Novel cytokine prodrugs

Abstract

To provide cytokine muteins and cytokine prodrugs, and methods of making and using same.SOLUTION: Provided is a prodrug comprising a cytokine moiety, a masking moiety and a carrier moiety, where the masking moiety is linked to the cytokine moiety to inhibit the biological activity of the cytokine moiety, the cytokine moiety is fused to the carrier moiety, the cytokine moiety comprises the IL-15 polypeptide or a functional fragment thereof, the masking moiety is fused to the cytokine moiety or the carrier moiety via a cleavable peptide linker, the masking moiety comprises the extracellular domain (ECD) of a receptor of the cytokine moiety, the carrier moiety is an antibody Fc domain or an antibody comprising knobs-into-holes mutations, and the cytokine moiety and the masking moiety are fused to different polypeptide chains of the antibody Fc domain or to the different heavy chains of the antibody.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Application No. 62 / 640,969, filed March 9, 2018; U.S. Provisional Application No. 62 / 643,104, filed March 14, 2018; U.S. Provisional Application No. 62 / 644,384, filed March 17, 2018; U.S. Provisional Application No. 62 / 644,577, filed March 18, 2018; U.S. Provisional Application No. 62 / 680,707, filed June 5, 2018; and U.S. Provisional Application No. 62 / 801,649, filed February 6, 2019, the contents of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Background of the Invention Interleukin-2 (IL-2) plays a central role in lymphocyte production, survival, and homeostasis. It has 133 amino acids and consists of four antiparallel amphipathic alpha helices that form a quaternary structure necessary for its function (Smith, Science 240:1169-76 (1988); Bazan, Science 257:410-13 (1992)).

[0003] IL-2 exerts its activity by binding to the IL-2 receptor (IL-2R), which consists of up to three subunits: α (CD25 or Tac antigen), β (CD122), and γ (γ c , common γ chain, or CD132) subunits result in a trimeric, high-affinity receptor for IL-2 (K D ~0.01 nM). The dimeric IL-2 receptor, consisting of β and γ subunits, confers intermediate affinity IL-2R (K D The α subunit alone forms a monomeric, low-affinity IL-2 receptor (K D~10 nM). See, e.g., Kim et al., Cytokine Growth Factor Rev. 17:349-66 (2006). The dimeric intermediate-affinity IL-2 receptor binds IL-2 with approximately 100-fold lower affinity than the trimeric high-affinity receptor, but both the dimeric and trimeric IL-2 receptors can transduce signals upon IL-2 binding (Minami et al., Annu Rev Immunol. 11:245-68 (1993)). Thus, although the α subunit has high-affinity binding of the receptor to IL-2, it may not be necessary for IL-2 signaling. However, the β and γ subunits are essential for IL-2 signaling (Krieg et al., Proc Natl Acad Sci. 107:11906-11 (2010)). This trimeric IL-2 receptor is expressed by CD4 + FoxP3 + Controllability T(T reg ) cells. reg T cells consistently express the highest levels of IL-2Rα (CD25) in vivo (Fontenot et al., Nature Immunol 6:1142-51 (2005)). The trimeric IL-2 receptor is also transiently induced in conventionally activated T cells, while in the resting state, these cells express only the dimeric IL-2 receptor.

[0004] Targeted muteins of IL-2 have been engineered to have increased or decreased binding affinity for CD25. Based on the published crystal structure of the IL-2 / IL-2R complex, the mutations are often placed in or near regions of IL-2 known to be in close proximity to CD25 (Wang et al., Science 310:1159-63 (2005)). IL-2 residues K35, R38, F42, K43, F44, Y45, E61, E62, K64, P65, E68, V69, L72, and Y107 are thought to contact CD25 (U.S. Patent No. 9,732,134).

[0005] To reduce the side effects of IL-2 treatment, researchers have mutated IL-2 to reduce its binding affinity to CD25. For example, WO2008 / 0034473 describes the mutations R38W and F42K, and WO2012 / 107417 describes a mutation at position 72. U.S. Patent Publication No. 2003 / 0124678 describes the introduction of the R38W mutation to eliminate the vascular permeability activity of IL-2. Heaton et al. (Cancer Res. 53:2597-602 (1993); U.S. Patent No. 5,229,109) describe the introduction of two mutations, R38A and F42K, to obtain an IL-2 mutein with reduced ability to induce secretion of pro-inflammatory cytokines from natural killer (NK) cells. EP2639241B1 describes the introduction of two mutations, R38A and F42K, to obtain an IL-2 mutein with reduced ability to induce secretion of pro-inflammatory cytokines from natural killer (NK) cells. reg IL-2 muteins are described that are at least 1000-fold less potent than endogenous IL-2 in activating cells, and have mutations selected from: 1) R38K, F42I, Y45N, E62L, and E68V; 2) R38A, F42I, Y45N, E62L, and E68V; 3) R38K, F42K, Y45R, E62L, and E68V; or 4) R38A, F42A, Y45A, and E62A. U.S. Patent Publication No. 2014 / 0328791 describes pegylated IL-2 with reduced affinity for CD25. Some IL-2 muteins are conjugated to antibodies targeting tumor antigens (e.g., CEA, FAP, and PD-L1). See, e.g., Klein et al., Oncoimmunology 6(3):e1277306 (2017); Soerensen et al., J Clin Onc. 36:15_suppl (2018); WO2017 / 220989; and U.S. Patent No. 9,206,260.

[0006] Interleukin-15 (IL-15) is a cytokine with structural similarity to IL-2. IL-15 binds to and signals through the IL-2Rβγ receptor, and is secreted by the mononuclear phagocyte system and other immune cells after viral infection. IL-15 induces proliferation of NK and other cells of the innate immune system and is involved in the killing of virus-infected and cancer cells.

