Masked cytokines and methods of use thereof

Abstract

To provide a cytokine-based cancer therapy that is more tumor site-selective, has improved PK and efficacy, and causes fewer side effects.SOLUTION: Provided is a prodrug comprising a cytokine moiety, a masking moiety, and a carrier moiety, wherein: a) the masking moiety binds to the cytokine moiety and inhibits a biological activity of the cytokine; b) the carrier moiety comprises an antigen binding moiety, wherein the antigen binding moiety binds to an antigen expressed on the surface of a target immune cell; and c) the masking moiety is directly or indirectly linked to the carrier moiety.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application also claims priority to U.S. Provisional Application No. 62 / 959,973, filed January 11, 2020; U.S. Provisional Application No. 63 / 027,138, filed May 19, 2020; U.S. Provisional Application No. 63 / 029,473, filed May 23, 2020; and U.S. Provisional Application No. 63 / 126,393, filed December 16, 2020. The disclosures of the foregoing priority applications are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing which is incorporated herein by reference in its entirety. The ASCII copy was created on January 11, 2021, is named 025471_WO008_SL.txt, and is 240,322 bytes in size. [Background technology]

[0003] Background of the Invention Interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21 play important roles in the proliferation, differentiation, and survival of immune cells. The receptors for these cytokines share a common gamma chain (γC), also known as CD132. Therefore, these cytokines are also referred to as common γC family cytokines.

[0004] IL-2 plays a central role in lymphocyte development, survival, and homeostasis. It has 133 amino acids and consists of four antiparallel, amphipathic alpha helices that form a quaternary structure essential for its function (Smith, Science (1988) 240:1169-76; Bazan, Science (1992) 257:410-13). IL-2 exerts its activity by binding to the IL-2 receptor (IL-2R), which consists of up to three individual subunits. The association of the α (CD25 or Tac antigen), β (CD122), and γC subunits results in a trimeric, high-affinity receptor for IL-2 (K DThe dimeric IL-2 receptor, consisting of β and γ subunits, is referred to as the intermediate affinity IL-2R (K D The α subunit alone forms a monomeric low-affinity IL-2 receptor (K D (approximately 10 nM). See, e.g., Kim et al., Cytokine Growth Factor Rev. (2006) 17:349-66. Although the dimeric intermediate-affinity IL-2 receptor binds IL-2 with an affinity approximately 100-fold lower than the trimeric high-affinity receptor, both the dimeric and trimeric IL-2 receptors can signal upon IL-2 binding (Minami et al., Annu Rev Immunol. (1993) 11:245-68).

[0005] IL-15 is a cytokine structurally similar to IL-2. IL-15 is secreted by mononuclear phagocytes and other immune cells after viral infection. IL-15 induces proliferation of natural killer (NK) and other cells of the immune system and participates in the killing of virus-infected and cancer cells. Like IL-2, IL-15 binds to the intermediate affinity receptor, the IL-2 receptor (IL-2R) β / γ complex, with a K of approximately 1 nM. D (Giri et al., EMBO J. (1994) 13:2822-30). IL-15 binds with much higher affinity to the IL-15 receptor (IL-15R) α (K D approximately 0.05nM). IL-15Rα can combine with the IL-2Rβ / γ complex to form the IL-15-specific, functional high-affinity (αβγ) receptor ((Minami et al., Annu. Rev. Immunol. (1993) 11:245-67; ​​Giri et al., J Leukoc Biol. (1995) 5745:763-6; and Lehours et al., (2000) 11:207-15).

[0006] IL-21 activates CD4 +It is produced by T cells, T follicular helper cells, and natural killer T (NKT) cells (Spolski and Leonard, Ann Rev Immunol. (2008) 26:57008). IL-21 has been shown to exert pleiotropic effects on the proliferation, differentiation, and cytotoxicity of various classes of lymphoid cells. More recently, IL-21 has been shown to inhibit the proliferation, differentiation, and cytotoxicity of various classes of lymphoid cells. + T-helper 17 (TH) is a subset of T cells that is associated with the development of inflammatory conditions and autoimmune diseases. 17 It has been shown that IL-21 plays an important role in the differentiation of IL-21 cells into IL-21Rα (Korn et al., Nature (2007) 448(7152):484-87; Nurieva et al., Nature (2007) 448(7152):480-83). The receptor complex of IL-21 consists of the dedicated chain IL-21Rα and the common chain γC (or Rγ). Human IL-21 binds to IL-21Rα with extremely high affinity (K D approximately 70 pM; Zhang et al., Biochem Biophys Res Commun. (2003) 300(2):291-6), while binding to IL-21Rγ is relatively low affinity (K D approximately 160 μM).

[0007] The above cytokines, their muteins, and fusion proteins have been explored for their potential as therapeutic agents, and recombinant IL-2 was the first cytokine approved for cancer treatment. However, these cytokine drugs and drug candidates have significant side effects. Furthermore, their in vivo half-lives are often short, even when present as antibody-cytokine fusion molecules, likely due to "PK precipitation" formed by the cytokine's receptor on immune cells. Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, there is a need to develop cytokine-based cancer therapies that are more tumor site selective, have improved PK and efficacy, and concomitantly cause fewer side effects. [Means for solving the problem]

[0009] Summary of the Invention The present invention provides prodrugs comprising a cytokine moiety, a masking moiety, and a carrier moiety, wherein the masking moiety binds to the cytokine moiety and inhibits biological activity of the cytokine; the carrier moiety comprises an antigen-binding moiety that binds to an antigen expressed on the surface of a target cell; and the masking moiety is linked to the carrier moiety without a cleavable peptide linker (e.g., indirectly linked to the carrier moiety via a non-cleavable peptide linker; or directly linked without a peptide linker).

[0010] In some embodiments, the prodrug is activated by cells that express both antigen and cytokine moiety receptors on the cell surface.In some embodiments, the biological activity of the prodrug is increased at least 2-fold, 5-fold, or 10-fold in diseased areas containing cells compared to areas that do not contain cells.In specific embodiments, the cytokine moiety receptor comprises two or more subunits.

[0011] In one embodiment, the antigen is selected from PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, CD25, CD16a, CD16b, NKG2D, NKP44, NKP30, CD19, CD20, CD30, CD38, BCMA, and signal regulatory protein alpha (SIRP alpha, designated CD172a).

[0012] In certain embodiments, the cytokine moiety comprises an IL-2 agonist polypeptide, an IL-7 agonist polypeptide, an IL-9 agonist polypeptide, an IL-15 agonist polypeptide, an IL-21 agonist polypeptide, an IL-1α agonist polypeptide, an IL-1β agonist polypeptide, an IL-4 agonist polypeptide, an IL-5 agonist polypeptide, an IL-6 agonist polypeptide, an IL-8 agonist polypeptide, an IL-10 agonist polypeptide, an IL-12 agonist polypeptide, an IL-17 agonist polypeptide, an IL-18 agonist polypeptide, an IL-22 agonist polypeptide, an IL-23 agonist polypeptide, an IL-31 agonist polypeptide, an IL-33 agonist polypeptide, an IL-36 agonist polypeptide, an interferon-alpha agonist polypeptide, an interferon gamma agonist polypeptide, a 4-1BB ligand, an OX-40 ligand, or a CD-40 ligand.

[0013] In certain embodiments, the cytokine portion comprises an IL-2 agonist polypeptide or an IL-15 agonist polypeptide, and the masking portion comprises the ECD of IL-2 receptor beta (IL-2Rβ ECD) or a functional fragment thereof. In further embodiments, the cytokine portion comprises an IL-21 agonist polypeptide, and the masking portion comprises a single-chain Fv (scFv) or single-domain antibody to IL-21. In other embodiments, the cytokine portion comprises an IL-2 agonist polypeptide, and the masking portion comprises an scFv or single-domain antibody to IL-2. In further embodiments, the cytokine portion comprises an IL-15 agonist polypeptide, and the masking portion comprises an scFv or single-domain antibody to IL-15. In other embodiments, the cytokine portion comprises an IL-15 agonist polypeptide, and the masking portion comprises the sushi domain of IL-15 receptor alpha (IL-15Rα sushi domain).

[0014] In certain embodiments, the cytokine portion comprises an IL-21 agonist polypeptide comprising SEQ ID NO: 1, or an amino acid sequence at least 90% identical thereto. In other embodiments, the cytokine portion comprises an IL-2 agonist polypeptide comprising SEQ ID NO: 6 or 62, or an amino acid sequence at least 95% identical thereto. In other embodiments, the cytokine portion comprises an IL-15 agonist polypeptide comprising SEQ ID NO: 7, or an amino acid sequence at least 95% identical thereto.

[0015] In certain embodiments, the masking moiety comprises the extracellular domain (ECD) of a cytokine receptor. In a specific embodiment, the cytokine moiety comprises an IL-7 agonist polypeptide, and the masking moiety comprises the ECD of IL-7 receptor alpha (IL-7Rα ECD) or a functional analog thereof. In a specific embodiment, the cytokine moiety is an IL-21 agonist polypeptide, and the masking moiety comprises the ECD of IL-21 receptor alpha (IL-21Rα ECD) or a functional analog thereof. In a specific embodiment, the masking moiety comprises an IL-21Rα ECD comprising an amino acid sequence selected from SEQ ID NOs: 12, 13, and 63-73, or at least 90% (e.g., at least 95%) identical thereto, or a functional analog thereof. In certain embodiments, the masking moiety comprises an IL-2Rβ ECD comprising SEQ ID NO: 11, or an amino acid sequence at least 95% identical thereto, or a functional analog thereof.

[0016] In certain embodiments, the masking moiety comprises an scFv that binds to the cytokine moiety and inhibits or prevents the interaction between the cytokine moiety and its receptor. In a specific embodiment, the masking moiety is an scFv that binds to human IL-21 and comprises the amino acid sequence of SEQ ID NO: 20 or 21. In a specific embodiment, the masking moiety comprises an scFv that binds to IL-2 and inhibits or prevents the interaction of IL-2 with IL-2Rα, IL-2Rβ, and / or IL-2Rγ, optionally wherein the scFv comprises the heavy chain CDRs and light chain CDRs of antibody 4E12B2D10. In certain embodiments, the scFv comprises the heavy and light chains of antibody 4E12B2D10. In a specific embodiment, the heavy and light chain amino acid sequences of 4E12B2D10 comprise SEQ ID NOs: 60 and 61, respectively. In certain embodiments, the masking moiety is an scFv that binds IL-2 and comprises SEQ ID NO: 22 or 23, or an amino acid sequence at least 95% identical to SEQ ID NO: 22 or 23. In a specific embodiment, the masking moiety comprises an scFv that binds IL-15 and inhibits or prevents the interaction of IL-15 with IL-2Rβ and / or the interaction of IL-15 with IL-2Rγ, optionally wherein the scFv comprises the heavy chain CDRs and light chain CDRs of anti-IL-15 antibody 146B7, 146H5, or 404E4, or optionally wherein the scFv comprises the heavy chain and light chain of anti-IL-15 antibody 146B7, 146H5, or 404E4, or optionally wherein the scFv comprises SEQ ID NO: 18 or 19. In certain embodiments, the scFv or Fab IL-15 antagonist comprises heavy chain CDRs 1-3 of an anti-IL-15 antibody selected from 146B7, 146H5, and 404E4; and light chain CDRs 1-3 of an anti-IL-15 antibody selected from 146B7, 146H5, and 404E4, all of which are described in WO2003 / 017935A2.

[0017] In a specific embodiment, the masking moiety comprises an scFv that binds to IL-21 and inhibits or disrupts the interaction between IL-21 and IL-21Rα and / or the interaction between IL-21 and IL-2Rγ. In a specific embodiment, the masking moiety comprises an scFv that binds to a cytokine selected from IL-2, IL-7, IL-9, IL-15, or IL-21 and inhibits or disrupts the interaction between the cytokine and IL-2Rγ.

[0018] In certain embodiments, the prodrug has a half-life in a non-human primate or human that is at least 20 times, at least 50 times, at least 100 times, at least 150 times, or at least 200 times longer than the corresponding wild-type cytokine.

[0019] In specific embodiments, the prodrug comprises two light chains having the amino acid sequence of SEQ ID NO:44 and two heavy chain polypeptide chains whose amino acid sequences comprise SEQ ID NOs:24 and 25; SEQ ID NOs:35 and 36; SEQ ID NOs:37 and 36; SEQ ID NOs:37 and 38; SEQ ID NOs:39 and 41; or SEQ ID NOs:42 and 43, respectively.

