IL-15 procytokine antibody fusion proteins

Abstract

The present invention provides activatable preprotein 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-15 protein, a protease cleavable linker, and an IL-15R [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 of U.S. Provisional Application No. 63 / 402,639, filed on August 31, 2022, under 35 U.S.C.§119(e), which is incorporated herein by reference in its entirety.

[0003] Statement Regarding Sequence Listing

[0004] The Sequence Listing XML related to this application is provided in XML file format and is hereby incorporated by reference into this specification. The name of the XML file containing the Sequence Listing XML is PRVA_016_01WO_ST26.xml. This XML file is approximately 212,188 bytes, was created on August 28, 2023, and was submitted electronically via the USPTO Patent Center. Background Field of Technology

[0006] The present invention relates to activatable pro - protein homodimers, which comprise two independent 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 - 15 protein, a protease - cleavable linker, and an IL - 15Rα protein. Also included are related pharmaceutical compositions and methods of use thereof.

[0007] Description of Related Art

[0008] Interleukin - 15 (IL - 15) immunotherapy has been shown to be useful in 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 therapies enhance anti - tumor T - cell responses and mediate anti - tumor activity (Dermani et al., J Cell Physiol. 234:1313 - 1325, 2019).

[0009] However, there are certain problems associated with most IL-15 therapies. For example, IL-15 has been shown to have a short half-life and may require high doses to achieve a biological response in vivo, resulting in clinical toxicity to patients and limited anti-tumor responses. IL-15 and IL-15 derivatives are being developed to improve therapeutic efficacy. However, there are significant drawbacks, including high serum Cmax that initially causes overactivation of the immune system, short PK due to the smaller molecular size of IL-15 (13-14 kD) or catabolism by a large number of immune cells expressing the IL-15 receptor of the IL-15 or IL-15Fc fusion protein, poor accumulation in the target tumor due to short PK, lack or inefficiency of tumor targeting, and undesired accumulation and immune activation activity in normal tissues. Nevertheless, IL-15 therapy remains effective, and there are strategies for addressing these and other drawbacks (see, for example, WO 2020 / 0123980). However, there is still a need in the art to improve such strategies.

[0010] Some embodiments of the present invention represent such an improvement by providing an anti-PD-1 / PD-L1 activatable proprotein comprising IL-15, which can be specifically targeted to the tumor microenviroment (TME) and activated therein.

[0011] Brief Overview

[0012] Some embodiments of the present invention include an activatable proprotein 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-15 protein, a second linker, and an IL-15Rα protein; wherein the hinge / Fc domain of the first polypeptide binds to the hinge / Fc domain of the second polypeptide, wherein the IL-15 protein of the first polypeptide binds to the IL-15Rα protein of the second polypeptide, and wherein the IL-15Rα of the first polypeptide binds to the IL-15 protein of the second polypeptide, wherein the binding masks the binding site of the IL-15 protein that would otherwise bind to the IL-15Rβ / γc chain 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, cemiplimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, toripalimab, dostarlimab, 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 having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 14, 15, 17, 19, 21, and 23, and the VL region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24.

[0027] In some embodiments, the Fab region specifically binds to human PD-L1 and optionally comprises the 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 VH CDR1, VH CDR2, and VH CDR3 regions shown in SEQ ID NO:25; and a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 regions shown in SEQ ID NO:26;

[0029] a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 regions shown in SEQ ID NO:27; and a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 regions shown in SEQ ID NO:28;

[0030] a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 regions shown in SEQ ID NO:29; and a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 regions shown in SEQ ID NO:30; or

[0031] a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 regions shown in SEQ ID NO:31; and a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 regions shown in SEQ ID NO:32;

[0032] a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 regions shown in SEQ ID NO:33; and a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 regions shown in SEQ ID NO:34;

[0033] a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 regions shown in SEQ ID NO:35; and a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 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 having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NO:25, 27, 29, 31, 33, 35, and 37, and the VL region correspondingly comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NO:26, 28, 30, 32, 34, 36, and 38.

[0036] In some embodiments, the Fab region specifically binds to human B7H3 and comprises a heavy chain variable (VH) region containing the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO:202; and a light chain variable (VL) region containing the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO:203. In some specific embodiments, the VH region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:202, and the VL region correspondingly comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:203.

[0037] In some embodiments, the Fc domain comprises the CH2 domain, CH3 domain, or CH2CH3 domain of an immunoglobulin, optionally wherein the immunoglobulin is from an immunoglobulin class selected from IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM. In some embodiments, the hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from Table F1, and wherein the Fc domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from Table F1. In some embodiments, the Fc domain is a modified Fc domain that does not bind or binds substantially non-specifically 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 an L234A / L235A (“LALA”) mutation and / or a P329A or P329G mutation (EU numbering).

[0038] In some embodiments, the IL-15 protein comprises, consists of, or consists essentially of 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 wherein the IL-15 protein comprises, consists of, or consists essentially of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO:79 and retaining the K86G and S162A mutations. In some embodiments, the IL-215 protein comprises or retains one or more amino acid substitutions at position D8, D22, E46, V49, I50, L66, and / or K86 as defined by SEQ ID NO:69 (mature human IL-15) and / or at S162 as defined by SEQ ID NO:68 (IL-15FL precursor). In some embodiments, the one or more amino acid substitutions are selected from D8N, D22K, E46K, V49D, I50D, L66E, K86G, and 162A, optionally in combination with K86G and S162A. In some embodiments, the one or more amino acid substitutions are a combination selected from K86G and 162A; V49D and S162A; I50D and S162A; L66E and S162A; D8N and S162A; V49D and S162A; E46K and S162A; E46K, E53K, and S162A; D22K, E46K, and S162A; and D22K, E46K, E53K, and S162A.

[0039] In some embodiments, the IL-15Rα protein comprises, consists of, or consists essentially of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to a sequence selected from Table S2, optionally wherein the IL-15Rα protein comprises, consists of, or consists essentially of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO:87 or 88 and retaining the T2A substitution. In some embodiments, the IL-15Rα protein comprises or retains one or more amino acid substitutions at positions R24, R26, and R35 as defined by SEQ ID NO:82 (IL-15Rα Sushi+). In some embodiments, the one or more amino acid substitutions are selected from R24E, R26E, and R35E. In some embodiments, the one or more amino acid substitutions are selected from the combination of R26E and R35E; and R24E, R26E, and R35. In some embodiments, the IL-15Rα protein comprises or retains an amino acid substitution at position T2 as defined by SEQ ID NO:82 (IL-15Rα Sushi+). In some embodiments, the amino acid substitution is T2A. In some embodiments, the IL-15α protein comprises SEQ ID NO:82 or 83 having the T2A substitution.

[0040] 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 an uncleavable or stable linker, and wherein the cleavable linker comprises a protease cleavage site, optionally wherein the cleavable linker is selected from Table S3.

[0041] In some embodiments, the protease cleavage site is cleavable by a protease selected from one or more of metalloproteases, serine proteases, cysteine proteases, and aspartic proteases. In some embodiments, the protease cleavage site is cleavable by a protease selected from one or more of MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, TEV protease, matriptase, uPA, FAP, podoplanin, 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.

[0042] In some embodiments, the Fab comprises SEQ ID NO:3 (VH) and the human IgG1 CH1 domain, and SEQ ID NO:4 (VL) and the CL domain (human κ); the Fc domain comprises the IgG1 hinge of SEQ ID NO:42, the modified human IgG1 CH2 domain of SEQ ID NO:57, and the human IgG1 CH3 domain of SEQ ID NO:58; the first linker is an 8-amino acid stable linker of SEQ ID NO: (178, where x is 2); the IL-15 protein comprises SEQ ID NO:79, optionally having K86G and S162A mutations; the second linker is a protease-cleavable linker of SEQ ID NO:90 or SEQ ID NO:201; and the IL-15Rα protein comprises SEQ ID NO:87, optionally having T2A mutations.

[0043] In some embodiments, cleavage of the second linker (optionally protease cleavage) exposes one or more binding sites of the IL-15 protein that bind to the IL-15Rβ / γc chain present on the surface of immune cells in vitro or in vivo. In some embodiments, the immune cells are selected from one or more of T cells, B cells, natural killer cells, monocytes, and macrophages.

[0044] In some embodiments, the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of the following amino acid sequences and VL / CL region polypeptides: the amino acid sequences have at least 80%, 85%, 90%, 95%, 98%, or 100% identity to the sequences (chain 1 and chain 2) selected from Table S4, and the VL / CL region polypeptides have at least 80%, 85%, 90%, 95%, 98%, or 100% identity to the corresponding sequences (chain 3 and chain 4) from Table S4, including where:

[0045] The first polypeptide and the second polypeptide comprise, consist of, or consist essentially of the following amino acid sequences and VL / CL region polypeptides: an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 136 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 137;

[0046] The first polypeptide and the second polypeptide comprise, consist of, or consist essentially of the following amino acid sequences and VL / CL region polypeptides: an amino acid sequence 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 comprise, consist of, or consist essentially of the following amino acid sequences and VL / CL region polypeptides: an amino acid sequence 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 comprise, consist of, or consist essentially of the following amino acid sequences and VL / CL region polypeptides: an amino acid sequence 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 comprise, consist of, or consist essentially of the following amino acid sequences and VL / CL region polypeptides: an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 100% identity to SEQ ID NO: 144 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98% or 100% identity to SEQ ID NO: 145;

[0050] The first polypeptide and the second polypeptide comprise, consist of, or consist essentially of the following amino acid sequences and VL / CL region polypeptides: an amino acid sequence 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;

[0051] The first polypeptide and the second polypeptide comprise, consist of, or consist essentially of the following amino acid sequences and VL / CL region polypeptides: an amino acid sequence 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; or

[0052] The first polypeptide and the second polypeptide comprise, consist of, or consist essentially of the following amino acid sequences and VL / CL region polypeptides: an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 100% identity to SEQ ID NO: 199 and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98% or 100% identity to SEQ ID NO: 200.

[0053] Certain activatable proprotein homodimers described herein are substantially in homodimer form in physiological solution, or under physiological conditions, optionally under in vivo conditions.

[0054] Also included are one or more recombinant nucleic acid molecules encoding the activatable proprotein homodimers described herein. In some embodiments, a first recombinant nucleic acid molecule encodes the VH / CH1 region of the Fab region, the hinge / Fc domain, the first linker, the IL-15 protein, the second linker, and the IL-15Rα protein, and a second nucleic acid molecule encodes the VL / CL region of the Fab region. Also included are one or more vectors comprising one or more of the recombinant nucleic acid molecules described herein. Certain embodiments include host cells comprising one or more of the recombinant nucleic acid molecules described herein, or one or more of the vectors described herein.

[0055] Certain embodiments include methods of producing an activatable proprotein, which include culturing the host cells described herein under culture conditions suitable for the expression of the activatable proprotein homodimer, and isolating the activatable proprotein from the culture.

[0056] Certain embodiments include methods of producing an activatable proprotein, which include culturing the host cells described herein under culture conditions suitable for the expression of the activatable proprotein homodimer, and isolating the activatable proprotein from the culture.

[0057] Also included are pharmaceutical compositions comprising the activatable proprotein homodimer described herein and a pharmaceutically acceptable carrier.

[0058] Certain embodiments relate to methods of treating a disease in a subject, and / or enhancing an immune response in a subject, which include administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein.

[0059] In some embodiments, the disease is cancer, e.g., cancer that expresses or overexpresses PD-L1 or B7H3. In some embodiments, the cancer is primary cancer or 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 myelogenous leukemia, acute myeloid leukemia or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, liver cancer (hepatocellular carcinoma), sarcoma, B cell malignancy, 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.

[0060] In certain embodiments, after administration, the activatable proprotein homodimer is activated by protease cleavage in cancer cells or cancer tissue or the tumor microenvironment (TME), which exposes one or more binding sites of the IL-15 protein that bind to the IL-15Rβ / 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 binds to the IL-15Rβ / γc chain present on the surface of immune cells in vitro or in vivo by the IL-15 protein. In some embodiments, the immune cells are selected from one or more of T cells, B cells, natural killer cells, monocytes, and macrophages.

[0061] In some embodiments, the 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, the 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.

[0062] In some embodiments, the pharmaceutical composition is administered to the subject by parenteral administration. In some embodiments, the parenteral administration is intravenous administration.

[0063] Certain embodiments include the use of the pharmaceutical composition described herein in the preparation of a medicament for treating a disease of a subject (optionally cancer (such as cancer expressing or overexpressing PD-L1)) and / or for enhancing the immune response of a subject. Certain embodiments include the pharmaceutical composition described herein for treating a disease of a subject (optionally cancer (such as cancer expressing or overexpressing PD-L1 or B7H3)) and / or for enhancing the immune response of a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figures 1A - 1B An exemplary structure of a single polypeptide (IgG-proIL-15 motif) is shown, which forms a homodimer (not shown) with a second polypeptide having the same structure. The IgG-proIL-15 motif consists of a high-affinity neutralizing antibody (e.g., without FcγR binding and with intact FcRn binding) and an IL-15 pro-cytokine module.

[0065] Figure 2 An exemplary structure of an activatable proprotein homodimer in its inactive (pro-cytokine) state is shown. The dashed line represents a cleavable linker. The intact IgG-proIL-15 construct shows no or little IL-15 activity because the IL-15Rβγ binding site is "masked" in this form.

[0066] Figure 3Illustrated is the "activation" of homodimeric IL-15 resulting from proteolytic cleavage of the protease-cleavable linker in the TME. The protease-cleavable linker between IL-15 and IL-15Rα is stable in peripheral blood but can be cleaved by tumor proteases in the TME, thereby releasing active IL-15 at the tumor site. This can reduce toxicity in peripheral blood and enhance anti-tumor activity.

[0067] Figures 4A - 4B Shown are the ELISA binding activities of (A) PD-1-proIL-15 to human PD-1 compared to PD-1IgG, and (B) PD-L1-proIL-15 to PD-L1 compared to PD-L1 IgG.

[0068] Figures 5A - 5B Shown is the proliferation of the human acute megakaryoblastic leukemia cell line M-07e induced by: (A) protease-activated PD-1-proIL-15 compared to human recombinant IL-15, or (B) protease-activated PD-L1-proIL-15 compared to human recombinant IL-15.

[0069] Figures 6A - 6D Shown are the results of STAT5 phosphorylation assays of intact and protease-activated PD-1-proIL-15 in resting PBMCs (CD4 T cells (A), CD8 T cells (B), regulatory T cells (C), and NK cells (D) of donor 1; legend on 6B).

[0070] Figures 7A - 7D Shown are the results of STAT5 phosphorylation assays of intact and protease-activated PD-L1-IL-15 in activated PBMCs (CD4 T cells (A), CD8 T cells (B), regulatory T cells (C), and NK cells (D) of donor 1; legend on 7B).

[0071] Figures 8A - 8D Shown are the results of STAT5 phosphorylation assays of intact and protease-activated PD-L1-IL-15 in resting PBMCs (CD4 T cells (A), CD8 T cells (B), regulatory T cells (C), and NK cells (D) of donor 2; legend on 8B).

[0072] Figure 9 Shown is the dose-dependent IFN-γ secretion of PBMCs from donor 1 after 3 days of in vitro stimulation with increasing concentrations of both intact protease-activated PD-1-proIL-15 and PD-L1-proIL-15.

[0073] Figure 10AShows the mean tumor volume measured over time in the A375-PBMC xenograft model, demonstrating the in vivo anti-tumor activity of B7H3-proIL-15 with different protease-cleavable linkers as a single agent in inhibiting tumor growth. Figure 10B Shows Figure 10A the mean tumor volume at day 22 of the same treatment in. Mice (n = 6) were injected i.v. at day 0, day 3, day 7, day 10, and day 14; results are expressed as mean ± S.E.M.

[0074] Figure 11A Shows the mean tumor volume measured over time in the A375-PBMC xenograft model, demonstrating the in vivo dose-dependent anti-tumor activity of PD-L1-proIL-15 as a single agent in inhibiting tumor growth. Figure 11B Shows the mean tumor volume measured over time in the A375-PBMC xenograft model, demonstrating the in vivo dose-dependent anti-tumor activity of PD-1-proIL-15 as a single agent in inhibiting tumor growth. Mice (n = 6 per group) were injected i.v. at day 0, day 7, and day 14; results are expressed as mean ± S.E.M.

[0075] Figure 12 Shows the mean tumor volume measured over time in the B16F10 syngeneic model, demonstrating the in vivo anti-tumor activity of mPD-1-proIL-15 (P55654367) as a single agent in inhibiting tumor growth. Mice (n = 6) were injected i.v. at day 0 and day 3; results are expressed as mean ± S.E.M.

[0076] Figure 13A Shows the concentration of PD-1-proIL-15 over time in cynomolgus monkey peripheral blood. Figures 13B - 13D Shows the expression of Ki67 in NK (B), CD4 (C), and CD8 (D) cells in cynomolgus monkey peripheral blood. Figures 13E - 13G Shows NK (E), CD4 + (F), and CD8 + (G) cell fold change in cell number over time in cynomolgus monkeys. Monkeys (n = 2) were injected i.v. with P53052037 at 10 mg / kg, Q2W. Circles = male; squares = female.

[0077] Figures 14A - 14DShows STAT5 phosphorylation in CD4 T cells (14A), CD8 T cells (14B), regulatory T cells (14C), and NK cells (14D) after treatment of resting PBMCs with rhIL-15 and full-length and activated PD-1-proIL-15. The proprotein form of P79772037 was unable to induce STAT5 phosphorylation in all tested cell subsets. The activated form of P79772037 (matrix metalloproteinase-2 cleaved) was equally effective in activating STAT5 phosphorylation in CD8 and CD4 T cells, but less potent in NK cells.

[0078] Figures 15A - 15G Shows the purification and characterization of PD-1-proIL-15 (P53052037, P79772037), PD-L1-proIL-15 (P53021942), and B7H3-proIL-15 (P40503699, P40743699). Figure 15A Shows the results of reduced SDS-PAGE analysis, Figure 15B Shows the results of non-reduced SDS-PAGE analysis. Figures 15C - 15G Shows the results of size exclusion chromatography (SEC-HPLC) analysis indicating high purity and homogeneity of the product (no significant amounts of aggregated or degraded products).

