylation in CD4 T cells and regulatory T cells.

[0514] STAT5 phosphorylation after treatment of preactivated human PBMCs with PD-1-proIL2. Frozen human PBMCs (SAILYBIO, donor 1) were recovered in RPMI-1640 supplemented with 10% FBS, 100U / ml penicillin and 100μg / ml streptomycin at 37°C in an atmosphere of 5% CO2 for 2 hours. For assays performed in preactivated PBMCs, 10-cm plates were coated with αCD3 (1μg / ml, BioLegend) overnight at 4°C. PBMCs suspended in RPMI1640 supplemented with soluble αCD28 (1μg / ml, BioLegend) were cultured for 3 days at 37°C in an atmosphere of 5% CO2. Preactivated PBMCs were adjusted to 2.6×10 6 The cell density was 4×10 cells / ml and 4×10 5 Cells / well were plated in 96-well plates.

[0515] After incubation with rhIL-2 or P41222037 for 15 min at 37°C, cells were immediately fixed with Cytofix buffer (BD Bioscience) to maintain phosphorylation status for 15 min on ice and stained with BD Pharmingen TM Wash once with 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 (Bd562442) at 4°C for 30 min. Wash cells once with 1× PBS and centrifuge cells at 500 g for 5 min. Permeabilize cells with pre-cooled Phosflow Perm buffer III (BD Bioscience) at 4°C for 30 min.

[0516] Prior to intracellular staining, cells were washed once with 1× PBS and centrifuged at 500 g for 5 min. They were then incubated with Foxp3 PE (BD 560046) and anti-Stat5 (pY694) Alexa Fluor 500 (400 μg / mL) at RT. 647 (BD 562076) was used to stain the cells for 40 min. TM The cells were washed twice with staining buffer (FBS) and centrifuged at 500 g for 5 min. Finally, each cell pellet was resuspended in 200 ul (per well) of BD Pharmingen staining buffer (FBS) for FACS data acquisition using flow cytometry. The STAT5 phosphorylation status in PBMC subpopulations after prodrug treatment was obtained and processed by CytoFLEX (Beckman).

[0517] Fig.25 Shown are STAT phosphorylation in CD4 T cells, CD8 T cells, and Treg cells following treatment with rhIL-2 or P41222037 of preactivated PBMCs from donor 1. Up to 300 nM of the intact P41222037 proprotein did not induce STAT5 phosphorylation in all cell subsets tested.

[0518] In general, constructs with shorter stabilizing linkers (e.g., 7 amino acids or less) between Fc and IL-2 in their proprotein form (such as the P41222037 proprotein) did not stimulate immunosuppressive CD4 T cells. reg and certain other immune cells and are therefore unexpectedly useful relative to constructs with longer stabilizing linkers (e.g., 8 amino acids or more) for treating diseases, such as cancer, that benefit from highly controlled activation of anti-tumor immune cell responses.

Claims

1. An activatable preprotein homodimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprise, in the N-terminal to C-terminal direction: A fragment antigen binding (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, wherein the IL-2 protein of the first polypeptide binds to the IL-2Rα protein of the second polypeptide, and wherein the IL-2Rα of the first polypeptide binds to the IL-2 protein of the second polypeptide, wherein the binding masks a binding site of the IL-2 protein that originally binds to IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of an immune cell in vitro or in vivo, And wherein the second linker is a cleavable linker, optionally wherein the first polypeptide and the second polypeptide comprise SEQ ID NO: 146, and further comprise an anti-PD-1 light chain of SEQ ID NO:

147.

2. The activatable proprotein homodimer according to claim 1, wherein the Fab region specifically binds to human PD-1, and optionally comprises a Fab region from an anti-PD-1 antibody selected from nivolumab, pembrolizumab, cemiplizumab, JTX-4014, spartalizumab, carrelizumab, sintilimab, tislelizumab, toripalizumab, dotalimumab, MGA012, AMP-22 and AMP-514.