[0020] In a specific embodiment, the prodrug comprises two light chains having the amino acid sequence of SEQ ID NO:50 and two heavy chain polypeptide chains whose amino acid sequences comprise SEQ ID NOs:51 and 54; SEQ ID NOs:51 and 55; SEQ ID NOs:51 and 56; SEQ ID NOs:52 and 54; SEQ ID NOs:53 and 58; SEQ ID NOs:53 and 59; or SEQ ID NOs:52 and 57, respectively.

[0021] In certain embodiments, the present invention provides a pharmaceutical composition comprising a prodrug of the present invention and a pharmaceutically acceptable excipient.

[0022] In certain embodiments, the present invention provides one or more polynucleotides encoding the prodrugs, expression vectors containing the polynucleotides, and host cells (e.g., mammalian host cells such as CHO, NS0, and 293T cells) containing the expression vectors. The present invention also provides methods for producing the prodrugs, comprising culturing host cells under conditions that allow for expression of the prodrug, and isolating (and also purifying) the prodrug.

[0023] Fc-based IL-21 prodrug molecules failed to bind to cells expressing the IL-21 receptor, such as Mino cells (Figure 2), confirming the masking of the cytokine moiety. Masked molecules with a PD-1 antibody as a carrier were also shown to have minimal activity on NK92 cells, which do not express PD-1 (Figure 3). Surprisingly, we observed that PD-1 antibody-based IL-21 prodrug molecules had significant activity on pre-activated Mino cells (Figures 4A and 4B). Similar results were observed with IL-2 prodrugs (Figures 6A and 6B) and IL-15 prodrugs (Figures 5A–C). Without intending to be bound by any theory, we hypothesize that when both the antigen and cytokine receptor are expressed on the same cells, the cis binding of the PD-1 antibody to the antigen and the cytokine moiety to the cytokine receptor, respectively, leads to the cancellation of the masking effect of the masking moiety. Therefore, we propose that prodrugs with a targeting moiety offer significant selectivity for cells expressing the antigen targeted by the carrier moiety over those without the antigen. Surprisingly, prodrugs can be constructed without the need for protease cleavage or removal of masking moieties.

[0024] The present invention provides prodrugs that can be selectively activated at disease sites without the need for protease cleavage or removal of the masking moiety. In one embodiment, the present invention provides novel cytokine prodrugs comprising a cytokine moiety (cytokine agonist polypeptide), a masking moiety, and a carrier moiety, wherein the masking moiety binds to the cytokine agonist polypeptide and inhibits the intended biological activity of the cytokine; the carrier moiety comprises an antigen-binding moiety; and the masking moiety is linked to the carrier moiety directly or indirectly via a non-cleavable peptide linker or without a peptide linker; and wherein the activity of the prodrug is higher for stimulating cells that express both the antigen targeted by the carrier moiety and the cytokine receptor than for stimulating cells that express the cytokine receptor but do not express the antigen targeted by the carrier moiety.

[0025] In one embodiment, the present invention provides novel cytokine prodrugs comprising a cytokine moiety (cytokine agonist polypeptide), a masking moiety, and a carrier moiety, wherein the masking moiety binds to the cytokine agonist polypeptide and inhibits the intended biological activity of the cytokine; the carrier moiety comprises an antigen-binding moiety; the masking moiety is linked to the carrier moiety directly or indirectly, with or without a non-cleavable peptide linker; and wherein the prodrug is activated at a disease site where cells expressing both the antigen targeted by the carrier moiety and a receptor for the cytokine are present, or the biological activity of the prodrug is increased by at least 2-fold, at least 5-fold, or at least 10-fold at a disease site where cells expressing both the antigen targeted by the carrier moiety and a receptor for the cytokine are present.

[0026] In some embodiments, the prodrug does not include any cleavable peptide linker.

[0027] In some embodiments, the carrier moiety comprises an antigen-binding moiety, wherein the antigen is expressed on an immune cell, hi some embodiments, the carrier moiety comprises an antigen-binding moiety, wherein the antigen is selected from PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, CD25, CD16a, and CD16b.

[0028] In certain embodiments, the cytokine portion comprises a cytokine selected from an IL-2 agonist polypeptide, an IL-7 agonist polypeptide, an IL-9 agonist polypeptide, an IL-15 agonist polypeptide, and an IL-21 agonist polypeptide. In certain embodiments, the cytokine is selected from an IL-1α agonist polypeptide, an IL-1β agonist polypeptide, an IL-4 agonist polypeptide, an IL-5 agonist polypeptide, an IL-6 agonist polypeptide, an IL-8 agonist polypeptide, an IL-10 agonist polypeptide, an IL-12 agonist polypeptide, an IL-15 agonist polypeptide, an IL-17 agonist polypeptide, an IL-18 agonist polypeptide, an IL-22 agonist polypeptide, an IL-23 agonist polypeptide, an IL-31 agonist polypeptide, an IL-33 agonist polypeptide, an IL-36 agonist polypeptide, an interferon-alpha agonist polypeptide, interferon gamma, a 4-1BB ligand, an OX-40 ligand, or a CD-40 ligand.

[0029] In some embodiments, the masking moiety is the extracellular domain (ECD) of a receptor for the cytokine. In some embodiments, the masking moiety comprises an antibody or a binding fragment of an antibody against the cytokine.

[0030] In certain embodiments, the cytokine is an IL-21 agonist polypeptide; and wherein the masking moiety is the extracellular domain of IL-21 receptor alpha (IL-21Rα ECD) or a functional analog thereof. In certain embodiments, the cytokine is an IL-2 agonist polypeptide or an IL-15 agonist polypeptide; and wherein the masking moiety is the extracellular domain of IL-2 receptor beta (IL-2Rβ ECD). In certain embodiments, the cytokine is an IL-21 agonist polypeptide; and wherein the masking moiety is a single chain Fv (scFv) or single domain antibody against IL-21.

[0031] In certain embodiments, the cytokine is an IL-2 agonist polypeptide; and wherein the masking moiety is a single chain Fv (scFv) or single domain antibody against IL-2.

[0032] In certain embodiments, the cytokine is an IL-15 agonist polypeptide; and wherein the masking moiety is a single-chain Fv (scFv) or single-domain antibody to IL-15. In certain embodiments, the cytokine moiety comprises an IL-15 agonist polypeptide, and wherein the chimeric molecule further comprises the sushi domain of IL-15 receptor alpha (IL-15Rα sushi domain).

[0033] In certain embodiments, the IL-21 agonist polypeptide comprises an amino acid sequence of SEQ ID NO: 1 or at least 90% identical to SEQ ID NO: 1. In certain embodiments, the masking moiety is IL-21Rα-ECD or a functional analog thereof, which comprises an amino acid sequence selected from SEQ ID NOs: 12, 63-72, and 73, or at least 90% identical to SEQ ID NO: 12. In certain embodiments, the masking moiety is an scFv that binds to human IL-21; and wherein the scFv comprises the amino acid sequence of SEQ ID NO: 20 or 21.

[0034] In certain embodiments, the IL-2 agonist polypeptide comprises an amino acid sequence of SEQ ID NO: 6 or 61 or at least 95% identical to SEQ ID NO: 6 or 61. In certain embodiments, the masking moiety comprises IL-2Rβ-ECD or a functional analog thereof; wherein the IL-2-Rβ-ECD comprises an amino acid sequence of SEQ ID NO: 11 or at least 95% identical to SEQ ID NO: 11. In certain embodiments, the masking moiety is an scFv that binds IL-2, wherein the scFv interferes with the interaction of IL-2 with IL-2Rα. In certain embodiments, the masking moiety is an scFv that binds IL-2, wherein the scFv interferes with the interaction of IL-2 with IL-2Rβ. In certain embodiments, the masking moiety is an scFv that binds IL-2, wherein the scFv interferes with the interaction of IL-2 with IL-2Rγ. In certain embodiments, the masking moiety is an scFv that binds IL-2, wherein the scFv comprises the same heavy chain CDRs and light chain CDRs as hybridoma 4E12B2D10. In certain embodiments, the masking moiety is an scFv that binds to IL-2, wherein the scFv comprises the amino acid sequence of SEQ ID NO:22, 23 or that is at least 95% identical to SEQ ID NO:22, 23.

[0035] In certain embodiments, the IL-15 agonist polypeptide comprises an amino acid sequence of SEQ ID NO:7 or at least 95% identical to SEQ ID NO:7. In certain embodiments, the IL-15 prodrug further comprises a sushi domain, which comprises an amino acid sequence of SEQ ID NO:8 or at least 95% identical to SEQ ID NO:8. In certain embodiments, the masking moiety comprises an IL-2Rβ-ECD or a functional analog thereof; wherein the IL-2-Rβ-ECD comprises an amino acid sequence of SEQ ID NO:11 or at least 95% identical to SEQ ID NO:11. In certain embodiments, the masking moiety is an scFv that binds to IL-15, wherein the scFv interferes with the interaction of IL-15 and IL-2Rβ. In certain embodiments, the masking moiety is an scFv that binds to IL-15, wherein the scFv interferes with the interaction of IL-15 and IL-2Rγ. In certain embodiments, the masking moiety is an scFv that binds to IL-15, wherein the scFv comprises the same heavy chain and light chain CDRs as IL-15 antibody 146B7, 146H5, or 404E4. In certain embodiments, the masking moiety is an scFv that binds to IL-15, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 18 or 19.

[0036] In certain embodiments, the human IL-21 agonist polypeptide comprises a mutation at one of positions selected from D18, Q19, E109, and K117 (numbering according to SEQ ID NO: 1). In a specific embodiment, the human IL-21 agonist polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, and 5.

[0037] In certain embodiments of the prodrug, the cytokine moiety and the masking moiety are fused to the carrier moiety via a non-cleavable peptide linker, such as one selected from SEQ ID NOs: 27-34.

[0038] In some embodiments of the prodrug, the carrier moiety is an IgG1 antibody (EU numbering) containing the mutations L234A and L235A ("LALA") or an IgG4 antibody containing the mutations 228P / L234A / L235A (PAA). Other mutations that reduce Fc functionality, such as those described by Tam et al., Antibodies (2017) 6(12):1-34, can also be introduced when the Fc domain or Fc of an antibody is used as the carrier moiety.

[0039] In a specific embodiment, the carrier moiety is an antibody comprising a knobs-into-hole mutation, wherein the cytokine moiety and the masking moiety are fused to different heavy chains of the antibody. In some embodiments, the knobs-into-hole mutation comprises a T366Y "knob" mutation in the polypeptide chain of the Fc domain or the heavy chain of the antibody and a Y407T "hole" mutation in the other polypeptide chain of the Fc domain or the other heavy chain of the antibody (EU numbering). In some embodiments, the knobs-into-hole mutation comprises a Y349C and / or T366W mutation in the CH3 domain of the "knob chain" and an E356C, T366S, L368A and / or Y407V mutation in the CH3 domain of the "hole chain" (EU numbering).

[0040] In certain embodiments, the carrier moiety is selected from the group consisting of guanylate cyclase C (GCC), carbohydrate antigen 19-9 (CA19-9), glycoprotein A33 (gpA33), mucin 1 (MUC1), carcinoembryonic antigen (CEA), insulin-like growth factor 1 receptor (IGF1-R), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), delta-like protein 3 (DLL3), delta-like protein 4 (DLL4), Epidermal growth factor receptor (EGFR), glypican-3 (GPC3), c-MET, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), nectin-4, Liv-1, glycoprotein NMB (GPNMB), prostate-specific membrane antigen (PSMA), Trop-2, carbonic anhydrase IX (CA9), endothelin B receptor (ETBR), six-transmembrane epithelial antigen of the prostate 1 (STEAP1), folate receptor and / or an antigen-binding fragment thereof that specifically binds to one or more antigens selected from receptor alpha (FR-α), SLIT and NTRK-like protein 6 (SLITRK6), carbonic anhydrase VI (CA6), ectonucleotide pyrophosphatase / phosphodiesterase family member 3 (ENPP3), mesothelin, trophoblast glycoprotein (TPBG), CD19, CD20, CD22, CD33, CD40, CD56, CD66e, CD70, CD74, CD79b, CD98, CD123, CD138, CD352, CD47, signal-regulatory protein alpha (SIRPα), PD1, claudin 18.2, claudin 6, 5T4, BCMA, PD-L1, PD-1, fibroblast activation protein alpha (FAP alpha), melanoma-associated chondroitin sulfate proteoglycan (MCSP), and epithelial cell adhesion molecule (EPCAM). In a specific embodiment, the carrier moiety is an antibody or fragment thereof that specifically binds to FAP alpha or 5T4.