[0079] Detailed description

[0080] Some embodiments of the invention relate to activatable proprotein homodimers that comprise two separate but identical polypeptides, each polypeptide comprising, in the N-terminal to C-terminal direction, a fragment antigen binding (Fab) region that specifically binds human PD-1 or human PD-L1 or human B7H3, a hinge / Fc domain, a stable linker, an IL-15 protein, a protease-cleavable linker, and an IL-15Rα protein. In some cases, the homodimer is formed by the following binding interactions: binding of the hinge / Fc domain of the first polypeptide to the hinge / Fc domain of the second polypeptide, and binding of each IL-15 protein of one polypeptide to each IL-15Rα protein of the other polypeptide. These binding interactions form an inactive (proprotein) homodimer by masking the binding sites of the IL-15 proteins that would otherwise bind to the IL-15Rβ / γc chains present on the surface of immune cells. The proprotein homodimer remains inactive or substantially inactive in plasma.

[0081] In some cases, the homodimer targets the tumor microenvironment (TME) via an anti-PD-1 or anti-PD-L1 Fab and is activated within the TME by exposure to a tumor-site protease that cleaves a protease-cleavable linker, thereby activating one or more IL-15 proteins (see, e.g., WO 2020 / 123980). This results in a synergistic effect between the anti-tumor activity of the anti-PD-1 / anti-PD-L1 Fab and the immune-stimulatory activity of the IL-15 protein. In some cases, the homodimer targets the tumor microenvironment (TME) via an anti-B7H3 Fab and is activated within the TME by exposure to a tumor-site protease that cleaves a protease-cleavable linker, thereby reactivating the one or more IL-15 proteins (see, e.g., WO2020 / 123980). This results in a synergistic effect between the anti-tumor activity of the anti-B7H3 Fab and the immune-stimulatory activity of the IL-15 protein.

[0082] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, materials, compositions, reagents, cells similar or equivalent to those described herein can be used in the practice or testing of the subject matter of this invention, the preferred methods and materials are described. All publications and references cited in this specification (including but not limited to patents and patent applications) are incorporated herein by reference in their entirety as if each individual publication or reference were specifically and separately indicated to be incorporated by reference in its entirety as fully set forth. Any patent application to which this application claims priority is also incorporated herein by reference in its entirety in the manner described above for publications and references.

[0083] Standard techniques are available for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions, or as commonly accomplished in the art or as described herein. These and related techniques and procedures can generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. Unless otherwise provided with specific definitions, the nomenclature used herein in connection with molecular biology, analytical chemistry, synthetic organic chemistry, medicine, and medicinal chemistry, as well as the laboratory procedures and techniques of these disciplines, are well known and commonly used in the art. Standard techniques are available for recombinant techniques, molecular biology, microbiology, chemical synthesis, chemical analysis, drug preparation, formulation, delivery, and treatment of patients.

[0084] For the purposes of this invention, the following terms are defined as follows.

[0085] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) grammatical object of the article. For example, "an element" includes "one element", "one or more elements" and / or "at least one element".

[0086] "About" means 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.

[0087] The terms "activatable proprotein", "proprotein", "activatable pro-cytokine", "pro-cytokine", "activatable prodrug" and "prodrug" are used interchangeably herein and refer to an activatable proprotein that contains at least one masking moiety and an active domain, or a derivative / variant thereof, as described herein. In one embodiment, the proprotein may also contain one or more protein domains.

[0088] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent (such as an antibody) and that can additionally be used in an animal to generate an antibody that can bind the antigen and an epitope. An antigen may have one or more epitopes. As used herein, the term "antigen" includes such substances that, under appropriate conditions, are capable of inducing an immune response to the substance and are capable of reacting with the products of the immune response. More broadly, the term "antigen" includes any substance to which an antibody binds, or any substance for which an antibody 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.

[0089] "Antagonist" refers to a biological structure or chemical agent that interferes with or otherwise attenuates the physiological action of the other agent or molecule. In some cases, the antagonist specifically binds the other agent or molecule. Complete and partial antagonists are included.

[0090] "Agonist" refers to a biological structure or chemical agent that increases or enhances the physiological action of another agent or molecule. In some cases, the agonist specifically binds another agent or molecule. Complete and partial agonists are included.

[0091] As used herein, the term "amino acid" is intended to refer to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimetics. For example, naturally occurring amino acids include the 20 (L)-amino acids utilized in protein biosynthesis, 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, etc., known to those of skill in the art. Amino acid analogs include modified forms of natural and non-natural amino acids. Such modifications can include, for example, the substitution or replacement of chemical groups and moieties on the amino acid, or the derivatization of the amino acid. Amino acid mimetics include, for example, organic structures that exhibit functional similarity properties, such as the charge and charge-spacing characteristics of a reference amino acid. For example, an organic structure that mimics arginine (Arg or R) will have a positive charge moiety located in a similar molecular space and having 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 in order to maintain optimal spacing and charge interactions of the amino acid or amino acid functional groups. Those of skill in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0092] As used herein, a subject "at risk" for developing a disease or adverse reaction may or may not have a detectable disease or disease symptom, and may or may not exhibit a detectable disease or disease symptom 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 the disease. A subject having one or more of these risk factors has a higher likelihood of developing the disease or adverse reaction than a subject without one or more of these risk factors.

[0093] "Biocompatible" means a material or compound that is generally not harmful to the biological functions of cells or a subject and does not cause any degree of unacceptable toxicity (including allergic and disease states).

[0094] The term "binding" refers to the direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bond interactions (including interactions such as salt bridges and water bridges).

[0095] "Coding sequence" means any nucleic acid sequence that contributes to encoding the polypeptide product of a gene. In contrast, the term "non-coding sequence" means any nucleic acid sequence that does not directly participate in encoding the polypeptide product of a gene.

[0096] Throughout the disclosure, unless the context requires otherwise, the words "comprise", "include" and "have" will be understood to imply the inclusion of the stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0097] "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 for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present, depending on whether they substantially affect the activity or action of the listed elements.

[0098] The terms "endotoxin-free" or "substantially endotoxin-free" generally refer to compositions, solvents, and / or containers that contain a maximum trace amount (e.g., an amount that has no clinically adverse physiological effects on a subject) of endotoxin (preferably undetectable amounts of endotoxin). Endotoxins are toxins associated with certain microorganisms such as bacteria (usually Gram-negative bacteria), although endotoxins can 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 major virulence characteristics of these bacteria's pathogenicity. Small amounts of endotoxin in the human body can cause fever, decreased blood pressure, inflammation, activation of blood clotting, and other adverse physiological effects.

[0099] Therefore, in drug production, it is generally necessary to remove most or all of the trace endotoxin from the drug product and / or drug container, because even small amounts of endotoxin can have side effects on the human body. Since decomposing most endotoxins usually requires temperatures above 300°C, a pyrogen 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-log reduction in endotoxin levels. Other methods for removing endotoxin are covered, including, for example, chromatography and filtration methods described herein and known in the art.

[0100] Endotoxin can be detected using conventional techniques known in the art. For example, the Limulus amoebocyte lysate assay using the blood of the horseshoe crab is a very sensitive assay for detecting the presence of endotoxin. In this test, very low levels of LPS can cause detectable coagulation of the Limulus lysate because a powerful enzymatic cascade amplifies the reaction. Endotoxin can also be quantified by enzyme-linked immunosorbent assay (ELISA). To achieve substantially endotoxin-free, 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 the active compound. Generally, 1 ng of lipopolysaccharide (LPS) is equivalent to about 1 - 10 EU.

[0101] The term "half maximal effective concentration" or "EC 50 " refers to the concentration at which the agent (e.g., a proprotein activator) described herein induces a reaction that is half way between the baseline and the maximum after a specified exposure time; thus, the EC 50 of a stepwise dose-response curve represents the concentration of the compound at which 50% of its maximum effect is observed. The EC 50 also represents the plasma concentration required to achieve 50% of the maximum effect in vivo. Similarly, "EC 90 " refers to the concentration of the agent or composition at which 90% of its maximum effect is observed for the agent or composition. "EC 90 " can be calculated from "EC 50 " and the Hill slope, or it can be determined directly from the data using conventional knowledge in the art. In some embodiments, the EC 50 of the agent (e.g., a proprotein activator) is 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, 25 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM or 500 nM. In some embodiments, the agent will have an EC50 Value

[0102] "Immune response" refers to any immune response originating from the immune system, including responses from the cellular and humoral, innate and adaptive immune systems. Exemplary cellular immune cells include, for example, lymphocytes, macrophages, T cells, B cells, NK cells, neutrophils, eosinophils, dendritic cells, mast cells, monocytes, and all of their subsets. Cellular responses include, for example, effector functions, cytokine release, phagocytosis, efferocytosis, transmigration, trafficking, proliferation, differentiation, activation, inhibition, cell-cell interactions, apoptosis, and the like. Humoral responses include, for example, IgG, IgM, IgA, IgE responses, and their corresponding effector functions.

[0103] The "half-life" of an agent such as a proprotein activator can refer to the time it takes for the agent to lose half of its pharmacological, physiological, or other activity relative to when it is administered into the serum or tissue of an organism or relative to any other defined time point. The "half-life" can also refer to the time it takes for the amount or concentration of the agent to be reduced to half of the starting amount administered into the serum or tissue of an organism relative to when it is administered into the serum or tissue of an organism or relative to any other defined time point. The half-life can be measured in serum and / or any one or more selected tissues.

[0104] The terms "modulate" and "alter" include "increase" or "enhance" as well as "decrease" or "reduce" in amounts or degrees that are typically statistically significant or physiologically significant relative to a control. An "increased" or "enhanced" amount is typically a "statistically significant" amount and can 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 the control. An "increased" or "enhanced" amount can also include an amount that is 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 the control. A "decreased" or "reduced" 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 relative to the control. A "decreased" or "reduced" amount can also include an amount that is 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000% or 5000% less than the control. Examples of comparing and "statistically significant" amounts are described herein.

[0105] The terms "polypeptide", "protein", and "peptide" are used interchangeably and refer to amino acid polymers of any specific length. The term "enzyme" includes polypeptide or protein catalysts. The terms include modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and addition or deletion of signal sequences. The term "polypeptide" or "protein" means one or more chains of amino acids, where each chain contains amino acids covalently linked by peptide bonds, and where the polypeptide or protein may contain multiple chains non-covalently and / or covalently linked together by peptide bonds (which have the sequence of a native protein (i.e., a protein produced by a naturally occurring, especially non-recombinant cell or a genetically engineered cell or a recombinant cell)), and includes molecules having the amino acid sequence of a native protein, or molecules having deletions, additions, and / or substitutions of one or more amino acids of the native sequence. In certain embodiments, a polypeptide is a "recombinant" polypeptide produced by a recombinant cell containing one or more recombinant DNA molecules, which are typically made up of a combination of heterologous polynucleotide sequences or polynucleotide sequences not originally present in the cell.

[0106] The terms "polynucleotide" and "nucleic acid" include mRNA, RNA, cRNA, cDNA, and DNA. The term generally refers to polymeric forms of nucleotides of at least 10 bases in length, ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. The term includes single-stranded and double-stranded forms of DNA. The terms "isolated DNA" and "isolated polynucleotide" and "isolated nucleic acid" 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 containing one or more coding sequences, but which is substantially separated or purified from the total genomic DNA of the species from which the DNA segment was obtained. Also included are non-coding polynucleotides (e.g., primers, probes, oligonucleotides) that do not encode polypeptides. Also included are recombinant vectors, including, for example, expression vectors, viral vectors, plasmids, cosmids, phagemids, phages, viruses, etc.

[0107] Additional coding or non-coding sequences may, but need not, be present in the polynucleotides described herein, and the polynucleotides may, but need not, be linked to other molecules and / or support materials. Thus, a polynucleotide or an expressible polynucleotide, regardless of the length of the coding sequence itself, may be combined with other sequences, such as expression control sequences.

[0108] The term "isolated" polypeptide or protein as used herein means a subject protein that (1) is free of at least some of the other proteins that are normally associated with it in nature, (2) is substantially free of other proteins from the same source, e.g., from the same species, (3) is expressed by cells from different species, (4) has been separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials that are associated with it in nature, (5) does not bind (by covalent or non-covalent interactions) to the protein moieties that are bound to the "isolated protein" in nature, (6) is operably bound (by covalent or non-covalent interactions) to a polypeptide that is not bound to it in nature, or (7) does not exist in nature. Such isolated proteins can be encoded by genomic DNA, cDNA, mRNA, or other RNA, can be of synthetic origin, or any combination thereof. In certain embodiments, the isolated protein is substantially free of proteins or polypeptides or other contaminants that are present in its native environment and that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or other uses).

[0109] In certain embodiments, the "purity" of any given agent (e.g., an activatable proprotein) in a composition can be defined. For example, certain compositions can contain an agent, such as a polypeptide agent, that has 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), measured, for example and without limitation, by high performance liquid chromatography (HPLC), which is a well-known form of column chromatography in biochemistry and analytical chemistry that is often used to separate, identify, and quantify compounds, on a protein basis or a weight-by-weight basis.

[0110] The term "reference sequence" generally refers to a nucleic acid coding sequence or an amino acid sequence that is compared to another sequence. All polypeptide and polynucleotide sequences described herein are included as reference sequences, including sequences described by name as well as sequences described in tables and sequence listings.

[0111] Certain embodiments include bioactive “variants” and “fragments” of the proteins / polypeptides described herein, as well as polynucleotides encoding them. A “variant” contains one or more substitutions, additions, deletions, and / or insertions relative to a reference polypeptide or polynucleotide (see, e.g., the tables and sequence listings). Variant polypeptides or polynucleotides contain amino acid or nucleotide sequences 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 the reference sequence described herein, and substantially retain the activity of that reference sequence. Also included are sequences consisting of or differing from the reference sequence by the addition, deletion, insertion, or substitution 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, and that substantially retain at least one activity of the reference sequence. In certain embodiments, the addition or deletion includes C-terminal and / or N-terminal additions and / or deletions.

[0112] As used herein, the term "sequence identity" or, for example, "a sequence that is 50% identical to" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis in a comparison window. Thus, the "percentage of sequence identity" can be calculated by the following process: comparing two optimally aligned sequences in a comparison window, determining the number of positions at which the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occurs in both sequences to yield 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 give the percentage of sequence identity. The optimal alignment of sequences for aligning the comparison window can be accomplished by computerized algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by visual inspection and the best alignment generated by any of a variety of methods selected (i.e., that yields the highest percentage of homology in the comparison window). Reference may also be made, for example, to the BLAST family of programs disclosed by Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[0113] The term "solubility" refers to the property of an agent provided herein (e.g., a proprotein activatable) to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as a concentration, or as the mass of solute per unit volume of solvent (grams of solute per kilogram of solvent, grams per deciliter (100 mL), mg / ml, etc.), molarity, molality, mole fraction, or other similar concentration descriptions. The maximum equilibrium amount of solute that can be dissolved per unit amount of solvent is the solubility of that solute in that solvent under specified conditions, including temperature, pressure, pH, and the nature of the solvent. In certain embodiments, solubility is measured at physiological pH or other pH values, e.g., 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). In certain embodiments, solubility is measured in water or a physiological buffer such as PBS or NaCl (with or without NaPO4). In a specific embodiment, solubility is measured at a 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

[0114] "Subject" or "subject in need" or "patient" or "patient in need" includes mammalian subjects, such as human subjects.

[0115] "Substantially" or "substantially" means almost entirely or completely, e.g., 95%, 96%, 97%, 98%, 99% or more of a given amount.

[0116] "Statistically significant" means that the result could not have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur if the null hypothesis were true. If the obtained p-value is less than the significance level, the null hypothesis is excluded. In simple cases, the significance level is defined as a p-value of 0.05 or less.

[0117] "Treatment response" means improvement of symptoms (whether or not sustained) based on the administration of one or more therapeutic agents.

[0118] As used herein, the terms "therapeutically effective amount", "therapeutic dose", "prophylactically effective amount", or "diagnostically effective amount" are the amount of an agent (e.g., a proprotein, an activated protein) that, upon administration, elicits the desired biological response.

[0119] 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 can be carried out prophylactically, after the onset of a pathological event, or after contact with a pathogen. Also included is "prophylactic" treatment, which can be used to reduce the rate of progression of a 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 denote the complete eradication, cure, or prevention of a disease or disorder or its associated symptoms.

[0120] The term "wild-type" refers to the gene or gene product (e.g., polypeptide) most frequently observed in a population and is thus arbitrarily designated as the "normal" or "wild-type" form of that gene.

[0121] Unless otherwise expressly stated, each embodiment in this specification applies to all other embodiments.

[0122] Activable proprotein homodimer

[0123] Certain embodiments relate to activatable proprotein homodimers that comprise a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprise, in an 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-15 protein, a second linker, and an IL-15Rα protein, wherein the hinge / Fc domain of the first polypeptide binds to the hinge / Fc domain of the second polypeptide, wherein the IL-15 protein of the first polypeptide binds to the IL-15Rα protein of the second polypeptide, and wherein the IL-15Rα of the first polypeptide binds to the IL-15 protein of the second polypeptide, wherein the binding masks the binding site in the IL-15 protein that would otherwise bind to the IL-15Rβ / γc chain present on the surface of immune cells in vitro or in vivo, and wherein the second linker is a cleavable linker.

[0124] As described above, the IL-15 protein interacts or binds to the IL-15Rα protein, for example, by non-covalent interactions or certain covalent bonds (such as disulfide bonds). In some cases, the binding of the IL-15 protein to the IL-15Rα protein sterically blocks or hinders the binding of the IL-15 protein to its cognate IL-15Rβ / γc receptor chain expressed on immune cells. Exemplary IL-15 proteins and IL-15Rα proteins are described elsewhere herein.

[0125] In some cases, the hinge / Fc domains of the first and second polypeptides dimerize together by at least one non-covalent interaction, at least one covalent bond (such as at least one disulfide bond), or any combination of non-covalent interactions and covalent bonds to further stabilize the activatable proprotein and / or further mask the binding of the IL-15 protein to its cognate receptor (such as the IL-15Rβ / γc receptor chain). However, typically, the hinge / Fc domains of the first and second polypeptides do not bind or dimerize together by peptide or amide bonds. In some embodiments, the hinge / Fc domains bind together to form a homodimer, i.e., a homodimer composed of two identical or nearly identical hinge / Fc domains. Thus, the hinge / Fc domains of the first and second polypeptides can be the same (or substantially the same) or different (such as a knob-in-hole structure). Exemplary hinge / Fc domains are described herein.