3. The activatable proprotein homodimer according to claim 1 or 2, wherein the Fab region specifically binds to human PD-1 and comprises a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO: 1, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO: 2; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO:3, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO:4; A heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions shown in SEQ ID NO:5, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions shown in SEQ ID NO:6; or a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO:7, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO:8; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO:9, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO:10; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO: 11, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO: 12; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO: 13, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO: 14; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO: 15, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO: 16; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO: 17, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO: 18; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO: 19, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO: 20; A heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions shown in SEQ ID NO: 21, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions shown in SEQ ID NO: 22; or A heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions shown in SEQ ID NO:23, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions shown in SEQ ID NO:

24.

4. The activatable proprotein homodimer of claim 3, wherein the VH region comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 14, 15, 17, 19, 21 and 23, and the VL region correspondingly comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24.

5. The activatable proprotein homodimer according to claim 1, wherein the Fab region specifically binds to human PD-L1, and optionally comprises a Fab region from an anti-PD-L1 antibody selected from atezolizumab, avelumab and durvalumab.

6. The activatable proprotein homodimer according to claim 1 or 5, wherein the Fab region specifically binds to human PD-L1 and comprises a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO:25, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO:26; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO:27, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO:28; A heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions shown in SEQ ID NO:29, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions shown in SEQ ID NO:30; or a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO:31, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO:32; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO:33, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO:34; A heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions shown in SEQ ID NO:35, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions shown in SEQ ID NO:36; or A heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions shown in SEQ ID NO:37, and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions shown in SEQ ID NO:

38.

7. The activatable proprotein homodimer of claim 6, wherein the VH region comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NOs: 25, 27, 29, 31, 33, 35 and 37, and the VL region correspondingly comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NOs: 26, 28, 30, 32, 34, 36 and 38.

8. The activatable proprotein homodimer of claim 1, wherein the Fab region specifically binds to human B7H3, and wherein the VH region comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 261, and the VL region correspondingly comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

262.

9. The activatable proprotein homodimer according to any one of claims 1 to 8, wherein 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.

10. The activatable proprotein homodimer of any one of claims 1 to 9, wherein the hinge comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from Table F1, and wherein the Fc domain comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from Table F1. 11 . The activatable proprotein homodimer according to any one of claims 1 to 10 , wherein the Fc domain is a modified Fc domain that does not bind or does not substantially bind to FcγR and maintains normal or substantially normal binding to FcRn.

12. The activatable proprotein homodimer of claim 11, wherein the modified Fc domain comprises a modified IgG1 CH2 domain having a L234A / L235A ("LALA") mutation and / or a P329A or P329G mutation (EU numbering).

13. The activatable proprotein homodimer according to any one of claims 1 to 11, wherein the IL-2 protein comprises, consists of, or consists essentially of the following amino acid sequence: An amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 100% identity to an amino acid sequence selected from Table S1, optionally amino acids 21-153 of SEQ ID NO:68 (full length wild-type human IL-2), optionally comprising a C145X (X is any amino acid) or C145S substitution as defined by SEQ ID NO:

68.

14. The activatable proprotein homodimer according to any one of claims 1 to 13, wherein the IL-2 protein comprises, consists of, or consists essentially of the following amino acid sequence: An amino acid sequence that is 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.

15. The activatable proprotein homodimer according to any one of claims 1 to 14, wherein the IL-2 protein comprises one or more substitutions selected from K35C, R38C, T41C, F42C, E61C and V69C as defined by SEQ ID NO:

69.

16. An activatable proprotein homodimer according to any one of claims 1 to 15, wherein the IL-2 protein of the first polypeptide forms a disulfide bond with the IL-2Rα protein of the second polypeptide, and wherein the IL-2Rα protein of the first polypeptide forms a disulfide bond with the IL-2 protein of the second polypeptide, optionally formed through one or more cysteines according to claim 14.

17. The activatable proprotein homodimer according to any one of claims 1 to 16, wherein 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.

18. The activatable proprotein homodimer of any one of claims 1-17, wherein 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 wherein the one or more amino acid substitutions are selected from T3A; 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; E68 A and E68V; and L72A, L72G, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K, including combinations thereof, optionally selected from the following combinations: 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.