[0041] In other embodiments, the invention provides polynucleotides encoding the prodrugs or fusion molecules, expression vectors containing the polynucleotides, and host cells (e.g., mammalian host cells such as CHO, NS0, and 293T cells) containing the expression vectors. The invention also provides methods for producing the prodrugs or fusion molecules, comprising culturing mammalian host cells under conditions that allow for expression of the prodrug or fusion molecule, and isolating the prodrug or fusion molecule.

[0042] The present invention also provides methods for treating cancer or infectious diseases or stimulating the immune system in a patient (e.g., a human patient) in need of treatment, comprising administering to the patient a therapeutically effective amount of a cytokine prodrug or pharmaceutical composition of the present invention. The patient may have, for example, a viral infection (e.g., HIV, HBV, HCV, or HPV infection) or a cancer selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer, and gastric cancer. Also provided herein are cytokine prodrugs for use in the methods for treating cancer or infectious diseases or stimulating the immune system; uses of cytokine prodrugs for the manufacture of medicaments for treating cancer or infectious diseases or stimulating the immune system in the methods; and articles of manufacture (e.g., kits) comprising one or more dosage units of the cytokine prodrugs.

[0043] Also provided herein are prodrugs for use in the methods to treat cancer or infectious diseases or stimulate the immune system; uses of the prodrugs in the methods to manufacture medicaments for treating cancer or infectious diseases or stimulating the immune system; and articles of manufacture (e.g., kits) comprising one or more dosage units of the prodrugs.

[0044] Other features, objects, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the present invention, is given by way of illustration only and not limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief explanation of the drawings]

[0045] [Figure 1A] Figure 1A illustrates a heterodimeric cytokine prodrug in which the carrier is an antibody. Figure 1A illustrates a prodrug in which the carrier is a typical antibody having two light chains and two heavy chains. [Figure 1B] Figure 1B shows a heterodimeric cytokine prodrug in which the carrier is an antibody. Figure 1B depicts a prodrug with a bispecific antibody as the carrier, where each antigen-binding moiety is a monomeric or single-domain antibody with one antigen-binding moiety comprising a Fab domain and a second antigen-binding moiety comprising an scFv. [Figure 1C] Figure 1C shows a heterodimeric cytokine prodrug in which the carrier is an antibody. Figure 1C shows a prodrug with a bispecific antibody as the carrier, in which each antigen-binding moiety is a dimer. [Figure 1D] Figure 1D shows a heterodimeric cytokine prodrug in which the carrier is an antibody. Figure 1D shows a prodrug with a dimeric Fc domain fused to one polypeptide chain and a cytokine peptide fused to the other polypeptide chain. The cytokine is fused to a masking moiety via the N-terminus. [Figure 1E] Figure 1E shows a heterodimeric cytokine prodrug in which the carrier is an antibody. Figure 1E illustrates a prodrug with a dimeric Fc domain fused to one polypeptide chain and a cytokine peptide fused to the other polypeptide chain. A masking moiety is fused to the cytokine peptide via the N-terminus.

[0046] [Figure 2]Figure 2 shows the binding of PD-1 antibodies, Fc-IL-21 fusion molecules, and control molecules to Mino cells. Binding was analyzed by FACS.

[0047] [Figure 3] Figure 3 shows the results of NK-92 cell-based biological activity assays of IL-21 prodrugs before and after activation with the protease MMP2 and control molecules. PW04-38 αPD1-IL21-a* and PW09-16 αPD1-IL21-a* are two batches of the same molecule, an anti-PD-1 antibody-based IL-21 with the IL-21R α-ECD as the masking moiety; PW05-68 αPD1-IL21-scFv is a PD-1 antibody-based IL-21 prodrug with an scFv as the masking moiety. The first control molecule, PW04-67 αPD1-IL21nomask, is an anti-PD-1 antibody-IL-21 fusion molecule with no mask and wild-type IL-21. Other control molecules, PW09-02 αPD1-IL21 mutein R9ER76A, are anti-PD-1 antibody-IL-21 fusion molecules with an unmasked IL-21 mutein with R9E and R76A amino acid substitutions (numbering according to SEQ ID NO: 1). PW04-38 αPD1-IL21-a*activating and PW09-16 αPD1-IL21-a*activating are both anti-PD-1 antibody-IL-21 wild-type fusion molecules in which the mask has been cleaved by a protease.

[0048] [Figure 4A] Figure 4 shows the results of a Mino cell-based biological activity assay of IL-21 prodrugs before and after activation with PD-1-protease MMP2 and control molecules. Figure 4A shows the results after 72 hours of incubation of Mino cells with cytokine fusion molecules before analysis. [Figure 4B] Figure 4B shows the results of a Mino cell-based biological activity assay of IL-21 prodrugs before and after activation with PD-1-protease MMP2 and control molecules. Figure 4B shows the results after 120 hours of incubation before analysis.

[0049] [Figure 5A] Figure 5A shows the results of Ki67 activation of CD4+ T cells in PBMCs after treatment with anti-PD-1 antibody (PD-1), scFv-masked Fc-IL-15 fusion molecule (215β), scFv-masked anti-PD1-antibody-IL-15 fusion molecule (215γ), unmasked Fc-IL-15 fusion molecule (215β ref), and unmasked PD1-antibody-IL-15 fusion molecule (PD1 / IL15 mutein (M2)). Figure 5B shows the results of Ki67 activation of CD4+ T cells lacking PD-1 expression. [Figure 5B] Figure 5B shows the results of Ki67 activation of CD4+ T cells in PBMCs after treatment with anti-PD-1 antibody (PD-1), scFv-masked Fc-IL-15 fusion molecule (215β), scFv-masked anti-PD1-antibody-IL-15 fusion molecule (215γ), unmasked Fc-IL-15 fusion molecule (215β ref), and unmasked PD1-antibody-IL-15 fusion molecule (PD1 / IL15 mutein (M2)). Figure 5B shows the results of Ki67 activation of CD4+ T cells with PD-1 expression. [Figure 5C] Figure 5C shows the results of Ki67 activation of CD4+ T cells in PBMCs after treatment with anti-PD-1 antibody (PD-1), scFv-masked Fc-IL-15 fusion molecule (215β), scFv-masked anti-PD1-antibody-IL-15 fusion molecule (215γ), unmasked Fc-IL-15 fusion molecule (215β ref), and unmasked PD1-antibody-IL-15 fusion molecule (PD1 / IL15 mutein (M2)). Figure 5C shows the EC50 values ​​of test articles in CD4+ T cells with and without PD-1 expression. The fold change in EC50 values ​​between CD4+ T cells with and without PD-1 expression is also shown.

[0050] [Figure 6A]Figure 6A shows the results of Ki67 activation of CD4+ T cells in PBMCs after treatment with anti-PD-1 antibody (PD-1), a negative control (IgG1 antibody), and an anti-PD1-antibody-IL-2 mutein fusion masked with the IL-2 receptor β extracellular domain (IL-2Rβ-ECD) (PD-1 / IL2V*). Figure 6B shows the results of Ki67 activation of CD4+ T cells expressing PD-1. [Figure 6B] Figure 6B shows the results of Ki67 activation of CD4+ T cells in PBMCs after treatment with anti-PD-1 antibody (PD-1), a negative control (IgG1 antibody), and an anti-PD1-antibody-IL-2 mutein fusion masked with the IL-2 receptor β extracellular domain (IL-2Rβ-ECD) (PD-1 / IL2V*). Figure 6B shows the results of Ki67 activation of CD4+ T cells with PD-1 expression.

[0051] [Figure 7] Figure 7 shows a novel mechanism of prodrug activation via "cis-binding." Target cells (right) express both the antigen targeted by the carrier moiety and a receptor for the cytokine moiety. Binding of the prodrug to the cell surface antigen brings the prodrug into proximity with the cell's cytokine receptor, allowing the cytokine receptor to efficiently compete with and displace the masking moiety, leading to cell signaling via the bound cytokine receptor. Protease cleavage of the masking moiety is not required to unmask and activate the prodrug. In cells that express the cytokine receptor but not the antigen (left), the cytokine moiety of the prodrug remains masked and inactive. DETAILED DESCRIPTION OF THE INVENTION

[0052] Detailed Description of the Invention As used herein and in the appended claims, the singular includes the plural unless the context clearly dictates otherwise.

[0053] A numerical value or parameter preceded by "about" herein includes (and describes) the numerical value or parameter itself. For example, "about X" includes "X." Furthermore, the use of "about" before any series of numerical values ​​includes "about" each numerical value in the series. For example, "about X, Y, or Z" means "about X, about Y, or about Z."

[0054] The term "antigen-binding portion" refers to a polypeptide or set of interacting polypeptides that bind to an antigen, including, but not limited to, an antibody (e.g., a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a bispecific or bispecific antibody, an anti-idiotypic antibody, or a bifunctional hybrid antibody) or an antigen-binding fragment thereof (e.g., an Fc-containing polypeptide such as Fab, Fab', F(ab'), Fv, disulfide-linked Fv, scFv, single-domain antibody (dAb) or bispecific antibody, single-chain antibody, and immunoadhesin). In certain embodiments, the antibody can be of any heavy chain isotype (e.g., IgG, IgA, IgM, IgE, or IgD) or subtype (e.g., IgG1, IgG2, IgG3, or IgG4). In certain embodiments, the antibody can be of any light chain isotype (e.g., kappa or lambda). Antibodies can be human, non-human (e.g., from mouse, rat, rabbit, goat, or other non-human animal), chimeric (e.g., having non-human variable regions and human constant regions), or humanized (e.g., having non-human CDRs and human framework and constant regions). In certain embodiments, the antibody is a derivatized antibody.

[0055] As used herein, the terms "towards," "binds to," or "specifically binds to" in the context of an interaction between two molecules refers to a binding that does not exceed 1000 nM (e.g., does not exceed 100 nM, 10 nM, or 1 nM; e.g., less than 1 nM, 1-10 nM, 10-100 nM, or 100-1000 nM) K D It is intended to have

[0056] The term "cytokine agonist polypeptide" refers to a wild-type cytokine or an analog thereof. An analog of a wild-type cytokine has the same biological specificity as the wild-type cytokine (e.g., binds to the same receptor and activates the same target cells), but the activity level of the analog may differ from that of the wild-type cytokine. An analog may be, for example, a mutein (i.e., mutant polypeptide) of a wild-type cytokine and may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten mutations relative to the wild-type cytokine.

[0057] The terms "cytokine antagonist," "masking moiety," or "cytokine mask" refer to a moiety (e.g., a polypeptide) that binds to a cytokine, thereby inhibiting the cytokine from binding to its receptor on the surface of a target cell and / or exerting its biological function while bound to the antagonist or mask. Examples of cytokine antagonists or masks include, but are not limited to, polypeptides derived from the extracellular domain of a cytokine that contact the cytokine.

[0058] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or composition sufficient to treat a specified disorder, condition, or disease (e.g., ameliorate, alleviate, reduce, and / or delay one or more of its symptoms). With respect to diseases such as cancer, an effective amount can be an amount sufficient to delay cancer development or progression (e.g., reduce tumor growth rate and / or delay or prevent tumor angiogenesis, metastasis, or cancer cell invasion into peripheral organs), reduce the number of epithelioid cells, induce cancer regression (e.g., shrink or eradicate tumors), and / or prevent or delay cancer onset or recurrence. An effective amount can be administered in one or more doses.

[0059] The term "functional analog" refers to a molecule that has the same biological specificity (eg, binding to the same ligand) and / or activity (eg, target cell activation or inhibition) as a reference molecule.

[0060] The term "fusion" or "fusion product" in reference to two polypeptide sequences refers to the linkage of the two polypeptide sequences via a backbone peptide bond. The two polypeptides can be fused directly or via a peptide linker that is one or more amino acids in length. A fusion polypeptide can be produced recombinantly from the coding sequences of each of the two fusion partners, with or without a sequence encoding a peptide linker between them. In certain embodiments, fusion includes chemical conjugation.

[0061] The term "pharmaceutically acceptable excipient," when used with reference to an ingredient in a composition, means that the excipient is suitable for administration to a subject, including a human subject, for treatment without undue adverse side effects to the subject and without affecting the biological activity of the active pharmaceutical ingredient (API).

[0062] The term "prodrug" refers to a therapeutic molecule that is inactive or has low activity in vitro or in the circulation, while its activity is increased at the disease site.

[0063] The term "subject" refers to a mammal, including, but not limited to, a human, a pet (eg, a dog or cat), a livestock (eg, a cow or horse), a rodent, or a primate.