[0126] As described above, the second linker comprises a cleavable linker, e.g., a linker that can be cleaved by a protease. In some cases, the first linker is a stable (e.g., physiologically stable) linker. In some cases, the first linker is also a cleavable linker, e.g., a linker cleavable by a protease. In some cases, the protease is expressed in the target tissue or cell (e.g., cancer tissue or cancer cell). In such cases, cleavage of the linker releases the masking moiety, relieves the steric hindrance of the IL-15 protein, and allows for selective activation of the IL-15 protein in diseased tissue or cells (e.g., TME) relative to normal or healthy tissue or cells. Such selective and local activation not only reduces the unnecessary consumption of the administered IL-15, thereby prolonging its half-life, but also enhances tissue penetration and reduces the undesirable systemic effects of IL-15, among other advantageous aspects. Exemplary linkers are described herein.

[0127] In some embodiments, homodimeric binding between the first polypeptide and the second polypeptide allosterically inhibits the binding of the IL-15 protein to its target (e.g., the cognate IL-15Rβ / γc receptor chain on the surface of immune cells). In these and related embodiments, the IL-15 moiety of the proprotein that can be activated shows no binding or substantially no binding to its target, or binds to its target 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% or 50% compared to the binding of the individual active domain or the IL-15 protein, 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.

[0128] In certain cases, anti-PD-1 or anti-PD-L1 or human B7H3 Fab not only enhances the targeting of the activatable IL-15 proprotein (or pro-cytokine) module to the TME, but also provides enhanced anti-cancer / immunostimulatory activity in addition to the activity of the IL-15 protein once the IL-15 protein is activated by protease cleavage of the second linker.

[0129] In a specific embodiment, the first and second polypeptides of the activatable pro-protein comprise an anti-PD-1 Fab (SEQ ID NO: 3 and 4) or an anti-PD-L1 Fab (SEQ ID NO: 25 and 26), as well as a human IgG1 CH1 domain and a CL domain (κ), an IgG1 hinge (SEQ ID NO: 42), a modified IgG1 Fc domain with LALA and P329A mutations (see the CH2 domain of SEQ ID NO: 57), an IgG1 CH3 domain (see the CH3 domain of SEQ ID NO: 58), a stable linker (e.g., an 8-amino acid linker such as GGGSGGGS; SEQ ID NO: 178), an IL-15 protein (SEQ ID NO: 69 or 79, optionally with K86G and S162A mutations), a protease-cleavable linker (e.g., GGGGGSPLGLSGRSDNQGGGGSGGGGS, SEQ ID NO: 90; or GGGSPLGLAGSGRSDNQGGSGGSGGS, SEQ ID NO: 201), and an IL-15Rα protein (SEQ ID NO: 87 or 88, optionally with T2A mutation), including active fragments and variants of the foregoing sequences, as described herein. The individual components of the exemplary activatable pro-protein are described in more detail herein.

[0130] Anti - PD - 1 and anti - PD - L1 and anti - B7H3 Fab regions. The activatable pro-protein 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 one constant domain and one variable domain each of the heavy and light chains of an immunoglobulin molecule, e.g., the VL / CL region binds to the VH / CH1 region and fuses at its C-terminus to the Fc domain (optionally via a linker or hinge) (hinge / Fc domain). The VL:VH and CL:CH1 regions of the Fab are typically bound together as a covalent heterodimer. 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, e.g., an IgA1, IgA2, IgG1, IgG2, IgG2, IgG3 or IgG4 CH1 domain. In some specific embodiments, the CL domain is a κ chain and the CH1 domain is an IgG1 domain.

[0131] In certain embodiments, the antibodies or Fab regions described herein include heavy and light chain CDR sets inserted between sets of heavy and light chain framework regions (FRs), which support the CDRs and define the spatial relationship of the CDRs relative to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of the heavy or light chain V region. Starting from the N-terminus of the heavy or light chain, these regions are referred to as "CDR1", "CDR2", and "CDR3", respectively. Thus, the antigen-binding site includes six CDRs, which include the CDR sets from each of the heavy and light chain V regions (VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3). A polypeptide containing a single CDR (e.g., CDR1, CDR2, or CDR3) is referred to herein as a "molecular recognition unit". Crystallographic analyses of many antigen-antibody complexes have demonstrated extensive contacts between the amino acid residues of the CDRs and the bound antigen, with the most extensive antigen contacts being with the heavy chain CDR3. Thus, the molecular recognition unit is primarily responsible for the specificity of the antigen-binding site.

[0132] As used herein, the term "FR set" refers to the four flanking amino acid sequences that are the framework for the CDR set of the heavy or light chain V region. Some FR residues can contact the bound antigen; however, the FRs are primarily responsible for folding the V region into the antigen-binding site, particularly the FR residues immediately adjacent to the CDRs. Within the FRs, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region folds into the binding site, the CDRs are displayed as protruding loop motifs that form the antigen-binding surface. It is generally believed that there are conserved structural regions of the FRs that influence the shape in which the CDR loops fold into certain "canonical" structures—regardless of the precise CDR amino acid sequence. In addition, certain FR residues are known to participate in noncovalent interdomain contacts that stabilize heavy and light chain interactions in the antibody.

[0133] In certain embodiments, the Fab region is humanized. These embodiments relate to chimeric molecules, which are typically prepared using recombinant techniques and have antigen-binding sites of immunoglobulins from non-human species and the remaining immunoglobulin structures of molecules based on human immunoglobulin structure and / or sequence. The antigen-binding site may comprise the entire variable domain fused to a constant domain, or may comprise only the CDRs grafted onto appropriate framework regions in the variable domain. The epitope-binding site may be wild-type or modified by substitution of one or more amino acids. This eliminates the constant region as an immunogen in humans, but the possibility of an immune response to the foreign variable region remains (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 those described in U.S. Patent No. 7,462,697.

[0134] Another approach not only focuses on providing constant regions of human origin, but also on modifying variable regions to reshape them into forms as close as possible to the human form. As is well known, the variable regions of both heavy and light chains contain three complementarity-determining regions (CDRs), which vary according to the target epitope and determine the binding ability, flanked by four framework regions (FRs), which are relatively conserved in a given species and are considered to be the scaffolds for the CDRs. When preparing non-human antibodies against a specific epitope, the variable regions can be "reshaped" or "humanized" by transplanting the CDRs derived from the non-human antibody onto the FRs present in the human antibody to be modified. The application of this method in various antibodies has been reported by Sato et al., Cancer Res. 53:851-856, 1993; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988; Kettleborough et al., Protein Engineering. 4:773-3783, 1991; Maeda et al., Human Antibodies Hybridoma 2:124-134, 1991; Gorman et al., PNAS USA. 88:4181-4185, 1991; Tempest et al., Bio / Technology 9:266-271, 1991; Co et al., PNAS USA. 88:2869-2873, 1991; Carter et al., PNAS USA. 89:4285-4289, 1992 and Co et al., J Immunol. 148:1149-1154, 1992. In some embodiments, the humanized antibody or Fab region retains all CDR sequences (e.g., a humanized mouse antibody or Fab that contains all six CDRs from a mouse antibody). In certain embodiments, the humanized antibody or Fab region has one or more CDRs (one, two, three, four, five, six) that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs of the original antibody.

[0135] The binding properties of the antibody and Fab region 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, wherein the equilibrium dissociation constant is about ≤10 -7 M or about 10 -8 M or in the range of ≤10 -7 M to about 10 -8within the range of M. In some embodiments, the equilibrium dissociation constant is about ≤ 10 -9 M or about ≤ 10 -10 M or within about ≤ 10 -9 M to about ≤ 10 -10 M. In certain illustrative embodiments, the affinity (Kd or EC 50 ) of the antibody or its antigen-binding fragment pair for the PD-1 or PD-L1 protein (to which it specifically binds) 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.

[0136] A molecule such as an antibody or Fab region is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell, substance, or particular epitope more frequently, more rapidly, for a longer duration, and / or with greater affinity than it reacts or binds to an alternative cell or substance or epitope. An antibody "specifically binds" or "preferentially binds" to a target molecule or epitope if the affinity, avidity, ease, and / or duration of binding of the antibody to the target molecule or epitope exceeds that of its binding to other substances or epitopes by, for example, a statistically significant amount. Generally, one member of a pair of molecules that exhibit specific binding has a region or cavity on its surface that specifically binds and is thus complementary to the specific spatial and / or polar organization of the other member of the pair of molecules. Thus, the members of the pair have the property of specifically binding to each other. For example, an antibody that specifically or preferentially binds to a particular epitope is an antibody that binds to that particular epitope with greater affinity, avidity, greater ease, and / or for a longer duration than it binds to other epitopes. It can also be understood from 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, in cases 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 various antigens carrying that epitope; for example, it can cross-react with many different forms of target antigens from multiple species sharing a common epitope.

[0137] Immunoconjugation generally refers to the type of non-covalent interaction that occurs, for example (by way of illustration and not limitation), between an immunoglobulin molecule and the antigen to which the immunoglobulin is specific due to electrostatic, ionic, hydrophilic and / or hydrophobic attraction or repulsion, steric forces, hydrogen bonding, van der Waals forces and other interactions. The strength or affinity of an immunoconjugation interaction can be expressed by the dissociation constant (Kd) of the interaction, where the smaller the Kd, the greater the affinity. The immunoconjugation properties of the selected polypeptide can be quantified using methods well known in the art. One such method requires measuring the rates of antigen-binding site / antigen complex formation and dissociation, where these rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Thus, both the "association rate constant" (Kon) and the "dissociation rate constant" (Koff) can be determined by calculating the concentrations and the actual association and dissociation rates. The ratio of Koff / Kon enables the elimination of all parameters unrelated to affinity and thus equals 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 an antibody for a PD-1 or PD-L1 protein or epitope can be, for example, from about 100 nanomolar concentration (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar concentration (pM), or from about 100 nM to about 1 femtomolar concentration (fM). As used herein, the term "affinity" refers to the resistance of a complex of two or more agents to dissociation upon dilution.

[0138] Programmed cell death protein 1 (PD-1; cluster of differentiation 279 (CD279)) refers to a protein expressed on the cell surface that regulates the immune response 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, inhibitory T cells). Thus, 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, for example, 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. Appl. 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, cemiplimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, MGA012, AMP-22, and AMP-514.

[0139] Programmed death ligand 1 (PD-L1) is a 40 kDa type I transmembrane protein that binds to its receptor PD-1 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, for example, U.S. Pat. Nos. 9,102,725; 9,393,301; 9,402,899; 9,439,962). For example, in some specific embodiments, the Fab region is from an anti-PD-L1 antibody selected from atezolizumab, avelumab, and durvalumab.

[0140] 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, the heavy chain variable region sequence comprising complementarity determining regions VHCDR1, VHCDR2, and VHCDR3 sequences, and the light chain variable region sequence comprising complementarity determining regions VLCDR1, VLCDR2, and VLCDR3 sequences. Tables P1 and P2 below provide exemplary VH, VHCDR1, VHCDR2, VHCDR3, VL, VLCDR1, VLCDR2, and VLCDR3 sequences.

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147] Thus, in certain embodiments, the anti-PD-1 Fab region thereof comprises a heavy chain variable (VH) region containing VHCDR1, VHCDR2, and VHCDR3 regions (underlined) from Table P1 and a corresponding light chain variable (VL) region containing VLCDR1, VLCDR2, and VLCDR3 regions (underlined) from Table P1. Also included are variants thereof that bind to human PD-1, e.g., variants having a total of 1, 2, 3, 4, 5, or 6 alterations in the combined CDR regions (e.g., the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and / or VLCDR3 sequences described herein). Exemplary "alterations" include amino acid substitutions, additions, and deletions. In certain embodiments, the anti-PD-1 Fab region comprises a VH region from Table P1 and a corresponding VL region from Table P1. In certain embodiments, the VH region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from Table P1, and the VL region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding sequence selected from Table P1. Also included are variants thereof that bind to human PD-1, e.g., variants having 1, 2, 3, 4, 5, 6 alterations in one or more framework regions. Exemplary "alterations" include amino acid substitutions, additions, and deletions.

[0148] Thus, in certain embodiments, its anti-PD-L1 Fab region comprises a heavy chain variable (VH) region containing the VH CDR1, VH CDR2, and VH CDR3 regions (underlined) from Table P2 and a corresponding light chain variable (VL) region containing the VL CDR1, VL CDR2, and VL CDR3 regions (underlined) from Table P2. Also included are variants thereof that bind human PD-L1, for example, variants having a total of 1, 2, 3, 4, 5, or 6 alterations in the combined CDR regions (such as the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 sequences described herein). Exemplary "alterations" include amino acid substitutions, additions, and deletions. In certain embodiments, the anti-PD-L1 Fab region comprises a VH region from Table P2 and a corresponding VL region from Table P2. In certain embodiments, the VH region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from Table P2, and the VL region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding sequence selected from Table P2. Also included are variants thereof that bind human PD-L1, for example, variants having 1, 2, 3, 4, 5, 6 alterations in one or more framework regions. Exemplary "alterations" include amino acid substitutions, additions, and deletions.

[0149] CD276 (B7H3) is an immune checkpoint molecule that is involved in regulating T cell-mediated immune responses and is expressed on some solid tumors. It plays a protective role in tumor cells, for example, by inhibiting natural killer cell-mediated cytolysis and potentially other anti-tumor immune responses. In some specific embodiments, the B7H3 is human B7H3 or a domain thereof. In certain embodiments, the anti-B7H3 Fab region specifically binds to the human B7H3 protein, for example, one or more human B7H3 domains selected from the Ig-like V-1 domain, Ig-like C2-1 domain, Ig-like V-2 domain, and Ig-like C2-2 domain. In some specific embodiments, the Fab region has a K of about 0.4 nM or 0.5 nM (400 or 500 pM) or lower D Specifically binds to human BH73.

[0150] In certain embodiments, the anti-B7H3 Fab region is characterized by or comprises a VH sequence and a VL sequence, the VH sequence comprising complementarity determining regions VHCDR1, VHCDR2, and VHCDR3 sequences, and the VL sequence comprising complementarity determining regions VLCDR1, VLCDR2, and VLCDR3 sequences. Exemplary VH, VHCDR1, VHCDR2, VHCDR3, VL, VLCDR1, VLCDR2, and VLCDR3 sequences are provided in Table P3 below.

[0151]

[0152] Thus, in certain embodiments, the anti-B7H3 Fab region thereof comprises a VH region comprising VHCDR1, VHCDR2, and VHCDR3 regions (underlined) from Table P3 and a corresponding VL region comprising VLCDR1, VLCDR2, and VLCDR3 regions (underlined) from Table P3. Also included are variants thereof that bind to human B7H3, e.g., variants having a total of 1, 2, 3, 4, 5, or 6 alterations in the combined CDR regions (e.g., the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and / or VLCDR3 sequences described herein). Exemplary "alterations" include amino acid substitutions, additions, and deletions. In certain embodiments, the anti-B7H3 Fab region comprises a VH region from Table P3 and a corresponding VL region from Table P3. In certain embodiments, the VH region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from Table P3, and the VL region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding sequence selected from Table P3. Also included are variants that bind to human B7H3, e.g., variants having 1, 2, 3, 4, 5, 6 alterations in one or more framework regions. Exemplary "alterations" include amino acid substitutions, additions, and deletions.

[0153] 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 improvements thereto. 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, E.A. et al., Sequences of Proteins of Immunological Interest. Fourth Edition US Department of Health and Human Services. 1987 and updates thereto.

[0154] It should be understood that any one or more of the foregoing anti-PD-1 Fab or anti-PD-L1 Fab can be combined with any additional component described herein (e.g., hinge / Fc domain, IL-15 protein, IL-15Rα protein, and linkers described herein) to produce one or more activatable proproteins.

[0155] Hinge / Fc domain. Some activatable proprotein homodimers contain a hinge / Fc domain. The hinge region (present in IgG, IgA, and IgD) serves as a flexible spacer that allows the Fab portion to move freely in space relative to the Fc domain. In contrast to the constant regions, the hinge region is structurally diverse, with the sequence and length varying between immunoglobulin classes and subclasses. The hinge region may also contain one or more glycosylation sites, including many structurally different types of sites for carbohydrate attachment. For example, IgA1 contains five glycosylation sites within a 17-amino acid segment of the hinge region, conferring significant resistance of the hinge region polypeptide to intestinal proteases. Residues in the hinge-proximal region of the CH2 domain can also influence the specificity of the interaction between an immunoglobulin and one or more of its corresponding Fc receptors (see, e.g., Shin et al., Intern. Rev. Immunol. 10:177-186, 1993).

[0156] 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. The "Fc domain" may also include one or more hinge regions of the immunoglobulin heavy chain constant region. In certain embodiments, the Fc domain does not contain one or more of the CH1, CL, VL, and / or VH regions of the immunoglobulin.

[0157] The Fc domain can be derived from the CH2 domain, CH3 domain, CH4 domain, and / or one or more hinge regions of any one or more immunoglobulin classes (including but not limited to IgA, IgD, IgE, IgG, IgM, including subclasses and combinations thereof). In some embodiments, the Fc domain is derived from an IgA immunoglobulin, including subclasses IgA1 and / or IgA2. In certain embodiments, the Fc domain is derived from an IgD immunoglobulin. In specific embodiments, the Fc domain is derived from an IgE immunoglobulin. In some embodiments, the Fc domain is derived from an IgG immunoglobulin, including subclasses IgG1, IgG2, IgG2, IgG3, and / or IgG4. In certain embodiments, the Fc domain is derived from an IgM immunoglobulin. Exemplary hinge and Fc domain sequences are provided in Table F1 below.

[0158]

[0159]

[0160]

[0161]

[0162] Thus, in some embodiments, the hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a hinge sequence selected from Table F1 (e.g., an IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4 hinge region selected from Table F1). In certain embodiments, the Fc domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence selected from Table F1 (e.g., an IgA1 CH2, CH3 or combined CH2CH3 sequence from Table F1, an IgA2 CH2, CH3 or combined CH2CH3 sequence, an IgD CH2, CH3 or combined CH2CH3 sequence, an IgE CH2, CH3, CH4 or combined CH2CH3 or CH2CH3CH4 sequence, an IgG1 CH2, CH3 or combined CH2CH3 sequence, an IgG2 CH2, CH3 or combined CH2CH3 sequence, an IgG3 CH2, CH3 or combined CH2CH3 sequence, an IgG4 CH2, CH3 or combined CH2CH3 sequence or an IgM CH2, CH3, CH4 or combined CH2CH3 or CH2CH3CH4 sequence). In certain embodiments, the hinge and the Fc domain are of the same Ig class.

[0163] In certain embodiments, the Fc domain is a modified Fc domain. Such modifications can be used to alter (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), its pharmacokinetic properties (e.g., stability or half-life, bioavailability, tissue distribution, volume of distribution, concentration, elimination rate constant, elimination rate, area under the curve (AUC), clearance, C max , t max , C min , fluctuations), its immunogenicity, its complement fixation or activation and / or CDC / ADCC / ADCP-related activities of the Fc region, as well as other properties described herein.