19. The activatable proprotein homodimer according to any one of claims 1 to 18, wherein the IL-2 protein comprises, consists of, or consists essentially of the following amino acid sequence: and SEQ ID NO:84 or 85 (human IL-2 mature sequence (26-153)) has an amino acid sequence of at least 80%, 85%, 90%, 95%, 98% or 100% identity, optionally wherein the IL-2 protein comprises or retains the R38D, K43E and C125S substitutions of SEQ ID NO:

85.

20. The activatable proprotein homodimer according to any one of claims 1 to 19, wherein the IL-2Rα protein comprises, consists of, or consists essentially of the following sequence: An amino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 100% identical to an amino acid sequence selected from Table S2, optionally amino acids 22-187 of SEQ ID NO:86 (full length wild-type human IL-2Rα), optionally wherein the IL-2Rα protein comprises, consists of or consists essentially of the following amino acid sequence: an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 100% identical to SEQ ID NO:90, including wherein the IL-2Rα protein retains D6R and / or E29K substitutions.

21. The activatable proprotein homodimer according to claim 20, wherein the IL-2Rα protein comprises one or more cysteine ​​substitutions selected from D4C, D6C, N27C, K38C, S39C, L42C, Y43C, I118C and H120C as defined by SEQ ID NO: 88 (human IL-2Rα Sushi 1 to Sushi2 domain), and / or a K38S substitution.

22. The activatable proprotein homodimer according to claim 20 or 21, wherein the IL-2Rα protein of the first polypeptide forms a disulfide bond with the IL-2 protein of the second polypeptide, and wherein 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 cysteines according to claim 20 and one or more cysteines in the IL-2 protein, optionally via one or more cysteines according to claim 14, optionally via The 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 are formed.

23. The activatable proprotein homodimer according to any one of claims 20-22, wherein the first IL-2Rα protein and the second IL-2Rα protein comprise an alanine substitution at position 49 and / or 68 as defined by SEQ ID NO:

88.

24. The activatable proprotein homodimer of any one of claims 1-23, wherein the hinge of the first polypeptide forms at least one or two disulfide bonds with the hinge of the second polypeptide.

25. An activatable preprotein homodimer according to any one of claims 1-24, wherein the first linker is a non-cleavable or stable linker of 7 or fewer amino acids in length (or 1, 2, 3, 4, 5, 6, 7 amino acids in length), and wherein the cleavable linker comprises a protease cleavage site, optionally wherein the cleavable linker is selected from Table S3, optionally SEQ ID NO:

93.

26. An activatable preprotein homodimer according to any one of claims 1-25, wherein the first linker is a non-cleavable or stable linker, optionally a stable linker of 4 amino acids, such as GGGS (SEQ ID NO: 188), and wherein the cleavable linker comprises a protease cleavage site, optionally wherein the cleavable linker is selected from Table S3, optionally SEQ ID NO:

93.

27. The activatable proprotein homodimer according to claim 26, wherein the protease cleavage site is capable of being cleaved by one or more proteases selected from the group consisting of metalloproteases, serine proteases, cysteine ​​proteases and aspartic proteases.

28. The activatable proprotein homodimer of claim 26 or 27, wherein the protease cleavage site is capable of being cleaved by one or more proteases selected from the group consisting of MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, TEV protease, matriptase, uPA, FAP, legumin, PSA, kallikrein, cathepsin A, and cathepsin B.

29. The activatable proprotein homodimer of any one of claims 1-28, wherein the first linker and / or the second linker is about 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 1-4, 1-3 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, optionally wherein the first linker is about 4 amino acids in length.

30. An activatable proprotein homodimer according to any one of claims 1-29, wherein the Fab comprises SEQ ID NO: 3 (VH) and a human IgG1 CH1 domain, and SEQ ID NO: 4 (VL) and a CL domain (human κ); 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.

31. An activatable proprotein homodimer according to any one of claims 1-30, wherein cleavage of the second linker, optionally protease cleavage of the second linker, exposes one or more binding sites of the IL-2 protein, which bind to the IL-2Rβ / γc chain present on the surface of the immune cell in vitro or in vivo, optionally wherein 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.