[0064] As used herein, "treatment" or "treating" refers to an approach to obtaining beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from a disease, reducing the extent of the disease, improving the disease state, stabilizing the disease (e.g., preventing or slowing disease worsening or progression), preventing or slowing disease spread (e.g., metastasis), preventing or slowing disease recurrence, providing partial or complete remission of the disease, reducing the dose of one or more other medications required to treat the disease, improving the patient's quality of life, and / or prolonging survival. The methods of the present invention contemplate any one or more of these aspects of treatment.

[0065] It is understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described thereunder.

[0066] I. Cytokine Prodrugs The present invention provides cytokine prodrugs that are more active at disease sites. The prodrugs comprise a cytokine agonist polypeptide (cytokine moiety), a carrier moiety, and a masking moiety. The cytokine moiety is linked to the carrier moiety, with or without a peptide linker (e.g., a non-cleavable peptide linker), and is masked (bound) by the masking moiety. The masking moiety can be linked to either the cytokine moiety or the carrier moiety, with or without a peptide linker (e.g., a non-cleavable peptide linker). Alternatively, the cytokine moiety is linked to the masking moiety, with or without a peptide linker (e.g., a non-cleavable peptide linker), which is then linked to the carrier moiety, with or without a peptide linker (e.g., a non-cleavable peptide linker).

[0067] The carrier moiety comprises an antigen-binding domain that binds to an antigen on a target cell (e.g., a tumor cell or an immune cell). In some embodiments, the carrier comprises an antibody. See, e.g., Figure 1A. In some embodiments, the carrier moiety comprises a bispecific antibody that binds to two different antigens, as illustrated in Figures 1B and 1C.

[0068] The masking moiety may comprise, for example, the extracellular domain (ECD) of a cytokine receptor, where the ECD is linked to the cytokine moiety (e.g., see Figures 1D and 1E) and / or the carrier moiety (e.g., see Figures 1A-C and 1E) via a non-cleavable linker. The mask binds to the cytokine moiety and inhibits its biological function. These prodrugs may bind to target cells via "cis-binding" of the cytokine receptor and antigen expressed on the cell surface, increasing the activity of the prodrug without cleavage and removal of the masking moiety. The cytokine moiety of the prodrug may increase activity at the target site (e.g., tumor site or surrounding environment) where both the antigen targeted by the carrier and the cytokine receptor are expressed on the same cell. Examples of such prodrugs are shown in Figures 1A-E, and the "cis-binding" mechanism of action is shown in Figure 7.

[0069] A. Cytokine Moiety of the Prodrug In some embodiments, the cytokine moiety comprises an IL-2 agonist polypeptide. In some embodiments, the IL-2 agonist polypeptide is an IL-2 mutein having the mutations R38S / F42A / Y45A / E62A or F42A / Y45A / L72G (numbering according to SEQ ID NO: 6). In some embodiments, the IL-2 muteins of the present invention can contain mutations at T3, D20, K35, R38, F42, F44, Y45, E62, E68, L72, A73, N88, N90, C125, and Q126 (numbering according to SEQ ID NO: 6). In some embodiments, the novel IL-2 muteins contain mutations at R38, F42, Y45, and A73 (numbering according to SEQ ID NO: 6).

[0070] In some embodiments, the cytokine portion of the prodrug comprises an IL-15 agonist polypeptide. In some embodiments, the human IL-15 polypeptide is selected from the group consisting of N1A, N1D, N4A, N4D, I6T, S7A, D8A, D8T, D8E, D8N, K10A, K10D, K11A, K11D, E46, V49, L45, S51, L52, D61A, D61N, T62L, T62A, E64A, E64L, E64K, E64Q, N65A, N65L, N65D, L66D, L66E, I67D, I67E, I68S, I68E, L69S, L69E, N72A, N72D, V63E, V63D, L66E, L66D, I67 E, I67D, Q108E, N112A, N1D / D61N, N1D / E64Q, N4D / D61N, N4D / E64Q, D8N / D61N, D8N / E64Q, D61N / E64Q, E64Q / Q108E, N1D / N4D / D8N, D61N / E64Q / N65D, N1D / D61N / E64Q, N1D / Q108E, N1D / D61N / E64Q / Q108E, N4D / D61N / E64Q / Q108E and D30N / E64Q / N65D (numbering according to SEQ ID NO: 7). In certain embodiments, the IL-15 prodrug further comprises a sushi domain. In certain embodiments, the sushi domain comprises an amino acid sequence of SEQ ID NO:8 or that is at least 95% identical to SEQ ID NO:8.

[0071] In certain embodiments, the cytokine moiety comprises an IL-21 agonist polypeptide. In certain embodiments, the IL-21 agonist polypeptide can be a wild-type IL-21 polypeptide, such as wild-type human IL-21 (e.g., SEQ ID NO: 1), or an IL-21 mutein derived from human IL-21, e.g., an IL-21 mutein having an amino acid sequence selected from SEQ ID NOs: 2-5. The IL-21 mutein can have significantly reduced affinity for IL-21Rα or IL-21RαRγ compared to wild-type IL-21. In certain embodiments, the IL-21 mutein has a binding affinity for high-affinity IL-2Rα that is 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 300-fold, 500-fold, 1,000-fold, or 10,000-fold lower compared to wild-type IL-21. Unless otherwise specified, all residue numbers for IL-21 and IL-21 muteins described herein are in accordance with the numbering system of SEQ ID NO: 1.

[0072] B. Masking Moieties of Prodrugs The masking moiety of the prodrug comprises a peptide or an antibody or antibody fragment that binds to the cytokine portion of the prodrug, masking the cytokine portion and inhibiting its biological function. In some embodiments, the masking moiety is operably linked to the remainder of the prodrug via a non-cleavable peptide linker.

[0073] For example, the prodrug comprises an IL-2 agonist polypeptide, and the masking moiety comprises a peptide or antibody, or antigen-binding fragment thereof, that binds to IL-2 and prevents IL-2 from binding to its cognate receptor. In certain embodiments, the masking moiety reduces the biological activity of the IL-2 moiety while masked. In certain embodiments, the IL-2 antagonist comprises an IL-2Rβ or IL-2Rγ extracellular domain, or a functional analog thereof, such as that derived from human IL-2Rβ or human IL-2Rγ (e.g., SEQ ID NO: 11 or 13). In certain embodiments, the IL-2 masking moiety comprises a peptide identified through peptide library screening. In certain embodiments, the IL-2 masking moiety comprises an antibody or fragment thereof that blocks the binding of IL-2 or an IL-2 mutein to the IL-2 receptor. In certain embodiments, the masking moiety comprises an scFv of an antibody against IL-2. In certain embodiments, the masking moiety comprises an amino acid sequence of SEQ ID NO: 22 or 23, or at least 95% identical to SEQ ID NO: 22 or 23.

[0074] In another example, the prodrug comprises an IL-21 agonist polypeptide, and the masking moiety comprises a peptide or antibody, or antigen-binding fragment thereof, that binds to IL-21 and prevents IL-21 from binding to its cognate receptor. In certain embodiments, the masking moiety reduces the biological activity of the IL-21 moiety while masked. In certain embodiments, the IL-21 antagonist comprises an IL-21Rα or IL-21Rγ extracellular domain, or a functional analog thereof, such as that derived from human IL-21Rα or human IL-21Rγ (e.g., SEQ ID NO: 12 or 13). In certain embodiments, the IL-21 masking moiety comprises a peptide identified via peptide library screening. In certain embodiments, the IL-21 masking moiety comprises an antibody or fragment thereof that blocks binding of IL-21 or an IL-21 mutein to the IL-21 receptor. In certain embodiments, the masking moiety comprises an scFv of an antibody against IL-21. In certain embodiments, the masking moiety comprises an amino acid sequence of SEQ ID NO: 20 or 21, or at least 95% identical to SEQ ID NO: 20 or 21.

[0075] In other examples, the prodrug comprises a masking moiety of any cytokine disclosed herein, including, but not limited to, a cytokine selected from an IL-7 agonist polypeptide, an IL-9 agonist polypeptide, an IL-15 agonist polypeptide, an IL-1α agonist polypeptide, an IL-1β agonist polypeptide, an IL-4 agonist polypeptide, an IL-5 agonist polypeptide, an IL-6 agonist polypeptide, an IL-8 agonist polypeptide, an IL-10 agonist polypeptide, an IL-12 agonist polypeptide, an IL-17 agonist polypeptide, an IL-18 agonist polypeptide, an IL-22 agonist polypeptide, an IL-23 agonist polypeptide, an IL-31 agonist polypeptide, an IL-33 agonist polypeptide, an IL-36 agonist polypeptide, an interferon-alpha agonist polypeptide, an interferon-gamma agonist polypeptide, a 4-1BB ligand, an OX-40 ligand, and a CD-40 ligand.

[0076] C. Carrier Moieties of Prodrugs The carrier moiety of the prodrug comprises an antigen-binding domain and optionally other domains, and the carrier moiety improves the PK profile, such as serum half-life, of the cytokine agonist polypeptide, and also targets the cytokine agonist polypeptide to a target site in the body, such as a tumor site.

[0077] 1. Antigen-binding domain of the carrier moiety The carrier moiety comprises an antigen-binding domain and can be an antibody or its antigen-binding fragment or an immunoadhesin. In some embodiments, the antigen-binding carrier moiety is a full-length antibody having two heavy chains and two light chains, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a disulfide-linked Fv fragment, a single-domain antibody, a nanobody, or a single-chain variable fragment (scFv). In some embodiments, the antigen-binding moiety is a bispecific antigen-binding moiety, capable of binding to two different antigens or two different epitopes of the same antigen. The antigen-binding moiety provides therapeutic efficacy that can be additive or synergistic to the cytokine agonist polypeptide.

[0078] The cytokine agonist polypeptide and its mask can be fused to the N-terminus or C-terminus of the light chain and / or heavy chain of the antigen-binding portion. For example, the IL-21 agonist polypeptide and its mask can be fused to an antibody heavy chain or its antigen-binding fragment, or an antibody light chain or its antigen-binding fragment. In some embodiments, one end of the IL-21 agonist polypeptide is fused to the C-terminus of one or both heavy chains of the antibody, and the IL-21 mask is fused to the other end of the IL-21 agonist polypeptide via a non-cleavable peptide linker. In some embodiments, the IL-21 agonist polypeptide is fused to the C-terminus of one heavy chain of the antibody, and the IL-21 mask is fused to the C-terminus of the other heavy chain of the antibody via a non-cleavable peptide linker, wherein the two heavy chains contain mutations that allow specific pairing of the two different heavy chains.

[0079] Strategies for forming heterodimers are well known (see, e.g., Spies et al., Mol Imm. (2015) 67(2)(A):95-106). For example, two heavy chain polypeptides of a prodrug can form stable heterodimers via "knob-into-hole" mutations. "Knob-into-hole" mutations are made to promote heterodimer formation of antibody heavy chains and are commonly used in the production of bispecific antibodies (see, e.g., U.S. Patent No. 8,642,745). For example, the Fc domain of an antibody can contain a T366W mutation in the CH3 domain of the "knob chain" and T366S, L368A, and / or Y407V mutations in the CH3 domain of the "hole chain." Additional interchain disulfide bridges between CH3 domains can also be used, for example, by introducing a Y349C mutation in the CH3 domain of the "knob chain" and an E356C or S354C mutation in the CH3 domain of the "hole chain" (see, e.g., Merchant et al., Nature Biotech (1998) 16:677-81). In other embodiments, the antibody portion can comprise a Y349C and / or T366W mutation in one of the two CH3 domains and an E356C, T366S, L368A and / or Y407V mutation in the other CH3 domain. In some embodiments, the antibody moiety may contain a Y349C and / or T366W mutation in one of the two CH3 domains and an S354C (or E356C), T366S, L368A, and / or Y407V mutation in the other CH3 domain, and an additional Y349C mutation in one of the CH3 domains and an additional E356C or S354C mutation in the other CH3 domain to form an interchain disulfide bridge (numbering is always according to the EU index of Kabat; Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Other knob-into-hole technologies, such as those described in EP1870459A1, may alternatively or additionally be used.Thus, another example of a knob-into-hole mutation in an antibody moiety is having R409D / K370E mutations in the CH3 domain of the "knob chain" and D399K / E357K mutations in the CH3 domain of the "hole chain" (EU numbering).