[0164] 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 endocytic trafficking in dendritic cells. The expression of FcγRI is upregulated by GM-CSF and gamma 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, PMN, macrophages, and mast cells. FcγIIb inhibits immunoreceptor tyrosine-based activation motif (ITAM)-mediated responses and is thus 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 PMN.

[0165] In a specific embodiment, the modified Fc domain comprises an L234A / L235A (“LALA”) mutation and / or a P329A or P329G mutation (EU numbering) (see, e.g., the CH2 domain of SEQ ID NO:57). In certain embodiments, the Fc domain or modified Fc domain maintains normal (wild-type) or substantially normal binding to the neonatal Fc receptor (FcRn). In a specific embodiment, the Fc region comprises the IgG1 hinge region of SEQ ID NO:42, the modified IgG1 CH2 domain of SEQ ID NO:57, and the IgG1 CH domain of SEQ ID NO:58.

[0166] It should be understood that any one or more of the foregoing hinge and Fc domains can be combined with any additional components described herein (e.g., anti-PD-1 Fab, anti-PD-L1 Fab, IL-15 protein, IL-15Rα protein, and the linkers described herein) to produce one or more activatable pro-proteins.

[0167] IL - 15 protein. The activatable proprotein described herein comprises at least one "IL-15 protein" (or interleukin-15 protein), including human IL-15 protein. IL-15 is a pleiotropic cytokine that has been shown to induce and regulate multiple immune functions. For example, IL-15 is crucial for lymphoid development, the peripheral maintenance of innate immune cells, and immune memory in T cells (primarily the natural killer (NK) and CD8+ T cell populations). Like IL-2, IL-15 binds to and signals through a complex composed of the IL-2 / IL-15 receptor β chain (CD122) and the common γ chain (γ-C, CD132).

[0168] IL-15 is a 14-15 kDa protein composed of a signal peptide (residues 1-29), a propeptide (residues 30-48), and an active mature protein (residues 46-162). Table S1 below provides an exemplary human IL-15 protein sequence.

[0169]

[0170]

[0171]

[0172] Thus, in certain embodiments, the IL-15 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 to a sequence selected from Table S1. In some embodiments, an "active" IL-15 protein or fragment or variant is characterized, for example, by its ability to bind to the IL-15Rβ / γc and / or IL-15Rα / β / γc receptor chains present on the surface of immune cells and stimulate downstream signaling activity in the absence of steric hindrance by the binding moieties described herein. Examples of downstream signaling activity include signaling mediated by IL-15 through Janus kinase 1 (Jak1) and the γc subunit Janus kinase 3 (Jak3), which results in phosphorylation and activation of the signal transducer and activator of transcription 3 (STAT3) and STAT5 pathways. Other examples include activation of Src family kinases (including Lck and Fyn), and subsequent activation of the PI3K and MAPK signaling pathways. In summary, IL-15 signaling stimulates a series of downstream pathways, leading to responses that play important roles in regulating the activation and proliferation of T cells and natural killer (NK) cells, and the survival of memory T cells.

[0173] In some specific embodiments, the IL-15 protein is the mature form of IL-15, or an active variant or fragment thereof, which comprises, consists of, or consists essentially of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to amino acids 49-162 of SEQ ID NO:68 (human IL-15FL precursor). Certain IL-15 proteins comprise, consist of, or consist essentially of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO:69 (mature human IL-15).

[0174] Certain IL-15 proteins comprise one or more amino acid substitutions determined relative to the exemplary amino acid sequences in Table S1. For example, in certain embodiments, the IL-15 protein comprises or retains one or more amino acid substitutions at position D8, D22, E46, V49, I50, L66, K86 as defined by SEQ ID NO:69 (mature human IL-15) and / or S162 as defined by SEQ ID NO:68 (IL-15FL precursor). Specific examples of substitutions are selected from one or more of D8N, D22K, E46K, V49D, I50D, L66E, K86G, and S162A, including combinations thereof (see Table S1). Exemplary combinations of substitutions are selected from V49D and S162A; I50D and S162A; L66E and S162A; D8N and S162A; V49D and S162A; E46K and S162A; E46K, E53K, and S162A; D22K, E46K, and S162A; and D22K, E46K, E53K, and S162A. In some specific embodiments, the IL-15 protein comprises the K86G and S162A mutations as defined by SEQ ID NO:69, e.g., a mature IL-15 protein having the K86G and S162A mutations (e.g., SEQ ID NO:79).

[0175] In some embodiments, the D8N substitution in IL-15 does not significantly reduce the binding affinity for IL-15Rα, but significantly reduces or completely eliminates IL-15 signaling activity. In some embodiments, the V49D substitution in IL-15 has a significantly lower (e.g., as low as about 1 / 13) binding affinity for IL-15Rα and retains about or at least about 90 - 100% of the IL-15 signaling activity. In some embodiments, the I50D substitution in IL-15 has a significantly lower (e.g., as low as about 1 / 100) binding affinity for IL-15Rα and retains about 10% of the IL-15 signaling activity. In some embodiments, the L66E substitution in IL-15 has a significantly lower (e.g., as low as about 1 / 15) binding affinity for IL-15Rα and does not retain or only retains very low IL-15 signaling activity.

[0176] It should be understood that any one or more of the foregoing IL-15 proteins can be combined with any additional components described herein (e.g., anti-PD-1 Fab, anti-PD-L1 Fab, hinge / Fc domain, IL-15Rα protein, and linkers described herein) to produce one or more activatable pro-proteins.

[0177] IL - 15Rα protein class The activatable pro-proteins described herein comprise at least one "IL-15Rα protein" (or interleukin-15 receptor-α protein), including the human IL-15Rα protein. The IL-15 receptor consists of the following three subunits: IL-15Rα, CD122, and CD132. IL-15Rα specifically binds IL-15 with very high affinity and is capable of binding IL-15 independently of the other subunits. Exemplary IL-15Rα protein sequences are provided in Table S2.

[0178]

[0179]

[0180] Thus, in certain embodiments, the IL-15Rα protein comprises, consists of, or consists essentially of the following amino acid sequences: an amino acid sequence selected from Table S2, or an active variant or fragment thereof having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to a sequence selected from Table S2 and that binds to the IL-15 protein. In some embodiments, the IL-15Rα protein comprises, consists of, or consists essentially of the following amino acid sequences: at least 80%, 85%, 90%, 95%, 98%, or 100% of the amino acid sequence of SEQ ID NO:80 (full-length wild-type human IL-15Rα) or a fragment thereof consisting of residues 31-205, 31-108, 31-95 of SEQ ID NO:80 (full-length wild-type human IL-15Rα) that binds to the IL-15 protein.

[0181] Certain IL-15Rα proteins contain one or more defined amino acid substitutions relative to the exemplary amino acid sequences in Table S2. For example, certain IL-15Rα proteins contain or retain one or more amino acid substitutions at positions T2A, R24, R26, and R35 as defined by SEQ ID NO:82 (IL-15Rα Sushi+), including combinations thereof. Exemplary combinations include R24E, R26E, and R35E, including combinations thereof. Exemplary combinations include R26E and R35E and R24E, R26E, and R35E. In a specific embodiment, the IL-15α protein comprises SEQ ID NO:82 or 83 having a T2A substitution, such as SEQ ID NO:87 or 88.

[0182] It should be understood that any one or more of the foregoing IL-15Rα proteins can be combined with any other component described herein (such as an anti-PD-1 Fab, an anti-PD-L1 Fab, a hinge / Fc domain, an IL-15 protein, and the linkers described herein) to produce one or more activatable proproteins.

[0183] Linker 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. In some cases, both the first and second linkers are cleavable linkers, e.g., cleavable linkers each comprising a protease cleavage site.

[0184] In some embodiments, the length of 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, or the length is 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.

[0185] In some embodiments, the cleavable linker comprises at least one protease cleavage site. Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., J. Gener. Virol. 78:699 - 722, 1997; and Scymczak et al., 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, podoplanin, PSA, kallikrein, cathepsin A, and cathepsin B.

[0186] Exemplary cleavable linker sequences are provided in Table S3.

[0187]

[0188]

[0189]

[0190] 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:150), -Gly-Gly-Arg-, -Gly-Arg-Gly-Asp-Asn-Pro- (SEQ ID NO:151), -Gly-Arg-Gly-Asp-Ser- (SEQ ID NO:152), -Gly-Arg-Gly-Asp-Ser-Pro-Lys- (SEQ ID NO:153), -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:154), -Ala-Ala-Pro-Leu- (SEQ ID NO:155), -Ala-Ala-Pro-Phe- (SEQ ID NO:156), -Ala-Ala-Pro-Ala- (SEQ ID NO:157), and -Ala-Tyr-Leu-Val- (SEQ ID NO:158).

[0191] The cleavable linker also includes amino acid sequences that can be cleaved by matrix metalloproteinases such as collagenase, stromelysin, and gelatinase. Illustrative examples of amino acid sequences cleavable by matrix metalloproteinases include, but are not limited to: -Gly-Pro-Y-Gly-Pro-Z- (SEQ ID NO:159), -Gly-Pro-, Leu-Gly-Pro-Z- (SEQ ID NO:160), -Gly-Pro-Ile-Gly-Pro-Z- (SEQ ID NO:161), and -Ala-Pro-Gly-Leu-Z- (SEQ ID NO:162), where Y and Z are amino acids. Illustrative examples of amino acid sequences cleavable by collagenase include, but are not limited to: -Pro-Leu-Gly-Pro-D-Arg-Z- (SEQ ID NO:163), -Pro-Leu-Gly-Leu-Leu-Gly-Z- (SEQ ID NO:164), -Pro-Gln-Gly-Ile-Ala-Gly-Trp- (SEQ ID NO:165), -Pro-Leu-Gly-Cys(Me)-His- (SEQ ID NO:166), -Pro-Leu-Gly-Leu-Tyr-Ala- (SEQ ID NO:167), -Pro-Leu-Ala-Leu-Trp-Ala-Arg- (SEQ ID NO:168), and -Pro-Leu-Ala-Tyr-Trp-Ala-Arg- (SEQ ID NO:169), where 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:170); an example of an amino acid sequence cleavable by gelatinase is -Pro-Leu-Gly-Met-Tyr-Ser-Arg- (SEQ ID NO:171).

[0192] The cleavable linker also includes amino acid sequences that can be cleaved by angiotensin converting enzyme, such as -Asp-Lys-Pro-, -Gly-Asp-Lys-Pro- (SEQ ID NO:172), and -Gly-Ser-Asp-Lys-Pro- (SEQ ID NO:173). The cleavable linker also includes amino acid sequences that can be degraded by cathepsin B, such as Val-Cit, Ala-Leu-Ala-Leu- (SEQ ID NO:174), Gly-Phe-Leu-Gly- (SEQ ID NO:175), and Phe-Lys.

[0193] In certain embodiments, the cleavable linker has a half-life of about or less than about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, or 96 hours, or any intermediate half-life, at pH 7.4 and 25 °C, e.g., at physiological pH and human body temperature (e.g., in vivo, in serum, in a given tissue).

[0194] Typically, at least one of the first or second linker is a non-cleavable linker. Exemplary non-cleavable linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., PNAS USA. 83:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180. Some specific non-cleavable linker sequences contain Gly, Ser, and / or Asn residues. Other near-neutral amino acids such as Thr and Ala can also be used in the peptide linker sequence if desired.

[0195] Certain exemplary non-cleavable linkers include the following Gly, Ser, and / or Asn-containing linkers: [G] x , [S] x , [N] x , [GS] x , [GGS] x , [GSS] x , [GSGS] x (SEQ ID NO:176), [GGSG] x (SEQ ID NO:177), [GGGS] x (SEQ ID NO:178), [GGGGS] x (SEQ ID NO:179), [GN] x , [GGN] x , [GNN] x , [GNGN] x (SEQ ID NO:180), [GGNG] x (SEQ ID NO:181), [GGGN] x (SEQ ID NO:182), [GGGGN] x (SEQ ID NO:183) linkers, where 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.

[0196] Other examples of non-cleavable linkers include the following amino acid sequences: Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO:184); 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:185); 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:186); 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:187); and Asn-Val-Asp-His-Lys-Pro-Ser-Asn-Thr-Lys-Val-Asp-Lys-Arg-(SEQ ID NO:188).

[0197] Other non-limiting examples of non-cleavable linkers include DGGGS (SEQ ID NO:189); TGEKP (SEQ ID NO:190) (see, e.g., Liu et al., PNAS. 94:5525-5530, 1997); GGRR (SEQ ID NO:191) (Pomerantz et al. 1995); (GGGGS) n(SEQ ID NO:192) (Kim et al., PNAS. 93:1156 - 1160, 1996); EGKSSGSGSESKVD (SEQ ID NO:193) (Chaudhary et al., PNAS. 87:1066 - 1070, 1990); KESGSVSSEQLAQFRSLD (SEQ ID NO:194) (Bird et al., Science. 242:423 - 426, 1988), GGRRGGGS (SEQ ID NO:195); LRQRDGERP (SEQ ID NO:196); LRQKDGGGSERP (SEQ ID NO:197); LRQKd(GGGS)2ERP (SEQ ID NO:198). In some specific embodiments, the linker comprises a Gly3 linker sequence, which contains three glycine residues. In some particular embodiments, a computer program capable of modeling the DNA binding site and the peptide itself can be used (Desjarlais & Berg, PNAS. 90:2256 - 2260, 1993; and PNAS. 91:11099 - 11103, 1994) or a flexible linker can be rationally designed by phage display methods.

[0198] In some embodiments, the linker comprises a spacer element and a cleavable element to make the enzyme responsible for cleavage more accessible to the cleavable element.

[0199] It should be understood that any one or more of the foregoing linkers can be combined with any one or more of the anti - PD - 1 Fab, anti - PD - L1 Fab, hinge / Fc domain, IL - 15 protein, and IL - 15Rα protein described herein to form an activatable pro - protein homodimer.

[0200] Exemplary activatable pro - protein sequences are provided in Table S4.

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209] Thus, in certain embodiments, the activatable proprotein comprises first and second polypeptides, the first and second polypeptides comprising, consisting of, or consisting essentially of the following amino acid sequences: an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to a sequence selected from Table S4 (i.e., chain 1 and chain 2), and a VL / CL region polypeptide having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to the corresponding sequence from Table S4 (i.e., chain 3 and chain 4).

[0210] Methods of Use and Pharmaceutical Compositions

[0211] Certain embodiments include methods of treating, ameliorating the symptoms of, and / or slowing the 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, which comprise administering to the subject at least one activatable proprotein as described herein. In certain specific embodiments, the disease is cancer. In some embodiments, the cancer expresses or overexpresses PD-L1 or B7H3.

[0212] In some embodiments, after administration, the activatable proprotein is activated by protease cleavage in a cell or tissue, which exposes the binding site for the IL-15 protein, which binds the IL-15Rβ / γc chain present on the surface of immune cells in vitro or in vivo, thereby generating an activated protein. In a particular embodiment, protease cleavage occurs in cancer cells or cancer tissue. Generally, the activated protein has at least one immunostimulatory IL-15 activity, e.g., by binding the IL-15Rβ / γc chain present on the surface of immune cells in vivo, thereby stimulating the 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.

[0213] In some embodiments, administration and activation of the activatable proprotein to produce the activated protein enhances the anti-cancer immune response in a subject by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more 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.

[0214] In certain embodiments, the activated anti-PD-1 Fab / IL-15 protein stimulates an enhanced (e.g., synergistically enhanced) anti-cancer immune response relative to the respective anti-PD-1 Fab (or respective anti-PD-1 antibody) alone and / or the respective IL-15 protein alone, e.g., enhanced up to 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-1 Fab / IL-15 protein stimulates enhanced (e.g., synergistically enhanced) cancer cell killing activity relative to the respective anti-PD-1 Fab (or respective anti-PD-1 antibody) alone and / or the respective IL-15 protein alone, e.g., enhanced up to 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.

[0215] In certain embodiments, the activated anti-PD-L1 Fab / IL-15 protein stimulates an enhanced (e.g., synergistically enhanced) anti-cancer immune response relative to the corresponding anti-PD-L1 Fab alone (or the corresponding anti-PD-L1 antibody) and / or the corresponding IL-15 protein alone. For example, the anti-cancer immune response is enhanced 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 relative to each individual component. In certain embodiments, the activated anti-PD-L1 Fab / IL-15 protein stimulates an enhanced (e.g., synergistically enhanced) cancer cell killing activity relative to the corresponding anti-PD-L1 Fab alone (or the corresponding anti-PD-L1 antibody) and / or the corresponding IL-15 protein alone. For example, the cancer cell killing activity is enhanced 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 relative to each individual component.

[0216] In certain embodiments, the activated anti-B7H3 Fab / IL-15 protein stimulates an enhanced (e.g., synergistically enhanced) anti-cancer immune response relative to the corresponding anti-B7H3 Fab alone (or the corresponding anti-B7H3 antibody) and / or the corresponding IL-15 protein alone. For example, the anti-cancer immune response is enhanced 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 relative to each individual component. In certain embodiments, the activated anti-B7H3 Fab / IL-15 protein stimulates an enhanced (e.g., synergistically enhanced) cancer cell killing activity relative to the corresponding anti-B7H3 Fab alone (or the corresponding anti-B7H3 antibody) and / or the corresponding IL-15 protein alone. For example, the cancer cell killing activity is enhanced 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 relative to each individual component.

[0217] In some embodiments, the disease is cancer, that is, the subject in need has or is suspected of having cancer. Accordingly, certain embodiments include methods of treating, ameliorating the symptoms of cancer or inhibiting the progression thereof in a subject in need, which include administering to the subject at least one proprotein activator as described herein. In certain embodiments, the cancer is primary cancer or metastatic cancer. In specific embodiments, the cancer is selected from one or more of the following cancers: 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 myelogenous leukemia, acute myeloid leukemia or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, liver cancer (hepatocellular carcinoma), sarcoma, B cell malignancy, 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.