32. The activatable proprotein homodimer of any one of claims 1-31, wherein the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of the following amino acid sequence and a VL / CL region polypeptide: The amino acid sequence is at least 80%, 85%, 90%, 95%, 98% or 100% identical to a sequence selected from Table S4 (chain 1 and chain 2), and the VL / CL region polypeptide is at least 80%, 85%, 90%, 95%, 98% or 100% identical to the corresponding sequence from Table S4 (chain 3 and chain 4), optionally wherein: The first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 138 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 140 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 142 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 146 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 148 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 152 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 154 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 156 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 158 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 209 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 211 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 213 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 215 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 217 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 219 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 221 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 223 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 225 and a VL / CL region polypeptide that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 226; the first polypeptide and the second polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 227 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 229 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 231 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 233 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 235 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 237 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 239 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 241 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 243 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 245 and a VL / CL region polypeptide that is 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 that is at least 80%, 85%, 90%, 95%, 98% or 100% identical to SEQ ID NO:247, and a VL / CL region polypeptide that is at least 80%, 85%, 90%, 95%, 98% or 100% identical to SEQ ID NO:

248.

33. The activatable proprotein homodimer according to any one of claims 1 to 32, which is substantially in the form of a homodimer in a physiological solution or under physiological conditions, optionally under in vivo conditions.

34. One or more recombinant nucleic acid molecules encoding the activatable proprotein homodimer according to any one of claims 1-33.

35. The one or more recombinant nucleic acid molecules of claim 34, wherein a first recombinant nucleic acid molecule encodes the VH / CH1 region of the Fab region, a hinge / Fc domain, a first linker, an IL-2 protein, a second linker, and an IL-2Rα protein, and wherein a second nucleic acid molecule encodes the VL / CL region of the Fab region.

36. One or more vectors comprising one or more recombinant nucleic acid molecules according to claim 34 or 35.

37. A host cell comprising one or more recombinant nucleic acid molecules according to claim 34 or 35 or one or more vectors according to claim 36.

38. A method for producing an activatable proprotein, the method comprising culturing the host cell according to claim 35 under culture conditions suitable for expressing an activatable proprotein homodimer, and isolating the activatable proprotein from the culture.

39. A pharmaceutical composition comprising the activatable proprotein homodimer according to any one of claims 1-33 and a pharmaceutically acceptable carrier.

40. A method of treating a disease in a subject, and / or a method of enhancing an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to claim 39.

41. The method of claim 40, wherein the disease is cancer, optionally a cancer that expresses or overexpresses PD-L1.

42. The method of claim 41, wherein the cancer is a primary cancer or a metastatic cancer and is selected from one or more of the following cancers: 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, liver cancer (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.

43. The method of any one of claims 40-42, wherein after administration, the activatable proprotein homodimer is activated by protease cleavage in cancer cells or cancer tissues or tumor microenvironment (TME), wherein the protease cleavage exposes one or more binding sites of the IL-2 protein that bind to the IL-2Rβ / γc chain present on the surface of immune cells in vitro or in vivo, thereby producing an activated protein.

44. The method of claim 43, wherein the activated protein binds to the IL-2Rβ / γc chain present on the surface of an immune cell in vitro or in vivo through the IL-2 protein.

45. The method of claim 44, wherein the immune cells are selected from one or more of T cells, B cells, natural killer cells, monocytes and macrophages.

46. ​​The method of any one of claims 40-45, wherein administration and activation of the activatable proprotein increases the anti-cancer immune response in the 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 relative to a control.

47. The method of any one of claims 40-46, wherein administration and activation of the activatable proprotein increases cancer cell killing in the 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 relative to a control.

48. The method of any one of claims 40-47, wherein the pharmaceutical composition is administered to the subject by parenteral administration.

49. The method of claim 48, wherein the parenteral administration is intravenous administration.

50. Use of the pharmaceutical composition according to claim 39 in the preparation of a medicament for treating a disease in a subject, optionally cancer (e.g., a cancer expressing or overexpressing PD-L1) and / or for enhancing an immune response in a subject.

51. A pharmaceutical composition according to claim 39, for use in treating a disease in a subject, optionally cancer (e.g., a cancer that expresses or overexpresses PD-L1), and / or for use in enhancing an immune response in a subject.

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