[0080] In one embodiment, the antigen-binding portion of the prodrug is an antibody containing L234A and L235A ("LALA") mutations in the Fc domain. The LALA mutations eliminate complement binding and fixation and Fcγ-dependent ADCC (see, e.g., Hezareh et al. J. Virol. (2001) 75 (24):12161-8). In a further embodiment, the LALA mutations are present in the antibody portion in addition to the knob-into-hole mutations.

[0081] In one embodiment, the antigen-binding portion is an antibody comprising M252Y / S254T / T256E ("YTE") mutations in the Fc domain. YTE mutations allow for simultaneous modulation of serum half-life, tissue distribution, and IgG1 activity (see Dall'Acqua et al., J Biol Chem. (2006) 281:23514-24; and Robbie et al., Antimicrob Agents Chemother. (2013) 57(12):6147-53). In a further embodiment, the YTE mutations are present in the antibody in addition to knob-into-hole mutations. In a specific embodiment, the antibody comprises YTE, LALA, and knob-into-hole mutations, or any combination thereof.

[0082] In a specific embodiment, the antigen-binding moiety is an antibody or antigen-binding fragment thereof that binds to an antigen on the surface of a target cell, such as an immune cell. Immune cells are well known in the art. Non-limiting examples of immune cells include T cells, NK cells, and macrophages. The antigen-binding moiety may have the ability to activate immune cells and enhance their anti-cancer activity. The antibody may or may not have ADCC activity. The antigen-binding moiety may also be conjugated to a cytotoxic drug. In some embodiments, the antigen-binding moiety may bind to PD-1, LAG-3, TIM-3, TIGIT, CTLA-4, or TGF-beta. In other embodiments, the antigen-binding moiety may bind to an antigen on the surface of a target cell, such as a tumor cell. For example, the antigen-binding moiety may bind to FAP alpha, 5T4, Trop-2, PD-L1, HER-2, EGFR, claudin 18.2, DLL-3, GCP3, or carcinoembryonic antigen (CEA).

[0083] In some embodiments, the antigen binding moiety is selected from the group consisting of guanylate cyclase C (GCC), carbohydrate antigen 19-9 (CA19-9), glycoprotein A33 (gpA33), mucin 1 (MUC1), insulin-like growth factor 1 receptor (IGF1-R), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), delta-like protein 3 (DLL3), delta-like protein 4 (DLL4), epidermal growth factor receptor (EGFR), glypican-3 (GPC3), c-MET, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), nectin-4, Liv-1, glycoprotein NMB (GPNMB), prostate-specific membrane antigen (PSMA), Trop-2, charcoal The antigen-binding moiety binds to acid anhydrase IX (CA9), endothelin B receptor (ETBR), six-transmembrane epithelial antigen of the prostate 1 (STEAP1), folate receptor alpha (FR-α), SLIT and NTRK-like protein 6 (SLITRK6), carbonic anhydrase VI (CA6), ectonucleotide pyrophosphatase / phosphodiesterase family member 3 (ENPP3), mesothelin, trophoblast glycoprotein (TPBG), CD19, CD20, CD22, CD33, CD40, CD56, CD66e, CD70, CD74, CD79b, CD98, CD123, CD138, CD352, CD47, signal-regulatory protein alpha (SIRPα), claudin 18.2, claudin 6, BCMA, or EPCAM. In one embodiment, the antigen-binding moiety binds to the epidermal growth factor (EGF)-like domain of DLL3. In some embodiments, the antigen-binding moiety binds to the Delta / Serrate / Lag2 (DSL)-like domain of DLL3. In some embodiments, the antigen-binding moiety binds to an epitope located after amino acid 374 of GPC3. In some embodiments, the antigen-binding moiety binds to heparin sulfate glycans of GPC3. In some embodiments, the antigen-binding moiety binds to claudin 18.2 but not claudin 18.1. In some embodiments, the antigen-binding moiety binds to claudin 18.1 with a binding affinity at least 10-fold weaker than that of claudin 18.2.

[0084] Exemplary antigen-binding portions bind to trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), anti-dPNAG antibody F598, and antigen-binding fragments thereof. In certain embodiments, the antigen-binding portion has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to trastuzumab, rituximab, brentuximab, cetuximab, or panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), anti-dPNAG antibody F598, or a fragment thereof. In certain embodiments, the antigen binding portion comprises an antibody heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the antibody heavy chain of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), anti-dPNAG antibody F598, or a fragment thereof. In some embodiments, the antigen-binding portion has an antibody light chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the antibody light chain of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), anti-dPNAG antibody F598, or a fragment thereof. The antigen-binding portion is fused to an IL-2 agonist polypeptide. In some embodiments, the antigen-binding portion comprises the six complementarity-determining regions (CDRs) of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33, anti-EGFR antibody mAb806, or anti-dPNAG antibody F598.

[0085] Several CDR designations are known in the art and are encompassed herein. One skilled in the art can easily determine CDRs based on the sequence of the heavy or light chain variable region. The "Kabat" CDR designations are based on sequence variability and are most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "Chothia" CDR designations refer to structural loops (Chothia & Lesk, J. Mol. Biol. (1987) 196:901-917). The "AbM" CDR designations represent a compromise between the Kabat CDRs and Chothia structural loops, and Oxford Molecular's AbM is used in antibody modeling software. The "Contact" CDR designations are based on analysis of available complex crystal structures. Residues from each of these CDRs are listed below in Table 1, with reference to the general antibody numbering scheme. Unless otherwise specified herein, antibody CDR amino acid numbering, including when made with reference to the Kabat, Chothia, AbM, or Contact schemes, refers to the Kabat numbering scheme described in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or more amino acids corresponding to shortening or insertion of framework regions (FRs) or CDRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues for a given antibody can be determined by aligning the regions of sequence homology with an antibody having a "standard" Kabat numbered sequence. [Table 1]

[0086] In some embodiments, the CDRs are "extended CDRs" and encompass regions that start or end according to different schemes. For example, the extended CDRs can be: L24-L36, L26-L34, or L26-L36 (VL-CDR1); L46-L52, L46-L56, or L50-L55 (VL-CDR2); L91-L97 (VL-CDR3); H47-H55, H47-H65, H50-H55, H53-H58, or H53-H65 (VH-CDR2); and / or H93-H102 (VH-CDR3).

[0087] In one embodiment, the antigen-binding portion binds to PDL1 and comprises a light chain or fragment thereof having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 45 and a heavy chain or fragment thereof having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 46. In one embodiment, the antigen-binding domain comprises CDR1, CDR2 and CDR3 from SEQ ID NO: 45 and CDR1, CDR2 and CDR3 from SEQ ID NO:46.

[0088] In one embodiment, the antigen-binding portion binds to PD-1 and comprises a light chain or fragment thereof having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 44, and a heavy chain or fragment thereof having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 47. In one embodiment, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 44 and CDR1, CDR2, and CDR3 from SEQ ID NO:47.

[0089] In one embodiment, the antigen-binding portion binds to PD-1 and comprises a light chain or fragment thereof having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 48, and a heavy chain or fragment thereof having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 49. In one embodiment, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 48 and CDR1, CDR2, and CDR3 from SEQ ID NO:49.

[0090] In some embodiments, the antigen-binding moiety comprises one, two, or three antigen-binding domains. For example, the antigen-binding moiety is bispecific and binds to two different antigens selected from the group consisting of CD3, HER2, HER3, EGFR, 5T4, FAP alpha, Trop-2, GPC3, VEGFR2, claudin 18.2, and PD-L1. In some embodiments, the bispecific antigen-binding moiety binds to two different epitopes of HER2. In other embodiments, the antigen-binding moiety is bispecific and binds to two different antigens selected from PD-1, PD-L1, CTLA-4, CD47, CD3, TIM-3, LAG-3, and TIGIT.

[0091] 2. Other domains in the carrier moiety The carrier moiety may also comprise other domains that are not antigen-binding, for example, an antibody Fc domain (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc), a polymer (e.g., PEG), albumin (e.g., human albumin) or a fragment thereof, or a nanoparticle may be used.

[0092] For example, a cytokine agonist polypeptide (e.g., IL-2, IL-21, IL-5, or any cytokine disclosed herein) and its antagonist can be fused to an antibody Fc domain to form an Fc fusion protein. In some embodiments, the cytokine agonist polypeptide is fused (directly or via a peptide linker) to the C-terminus or N-terminus of one Fc domain polypeptide chain, and the cytokine mask is fused via a cleavable peptide linker to the corresponding C-terminus or N-terminus of the other Fc domain polypeptide chain, wherein the two Fc domain polypeptide chains contain mutations that enable specific pairing of the two different Fc chains. In some embodiments, the Fc domain contains the hole-into-hole mutation described above. In further embodiments, the Fc domain can also contain the YTE and / or LALA mutations described above.

[0093] The carrier moiety of the prodrug can comprise albumin (e.g., human serum albumin) or a fragment thereof. In certain embodiments, the carrier moiety comprises an albumin fragment (e.g., a human serum albumin fragment) that is about 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 120 or more, 140 or more, 160 or more, 180 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, or 550 or more amino acids in length. In some embodiments, the albumin fragment is about 10 to about 584 amino acids in length (e.g., about 10 to about 20, about 20 and about 40, about 40 and about 80, about 80 and about 160, about 160 and about 250, about 250 and about 350, about 350 and about 450, or about 450 and about 550 amino acids in length). In some embodiments, the albumin fragment comprises a Sudlow I domain or a fragment thereof or a Sudlow II domain or a fragment thereof.

[0094] D. Prodrug Linker Elements The cytokine agonist polypeptide can be fused to a carrier moiety with or without a peptide linker. The peptide linker is non-cleavable. In certain embodiments, the peptide linker is selected from SEQ ID NOs: 27-34. In a specific embodiment, the peptide linker comprises the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 30).

[0095] The masking moiety may be fused to the cytokine moiety or carrier via a non-cleavable linker or without a peptide linker.

[0096] II. Pharmaceutical Composition Pharmaceutical compositions containing the prodrugs and muteins (i.e., active pharmaceutical ingredients or API) of the present invention can be prepared as lyophilized formulations or aqueous solutions by mixing the API having the desired purity with one or more optional pharmaceutically acceptable additives (see, e.g., Remington's Pharmaceutical Sciences, 16th Edition., Osol, A. Ed. (1980)). Pharmaceutically acceptable additives (or carriers) are non-toxic to recipients at commonly used dosages and concentrations, and include, for example, buffers including phosphate, citric acid, succinate, histidine, acetic acid, or other inorganic or organic acids or salts thereof; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (approximately 1 0 residues or less) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including sucrose, glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG).

[0097] Buffers are used to control the pH within a range that optimizes therapeutic efficacy, particularly when stability is pH-dependent. The buffer is preferably present at a concentration ranging from about 50 mM to about 250 mM. Suitable buffers for use in the present invention include both organic and inorganic acids and their salts, such as citric acid, phosphoric acid, succinic acid, tartaric acid, fumaric acid, gluconic acid, oxalic acid, lactic acid, and acetic acid. Additionally, buffers may include histidine and trimethylamine salts such as Tris.

[0098] Preservatives are used to retard microbial growth and are generally present in the range of 0.2% to 1.0% (w / v). Suitable preservatives for use in the present invention include octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0099] Tonicity agents, sometimes known as "stabilizers," are used to adjust or maintain the liquid tonicity of a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often referred to as "stabilizers" because they can interact with the charged groups on amino acid side chains, thereby reducing the likelihood of inter- and intramolecular interactions. Tonicity agents can be present in amounts ranging from 0.1% to 25% by weight or more, preferably 1% to 5% by weight, taking into account the relative amounts of other components. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0100] The non-ionic surfactant or detergent (also known as a "wetting agent") is present to aid in solubilizing the therapeutic agent, protect the therapeutic protein against agitation-induced aggregation, and allow the formulation to be subjected to shear surface stresses without causing denaturation of the active therapeutic protein or antibody. The non-ionic surfactant is present in a range of about 0.05 mg / ml to about 1.0 mg / ml, preferably about 0.07 mg / ml to about 0.2 mg / ml.

[0101] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), and pluronics. (登録商標) Polyol, TRITON (登録商標) , polyoxyethylene sorbitan monoether (TWEEN (登録商標) -20, TWEEN (登録商標) -80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic surfactants that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic surfactants include benzalkonium chloride or benzethonium chloride.

[0102] The choice of pharmaceutical carrier, excipient or diluent can be selected taking into consideration the intended route of administration and standard procedures. The pharmaceutical composition can further comprise any suitable binder, lubricant, suspending agent, coating agent or solubilizing agent.