[0218] In some embodiments, as described above, the cancer is metastatic cancer. In addition to the cancers described above, exemplary metastatic cancers include, but are not limited to, bladder cancer that has metastasized to bone, liver, and / or lung; breast cancer that has metastasized to bone, brain, liver, and / or lung; colorectal cancer that has metastasized to liver, lung, and / or peritoneum; renal cancer that has metastasized to adrenal gland, bone, brain, liver, and / or lung; lung cancer that has metastasized to adrenal gland, bone, brain, liver, and / or other lung sites; melanoma that has metastasized to bone, brain, liver, lung, and / or skin / muscle; ovarian cancer that has metastasized to liver, lung, and / or peritoneum; pancreatic cancer that has metastasized to liver, lung, and / or peritoneum; prostate cancer that has metastasized to adrenal gland, bone, liver, and / or lung; gastric cancer that has metastasized to liver, lung, and / or peritoneum; thyroid cancer that has metastasized to bone, liver, and / or lung; and uterine cancer that has metastasized to bone, liver, lung, peritoneum, and / or vagina; and so on.

[0219] In certain embodiments, as described herein, the cancer expresses or overexpresses PD-L1 or B7H3. The expression levels of PD-L1 and B7H3 in tissue (e.g., cancer tissue) samples can be determined by a variety of methods. For example, the PD-L1 or B7H3 protein level can be determined by immunohistochemistry (IHC), which includes chromogenic or fluorescent IHC, enzyme-linked immunosorbent assay (ELISA), or western blotting of human proteins or genes, as well as other assays. The PD-L1 or B7H3 mRNA level can be measured, for example, by RT-PCR, such as quantitative competitive (QC) RT-PCR, and other techniques known in the art. Accordingly, certain embodiments include the step of determining or detecting or measuring the levels of PD-L1 and / or B7H3 in a tissue sample from a subject in need thereof. Also included is the step of comparing the level of PD-L1 in the tissue sample to a control or reference level. Certain embodiments include the step of determining the levels of PD-L1 and / or B7H3 in a cancer tissue sample (e.g., a biopsy) from a subject, and if the cancer tissue from the subject expresses or overexpresses PD-L1 or B7H3, administering an activatable proprotein homodimer.

[0220] The methods for treating cancer can be combined with other treatment modalities. For example, the combination therapies described herein can be administered to a subject before, during, or after other therapeutic interventions, which include symptomatic care, radiotherapy, surgery, transplantation, hormone therapy, photodynamic therapy, antibiotic therapy, or any combination thereof. Symptomatic care includes administering corticosteroids to reduce cerebral edema, headache, cognitive dysfunction, and vomiting, and administering anticonvulsants to reduce seizures. Radiotherapy includes whole brain irradiation, fractionated radiotherapy, and radiosurgery, such as stereotactic radiosurgery, which can be further combined with conventional surgery.

[0221] Accordingly, certain embodiments include combination therapies for treating cancer, including methods for treating to improve the symptoms of cancer or inhibit cancer progression in a subject in need thereof, which include administering to the subject a combination of at least one activatable proprotein described herein with at least one additional agent (e.g., a chemotherapeutic agent, a hormone therapeutic agent, and / or a kinase inhibitor). In some embodiments, administering at least one activatable proprotein enhances the susceptibility of the cancer to the additional agent (e.g., a chemotherapeutic agent, a hormone therapeutic agent, and / or a kinase inhibitor) by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, or more.

[0222] Some 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), antimicrotubule agents, and the like.

[0223] Examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, mechlorethamine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (such as N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozocin), tetrazines (such as dacarbazine, mitozolomide, and temozolomide), aziridines (such as thiotepa, mitomycin, and diaziquone (AZQ)), cisplatin and its derivatives (such as carboplatin and oxaliplatin), and non-classical alkylating agents (optionally, procarbazine and hexamethylmelamine).

[0224] Examples of antimetabolites include antifolates (such as methotrexate and pemetrexed), fluoropyrimidines (such as 5-fluorouracil and capecitabine), deoxynucleoside analogs (such as ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), and mercaptopurines (such as thioguanine and mercaptopurine);

[0225] Examples of cytotoxic antibiotics include anthracyclines (such as doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycin, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mebanazine, and aclarubicin.

[0226] Examples of antimicrotubule agents include taxanes (such as paclitaxel and docetaxel) and vinca alkaloids (such as vinblastine, vincristine, vindesine, and vinorelbine).

[0227] One of ordinary skill in the art will understand that the various chemotherapeutic agents described herein can be combined with any one or more of the proproteins that can be activated described herein and used according to any one or more of the methods or compositions described herein.

[0228] Some combination therapies use at least one hormonal therapeutic agent. General examples of hormonal therapeutic agents include hormone agonists and hormone antagonists. Specific examples of hormone agonists include progestins (progesterone), corticosteroids (e.g., prednisone, methylprednisolone, dexamethasone), insulin-like growth factors, VEGF-derived angiogenic factors and lymphangiogenic factors (e.g., VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factors (FGF), galectins, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, androgens, estrogens, 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 (SERM; e.g., tamoxifen, raloxifene, toremifene) and antiandrogens (e.g., flutamide, bicalutamide, nilutamide).

[0229] Also included are hormone pathway inhibitors, such as antibodies against hormone receptors. Examples include inhibitors of IGF receptors (e.g., IGF-IR1) such as cetuximab, daratumumab, figitumumab, ganitumab, esmoltuzumab, and lobatuzumab; inhibitors of vascular endothelial growth factor receptor 1, 2, or 3 (VEGFR1, VEGFR2, or VEGFR3), such as ramucirumab, bevacizumab, alacizumab pegol, ramucirumab; inhibitors of TGF-β receptors R1, R2, and R3, such as fresolimumab and metelimumab; inhibitors of c-Met, such as necitumumab; inhibitors of EGF receptor, such as cetuximab, depatuxizumab mafodotin, fotemustine, imetelstat, enzastaurin, matuzumab, zalutumumab, necitumumab, panitumumab, tomuzotuximab, and zalutumumab; FGF receptor inhibitors, such as aprutumab ixadotin and bemarituzumab; and PDGF receptor inhibitors, such as olaparib and tovoretumab.

[0230] One of ordinary skill in the art will understand that the various hormonal therapeutic agents described herein can be combined with any one or more of the various proproteins that can be activated described herein and used according to any one or more of the methods or compositions described herein.

[0231] Certain combination therapies use at least one kinase inhibitor, including tyrosine kinase inhibitors. Examples of kinase inhibitors include, but are not limited to, adavosertib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mulitinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemurafenib.

[0232] Those skilled in the art will understand that the various kinase inhibitors described herein can be combined with any one or more of the various proproteins that can be activated described herein and used according to any one or more of the methods or compositions described herein.

[0233] In some embodiments, the methods and pharmaceutical compositions described herein extend the median survival time of a 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 a subject by 1 year, 2 years, 3 years, or longer. In some embodiments, the methods and pharmaceutical compositions 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.

[0234] 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 of at least 10%, 20%, 30%, 40%, 50%, or more in tumor mass) or by altered (e.g., statistically significant decrease) scan dimensions. In certain embodiments, the methods and therapeutic compositions described herein are sufficient to cause stable disease.

[0235] In certain embodiments, the methods and therapeutic compositions described herein are sufficient to cause a clinically relevant reduction in the symptoms of a particular disease indication known to a skilled clinician.

[0236] For in vivo applications, as described above, for treating human or non-human mammalian diseases or for testing, the agents described herein are typically incorporated into one or more therapeutic or pharmaceutical compositions (including veterinary therapeutic compositions) prior to administration.

[0237] Accordingly, as described herein, certain embodiments relate to a pharmaceutical or therapeutic composition comprising at least one activatable proprotein. In some cases, the pharmaceutical or therapeutic composition comprises one or more activatable proproteins described herein and a pharmaceutically or physiologically acceptable carrier or excipient. Certain pharmaceutical or therapeutic compositions further comprise at least one additional agent, such as, for example, a chemotherapeutic agent, a hormonal therapeutic agent, and / or a kinase inhibitor as described herein.

[0238] Some therapeutic compositions comprise (and certain methods utilize) only one activatable proprotein. Certain therapeutic compositions comprise (and certain methods utilize) a mixture of at least two, three, four, or five different activatable proproteins.

[0239] In certain embodiments, the pharmaceutical or therapeutic composition comprising at least one activatable proprotein is substantially pure on a protein or weight-by-weight basis, e.g., the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein or weight-by-weight basis.

[0240] In some embodiments, as is known in the art, the activatable proproteins described herein do not form aggregates, have a desired solubility, and / or have immunogenic characteristics suitable for humans. Accordingly, in some embodiments, the therapeutic composition comprising the activatable proprotein is substantially aggregate-free. For example, certain compositions comprise less than about 10% (by protein) high molecular weight aggregated protein, or less than about 5% high molecular weight aggregated protein, or less than about 4% high molecular weight aggregated protein, or less than about 3% high molecular weight aggregated protein, or less than about 2% high molecular weight aggregated protein, or less than about 1% high molecular weight aggregated protein. Some compositions comprise an activatable proprotein that is at least about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% monodisperse according to its apparent molecular weight.

[0241] 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 agent.

[0242] To prepare a therapeutic or pharmaceutical composition, an effective or 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 and suitable for the 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 contain, for example, a sterile diluent (such as water), a salt solution (such as phosphate buffered saline; PBS), a fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; an antimicrobial agent (such as benzyl alcohol and methylparaben); an antioxidant (such as ascorbic acid and sodium bisulfite) and a chelating agent (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as acetate, citrate and phosphate). If administered intravenously (e.g., by IV infusion), suitable carriers include normal saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents (such as glucose, polyethylene glycol, propylene glycol and mixtures thereof).

[0243] The agents described herein can be administered in pure form or in the form of a suitable therapeutic or pharmaceutical composition by any acceptable mode of administration of agents for similar uses. The therapeutic or pharmaceutical composition can be prepared by combining a composition containing the agent with a suitable physiologically acceptable carrier, diluent or excipient, and can be formulated into preparations in solid, semi-solid, liquid or gaseous forms 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 may or may not be present in the composition.

[0244] 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.

[0245] The carrier can include, for example, a pharmaceutically or physiologically acceptable carrier, excipient, or stabilizer, which is non-toxic to the cells or mammals in contact therewith at the dosages and concentrations used. Physiologically acceptable carriers are generally aqueous pH-buffered 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 dextrin; 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 poloxamers (PLURONICS TM ), etc.

[0246] In some embodiments, one or more agents can be encapsulated in, for example, microcapsules prepared by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules, respectively), 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., Editor, (1980). One or more particles or liposomes can also contain other therapeutic or diagnostic agents.

[0247] The precise dosage and duration of treatment are a function of the disease being treated and can be determined empirically using known testing protocols or by testing the composition in model systems known in the art and extrapolating therefrom. Controlled clinical trials can also be conducted. The dosage can also vary with the severity of the disease to be alleviated. The pharmaceutical composition is generally formulated and administered to achieve a therapeutically useful effect while minimizing undesirable side effects. The composition can be administered in a single dose or divided into multiple smaller doses and administered at different time intervals. For any particular subject, the specific dosage regimen can be adjusted over time according to individual needs.

[0248] Accordingly, exemplary routes of administration of these and related therapeutic or pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal routes. As used herein, the term "parenteral" includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, or infusion techniques. Therapeutic or pharmaceutical compositions according to certain embodiments of the present invention are formulated to permit the active ingredient(s) contained therein to be bioavailable upon administration of the composition to a subject or patient. The composition to be administered to a subject or patient may take the form of one or more dosage units, where, for example, a tablet may be a single dosage unit, and a container of a dosage in the form of an aerosol as described herein may contain multiple dosage units. The actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). For treating a target disease or disorder, the composition to be administered typically contains a therapeutically effective amount of the agent(s) described herein.

[0249] Therapeutic or pharmaceutical compositions may be in solid or liquid form. In one embodiment, one or more carriers are particulate, and thus the composition is, for example, in the form of a tablet or powder. One or more carriers may be liquid, and the composition may be, for example, an oral oil, an injectable solution, or an aerosol, which may be used, for example, for inhalation administration. When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, where semi-solid, semi-liquid, suspension, and gel forms are included within the forms considered solid or liquid herein. Certain embodiments include sterile injectable solutions.

[0250] As a solid composition for oral administration, the pharmaceutical composition may be formulated as a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer, etc. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch, lactose, or dextrin; disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate, or orange flavoring; and coloring agents. When the pharmaceutical composition is in capsule form, for example, a gelatin capsule, it may further contain a liquid carrier such as polyethylene glycol or an oil in addition to the substances of the above types.

[0251] A therapeutic or pharmaceutical composition may be in liquid form, such as an elixir, syrup, solution, emulsion or suspension. As two examples, the liquid can be used for oral administration or administration by injection. When intended for oral administration, in addition to the compounds of the present invention, preferred compositions also contain one or more of sweetening agents, preservatives, dyes / colorants and flavoring agents. In compositions intended for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizers and isotonic agents may be included.

[0252] 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 of glycerol which can be used as solvents or suspending media, polyethylene glycols, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffering agents such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or glucose. Parenteral preparations may be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0253] Liquid therapeutic or pharmaceutical compositions for parenteral or oral administration should contain an amount of the agent such that a suitable dosage can be obtained. Generally, the amount is at least 0.01% of the target agent in the composition. When intended for oral administration, the amount may vary between 0.1% and about 70% by weight of the composition. Certain oral therapeutic or pharmaceutical compositions contain from about 4% to about 75% of the target agent. In certain embodiments, therapeutic or pharmaceutical compositions and formulations are prepared such that, prior to dilution, a parenteral dosage unit contains from 0.01% to 10% by weight of the target agent.

[0254] A therapeutic or pharmaceutical composition may be used for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel matrix. For example, the matrix may comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifying and stabilizing agents. Thickeners may be present in therapeutic or pharmaceutical compositions for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.

[0255] A therapeutic or pharmaceutical composition may 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.

[0256] Therapeutic or pharmaceutical compositions can include various substances that alter the physical form of solid or liquid dosage units. For example, the composition can include substances that form a coating shell around the active ingredient. Substances that form the coating shell are generally inert and can be selected from, for example, sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient can be encapsulated in a gelatin capsule. Therapeutic or pharmaceutical compositions in solid or liquid form can include components that bind to the agent, thereby facilitating the delivery of the compound. Suitable components that can play this role include monoclonal or polyclonal antibodies, one or more proteins, or liposomes.

[0257] Therapeutic or pharmaceutical compositions can consist essentially of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of a pressurized package. Delivery can be effected by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredient. The aerosol can be delivered in a single-phase, two-phase, or three-phase system to deliver one or more active ingredients. Delivery of the aerosol includes the necessary container, activator, valve, sub-container, etc., which together can form a kit. A person of ordinary skill in the art can determine the preferred aerosol without undue experimentation.

[0258] 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 microencapsulation delivery systems, and biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and other polymers known to a person of ordinary skill in the art.

[0259] Therapeutic or pharmaceutical compositions can be prepared by methods well known in the pharmaceutical art. For example, a therapeutic or pharmaceutical composition intended to be administered by injection can contain one or more of salts, buffers, and / or stabilizers, together with sterile distilled water to form a solution. Surfactants can be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that interact non-covalently with the agent to facilitate the dissolution or homogeneous suspension of the agent in an aqueous delivery system.

[0260] A therapeutic 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; the metabolic stability and duration of action of the compound; the age, body weight, general health, sex and diet of the subject; the mode and time of administration; the rate of excretion; drug combinations; the severity of the particular disorder or disease; and the subject being treated. In some cases, the therapeutically effective daily dose is from about 0.001 mg / kg (i.e., about 0.07 mg) to about 100 mg / kg (i.e., about 7.0 g) for a 70 kg mammal; preferably, the therapeutically effective dose is from about 0.01 mg / kg (i.e., about 0.7 mg) to about 50 mg / kg (i.e., about 3.5 g) for a 70 kg mammal; more preferably, the therapeutically effective dose is from about 1 mg / kg (i.e., about 70 mg) to about 25 mg / kg (i.e., about 1.75 g) for a 70 kg mammal. In some embodiments, the therapeutically effective dose is administered once a week, once every two weeks or once a month. In a specific embodiment, for example, the therapeutically effective dose is administered 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 at a dose of 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.

[0261] The combination therapies described herein can include administering a single pharmaceutical dosage form that contains a proprotein activator and an additional therapeutic agent (e.g., a chemotherapeutic agent, a hormonal therapeutic agent, a kinase inhibitor), and administering a composition that contains a proprotein activator and an additional therapeutic agent in its separate pharmaceutical dosage form. For example, the proprotein activator and the additional therapeutic agent can be administered together to a subject in a single oral dosage composition (such as a tablet or a capsule), or each agent can be administered in a separate oral dosage formulation. Similarly, the proprotein activator and the additional therapeutic agent can be administered together to a subject in a single parenteral dosage composition (such as in a saline solution or other physiologically acceptable solution), or each agent can be administered in a separate parenteral dosage formulation. As another example, for cell-based therapies, the proprotein activator can be mixed with the cells prior to administration, administered as part of a separate composition, or both. When separate dosage formulations are used, the compositions can 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 such regimens.

[0262] Also included is a patient care kit, which comprises (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 certain kits, (a) and (b) are in separate therapeutic compositions. In some kits, (a) and (b) are in the same therapeutic composition.

[0263] The kits herein may also include one or more additional therapeutic agents or other components suitable or desired for the indication being treated or the desired diagnostic application. The kits herein may also include one or more syringes or other components necessary or desired to facilitate the intended mode of delivery (e.g., a stent, an implantable reservoir, etc.).

[0264] In some embodiments, the patient care kit comprises separate containers, dividers, or compartments for one or more compositions and one or more information materials. For example, one or more compositions may be contained in bottles, vials, or syringes, and one or more information materials may be packaged together in association 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 with the information material attached in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of separate containers, each containing one or more unit dosage forms (e.g., the dosage forms described herein) of the activatable proprotein and optionally at least one additional therapeutic agent. For example, the kit includes a plurality of syringes, ampoules, foil packs, or blister packs, each containing a single unit dose of the activatable proprotein and optionally at least one additional therapeutic agent. The containers of the kit may be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light - impermeable.

[0265] The patient care kit optionally includes a device suitable for the 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 is a method of providing the kit, for example, by combining the components described herein.

[0266] Expression and purification system

[0267] Certain embodiments include methods and related compositions for expressing and purifying the activatable proproteins described herein. Such recombinant activatable proproteins can be readily prepared using standard protocols, such as those described in 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, an activatable proprotein can be prepared by a procedure comprising one or more of the following steps: (a) preparing one or more vectors or constructs that contain one or more polynucleotide sequences encoding the first and second polypeptides described herein and the VL / CL regions of the anti-PD-1 Fab region or anti-PD-L1 Fab region or anti-B7H3 Fab region described herein, the polynucleotide sequences being 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 regions, which associate to form the activatable proprotein; and (d) isolating the activatable proprotein from the host cells.

[0268] To express the desired polypeptides, the nucleotide sequences encoding the first and / or second polypeptide chains of the activatable proprotein can be inserted into one or more suitable expression vectors, i.e., one or more vectors that contain the essential elements for transcription and translation of the inserted coding sequences. Methods well known to those skilled in the art can be used to construct expression vectors containing the sequences encoding the target polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic 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).

[0269] 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 phage, 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.

[0270] "Control elements" or "regulatory sequences" present in the expression vector are those non-translated regions of the vector - enhancers, promoters, 5' and 3' untranslated regions - which interact with host cell proteins for transcription and translation. The strength and specificity of such elements can vary. Depending on the vector system and host used, many suitable transcriptional and translational 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 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 generate cell lines containing multiple copies of the sequence encoding the polypeptide, vectors based on SV40 or EBV can be advantageously used together with a suitable selectable marker.

[0271] In bacterial systems, depending on the intended use of the expressed polypeptide, many expression vectors can be chosen. For example, when large amounts are needed, vectors that direct 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 ligated in-frame into the vector with the amino-terminal Met of β-galactosidase and the subsequent 7 residues, such that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264:5503-5509 (1989)); and so on. pGEX vectors (Promega, Madison, Wis.) can also be used to express foreign polypeptides in the form of fusion proteins with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption to glutathione-agarose beads and then elution in the presence of free glutathione. Proteins prepared in such systems can be designed to include heparin, thrombin, or factor XA protease cleavage sites such that the cloned target polypeptide can be released from the GST moiety at will.

[0272] Certain embodiments employ E. coli-based expression systems (see, for example, Structural Genomics Consortium et al., Nature Methods. 5:135-146, 2008). These and related embodiments can rely in part or in whole on ligation-independent cloning (LIC) to generate suitable expression vectors. In specific embodiments, protein expression can be controlled by T7 RNA polymerase (e.g., the pET vector series). These and related embodiments can utilize the expression host strain BL21(DE3) (a λDE3 lysogen of BL21 that supports T7-mediated expression and lacks the lon and ompT proteases) to enhance 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 the ROSETTA TM (DE3) and Rosetta 2(DE3) strains. Cell lysis and sample handling can also be improved using reagents sold under the trademarks nuclease and protein extraction reagents. For cell culture, autoinduction media can enhance the efficiency of many expression systems, including high-throughput expression systems. This type of media (e.g., OVERNIGHT EXPRESS TMAn auto-induction system gradually initiates protein expression through metabolic shift without adding artificial inducers such as IPTG. Some specific embodiments use hexahistidine tags (such as those sold under the trade name 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 an affinity tag (see, for example, Shimp et al., Protein Expr Purif. 50:58-67, 2006). As another example, some embodiments can use a cold-shock-induced high-yield production system in E. coli because overexpression of proteins in E. coli at low temperatures increases their solubility and stability (see, for example, Qing et al., Nature Biotechnology. 22:877-882, 2004).

[0273] Also included is a high-density bacterial fermentation system. For example, high cell density cultivation of Ralstonia eutropha allows production of proteins at cell densities exceeding 150 g / L, and the expression titer of recombinant proteins exceeds 10 g / L.

[0274] In Saccharomyces cerevisiae, many vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For a review, see Ausubel et al. (ibid.) and Grant et al., Methods Enzymol. 153:516-544 (1987). Also included is the Pichia pastoris expression system (see, for example, Li et al., Nature Biotechnology. 24, 210-215, 2006; and Hamilton et al., Science, 301:1244, 2003). Some embodiments include yeast systems engineered to selectively glycosylate proteins, including yeast with a humanized N-glycosylation pathway and the like (see, for example, Hamilton et al., Science. 313:1441-1443, 2006; Wildt et al., Nature Reviews Microbiol. 3:119-28, 2005; and Gerngross et al., Nature-Biotechnology. 22:1409 -1414, 2004; U.S. Patent Nos. 7,629,163, 7,326,681, and 7,029,872). By way of example only, recombinant yeast cultures can be grown in Fernbach flasks or in 15 L, 50 L, 100 L, and 200 L fermenters and the like.

[0275] In the case of using a plant expression vector, the expression of a sequence encoding a polypeptide can be driven by any one of a number of promoters. For example, viral promoters such as the 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 can be used (Coruzzi et al., EMBO J. 3:1671-1680 (1984); Broglie et al., Science 224:838-843 (1984); and Winter et al., Results Probl. Cell Differ. 17:85-105 (1991)). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in many readily available reviews (e.g., see Hobbs in McGraw Hill, Yearbook of Science and Technology, pp. 191-196 (1992)).

[0276] Insect systems can also be used to express the target polypeptide. For example, in one such system, the Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes in Spodoptera frugiperda cells or Trichoplusia ni cells. The sequence encoding the polypeptide can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Successful insertion of the polypeptide-encoding sequence will inactivate the polyhedrin gene and generate a recombinant virus lacking the capsid protein. The recombinant virus can then be used to infect, for example, Spodoptera frugiperda cells or Trichoplusia ni cells in which the target polypeptide can be expressed (Engelhard et al., Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227 (1994)). Also included are baculovirus expression systems, including those utilizing SF9, SF21, and T. ni cells (see, e.g., Murphy and Piwnica-Worms, Curr Protoc Protein Sci. Chapter 5: Unit 5.4, 2001). Insect systems can provide post-translational modifications similar to those of mammalian systems.

[0277] In mammalian host cells, many virus-based expression systems are generally available. For example, in the case of using adenovirus as an expression vector, a sequence encoding a target polypeptide can be ligated into an adenovirus transcriptional / translational complex consisting of a late promoter and a tripartite leader sequence. Insertion into the non-essential E1 or E3 region of the viral genome can be used to obtain a live virus capable of expressing the polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. U.S.A. 81:3655-3659 (1984)). In addition, transcriptional enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

[0278] 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); dog kidney cells (MDCK, ATCC CCL34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. 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 (edited by B.K.C Lo, Humana Press, Totowa, N.J., 2003), pp. 255-268. Some preferred mammalian cell expression systems include expression systems based on CHO and HEK293 cells. Mammalian expression systems can utilize adherent cell lines, e.g., in T-flasks, roller bottles or cell factories or suspension culture, e.g., in 1L and 5L spinner flasks, 5L, 14L, 40L, 100L and 200L stirred tank bioreactors, or 20 / 50L and 100 / 200L WAVE bioreactors, and in other reactors known in the art.

[0279] Also included is cell-free expression of proteins. These and related embodiments generally utilize purified RNA polymerase, ribosomes, tRNA, and ribonucleotides; these reagents can be produced by extraction from cells or cell-based expression systems.

[0280] Certain initiation signals can also be used to achieve more efficient translation of a sequence encoding a target polypeptide. Such signals include the ATG initiation codon and adjacent sequences. When the sequence encoding the polypeptide, its initiation codon, and the upstream sequence are inserted into a suitable expression vector, additional transcriptional or translational control signals may not be required. However, when only the coding sequence or a portion thereof is inserted, exogenous translational control signals including the ATG initiation codon should be provided. In addition, the initiation codon should be in the correct reading frame to ensure translation of the entire inserted fragment. The exogenous translational elements and initiation codons can have various sources, including natural and synthetic. The expression efficiency can be enhanced by including enhancers suitable for the particular cell system used, such as those described in the literature (Scharf. Results Probl. Cell Differ. 20:125-162 (1994)).

[0281] Furthermore, the host cell line can be selected based on its ability to regulate the expression of the inserted sequence or to process the expressed protein in the desired manner. Such modifications of the polypeptide include, but are not limited to, post-translational modifications such as acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves the "prepro" form of the protein can also be used to facilitate proper insertion, folding, and / or function. In addition to bacterial cells, different host cells can be selected, such as yeast, CHO, HeLa, MDCK, HEK293, and W138, which have or even lack specific cellular and characteristic mechanisms for such post-translational activities to ensure proper modification and processing of the foreign protein.

[0282] For long-term high-yield production of recombinant proteins, stable expression is generally preferred. For example, a cell line stably expressing the target polynucleotide can be transformed with an expression vector that can contain a viral origin of replication and / or endogenous expression elements and a selectable marker gene on the same vector or a separate vector. After 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 selectable marker is to confer resistance to the selection, and its presence allows the growth and recovery of cells that have successfully expressed the introduced sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques suitable for the cell type. Transient production can also be used, such as by transient transfection or infection. Exemplary mammalian expression systems suitable for transient production include HEK293- and CHO-based systems.

[0283] Many selection systems are available for the recovery of transformed or transduced cell lines. These selection systems include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223-232 (1977)) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817-823 (1990)) genes, which can be used for tk- cells or aprt- cells, respectively. In addition, antimetabolite, antibiotic, or herbicide resistance can also be used as a basis for selection; for example, dhfr, which confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. U.S.A. 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 phosphinotricin acetyltransferase, respectively (Murry, ibid.). Additional selectable genes have been described, such as trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. U.S.A. 85:8047-51 (1988)). The use of 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 has become widespread not only for the identification of transformants, but also for the quantification of the amount of transient or stable protein expression attributable to a particular vector system (see, for example, Rhodes et al., Methods Mol. Biol. 55:121-131 (1995)).

[0284] Also included are high-throughput protein production systems, or microproduction systems. Certain aspects can utilize, for example, a hexahistidine fusion tag for protein expression and purification on the surface of a metal chelator-modified slide or on MagneHis nickel particles (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., MethodsMolBiol. 498:229-44, 2009).

[0285] A variety of protocols for detecting and measuring the expression of a product encoded by a polynucleotide using a binding agent or antibody, such as a polyclonal or monoclonal antibody specific for the product, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), Western blot, radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). These and other assays are described, among other places, in Hampton et al., Serological Methods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med. 158:1211-1216 (1983).

[0286] A variety of labels and conjugation techniques are known to those of skill in the art and can be used in a variety of nucleic acid and amino acid assays. Methods for generating labeled hybridization or PCR probes for detecting sequences related to polynucleotides include oligolabeling, nick translation, end-labeling, or PCR amplification using labeled nucleotides. Alternatively, a sequence or any portion thereof can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by adding a suitable RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures can be carried out using a variety of commercially available kits. Suitable reporter molecules or labels that can be used include radionuclides, enzymes, fluorescers, chemiluminescers, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

[0287] Host cells transformed with one or more target polynucleotide sequences can be cultured under conditions suitable for expressing the protein and recovering the protein from the cell culture. Certain specific embodiments utilize serum-free cell expression systems. Examples include HEK293 cells and CHO cells that can be grown in serum-free media (see, for example, Rosser et al., Protein Expr. Purif. 40:237-43, 2005; and U.S. Patent No. 6,210,922).

[0288] Depending on the sequence and / or vector used, the activatable proprotein produced by the recombinant cell can be secreted or contained intracellularly. As will be understood by those skilled in the art, an expression vector containing a polynucleotide can be designed to contain a signal sequence that directs the encoded polypeptide to be secreted through the prokaryotic or eukaryotic cell membrane. Other recombinant constructs can be used to ligate the sequence encoding the target polypeptide to the nucleotide sequence encoding a polypeptide domain that will facilitate the purification and / or detection of the soluble protein. Examples of such domains include cleavable and non-cleavable affinity purification and ec50 tags, such as avidin, FLAG tag, polyhistidine tag (e.g., 6xHis), cMyc tag, V5 tag, glutathione S-transferase (GST) tag, etc.

[0289] The proteins produced by the recombinant cells can be purified and characterized according to a variety of techniques known in the art. Exemplary systems for performing protein purification and analyzing protein purity include fast protein liquid chromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems), high pressure liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemical methods for purification include ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradient, 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 blue staining, silver staining), immunoblotting, Bradford, and ELISA, which can be used at any step in the production or purification process and are commonly used to measure the purity of protein compositions.

[0290] Also included are methods for concentrating the activatable proprotein, and compositions comprising the concentrated soluble activatable proprotein. In some aspects, such a concentrated solution of at least one activatable proprotein contains a protein at a concentration of about or at least about 5 mg / mL, 8 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or higher.

[0291] In some aspects, such compositions can be substantially monodisperse, meaning that when evaluated, for example, by size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation, the activatable proprotein is present predominantly (i.e., at least about 90% or more) in one apparent molecular weight form.

[0292] In some aspects, such compositions have a purity of at least about 90% (based on protein), or in some aspects, have a purity of at least about 95%, or in some embodiments, have a purity of at least 98%. The purity can be determined by any conventional analytical method known in the art.

[0293] In some aspects, compared to the total amount of protein present, 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% high molecular weight aggregate content. 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).

[0294] Examples of concentration methods contemplated herein include lyophilization, which is typically employed when the solution contains little or no other soluble components other than the target protein. Lyophilization is usually carried out after HPLC runs and can remove most or all of the volatile components from the mixture. Also included are ultrafiltration techniques, which typically use one or more selectively permeable membranes to concentrate protein solutions. The membranes allow water and small molecules to pass through and retain the protein; the solution can be forced against the membrane by techniques such as mechanical pumps, air pressure, or centrifugation.

[0295] In certain embodiments, as measured according to conventional techniques in the art, the activatable proprotein in the composition has a purity of at least about 90%. In certain embodiments, such as diagnostic compositions or certain pharmaceutical or therapeutic compositions, the activatable proprotein composition has a purity of at least about 95% or at least about 97% or 98% or 99%. In some embodiments, such as when used as a reference or research reagent, the activatable proprotein can have a lower purity and can have a purity of at least about 50%, 60%, 70%, or 80%. The purity can be measured overall or relative to selected components (such as other proteins), for example, purity based on protein.

[0296] 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 techniques being such as and related techniques that utilize surface plasmon resonance (SPR), which is an optical phenomenon enabling real-time detection of unlabeled interactants. Biosensors based on SPR can be used for determination of activity concentration, screening, and characterization of affinity and kinetics. The presence or level of one or more bioactivities can be measured according to cell-based assays, including those that utilize at least one IL-15 receptor, which is optionally functionally coupled to a readout or indicator, such as a fluorescent or luminescent indicator of bioactivity, as described herein.

[0297] In certain embodiments, as described above, the activatable proprotein composition is substantially endotoxin-free, including, for example, about 95% endotoxin-free, preferably about 99% endotoxin-free, more preferably about 99.99% endotoxin-free. As described herein, the presence of endotoxin can be detected according to conventional techniques in the art. In a specific embodiment, the activatable proprotein composition is prepared by eukaryotic cells such as mammalian or human cells in a substantially serum-free medium. In certain embodiments, as described herein, the endotoxin content of the activatable proprotein composition is less than about 10 EU / mg of activatable proprotein, or less than about 5 EU / mg of activatable proprotein, less than about 3 EU / mg of activatable proprotein, or less than about 1 EU / mg of activatable proprotein.

[0298] In certain embodiments, the activatable proprotein composition comprises less than about 10% wt / wt of high molecular weight aggregates, or less than about 5% wt / wt of high molecular weight aggregates, or less than about 2% wt / wt of high molecular weight aggregates, or less than about 1% wt / wt of high molecular weight aggregates.

[0299] Also included are protein-based analytical assays and methods that can be used to evaluate characteristics such as protein purity, size, solubility, and degree of aggregation. Protein purity can be evaluated by a variety of methods. For example, purity can be evaluated based on primary structure, higher-order structure, size, charge, hydrophobicity, and glycosylation. Examples of methods for evaluating primary structure include N-terminal and C-terminal sequencing and peptide mapping (see, for example, Allen et al., Biologicals. 24:255-275, 1996). Examples of methods for evaluating higher-order structure include circular dichroism (see, for example, Kelly et al., Biochim Biophys Acta. 1751:119-139, 2005), fluorescence spectroscopy (see, for example, Meagher et al., J. Biol. Chem. 273:23283-89, 1998), FT-IR, amide hydrogen-deuterium exchange kinetics, differential scanning calorimetry, NMR spectroscopy, immunoreactivity with conformation-sensitive antibodies. Higher-order structure can also be evaluated as a function of various parameters such as pH, temperature, or added salt. Examples of methods for evaluating 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 evaluated 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 evaluated by, for example, mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.

[0300] As described above, certain embodiments include using SEC-HPLC to evaluate protein characteristics such as purity, size (e.g., size homogeneity), or degree of aggregation, and / or purifying proteins and other uses. SEC, which also includes gel filtration chromatography (GFC) and gel permeation chromatography (GPC), refers to a chromatographic method in which molecules in solution are separated in a porous material based on their size, or more specifically, based on their hydrodynamic volume, diffusion coefficient, and / or surface properties. This method is commonly used to separate biomolecules and to determine the molecular weight and molecular weight distribution of polymers. Generally, a biological or protein sample (such as a protein extract produced according to the protein expression methods provided herein and known in the art) is loaded into a selected size exclusion column having a defined stationary phase (porous material) (preferably a phase that does not interact with the proteins in the sample). In some aspects, the stationary phase consists of inert particles in a dense three-dimensional matrix packed inside 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 smaller than a certain size to enter. Thus, large particles are excluded from these pores and channels, and their limited interaction with the stationary phase causes them to elute as a "fully excluded" peak at the beginning of the experiment. Smaller molecules that can fit into the pores are removed from the flowing mobile phase, and the time they spend immobilized in the pores of the stationary phase depends in part on the depth to which they penetrate into the pores. Their removal from the mobile phase flow causes them to take longer to elute from the column and results in separation between the particles based on their size differences. A given size exclusion chromatography column has a range of molecular weights that can be separated. Generally, molecules larger than the upper limit will not be captured by the stationary phase, molecules smaller than the lower limit will enter the solid phase completely 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.

[0301] For example, Anicetti et al. (Trends in Biotechnology. 7:342-349, 1989) also discussed protein purity for clinical applications. Recently, techniques for analyzing protein purity include, but are not limited to, LabChip GXII (an automated platform for rapid analysis of proteins and nucleic acids), which provides high-throughput analysis of protein titer, sizing, and purity. In certain non-limiting embodiments, clinically gradeable pro-proteins can be obtained by utilizing a combination of chromatographic materials and other methods in at least two orthogonal steps (see, e.g., Therapeutic Proteins: Methods and Protocols. Volume 308, edited by Smales and James, Humana Press Inc., 2005). Generally, protein agents (such as pro-proteins) are substantially endotoxin-free, as measured according to techniques known in the art and described herein.