[0103] Depending on the different delivery systems, there may be different composition / formulation requirements. For example, the pharmaceutical compositions useful in the present invention may be formulated for administration using a minipump or by a mucosal route, for example, as a nasal spray or an aerosol for inhalation, or as an ingestible solution, or parenterally, where the composition is formulated in an injectable form for delivery, for example, by an intravenous, intramuscular, or subcutaneous route.

[0104] In some embodiments, the pharmaceutical composition of the present invention is a lyophilized protein formulation. In other embodiments, the pharmaceutical composition may be an aqueous liquid formulation.

[0105] III. Treatment method Cytokine prodrugs and fusion molecules can be used to treat diseases depending on the antigen bound by the antigen-binding domain. In some embodiments, cytokine prodrugs or fusion molecules are used to treat cancer. In some embodiments, cytokine prodrugs or fusion molecules are used to treat infections.

[0106] In certain embodiments, the method of treating a disease (e.g., cancer, a parasitic infection, a viral infection, or a bacterial infection) in a subject comprises administering to the subject an effective amount of a cytokine prodrug or a fusion molecule.

[0107] In some embodiments, cancer is solid cancer.In some embodiments, cancer is blood cancer or solid tumor.The examples of cancer that can be treated include but are not limited to leukemia, lymphoma, kidney cancer, bladder cancer, urinary tract cancer, cervical cancer, brain cancer, head and neck cancer, skin cancer, uterine cancer, testicular cancer, esophageal cancer, liver cancer, colorectal cancer, gastric cancer, squamous cell carcinoma, prostate cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer, bile duct cancer, breast cancer and ovarian cancer.

[0108] In some embodiments, the cytokine prodrug or fusion molecule is used to treat viral infection. In some embodiments, the virus causing viral infection is hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus (HIV) or human papillomavirus (HPV). In some embodiments, the antigen-binding moiety binds to viral antigen.

[0109] In some embodiments, the cytokine prodrug or fusion molecule is used to treat a bacterial infection, such as sepsis. In some embodiments, the bacteria causing the bacterial infection are drug-resistant bacteria. In some embodiments, the antigen-binding moiety binds to a bacterial antigen.

[0110] Generally, the dosage and administration route of this pharmaceutical composition are determined by standard method according to the physique and condition of the patient.In some embodiments, pharmaceutical composition is administered to patient by any route, including oral, transdermal, inhalation, intravenous, intraarterial, intramuscular, directly applied to wound site, applied to surgical site, intraperitoneal, suppository, subcutaneous, intradermal, transdermal, spray, intrapleural, intraventricular, intraarticular, intraocular, intracranial or intrathecal.In some embodiments, composition is administered to patient intravenously.

[0111] In some embodiments, the dosage of pharmaceutical composition is single administration or repeated administration.In some embodiments, the dosage is administered to the subject once a day, twice a day, three times a day, or four times a day or more.In some embodiments, it is administered about once a week or more (for example, about 2, 3, 4, 5, 6 or 7 times or more).In some embodiments, it is administered about once a week or more (for example, about 2, 3, 4, 5, 6 or 7 times or more).In some embodiments, it is administered every week, every 2 weeks, every 3 weeks, every 4 weeks, every 2 weeks out of 3 weeks, or every 3 weeks out of 4 weeks.In some embodiments, it is administered multiple doses over several days, weeks, months or years.In some embodiments, the course of treatment is about once or more (for example, about 2, 3, 4, 5, 7, 10, 15 or 20 times or more).

[0112] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. In the event of a conflict, the present specification, including definitions, will control. In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization, as described individually, are well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly practiced in the art or as described herein. Furthermore, singular terms include pluralities, and plural terms include the singular, unless the context otherwise requires. Throughout this specification and the embodiments, the terms "having" and "including" or variations such as "having," "having," "including," or "comprising" should be interpreted to imply the inclusion of the recited integer or group of integers, but not the exclusion of any integer or group of integers. It should be understood that the embodiments and variations of the invention described herein include embodiments and variations that "consist of" and / or "consist essentially of." All publications and other references mentioned herein are incorporated herein by reference in their entirety. Although certain documents are cited herein, this citation does not constitute an admission that these documents form part of the general knowledge in the art.

[0113] IV. Exemplary Embodiments Additionally, specific embodiments of the present invention are described below. These embodiments are intended to illustrate the compositions and methods described in this disclosure and are not intended to limit the scope of the invention. 1. A prodrug comprising a cytokine moiety, a masking moiety, and a carrier moiety, wherein: a) the masking moiety binds to the cytokine moiety and inhibits the intended biological activity of the cytokine; b) the carrier moiety comprises an antigen-binding moiety that binds to an antigen expressed on the surface of a cell; and c) the masking moiety is linked to the carrier moiety directly or indirectly via a non-cleavable peptide linker or without a peptide linker; wherein the activity of the prodrug is greater for stimulating cells that express both the antigen targeted by the carrier moiety and a receptor for the cytokine than for stimulating cells that express a receptor for the cytokine but do not express the antigen targeted by the carrier moiety; Prodrug. 2. A prodrug comprising a cytokine moiety, a masking moiety, and a carrier moiety, wherein: a) the masking moiety binds to the cytokine moiety and inhibits the intended biological activity of the cytokine; b) the carrier moiety comprises an antigen-binding moiety that binds to an antigen expressed on the surface of a cell; and c) the masking moiety is linked to the carrier moiety directly or indirectly via a non-cleavable peptide linker or without a peptide linker; wherein the prodrug is activated at the disease site where cells expressing both the antigen targeted by the carrier moiety and a receptor for the cytokine are present, or the biological activity of the prodrug is increased by at least 2-fold, at least 5-fold, or at least 10-fold at the disease site where cells expressing both the antigen targeted by the carrier moiety and a receptor for the cytokine are present. Prodrug. 3. The prodrug of embodiment 1 or 2, wherein the receptor for the cytokine moiety comprises two or more subunits. 4. The prodrug of embodiment 1 or 2, wherein the carrier comprises an antigen-binding moiety, wherein the antigen is expressed on an immune cell. 5. The prodrug of embodiment 1 or 2, wherein the carrier moiety comprises an antigen-binding moiety, wherein the antigen is selected from PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, CD25, CD16a, CD16b, NKG2D, NKP44, NKP30, CD19, CD20, CD38 and BCMA. 6. The prodrug of any of embodiments 1-5, wherein the cytokine moiety comprises a cytokine selected from an IL-2 agonist polypeptide, an IL-7 agonist polypeptide, an IL-9 agonist polypeptide, an IL-15 agonist polypeptide, and an IL-21 agonist polypeptide. 7. The prodrug of any of embodiments 1-5, wherein the cytokine is selected from IL-1α agonist polypeptides, IL-1β agonist polypeptides, IL-4 agonist polypeptides, IL-5 agonist polypeptides, IL-6 agonist polypeptides, IL-8 agonist polypeptides, IL-10 agonist polypeptides, IL-12 agonist polypeptides, IL-17 agonist polypeptides, IL-18 agonist polypeptides, IL-22 agonist polypeptides, IL-23 agonist polypeptides, IL-31 agonist polypeptides, IL-33 agonist polypeptides, IL-36 agonist polypeptides, interferon-alpha agonist polypeptides, interferon-gamma agonist polypeptides, 4-1BB ligand, OX-40 ligand, and CD-40 ligand. 8. The prodrug of any of embodiments 1-7, wherein the masking moiety is the extracellular domain (ECD) of a receptor for a cytokine. 9. The prodrug of any of embodiments 1-6, wherein the cytokine is an IL-7 agonist polypeptide; and wherein the masking moiety is the extracellular domain of IL-7 receptor alpha (IL-7Rα ECD) or a functional analog thereof. 10. The prodrug of any of embodiments 1-6, wherein the cytokine is an IL-21 agonist polypeptide; and wherein the masking moiety is the extracellular domain of IL-21 receptor alpha (IL-21Rα ECD) or a functional analog thereof. 11. The prodrug of any of embodiments 1-6, wherein the cytokine is an IL-2 agonist polypeptide or an IL-15 agonist polypeptide; and wherein the masking moiety is the extracellular domain of IL-2 receptor β (IL-2Rβ ECD). 12. The prodrug of any of embodiments 1-6, wherein the cytokine is an IL-21 agonist polypeptide; and wherein the masking moiety is a single-chain Fv (scFv) or single-domain antibody against IL-21. 13. The prodrug of any of embodiments 1-6, wherein the cytokine is an IL-2 agonist polypeptide; and wherein the masking moiety is a single-chain Fv (scFv) or single-domain antibody against IL-2. 14. The prodrug of any of embodiments 1-6, wherein the cytokine is an IL-15 agonist polypeptide; and wherein the masking moiety is a single-chain Fv (scFv) or single-domain antibody against IL-15. 15. The prodrug of any of embodiments 1-6, 11 and 14, wherein the cytokine moiety comprises an IL-15 agonist polypeptide, and wherein the fusion molecule further comprises the sushi domain of IL-15 receptor alpha (IL-15Rα sushi domain). 16. The prodrug of any of embodiments 10 or 12, wherein the IL-21 agonist polypeptide comprises an amino acid sequence of SEQ ID NO:1 or that is at least 90% identical to SEQ ID NO:1. 17. The prodrug of embodiment 10 or 16, wherein the masking moiety is IL-21Rα-ECD or a functional analog thereof, which comprises an amino acid sequence selected from SEQ ID NOs: 12, 63-72 and 73 or which is at least 90% identical to SEQ ID NO: 12. 18. The prodrug of embodiment 12 or 16, wherein the masking moiety is an scFv that binds to human IL-21; and wherein the scFv comprises the amino acid sequence of SEQ ID NO: 20 or 21. 19. The prodrug of embodiment 11 or 13, wherein the IL-2 agonist polypeptide comprises an amino acid sequence of SEQ ID NO: 6 or 61 or at least 95% identical to SEQ ID NO: 6 or 61. 20. The prodrug of embodiment 11 or 14, wherein the IL-15 agonist polypeptide comprises an amino acid sequence of SEQ ID NO:7 or at least 95% identical to SEQ ID NO:7. 21. The prodrug of embodiment 11, 19 or 20, wherein the masking moiety comprises IL-2Rβ-ECD or a functional analog thereof; wherein IL-2-Rβ-ECD comprises the amino acid sequence of SEQ ID NO: 11 or at least 95% identical to SEQ ID NO: 11. 22. The prodrug of any of embodiments 6, 13, or 19, wherein the masking moiety is an scFv that binds to IL-2, wherein the scFv inhibits or disrupts the interaction of IL-2 with IL-2Rα, the interaction of IL-2 with IL-2Rβ, and / or the interaction of IL-2 with IL-2Rγ. 23. The prodrug of any of embodiments 6, 14, 15, or 20, wherein the masking moiety is an scFv that binds to IL-15, wherein the scFv inhibits or disrupts the interaction of IL-15 with IL-2Rβ and / or IL-2 with IL-2Rγ. 24. The prodrug of any of embodiments 6, 12, or 16, wherein the masking moiety is an scFv that binds to IL-21, wherein the scFv inhibits or disrupts the interaction of IL-21 with IL-21Rα and / or IL-21 with IL-2Rγ. 25. The prodrug of embodiment 6, wherein the masking moiety is an scFv that binds to a cytokine selected from IL-2, IL-7, IL-9, IL-15, or IL-21; wherein the scFv inhibits or disrupts the interaction between the cytokine and IL-2Rγ; and wherein the prodrug has a half-life in a non-human primate or a human that is at least 20-fold, at least 50-fold, at least 100-fold, at least 150-fold, or at least 200-fold longer than the corresponding wild-type cytokine. 26. A prodrug comprising a carrier moiety, a cytokine moiety, and a masking moiety, wherein: a) the cytokine moiety is selected from an IL-2 agonist polypeptide, an IL-7 agonist polypeptide, an IL-9 agonist polypeptide, an IL-15 agonist polypeptide, or an IL-21 agonist polypeptide; b) the masking moiety is an scFv that binds to the cytokine moiety; c) the scFv inhibits or interferes with the interaction between the cytokine and IL-2Rγ; and d) A prodrug, wherein the prodrug has a half-life in a non-human primate or human that is at least 20 times, at least 50 times, at least 100 times, at least 150 times, or at least 200 times longer than the wild-type cytokine. 27. scFv binds cytokines with a K of 1–10 nM D 26. The prodrug of any of embodiments 22 to 25, wherein 28. scFv binds cytokines with a K of 10–100 nM D 26. The prodrug of any of embodiments 22 to 25, wherein 29. scFv binds cytokines with a K of 100–1000 nM D 26. The prodrug of any of embodiments 22 to 25, wherein 30. The prodrug of embodiment 13 or 19, wherein the masking moiety is an scFv that binds to IL-2, wherein the scFv comprises the same heavy chain CDRs and light chain CDRs as hybridoma 4E12B2D10. 31. The prodrug of embodiment 13 or 19, wherein the masking moiety is an scFv that binds to IL-2, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 22 or 23 or at least 95% identical to SEQ ID NO: 22 or 23. 32. The prodrug of embodiment 14 or 20, wherein the masking moiety is an scFv that binds to IL-15, wherein the scFv comprises the same heavy chain CDRs and light chain CDRs as the IL-15 antibody 146B7, 146H5, or 404E4. 33. The prodrug of embodiment 14 or 20, wherein the masking moiety is an scFv that binds to IL-15, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 18 or 19. 34. The prodrug of any of embodiments 1-32, wherein the prodrug does not comprise a cleavable peptide linker. 35. The prodrug comprises two light chains having the amino acid sequence of SEQ ID NO: 44 and two light chains having the amino acid sequences of SEQ ID NO: 45 and SEQ ID NO: 46, respectively. SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 41 or SEQ ID NOs: 42 and 43 and two heavy polypeptide chains comprising: The prodrug of embodiment 1. 36. The prodrug comprises two light chains having the amino acid sequence of SEQ ID NO: 50 and two light chains having the amino acid sequence of SEQ ID NOs: 51 and 54, SEQ ID NOs: 51 and 55, SEQ ID NOs: 51 and 56, SEQ ID NOs: 52 and 54, SEQ ID NOs: 53 and 58, SEQ ID NOs: 53 and 59 or SEQ ID NOs: 52 and 57 and two heavy polypeptide chains comprising: The prodrug of embodiment 1. 37. A pharmaceutical composition comprising the prodrug of any of Embodiments 1-36 and a pharmaceutically acceptable excipient. 38. One or more polynucleotides encoding the prodrug of any of embodiments 1 to 36. 39. One or more expression vectors comprising one or more polynucleotides of embodiment 38. 40. A host cell comprising the vector of embodiment 39. 41. A method for producing a prodrug of any of embodiments 1-36, comprising: Culturing the host cell of embodiment 46, which is a mammalian cell, under conditions that allow expression of the prodrug; and Isolating the prodrug A method comprising: 42. A method for treating cancer, an autoimmune disease or an infectious disease or stimulating the immune system in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of embodiment 37. 43. The prodrug of any of embodiments 1-36 for use in treating cancer, an autoimmune disease, or an infectious disease, or stimulating the immune system, in a patient in need thereof. 44. Use of a prodrug of any of embodiments 1-36 for the manufacture of a medicament for the treatment of cancer, an autoimmune disease or an infectious disease, or for stimulating the immune system in a patient in need thereof. 45. The method of embodiment 41, the prodrug for use of claim 42, or the fusion molecule for use of claim 43, or the use of claim 44, wherein the patient has HIV, HBV, HCV, or HPV infection; an autoimmune disease selected from lupus, type 1 diabetes, psoriasis, dermatomyositis, GvHD, or rheumatoid arthritis; or a cancer selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer, and gastric cancer. 46. ​​A prodrug comprising a carrier moiety, a cytokine moiety, and a masking moiety, wherein: a) the cytokine moiety is selected from an IL-2 agonist polypeptide, an IL-7 agonist polypeptide, an IL-9 agonist polypeptide, an IL-15 agonist polypeptide, or an IL-21 agonist polypeptide; b) the masking moiety is an scFv that binds to the cytokine moiety; c) the scFv inhibits or interferes with the interaction between the cytokine and IL-2Rγ; and d) Wherein the prodrug has an area under the curve (AUC) in a non-human primate or human that is at least 20-fold, at least 50-fold, at least 100-fold, at least 150-fold, or at least 200-fold greater than the wild-type cytokine.

[0114] In order that this invention may be better understood, the following examples are set forth, which are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way. [Example]

[0115] The materials and methods for the experiments described in Examples 1 and 2 are as follows.

[0116] SEC-HPLC analysis SEC-HPLC was performed on an Agilent 1100 series HPLC system with a TSKgel G3000SWXL column (7.8 mm ID x 30 cm, 5 μm particle size) ordered from Tosoh Bioscience. Up to 100 μl of sample was loaded. The column was run with a buffer containing 200 mM K3PO4, 250 mM KCl, pH 6.5. The flow rate was 0.5 ml / min. The column was run at room temperature.

[0117] Proteolytic treatment Proteases, human MMP2, human MMP9, mouse MMP2, and mouse MMP9, were purchased from R&D Systems. Protease digestion was performed by incubating 10–50 μg of prodrug with 1 μg of human MMP2, human MMP9, mouse MMP2, or mouse MMP9 in HBS buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) containing 2 mM CaCl and 10 μM ZnCl at 37°C for 12 hours.

[0118] Cell-based activity assays The prodrugs and control samples before protease digestion were tested by cell-based activity assay. Briefly, NK92 cells were grown in RPMI-1640 medium supplemented with L-glutamine, 10% fetal bovine serum, 10% non-essential amino acids, 10% sodium pyruvate, and 55 μM beta-mercaptoethanol. NK92 cells were nonadherent and cultured at 1×10 in medium with 100 ng / ml IL-2. 5 ~1×10 6 NK92 cells were maintained at a density of 5×10 cells / ml. Generally, cells were split twice a week. For bioassays, cells were best used 48 hours or later after passage. IL-21 functional activity was measured using NK92 cells at a density of 5×10 4 This was determined by culturing cells / well with serially diluted samples in the presence of a constant amount of IL-2 for 2 days, and then the supernatants were assayed for interferon-γ by ELISA.

[0119] Mino IL-21 viability assay The Mino cell viability assay is performed according to the following protocol: a) Serial dilutions of test articles are performed in 50 μL assay medium (RPMI 1640, 10% fetal bovine serum, NEAA, sodium pyruvate, 55 μM b-mercaptoethanol) in 96-well tissue culture plates. b) Add 20,000 Mino cells / well in 50 μL assay medium. c) Incubate for 3 or 5 days. d) Add 100 μL / well Cell Titer Glo (Promega). Cell Titer-Glo provides an index of cell viability by providing a quantitative assessment of ATP. e) Measuring luminescence.

[0120] Ki67 activation assay in primary PBMC cells Human PBMCs were stimulated with 100 ng / mL of the anti-CD3 antibody, OKT3, for two days, followed by three washes and then resting for three days in customary cell culture medium in a 37°C, 5% CO2 incubator. After this stimulation / rest period, PBMCs were seeded at 200K cells / well in 96-well plates and treated with various concentrations of test articles as indicated for five days, followed by PD-1 + CD4 + and subjected to cell staining and flow cytometry analysis for Ki67 in the PD-1-CD4+ cell population (all Abs used for cell staining were purchased from BD).

[0121] Example 1: Construction of an anti-PD-1 antibody-IL-21 prodrug fusion molecule An anti-PD-1 antibody-based IL-21 prodrug was constructed using a first heavy chain polypeptide chain (having the amino acid sequence set forth in SEQ ID NO:42), a second heavy chain polypeptide chain (having the amino acid sequence set forth in SEQ ID NO:76), and two identical light chains (having the amino acid sequence set forth in SEQ ID NO:44). The molecule was transiently expressed and purified (lot# PW04-38).

[0122] A second PD-1 antibody-based IL-21 prodrug with an scFv as the masking moiety was also expressed and purified (Lot #PW05-68).

[0123] Additionally, as a control, an unmasked anti-PD-1 antibody-IL-21 fusion molecule was also expressed and purified (Lot #PW05-67).

[0124] In addition, a second control, unmasked anti-PD-1 antibody-IL-21 mutein (R9ER76A) fusion molecule was also expressed and purified (Lot #PW09-02).

[0125] Example 2: Biological activity of anti-PD-1 antibody-IL-21 prodrug fusion molecules The binding of the prodrug molecules and several control molecules to Mino cells was tested by FACS. The results in Figure 2 show that both the PD-1 antibody and the Fc-IL-21 fusion molecule were able to bind to Mino cells, indicating that Mino cells express receptors for both PD-1 and IL-21. The results also show that neither of the Fc-based IL-21 prodrug molecules bound to the cells, indicating that the IL-21 cytokine moiety was masked by the corresponding masking moiety. However, the PD-1 antibody-based IL-21 prodrug molecule and fusion molecule were able to bind to Mino cells.

[0126] The cell-based activity of the molecules was evaluated using NK92 cells, a natural killer (NK) cell line. The results are shown in Figure 3. The data indicated that, without activation, the prodrug molecule with the IL-21α ECD as the masking moiety (Lot# PW04-38) had minimal activity, while the prodrug with the scFv as the masking moiety (Lot# PW05-68) had approximately 1000-fold less activity than the one without the masking moiety (PW04-67). The data also indicate that the bioassay activity of the prodrugs was significantly enhanced by treatment with the protease MMP2.

[0127] To test the tumoricidal activity of the fusion molecules, we used Mino cells, a mantle cell lymphoma cell line that expresses both PD-1 and IL-21 receptors (Harington et al., Leuk Lymphoma (2019) 60(10):2498-2507 and Gelebart et al., Leukemia (2009) 23:1836-1846). The Mino viability assay results are shown in Figures 4A and 4B. Surprisingly, the anti-PD-1 antibody-based IL-21 prodrugs (Lots # PW04-38 and PW05-68) had substantial activity prior to activation, whereas the control molecule (PD-1 antibody-IL-21R9E / R76A fusion molecule, Lot # PW09-02) had little to no activity. The results indicate that the anti-PD-1 IL-21 fusion molecule was activated through "cis binding," i.e., binding to both the PD-1 and IL-21 receptors. The cis binding of the PD-1 antibody to the PD-1 antigen on the cell surface and the cytokine to its receptor on the same cell surface did not destroy the masking effect of the masking moiety. Therefore, it is predicted that prodrugs without a cleavable peptide linker can be "activated" at disease sites, such as tumors, because local immune cells can express both the antigen targeted by the carrier and the receptor that binds to the cytokine moiety (e.g., IL-21).

[0128] Example 3: Construction of anti-PD-1 antibody-IL-15 prodrug fusion molecule A series of anti-PD-1 antibody-based IL-15 prodrugs were constructed in which the IL-15 moiety was (i) masked with scFv 215β, (ii) masked with scFv 215γ, (iii) unmasked (215β Ref), or (iv) mutated but unmasked (PD1 / IL15 mutein (M2)). The anti-PD-1 antibodies in these prodrugs comprise two identical light chains having the amino acid sequence of SEQ ID NO:44 and two identical heavy chains having the amino acid sequence of SEQ ID NO:47.

[0129] The Fc-based prodrug 215β is a heterodimer comprising amino acid sequences SEQ ID NOs: 16 and 17.

[0130] The anti-PD-1 antibody-based prodrug 215γ comprises two identical light chains (SEQ ID NO:44), a first heavy chain fusion polypeptide chain having the amino acid sequence of SEQ ID NO:37, and a second heavy chain fusion polypeptide chain having the amino acid sequence of SEQ ID NO:77.

[0131] 215β Ref is an IL-15 mutein (D30N / E64Q / N65D)-Fc fusion molecule.

[0132] PD1 / IL15 mutein (M2) is an anti-PD-1-antibody fusion with an IL-15 mutein, where the IL-15 agonist contains the mutations E46G, V49R, E64Q, D30N and N1G.

[0133] All samples were expressed in CHO cells and purified using Protein A affinity chromatography followed by an additional chromatography step.

[0134] Example 4: Biological activity of anti-PD-1 antibody-IL-15 prodrug fusion molecules IL-15 prodrugs were tested for their ability to stimulate Ki67 expression in PBMC cells obtained from human donors. Figures 5A-5C show the CD4 expression in PBMCs after treatment. + The results of Ki67 activation of T cells are shown in Figure 5A. + Figure 5B shows the results of Ki67 activation of T cells. + The results of Ki67 activation of T cells are shown in Figure 5C. CD4 T cells with and without PD-1 expression are shown in Figure 5D. + EC of test article on T cells 50 The values ​​are also shown. + ECs between T cells 50 The fold change in values ​​is also shown. Surprisingly, the data show that 215γ is a potent inhibitor of PD-1-expressing CD4 +We showed that cells expressing both the antigen PD-1 and the cytokine IL-15 had significantly higher Ki67 activity than those without PD-1 expression, indicating that the prodrug 215γ could activate cells expressing both the antigen PD-1 and the cytokine IL-15 receptors without protease cleavage.