[0302] Protein solubility assays are also included. Such assays can be used, for example, to determine optimal growth and purification conditions for recombinant production, to optimize the selection of one or more buffers, and to optimize the selection of pro-proteins and their variants. Solubility or aggregation can be evaluated according to various parameters, including temperature, pH, salt, and the presence of other additives. Examples of solubility screening assays include, but are not limited to, microplate-based methods that use turbidity or other metrics as an endpoint to measure protein solubility, high-throughput assays for analyzing the solubility of purified recombinant proteins (see, e.g., Stenvall et al., Biochim Biophys Acta. 1752:6-10, 2005), assays that use structural complementarity of genetically tagged proteins to monitor and measure protein folding and solubility in vivo (see, e.g., Wigley et al., Nature Biotechnology. 19:131-136, 2001), and electrochemical screening of the solubility of recombinant proteins in Escherichia coli using scanning electrochemical microscopy (SECM) (see, e.g., Nagamine et al., Biotechnology and Bioengineering. 96:1008-1013, 2006), among others. Pro-proteins with increased solubility (or decreased aggregation) can be identified or selected according to conventional techniques in the art, including simple in vivo assays of protein solubility (see, e.g., Maxwell et al., Protein Sci. 8:1908-11, 1999).

[0303] Protein solubility and aggregation can also be measured by dynamic light scattering techniques. Aggregation is a general term encompassing several types of interactions or characteristics, including soluble / insoluble, covalent / non-covalent, reversible / irreversible, and native / denatured interactions and characteristics. For protein therapeutics, the presence of aggregates is generally considered undesirable because of concerns that aggregates may elicit immunogenic responses (e.g., small aggregates) or may cause adverse events upon administration (e.g., microparticles). Dynamic light scattering refers to a technique that can be used to determine the size distribution characteristics of small particles in a suspension or polymers such as proteins in solution. This technique is also known as photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QELS) and uses scattered light to measure the diffusion rate of protein particles. Due to the Brownian motion of molecules and particles in solution, fluctuations in the scattered 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 applications that rely on real-time monitoring of changes at elevated temperatures. Accordingly, certain embodiments include using dynamic light scattering to analyze the solubility and / or presence of aggregates in samples containing an activatable proprotein of the invention.

[0304] Although the foregoing embodiments have been described in considerable detail for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are for illustrative purposes only and not limiting. Those skilled in the art will readily recognize various non-critical parameters that can be altered or modified to produce substantially similar results. Examples

[0305] Example 1A. Preparation of PD-L1-proIL-15 Fusion Protein

[0306] Plasmids encoding PD-L1-proIL-15 or anti-human PD-L1 were constructed by standard gene synthesis followed by subcloning into the pTT5 expression vector. Figure 1- Figure 2 depicts a schematic illustration of an exemplary IgG-proIL-15 fusion protein form.

[0307] Exemplary proteins in the PD-L1-proIL-15 form include P53021942 (SEQ ID NO: 144 and 145). Exemplary anti-human PD-L1 proteins include P40751942 (SEQ ID NO: 140 and 141).

[0308] The PD-L1-proIL-15 fusion protein or anti-human PD-L1 was produced by transient transfection in Expi293 cells and purified by one-step purification on 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 an Agilent 1260 according to the manufacturer's instructions. The purified protein showed high purity on SDS-PAGE gels and good homogeneity based on the HPLC results.

[0309] Example 1B Preparation of PD-1-proIL-15 Fusion Protein

[0310] Plasmids encoding PD-1-proIL-15 or anti-human PD-1 were constructed by standard gene synthesis followed by subcloning into the pTT5 expression vector. Figure 1- Figure 2 depicts a schematic diagram of an exemplary IgG-proIL-15 fusion protein form.

[0311] Exemplary proteins in the PD-1-proIL-15 form include P53052037 (SEQ ID NO: 146 and 147) and P79772037 (SEQ ID NO: 199 and 200). Exemplary proteins in the murine surrogate mPD-1-proIL-15 form include P55654367 (SEQ ID NO: 148 and 149). Exemplary proteins of anti-human PD-1 include P42412037 (SEQ ID NO: 142 and 143).

[0312] The PD-1-proIL-15 fusion protein or anti-human PD-1 was produced by transient transfection in Expi293 cells and purified by one-step purification on 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 an Agilent 1260 according to the manufacturer's instructions. The purified protein showed high purity on SDS-PAGE gels and good homogeneity based on the HPLC results.

[0313] Example 1C Preparation of B7H3-proIL-15 Fusion Protein

[0314] Plasmids encoding B7H3-proIL-15 with different protease-cleavable linkers were constructed by standard gene synthesis followed by subcloning into the pTT5 expression vector. Figure 1- Figure 2 depicts a schematic diagram of an exemplary IgG-proIL-2 fusion protein form.

[0315] Exemplary proteins of the B7H3-proIL-15 form with different protease-cleavable linkers include P40503699 (SEQ ID NO:136 and 137), P40743699 (SEQ ID NO:138 and 139).

[0316] The B7H3-proIL-15 fusion protein was produced by transient transfection in Expi293 cells and purified by one-step purification on 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 a Nanofilm SEC-250 column (Sepax) and an Agilent 1260 according to the manufacturer's instructions. The purified protein showed high purity on SDS-PAGE gels and good homogeneity based on the HPLC results.

[0317] Figures 15A - 15G Purification and characterization of PD-1-proIL-15 (P53052037, P79772037), PD-L1-proIL-15 (P53021942) and B7H3-proIL-15 (P40503699, P40743699) are shown. Figure 15A The results of reduced SDS-PAGE analysis are shown, Figure 15B The results of non-reduced SDS-PAGE analysis are shown. Figures 15C - 15G The results of size exclusion chromatography (SEC-HPLC) analysis are shown, indicating high purity and homogeneity of the product (no significant amounts of aggregated or degraded products).

[0318] Example 2A Binding of PD-1-proIL-15 to Human PD-1

[0319] The binding activities of P53052037 and P42412037 were determined by ELISA. At 4 °C, microtiter plates were coated overnight with 100 μl of 2 μg / ml streptavidin. The next day, the plates were washed with PBS and blocked with 2% BSA (2% bovine serum albumin in PBS). 100 μl of 2 μg / ml biotinylated huPD-1 was added to the corresponding wells and incubated for 1 hr at room temperature to capture the biotinylated protein. The plates were washed successively with PBST (0.01% Tween in PBS) and PBS.

[0320] The indicated samples (PD-1IgG or PD-1-proIL-15) used in the ELISA assay were prepared in 2% BSA at an initial concentration of 10 μg / ml, followed by serial 1 / 3 dilutions. 100 μl of the diluted protein was added to the corresponding wells and incubated for 1 h at room temperature. The plates were washed successively with PBST and PBS.

[0321] Bound antibodies were detected with a peroxidase-conjugated anti-human IgG secondary antibody (Jackson Immunoresearch). The plates were washed successively with PBST and PBS. 90 μl of TMB substrate was added to each well and incubated for 5 min at room temperature in the dark. The reaction was terminated with 45 μl of 2 M sulfuric acid, and the absorbance was read at 450 nm. The data were analyzed by Prism.

[0322] As Figure 4A shown, PD-1-proIL-15 and the corresponding PD-1IgG bound to human PD-1 similarly.

[0323] Example 2B Binding of PD-L1-proIL-15 to Human PD-L1

[0324] The binding activities of P53021942 and P40751942 were determined by ELISA. At 4 °C, microtiter plates were coated overnight with 100 μl of 2 μg / ml streptavidin. The next day, the plates were washed with PBS and blocked with 2% BSA (2% bovine serum albumin in PBS). 100 μl of 2 μg / ml biotinylated huPD-L1 was added to the corresponding wells and incubated for 1 hr at room temperature to capture the biotinylated protein. The plates were washed successively with PBST (0.01% Tween in PBS) and PBS.

[0325] Prepare the specified samples (PD-L1 IgG or PD-L1-proIL-15) used in ELISA assays in 2% BSA at an initial concentration of 10 μg / ml, followed by 1 / 3 serial dilutions. Add 100 μl of the diluted protein to the corresponding wells and incubate for 1 h at room temperature. Wash the plates sequentially with PBST and PBS.

[0326] Detect the bound antibodies with peroxidase-conjugated anti-human IgG secondary antibody (Jackson Immunoresearch). Wash the plates sequentially with PBST and PBS. Add 90 μl of TMB substrate to each well and incubate for 5 min at room temperature in the dark. Terminate the reaction with 45 μl of 2 M sulfuric acid and read the absorbance at 450 nm. Analyze the data by Prism.

[0327] As Figure 4B shown, PD-L1-proIL-15 and the corresponding PD-L1 IgG bind to human PD-L1 similarly.

[0328] Example 2C Proliferation of M07-e with PD-1-proIL-15 and PD-L1-proIL-15

[0329] Culture human M-07e cells (expressing human IL-15Rβ / γ) in RPMI 1640 supplemented with 20% fetal bovine serum (FBS) and 10% 5637 cell culture supernatant. To measure cytokine-dependent cell proliferation, harvest M-07e cells during the logarithmic growth phase and wash twice with PBS. Seed 90 μl of the cell suspension (2×10 4 cells / well) into 96-well plates and incubate for 4 h at 37 °C and 5% CO2 in assay medium (RPMI 1640 supplemented with 10% FBS) for cytokine starvation. Prepare the IL-15, P53052037 or P53021942 protein samples used in the assay at an initial concentration (30 nM for IL-15, 810 nM for P53052037 / P53021942 and 270 nM for MMP2-activated fusion protein) in assay medium, then perform 1 / 3 serial dilutions. Add 10 μl of the diluted protein to the corresponding wells and incubate for 72 h at 37 °C and 5% CO2. Perform a colorimetric assay using a Cell Counting Kit-8 (CCK-8, Dojindo, CK04) to measure the amount of viable cells.

[0330] Figure 5AIt was shown that PD-1-proIL-15 could not induce the proliferation of M07-e even at the highest concentration. PD-1-proIL-15 cleaved by MMP-2 restored part of the IL-15 activity and exhibited an activity as low as approximately 1 / 20 of wild-type IL-15. Figure 5B It was shown that PD-L1-proIL-15 had little activity at the highest concentration of 810 nM. In contrast, PD-L1-proIL-15 cleaved by MMP-2 induced the proliferation of M07-e cells with an efficiency as high as that of PD-1-proIL-15.

[0331] Example 3A Cell activation of resting and activated PBMCs with PD-1-proIL15 (pSTAT5 assay)

[0332] Frozen human PBMCs (SAILYBIO, donor 1) were recovered in RPMI-1640 supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin for approximately 2 hours at 37°C in an atmosphere of 5% CO2. For the analysis performed on pre-activated PBMCs, 10-cm plates were coated with αCD3 (1 μg / ml, BioLegend) overnight at 4°C. The recovered PBMCs were suspended in RPMI1640 supplemented with soluble αCD28 (1 μg / ml, BioLegend) and cultured for 3 days at 37°C in an atmosphere of 5% CO2. The resting or pre-activated PBMCs were adjusted to 2.6x10 6 cells / ml and seeded at 4x10 5 cells per well in a 96-well plate. After incubation with rhIL-15 or P53052037 at 37°C for 15 min, the cells were immediately fixed with Cytofix buffer (BD Bioscience) to maintain the phosphorylated state on ice for 15 min and washed once with BD Pharmingen TM staining buffer (FBS).

[0333] 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) at 4 °C for 30 min. 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) at 4 °C for 30 min. Before starting intracellular staining, cells were washed once with 1×PBS and centrifuged at 500 g for 5 min to collect the pellet. Then cells were stained with Foxp3 PE (BD 560046) and anti-Stat5 (pY694) Alexa 647 (BD 562076) for 40 min at RT. Cells were washed twice with BD Pharmingen TM 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 PBMC subsets after prodrug treatment was obtained and processed by CytoFLEX (Beckman).

[0334] Figures 6A - 6D Shown are STAT phosphorylations in CD4 T cells ( Figure 6A ), CD8 T cells ( Figure 6B ), regulatory T cells ( Figure 6C ), and NK cells ( Figure 6D ) upon treatment of resting PBMCs from donor 1 with rhIL-15 and intact and protease-activated PD-1-proIL-15. The masked or inactive pro-cytokine form of P53052037 could not induce STAT5 phosphorylation in all tested cell subsets. The protease-activated form of P53052037 (cleaved by matrix metalloproteinase-2) was equally effective in activating STAT5 phosphorylation in CD8 and CD4 T cells but was less potent in NK cells. Notably, pre-blockade of the PD-1 receptor with the parental anti-PD-1 antibody reduced the potency of P53052037 (activated form) on PD-1-expressing T cells to less than 1 / 20. These data confirm that the preferential cis-targeting of PD-1-proIL-15 leads to a significant enhancement in potency on PD-1-expressing T cells.

[0335] Figures 7A - 7DShows STAT5 phosphorylation in CD4 T cells (7A), CD8 T cells (7B), regulatory T cells (7C), and NK cells (7D) after pre-activation of PBMCs from donor 1. Data confirm that PD-1 binding enhances the potency of P53052037 (PD-1-proIL-15 fusion protein).

[0336] Figures 14A - 14D Shows STAT5 phosphorylation in CD4 T cells (14A), CD8 T cells (14B), regulatory T cells (14C), and NK cells (14D) after treatment of resting PBMCs with rhIL-15 and intact and activated PD-1-proIL-15. The pro-protein form of P79772037 could not induce STAT5 phosphorylation in all tested cell subsets. The activated form of P79772037 (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. Similar to P53052037, pre-blocking the PD-1 receptor with the parental anti-PD1 antibody reduced the potency of P79772037 (activated form) on PD-1-expressing T cells, demonstrating that cis-binding of P79772037 to PD-1 and IL-2βγ on the same cell surface significantly enhances the potency of P79772037.

[0337] Example 3B Cell activation of resting PBMCs with PD-L1-proIL-15 (pSTAT5 assay)

[0338] Frozen human PBMCs (SAILYBIO, donor 2) were recovered in RPMI-1640 supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin for approximately 2 hours at 37°C in an atmosphere of 5% CO2. The PBMCs were adjusted to 2.6×10 6 cells / ml and plated in 96-well plates at a density of 4×10 5 cells per well. The recovered PBMCs were incubated with rhIL-15 and P53021942 (intact and activated forms) at 37°C for 15 min. After incubation, the cells were immediately fixed with Cytofix buffer (BD Bioscience) to maintain the phosphorylated state on ice for 15 min and washed once with BD Pharmingen TM Staining buffer (FBS).

[0339] 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) at 4 °C for 30 min. 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) at 4 °C for 30 min. Before starting intracellular staining, cells were washed once with 1×PBS and centrifuged at 500 g for 5 min to collect the pellet. Then cells were stained with Foxp3 PE (BD 560046) and anti-Stat5 (pY694) Alexa 647 (BD 562076) for 40 min at RT. Cells were washed twice with BD Pharmingen TM 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 PBMC subsets before and after cytokine treatment was obtained and processed by CytoFLEX (Beckman).

[0340] Figures 8A - 8D STAT5 phosphorylation in CD4 T cells (8A), CD8 T cells ( Figure 8B ), regulatory T cells ( Figure 8C ), and NK cells ( Figure 8D ) is shown upon treatment of resting PBMCs from donor 2 with rhIL-15 and intact and protease-activated PD-L1-proIL-15. The masked or inactive pro-cytokine form of P53021942 could not induce STAT5 phosphorylation in all tested cell subsets. The activated form of P53021942 (cleaved by matrix metalloproteinase-2) was equally effective in activating STAT5 phosphorylation in CD8 and CD4 T cells, but was less potent in NK cells. Notably, pre-blocking the PD-L1 receptor with the parental anti-PD-L1 antibody reduced the potency of P53021942 (activated form) to less than 1 / 100 in PD-L1-expressing CD4+ T cells, CD8+ T cells, and Treg cells. These data confirm that PD-L1 binding enhances the potency of IL-15.

[0341] Example 3C Activated PD-1 / L1-proIL-15 Stimulates IFNγ Secretion by PBMC

[0342] Cryopreserved PBMCs from Donor 1 were thawed with soluble αCD3 added at 1 μg / ml and plated at 4e5 cells / well in 96-well plates with increasing concentrations of parental PD-1 / L1 antibody or PD-1 / PD-L1-proIL-15 fusion protein (both intact and activated forms). After incubation at 37 °C for 3 days, IFNγ secretion in the culture supernatants was analyzed by Human IFN-γ ELISA Set (Biolegend Cat. 430104). A pre-coated capture antibody plate was prepared as described in the manufacturer's instructions, and 100 μl of diluted standards or samples were added to the corresponding wells and incubated at room temperature for 2 h. The plate was washed 4 times with wash buffer, 100 μl of diluted detection antibody solution was added to each well, and the plate was incubated with shaking at room temperature for 30 min. Then the plate was washed 5 times with wash buffer, 100 μl of freshly mixed TMB substrate solution was added to each well, and the plate was incubated in the dark at room temperature for 20 min. The reaction was terminated with 100 μl of stop solution, and the absorbance was read at 450 nm. Data were analyzed by Prism.

[0343] As Figure 9 shown, activated PD-1 / L1-proIL-15 stimulates IFNγ production by PBMC cells, while the intact pro-cytokine does not. In contrast, PD-1 / L1 blockade alone does not induce any significant level of IFNγ secretion.

[0344] Example 4 In Vivo Efficacy of B7H3-proIL-15 with Different Protease-Cleavable Linkers in the A375-PBMC Xenograft Model

[0345] The anti-tumor efficacy of B7H3-proIL-15 with different protease-cleavable linkers as a single agent was tested in the A375-PBMC xenograft model.

[0346] 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 maintained in vitro as a suspension 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.

[0347] Female NCG mice (GemPharmatech Co., Ltd., Nanjing, China), 8 to 10 weeks old at the start of the experiment, were maintained under specific pathogen-free conditions with a 12h light / 12h dark daily cycle. The mice were kept in individually ventilated cages at a constant temperature (20 to 26 °C) and humidity (40 to 70%), with ≤6 animals per cage. Throughout the study period, the animals had free access to irradiated and sterilized dry pellet food and sterile drinking water. All procedures related to animal handling, care, and treatment in this study were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Viva before the experiment. After arrival, the animals were maintained for at least 3 days to acclimatize to the new environment. During routine monitoring, the animals were examined for any effects of tumor growth on normal behavior (such as activity, food and water consumption (by visual inspection only), weight gain / loss (weight measured twice a week), dulling of eyes / hair), and any other abnormal effects.