[0135] Example 5: Anti-PD-1 antibody-IL-2 prodrug fusion molecule An anti-PD-1 antibody-based IL-2 prodrug was similarly constructed. The prodrug, PD-1 / IL-2v*, contained two identical light chains (SEQ ID NO: 44) and two heavy chain fusion polypeptides (SEQ ID NOs: 24 and 25, respectively). The IL-2 moiety was an IL-2 mutein, and the mask in the prodrug was the IL-2 receptor β extracellular domain. The IL-2 prodrug was evaluated for its ability to stimulate Ki67 expression in PBMC cells obtained from human donors.

[0136] Figures 6A and 6B show CD4 counts from PBMCs after treatment with anti-PD-1 antibody (PD-1), negative control, or PD-1 / IL-2v*. + The results of Ki67 activation in T cells are shown. The negative control was an IgG1 antibody against human claudin 18.2. All samples were expressed in CHO cells and purified using protein A affinity chromatography followed by an additional chromatography step. Figure 6A shows the results of Ki67 activation in T cells. + Figure 6B shows the results of Ki67 activation in T cells. + Results of Ki67 activation in T cells. Surprisingly, the data show that the prodrug PD-1 / IL2V* inhibits PD-1-expressing CD4 + We showed that PD-1 / IL-2v* had significantly higher Ki67-inducing activity than cells expressing neither the antigen PD-1 nor the cytokine IL-2. These results indicate that PD-1 / IL-2v* can activate cells expressing both the receptors for the antigen PD-1 and the cytokine IL-2 without protease cleavage.

[0137] Taken together, the above data demonstrate that cytokine prodrugs can be activated at sites where cells express both the cytokine receptor and the antigen targeted by the carrier moiety are present. It was surprising that prodrugs bearing carriers with antigen-binding moieties could be activated without protease cleavage. Without intending to be bound by theory, we predict that the antigen-binding domain of the carrier moiety targets the prodrug molecule to the surface of cells expressing the antigen, thereby allowing the cytokine receptor surface of the cells to efficiently compete with the masking moiety of the prodrug. This process, as shown in Figure 7, allows protease-free activation of the prodrug via displacement of the masking moiety by the cytokine receptor on the cell surface.

[0138] Cells such as exhausted Teff cells express both antigens (e.g., PD-1) and cytokine receptors (e.g., IL-2Rβ and IL-2Rγ). Exhausted Teff cells are generally localized in the tumor microenvironment, but are also present in normal tissues. This differential distribution of cells expressing antigens and cytokine receptors allows for disease-site-specific activation of prodrugs. Such target specificity also enhances the safety of cytokine therapy.

[0139] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] Table 9 Table 10 Table 11 Table 12 Table 13 Table 14

Claims

1. A prodrug comprising a cytokine moiety, a masking moiety and a carrier moiety, wherein: a) a masking moiety that binds to the cytokine moiety and inhibits the biological activity of the cytokine; b) the carrier moiety comprises an antigen-binding moiety, where the antigen-binding moiety binds to an antigen expressed on the surface of a target cell; and c) the masking moiety is linked to the carrier moiety directly or indirectly via a non-cleavable peptide linker or without a peptide linker; Prodrug.

2. 2. The prodrug of claim 1, wherein the prodrug is activated by cells expressing both the antigen and the cytokine moiety receptors on the cell surface, optionally wherein the biological activity of the prodrug is increased by at least 2-fold, 5-fold, or 10-fold in diseased sites containing the cells compared to sites not containing the cells.

3. 3. The prodrug of claim 1 or 2, wherein the receptor for the cytokine moiety comprises two or more subunits.

4. The prodrug of any one of claims 1 to 3, wherein the antigen is selected from PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, CD25, CD16a, CD16b, NKG2D, NKP44, NKP30, CD19, CD20, CD30, CD38, BCMA, and signal regulatory protein alpha (SIRP alpha).

5. The cytokine portion may be an IL-2 agonist polypeptide, an IL-7 agonist polypeptide, an IL-9 agonist polypeptide, an IL-15 agonist polypeptide, an IL-21 agonist polypeptide, an IL-1α agonist polypeptide, an IL-1β agonist polypeptide, an IL-4 agonist polypeptide, an IL-5 agonist polypeptide, an IL-6 agonist polypeptide, an IL-8 agonist polypeptide, an IL-10 agonist polypeptide, an IL-12 agonist polypeptide, ...1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL-1β agonist polypeptide, an IL 5. The prodrug of any one of claims 1 to 4, comprising an IL-17 agonist polypeptide, an IL-18 agonist polypeptide, an IL-22 agonist polypeptide, an IL-23 agonist polypeptide, an IL-31 agonist polypeptide, an IL-33 agonist polypeptide, an IL-36 agonist polypeptide, an interferon-alpha agonist polypeptide, an interferon-gamma agonist polypeptide, a 4-1BB ligand, an OX-40 ligand, or a CD-40 ligand.

6. The prodrug of any one of claims 1 to 5, wherein the masking moiety comprises an ECD of a cytokine receptor or an antigen-binding domain that specifically binds to a cytokine.

7. The prodrug of any one of claims 1 to 6, i) the cytokine portion comprises an IL-7 agonist polypeptide and the masking portion comprises the extracellular domain (ECD) of IL-7 receptor alpha (IL-7Rα ECD) or a functional analog thereof; ii) the cytokine portion comprises an IL-21 agonist polypeptide and the masking portion comprises the ECD of IL-21 receptor alpha (IL-21Rα ECD) or a functional analog thereof; iii) the cytokine portion comprises an IL-2 agonist polypeptide or an IL-15 agonist polypeptide, and the masking portion comprises the ECD of IL-2 receptor β (IL-2Rβ ECD) or a functional analog thereof; iv) the cytokine portion comprises an IL-21 agonist polypeptide and the masking portion comprises a single chain Fv (scFv) or single domain antibody against IL-21; v) the cytokine portion comprises an IL-2 agonist polypeptide and the masking portion comprises an scFv or single domain antibody against IL-2; vi) the cytokine portion comprises an IL-15 agonist polypeptide and the masking portion comprises an scFv or single domain antibody against IL-15; or vii) the cytokine portion comprises an IL-15 agonist polypeptide, and the masking portion comprises the sushi domain of the IL-15 receptor alpha (IL-15Rα sushi domain); Prodrug.

8. 8. The prodrug of any of claims 5 to 7, wherein the cytokine portion comprises an IL-2 agonist polypeptide comprising SEQ ID NO: 6 or 62 or an amino acid sequence at least 95% identical thereto.

9. The prodrug of any of claims 5 to 7, wherein the cytokine portion comprises an IL-15 agonist polypeptide comprising SEQ ID NO: 7 or an amino acid sequence at least 95% identical thereto.

10. 10. The prodrug of any of claims 5-9, wherein the cytokine portion comprises an IL-2 or IL-15 agonist polypeptide and the masking portion comprises an IL-2Rβ ECD or a functional analog thereof, optionally wherein the IL-2Rβ ECD comprises SEQ ID NO:11 or an amino acid sequence at least 95% identical thereto.

11. The cytokine portion comprises an IL-2 agonist polypeptide, and the masking portion comprises an scFv against IL-2, optionally inhibiting or disrupting the interaction between the IL-2 cytokine portion and one or more of IL-2Rα, IL-2Rβ, and IL-2Rγ, and optionally, wherein the scFv HCDR1-3 and LCDR1-3 or heavy and light chain variable domains of antibody 4E12B2D10, anti-IL-2 scFv1 or anti-IL-2 scFv2, or An amino acid sequence comprising SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 60 and 61 or an amino acid sequence at least 95% identical thereto The prodrug of any one of claims 5 to 9, comprising:

12. The cytokine portion comprises an IL-15 agonist polypeptide, and the masking portion comprises an scFv against IL-15, optionally inhibiting or disrupting the interaction between the IL-15 cytokine portion and one or more of IL-15Rα, IL-2Rβ, and IL-2Rγ, and optionally, wherein the scFv HCDR1-3 and LCDR1-3 or heavy and light chain variable domains of antibody 146B7, anti-IL-15 scFv1 or anti-IL-15 scFv2, or An amino acid sequence comprising SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NOs: 83 and 84 or an amino acid sequence at least 95% identical thereto The prodrug of any one of claims 5 to 9, comprising:

13. The prodrug of any of claims 5 to 7, wherein the cytokine portion comprises an IL-21 agonist polypeptide comprising any of SEQ ID NOs: 1 to 5 or an amino acid sequence at least 95% identical thereto.

14. 14. The prodrug of any of claims 5-7 or 13, wherein the cytokine portion comprises an IL-21 agonist polypeptide and the masking portion comprises an IL-21Rα ECD or a functional analog thereof or an IL-21Rγ ECD or a functional analog thereof, optionally wherein the masking portion comprises any of SEQ ID NOs: 12, 13 or SEQ ID NOs: 63-73 or an amino acid sequence at least 95% identical thereto.

15. the cytokine portion comprises an IL-21 agonist polypeptide, and the masking portion comprises an scFv against human IL-21, optionally inhibiting or disrupting the interaction between the IL-21 cytokine portion and IL-21Rα and / or IL-21Rγ, and optionally, wherein the scFv is HCDR1-3 and LCDR1-3 or heavy and light chain variable domains of anti-IL-21 scFv1 or anti-IL-21 scFv2, or An amino acid sequence comprising SEQ ID NO: 20 or 21 or an amino acid sequence at least 95% identical thereto. The prodrug of any one of claims 5 to 7 or 13, comprising:

16. The masking part an scFv against a cytokine selected from IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, optionally inhibiting or disrupting the interaction between the cytokine and IL-2Rγ; comprising an ECD of a cytokine receptor α or γ selected from IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21; The prodrug of any one of claims 1 to 15.

17. The prodrug comprises an anti-PD-1 antibody moiety, wherein the antibody moiety comprises two light chains comprising SEQ ID NO: 44 and two light chains comprising SEQ ID NO: 45, each of which comprises a PD-1 antibody moiety. SEQ ID NOs: 24 and 25, SEQ ID NOs: 24 and 26 or 74, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 36, SEQ ID NOs: 37 and 38 or 75, SEQ ID NOs: 39 and 41 or 74 or SEQ ID NOs: 42 and 43 or 74 two heavy chains comprising The prodrug of any one of claims 1 to 16, comprising:

18. The prodrug comprises an anti-CTLA4 antibody moiety, wherein the antibody moiety comprises two light chains comprising SEQ ID NO: 50 and two light chains comprising SEQ ID NO: 51, respectively. SEQ ID NOs: 51 and 54, SEQ ID NOs: 51 and 55, SEQ ID NOs: 51 and 56, SEQ ID NOs: 52 and 54, SEQ ID NOs: 53 and 58, SEQ ID NOs: 53 and 59 or SEQ ID NOs: 52 and 57 two heavy chains comprising The prodrug of any one of claims 1 to 17, comprising:

19. A pharmaceutical composition comprising the prodrug of any one of claims 1 to 18 and a pharmaceutically acceptable excipient.

20. One or more polynucleotides encoding the prodrug of any one of claims 1 to 18.

21. 21. One or more expression vectors comprising one or more polynucleotides of claim 20.

22. 22. A host cell, optionally a mammalian cell, comprising the vector of claim 21.

23. A method for producing the prodrug of any one of claims 1 to 18, comprising: a) culturing the host cell of claim 22, which is a mammalian cell, under conditions that allow expression of the prodrug; and b) Isolating the prodrug from the cell culture A method comprising:

24. 20. A method of treating cancer, an autoimmune disease, or an infectious disease or stimulating the immune system in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a prodrug of any of claims 1-18 or a pharmaceutical composition of claim 19.

25. 20. The prodrug of any of claims 1 to 18 or the pharmaceutical composition of claim 19 for use in treating cancer, an autoimmune disease or an infectious disease or for stimulating the immune system in a patient in need thereof.

26. 20. Use of a prodrug of any of claims 1 to 18 for the manufacture of a medicament for the treatment of cancer, an autoimmune disease or an infectious disease, or for stimulating the immune system in a patient in need thereof.

27. The patient HIV, HBV, HCV or HPV infection, an autoimmune disease selected from lupus, type 1 diabetes, psoriasis, dermatomyositis, GvHD, or rheumatoid arthritis; Cancer selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer, or gastric cancer 27. The method of claim 24, the prodrug or pharmaceutical composition for the use of claim 25 or the use of claim 26,

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