[0348] 4×10 6 A375 cells and 4×10 5 hPBMCs (co-cultured with A375) were subcutaneously inoculated into the right flank of the mice in 0.2 ml HBSS containing 0.1 ml Matrigel (1:1) for tumorigenesis. One week after tumor cell inoculation, the mice were injected i.v. with B7H3-proIL-15. Mice in the vehicle group were injected with PBS. Tumor volume was measured in two dimensions using calipers, and the volume was expressed in mm 3 as follows: V = 0.5 a × b 2 , where a and b are the longest and shortest diameters of the tumor, respectively. Then the tumor volume was 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.

[0349] TGI% = (1 - (T n - T0) / (V n - V0)) × 100%

[0350] T / C% = (T n / T0) / (V n / V0) × 100%

[0351] In the formula, T n and V n represent the tumor volumes of the treatment group and the vehicle control group on the nth day after the start of treatment, respectively. T0 and V0 represent the tumor volumes of the corresponding groups on the day of grouping. The results were analyzed using Prism GraphPad.

[0352] Since the protease-cleavable linker in P40503699 is more readily cleaved than the protease-cleavable linker in P40743699, Figures 10A - 10B and Table 1 show that in the A375-PBMC xenograft model, P40503699 showed better anti-tumor activity than P40743699 at 0.3 mg / kg, and 1 / 6 of the mice treated with P40503699 showed complete remission.

[0353]

[0354]

[0355] Example 5

[0356] In vivo efficacy of PD-L1-proIL-15 and PD-L1-proIL-15 in the A375-PBMC xenograft model

[0357] The anti-tumor efficacy of PD-L1-proIL-15 and PD-1-proIL-15 was tested in the A375-PBMC xenograft model. 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.

[0358] Female NCG mice (GemPharmatech Co., Ltd., Nanjing, China), 8 to 10 weeks old at the start of the experiment, were maintained under specific pathogen-free conditions with a 12 h light / 12 h dark daily cycle. The mice were kept in individually ventilated cages at a constant temperature (20 to 26 °C) and humidity (40 to 70%), with ≤6 animals per cage. Throughout the study, animals had free access to irradiated sterilized dry pellet food and sterile drinking water. Prior to conduct, all procedures related to animal handling, care, and treatment in this study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Viva. Upon arrival, the animals were maintained for at least 3 days to acclimatize to the new environment. During routine monitoring, the animals were examined for any effects of tumor growth on normal behavior (such as motility, food and water consumption (by visual inspection only), weight gain / loss (body weight measured twice a week), dulling of eyes / hair), and any other abnormal effects.

[0359] 3.5×10 6 A375 cells and 3.5×10 5hPBMCs were used for tumorigenesis. One week after tumor cell inoculation, mice were injected i.v. with PD-L1-proIL-15 or PD-1-proIL-15. Mice in the vehicle group were injected with PBS. Tumor volume was measured in two dimensions using calipers, and the volume was expressed in mm 3 as: V = 0.5 a × 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.

[0360] TGI% = (1 - (T n - T0) / (V n - V0)) × 100%

[0361] T / C% = (T n / T0) / (V n / V0) × 100%

[0362] In the formulas, T n and V n represent the tumor volumes of the treatment group and the vehicle control group on the nth day after the start of treatment, respectively. T0 and V0 represent the tumor volumes of the corresponding groups on the day of grouping. The results were analyzed using Prism GraphPad.

[0363] Figure 11A And Table 2 showed that in the A375-PBMC xenograft model, PD-L1-proIL-15 mediated a dose-dependent anti-tumor efficacy, Figure 11B and Table 2 showed that PD-1-proIL-15 also mediated a dose-dependent anti-tumor efficacy in the A375-PBMC xenograft model.

[0364]

[0365] Example 6

[0366] In vivo efficacy of mPD-1-proIL-15 in the B16F10 syngeneic model

[0367] The anti-tumor efficacy of mPD-1-proIL-15 was tested in the B16F10 syngeneic model. B16F10 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.

[0368] C57 / B6 mice (6 to 10 weeks old at the start of the experiment, Shanghai Lingchang Biotechnology Co., Ltd., Shanghai, China) were maintained under specific pathogen-free conditions with a 12h light / 12h dark daily cycle. The mice were kept in individually ventilated cages at a constant temperature (20 to 26 °C) and humidity (40 to 70%), with ≤6 animals per cage. Throughout the study period, the animals had free access to irradiated and sterilized dry pellet food and sterile drinking water. Prior to the experiment, all procedures related to animal handling, care, and treatment in this study were conducted according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Viva. Upon arrival, the animals were maintained for at least 3 days to acclimatize to the new environment. During routine monitoring, the animals were examined for any effects of tumor growth on normal behavior (such as activity, food and water consumption (by visual inspection only), weight gain / loss (weight measured twice a week), dull eyes / hair), and any other abnormal effects.

[0369] 2×10 5 B16F10 cells in 0.1 ml HBSS were subcutaneously inoculated into the right flank of the mice for tumorigenesis. Eight days after tumor cell inoculation, the mice were injected i.v. with mPD-1-proIL-15. The mice in the vehicle group were injected with PBS. Tumor volume was measured in two dimensions using calipers, and the volume was expressed in mm 2 using the formula V = 0.5a × b 3 , where a and b are the longest and shortest diameters of the tumor, respectively. Then the tumor volume was 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.

[0370] TGI% = (1 - (T n - T0) / (V n - V0)) × 100%

[0371] T / C% = (T n / T0) / (V n / V0) × 100%

[0372] In the formula, T n and V n represent the tumor volumes of the treatment group and the vehicle control group on the nth day after the start of treatment, respectively. T0 and V0 represent the tumor volumes of the corresponding groups on the day of grouping. The results were analyzed using Prism GraphPad.

[0373] Figure 12As shown in Table 3, in the B16F10 syngeneic gene model, mPD-1-proIL-15 significantly inhibited tumor growth at a dose of 1.2 mg / kg.

[0374]

[0375] Example 7

[0376] Biological Activity of PD-1-proIL-15 in Cynomolgus Monkeys

[0377] The in vivo biological activity of PD-1-proIL-15 was evaluated in cynomolgus monkeys. One male and one female cynomolgus monkey received 2 repeated doses of 10 mg / kg P53052037.

[0378] Blood samples for pharmacodynamic studies in cynomolgus monkeys were collected before dosing and at 1 h, 6 h, 24 h, 48 h, 72 h, and 96 h after the first and second doses. The concentration of P53052037 was determined using ELISA, with PD-1 as the capture antibody and HRP-conjugated anti-human IgG as the detection antibody. Figure 13A The concentration of P53052037 in cynomolgus monkey serum demonstrated that the T 1 / 2 was prolonged compared to IL-15.

[0379] Blood samples for analysis of immune cell subsets in cynomolgus monkeys were collected before dosing and at 4 days, 8 days, 15 days, 18 days, 22 days, 24 days, and 29 days after dosing. Cells were stained with a staining buffer including CD45 (clone D058-1283, BD Pharmingen), CD3 (clone SP34-2, BD Pharmingen), CD4 (clone L200, BD Pharmingen), CD8 (clone RPA-T8, BioLegend), and CD56 (clone HCD56, BioLegend) at 4°C for 30 min. The cells were then fixed and permeabilized with transcription factor buffer (eukaryotic transcription factor buffer, BioLegend), and then incubated with Ki67 (clone B56, BD Pharmingen) at room temperature for 45 min.

[0380] Flow cytometry (CytoFLEX S, Beckman coulter) was used to gate NK, CD4 + T cells and CD8 + CD45 in T cells + CD3 - CD56 + cells (NK cells), CD45 + CD3+ CD4 + cells (CD4 T cells), CD45 + CD3 + CD8 + cells (CD8 T cells) and Ki67 + cells to analyze the sample. Figures 13B - 13G showed that after treatment with P53052037 at 10 mg / kg, the expression of Ki67 and the fold change in the number of cells in NK, CD4 + T cells and CD8 + T cells increased, and then decreased to normal levels over time. After treatment with P53052037 at 10 mg / kg, all monkeys had good tolerance.

Claims

1. An activatable proprotein 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-15 protein, a second linker, and an IL-15Rα protein, wherein the hinge / Fc domain of the first polypeptide binds to the hinge / Fc domain of the second polypeptide, wherein the IL-15 protein of the first polypeptide binds to the IL-15Rα protein of the second polypeptide, and wherein the IL-15Rα of the first polypeptide binds to the IL-15 protein of the second polypeptide, wherein the binding masks the binding site of the IL-15 protein, which originally binds to the IL-15Rβ / γc chain present on the surface of immune cells in vitro or in vivo, and wherein the second linker is a cleavable linker.

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

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

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 according to claim 6, wherein the VH region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with 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 having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from SEQ ID NOs: 26, 28, 30, 32, 34, 36 and 38.

8. The activatable proprotein homodimer according to claim 1, wherein the Fab region specifically binds to human B7H3 and comprises a heavy chain variable (VH) region and a light chain variable (VL) region, the heavy chain variable region comprising the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO:202, and the light chain variable region comprising the VLCDR1, VLCDR2, and VLCDR3 shown in SEQ ID NO:

203.

9. The activatable proprotein homodimer according to claim 8, wherein the VH region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:202, and the VL region correspondingly comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:

203.

10. The activatable proprotein homodimer according to any one of claims 1-9, wherein the Fc domain comprises the CH2 domain, CH3 domain or 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.

11. The activatable proprotein homodimer according to any one of claims 1-10, wherein the hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence selected from Table F1, and wherein the Fc domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence selected from Table F1.

12. The activatable proprotein homodimer according to any one of claims 1-11, wherein the Fc domain is a modified Fc domain that does not bind or substantially does not bind to FcγR and retains normal or substantially normal binding to FcRn.

13. The activatable proprotein homodimer according to claim 12, wherein the modified Fc domain comprises a modified IgG1 CH2 domain having an L234A / L235A (“LALA”) mutation and / or a P329A or P329G mutation (EU numbering).

14. The activatable proprotein homodimer according to any one of claims 1-11, wherein the IL-15 protein comprises, consists of or consists essentially of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 100% identity to a sequence selected from Table S1: optionally Wherein the IL-15 protein comprises, consists of, or consists essentially of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 79 and retaining the K86G and S162A mutations.

15. The activatable proprotein homodimer according to claim 14, wherein the IL-15 protein comprises or retains one or more amino acid substitutions at positions D8, D22, E46, V49, I50, L66 and / or K86 as defined by SEQ ID NO:69 (mature human IL-15) and / or at position S162 as defined by SEQ ID NO:68 (IL-15FL precursor).

16. The activatable proprotein homodimer according to claim 15, wherein the one or more amino acid substitutions are selected from D8N, D22K, E46K, V49D, I50D, L66E, K86G and 162A, optionally a combination of K86G and S162A.

17. The activatable proprotein homodimer according to claim 16, wherein the one or more amino acid substitutions are selected from the group consisting of K86G and I62A, V49D and S162A; I50D and S162A; L66E and S162A; D8N and S162A; V49D and S162A; E46K and S162A; E46K, E53K and S162A; D22K, E46K and S162A; and the combination of D22K, E46K, E53K and S162A.

18. The activatable proprotein homodimer according to any one of claims 1-17, wherein the IL-15Rα protein comprises, consists of, or consists essentially of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 100% identity to a sequence selected from Table S2, optionally wherein the IL-15Rα protein comprises, consists of, or consists essentially of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 100% identity to SEQ ID NO: 87 or 88 and retaining the T2A substitution.

19. The activatable proprotein homodimer according to claim 18, wherein the IL-15Rα protein comprises or retains one or more amino acid substitutions at positions R24, R26 and R35 as defined by SEQ ID NO: 82 (IL-15Rα Sushi+).

20. The activatable proprotein homodimer according to claim 19, wherein the one or more amino acid substitutions are selected from R24E, R26E and R35E.

21. The activatable proprotein homodimer according to claim 20, wherein the one or more amino acid substitutions are selected from the combination of R26E and R35E; and R24E, R26E and R35.

22. The activatable proprotein homodimer according to any one of claims 18-20, wherein the IL-15Rα protein comprises or retains an amino acid substitution at position T2 as defined by SEQ ID NO: 82 (IL-15Rα Sushi+).

23. The activatable proprotein homodimer according to claim 22, wherein the amino acid substitution is T2A.

24. The activatable proprotein homodimer according to claim 23, wherein the IL-15α protein comprises SEQ ID NO: 82 or 83 having the T2A substitution.

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

26. The activatable pro-protein homodimer according to any one of claims 1-25, wherein the first linker is an uncleavable or stable linker, and wherein the cleavable linker comprises a protease cleavage site, optionally wherein the cleavable linker is selected from Table S3.

27. The activatable pro-protein homodimer according to claim 26, wherein the protease cleavage site is cleavable by a protease selected from one or more of metalloproteases, serine proteases, cysteine proteases, and aspartic proteases.

28. The activatable pro-protein homodimer according to claim 26 or 27, wherein the protease cleavage site is cleavable by a protease selected from one or more of MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, TEV protease, matriptase, uPA, FAP, podoplanin, PSA, kallikrein, cathepsin A, and cathepsin B.

29. The activatable pro-protein homodimer according to any one of claims 1-28, wherein the length of 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 or has a length of 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.

30. An activatable pro-protein 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 kappa); the Fc domain comprises an IgG1 hinge of SEQ ID NO:42, a modified human IgG1 CH2 domain of SEQ ID NO:57, and a human IgG1 CH3 domain of SEQ ID NO:58; the first linker is an 8-amino acid stable linker of SEQ ID NO: (178, where x is 2); the IL-15 protein comprises SEQ ID NO:79, optionally having K86G and S162A mutations; the second linker is a protease-cleavable linker of SEQ ID NO:90 or SEQ ID NO:201; and the IL-15Rα protein comprises SEQ ID NO:87, optionally having T2A mutations.

31. An activatable pro-protein 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 in the IL-15 protein that bind to the IL-15Rβ / γc chain present on the surface of the immune cells in vitro or in vivo.

32. An activatable pro-protein homodimer according to claim 31, wherein the immune cells are selected from one or more of T cells, B cells, natural killer cells, monocytes, and macrophages.

33. An activatable pro-protein homodimer according to any one of claims 1-32, wherein the first polypeptide and the second polypeptide comprise the following amino acid sequences and VL / CL region polypeptides, consist of the amino acid sequences and the VL / CL region polypeptides, or consist essentially of the amino acid sequences and the VL / CL region polypeptides: The amino acid sequence has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to a sequence (chain 1 and chain 2) selected from Table S4, and the VL / CL region polypeptide has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to the corresponding sequence (chain 3 and chain 4) from Table S4, optionally wherein: The first polypeptide and the second polypeptide comprise, consist of, or consist essentially of the following amino acid sequence and VL / CL region polypeptide: the amino acid sequence has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 136, and the VL / CL region polypeptide has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 137; The first polypeptide and the second polypeptide comprise, consist of, or consist essentially of the following amino acid sequence and VL / CL region polypeptide: the amino acid sequence has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 138, and the VL / CL region polypeptide has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 139; The first polypeptide and the second polypeptide comprise, consist of, or consist essentially of the following amino acid sequence and VL / CL region polypeptide: the amino acid sequence has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 140, and the VL / CL region polypeptide has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 141; The first polypeptide and the second polypeptide comprise, consist of, or consist essentially of the following amino acid sequence and VL / CL region polypeptide: the amino acid sequence has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 142, and the VL / CL region polypeptide has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 143; The first polypeptide and the second polypeptide comprise, consist of, or consist essentially of the following amino acid sequence and VL / CL region polypeptide: the amino acid sequence has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 144, and the VL / CL region polypeptide has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 145; The first polypeptide and the second polypeptide comprise, consist of, or consist essentially of the following amino acid sequence and VL / CL region polypeptide: the amino acid sequence has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 146, and the VL / CL region polypeptide has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 147; The first polypeptide and the second polypeptide comprise, consist of, or consist essentially of the following amino acid sequence and VL / CL region polypeptide: the amino acid sequence has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 148, and the VL / CL region polypeptide has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 149; or The first polypeptide and the second polypeptide comprise, consist of, or consist essentially of the following amino acid sequence and VL / CL region polypeptide: the amino acid sequence has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 199, and the VL / CL region polypeptide has at least 80%, 85%, 90%, 95%, 98%, or 100% identity to SEQ ID NO: 200; 34. An activatable pro-protein homodimer according to any one of claims 1-33, which is substantially in the homodimer form in a physiological solution or under physiological conditions, optionally under in vivo conditions.

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

36. The one or more recombinant nucleic acid molecules according to claim 35, wherein the first recombinant nucleic acid molecule encodes the VH / CH1 region of the Fab region, the hinge / Fc domain, the first linker, the IL-15 protein, the second linker, and the IL-15Rα protein, and wherein the second nucleic acid molecule encodes the VL / CL region of the Fab region.

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

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

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

40. A pharmaceutical composition comprising an activatable proprotein homodimer according to any one of claims 1-34 and a pharmaceutically acceptable carrier.

41. 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 the pharmaceutical composition according to claim 40.

42. The method according to claim 41, wherein the disease is cancer, optionally cancer expressing or overexpressing PD-L1 or B7H3.

43. The method according to claim 42, wherein the cancer is primary cancer or metastatic cancer and is selected from one or more of the following cancers: 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 myelogenous leukemia, acute myeloid leukemia or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, liver cancer (hepatocellular carcinoma), sarcoma, B cell malignancy, 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.

44. The method according to any one of claims 41-43, wherein after administration, the activatable pro-protein homodimer is activated by proteolytic cleavage in cancer cells or cancer tissues or the tumor microenvironment (TME), and the proteolytic cleavage exposes one or more binding sites of the IL-15 protein that bind to the IL-15Rβ / γc chain present on the surface of immune cells in vitro or in vivo, thereby generating an activated protein.

45. The method according to claim 44, wherein the activated protein binds to the IL-15Rβ / γc chain present on the surface of immune cells by the IL-15 protein in vitro or in vivo.

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

47. The method according to any one of claims 41-43, wherein the administration and activation of the activatable pro-protein 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.

48. The method according to any one of claims 41-47, wherein the administration and activation of the activatable pro-protein increases the 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.

49. The method according to any one of claims 41-48, wherein the pharmaceutical composition is administered to the subject by parenteral administration.

50. The method according to claim 49, wherein the parenteral administration is intravenous administration.

51. Use of the pharmaceutical composition according to claim 40 in the preparation of a drug for treating a disease of a subject, optionally cancer (such as cancer expressing or overexpressing PD-L1) and / or for enhancing the immune response in a subject.

52. The pharmaceutical composition according to claim 40, which is for treating a disease in a subject, optionally cancer (such as cancer expressing or overexpressing PD-L1 or B7H3), and / or for enhancing the immune response in a subject.

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