[0007] Unfortunately, the side effects of current IL-2 and IL-15 drug candidates are significant, limiting the dosage of these cytokines. Furthermore, activation of T cells and other immune cells is not site-specific. Also, PK sinkers of IL-2 mutant proteins may exist, although their affinity for CD25 is significantly reduced. Thus, there is a need to develop improved cytokine therapies that are site-specific in activating immune cells and exhibit efficacy but reduced side effects. Summary of the Invention

[0008] The present disclosure provides prodrugs comprising a cytokine moiety, a masking moiety, and a carrier moiety, wherein the masking moiety binds to the cytokine moiety and inhibits a biological activity of the cytokine moiety (e.g., inhibits the cytokine moiety from binding to its receptor in a target cell or reduces one or more biological activities of the cytokine moiety), the cytokine moiety is fused to the carrier moiety, and the masking moiety is fused to the cytokine moiety or the carrier moiety via a cleavable peptide linker. In certain embodiments, the masking moiety comprises the extracellular domain (ECD) of a receptor for the cytokine moiety.

[0009] In certain embodiments, the cytokine portion is a wild-type human cytokine or a mutein thereof, e.g., a human IL-2 agonist polypeptide, e.g., one comprising SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1. In certain embodiments, the human IL-2 agonist polypeptide comprises one or more mutations at positions selected from T3, K35, R38, F42, Y45, E62, E68, L72, A73, N88, C125, and Q126 (numbered according to SEQ ID NO: 1). In certain embodiments, the human IL-2 agonist polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 8-17, 19-33, 36, 37, and 39-46.

[0010] In certain embodiments, the masking moiety of the prodrug of the present invention comprises the ECD of human IL-2Rβ or a functional analog thereof. In further embodiments, the masking moiety includes (i) two copies of the ECD of human IL-2Rβ or a functional analog thereof fused together via a peptide linker, or (ii) the ECD of human IL-2Rβ or a functional analog thereof fused to the ECD of human IL-2Rγ or a functional analog thereof via a peptide linker. In certain embodiments, the ECD of human IL-2Rγ or a functional analog thereof comprises SEQ ID NO:6 or an amino acid sequence at least 90% identical to SEQ ID NO:6. In certain embodiments, the ECD of human IL-2Rβ or a functional analog thereof comprises SEQ ID NO:3, 4, or 5, or an amino acid sequence at least 90% identical to SEQ ID NO:3, 4, or 5.

[0011] In certain embodiments, the cytokine portion of the prodrug of the invention is a human IL-15 agonist polypeptide. The human IL-15 agonist polypeptide comprises SEQ ID NO:2 or an amino acid sequence at least 90% identical to SEQ ID NO:2. In certain embodiments, the IL-15 agonist polypeptide comprises or further comprises (i) an IL-15Rα sushi domain comprising SEQ ID NO:7, or (ii) an amino acid sequence at least 90% identical to SEQ ID NO:7. In certain embodiments, the IL-15 masking domain comprises human IL-2Rβ or a functional analog thereof, or the ECD or functional analog of human IL-2Rγ. In certain embodiments, the IL-15 masking domain comprises SEQ ID NO:3, 4, 5, or 6, or an amino acid sequence at least 90% identical to SEQ ID NO:3, 4, 5, or 6.

[0012] In certain embodiments, the prodrug further includes a second effector polypeptide, such as (i) a human IL-2 agonist polypeptide comprising a mutation at position 126 (numbering according to SEQ ID NO: 1), or (ii) a CCL19 polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 123.

[0013] In certain embodiments of the prodrug of the invention, the cytokine moiety is fused to the carrier moiety via a non-cleavable peptide linker (eg, one selected from SEQ ID NOs: 47-51).

[0014] In certain embodiments of the prodrug of the present invention, the cleavable peptide linker that directly or indirectly (e.g., via the cytokine moiety) attaches the masking moiety to the carrier moiety includes a substrate sequence for urokinase-type plasminogen activator (uPA), matrix metallopeptidase (MMP) 2, or MMP 9. In further embodiments, the cleavable peptide linker includes sequences for (i) both uPA and MMP 2, (ii) both uPA and MMP 9, or (iii) uPA, MMP 2, and MMP 9. In certain embodiments, the cleavable peptide linker includes an amino acid sequence selected from SEQ ID NOs: 18, 34, 35, 38, 52-121, and 217. In certain embodiments, the cleavable peptide linker is cleavable by one or more proteases localized at a tumor site or its surrounding environment, and the cleavage results in activation of the prodrug at the tumor site or its surrounding environment.

[0015] In some embodiments of the prodrugs of the present invention, the carrier moiety is a PEG molecule, albumin (e.g., human serum albumin) or a fragment thereof, an antibody Fc domain, or an antibody or antigen-binding fragment thereof. In certain embodiments, the carrier moiety is an antibody Fc domain or antibody comprising the mutations L234A and L235A ("LALA") (EU numbering). In certain embodiments, the carrier moiety is an antibody Fc domain or antibody comprising knob-into-hole mutations, and the cytokine moiety and masking moiety are fused to different polypeptide chains of the antibody Fc domain or different heavy chains of the antibody. In certain embodiments, the cytokine moiety and masking moiety are fused to the C-terminus of two different polypeptide chains of the Fc domain or the C-terminus of two different heavy chains of the antibody. In other embodiments, the cytokine moiety and masking moiety are fused to the N-terminus of two different polypeptide chains of the Fc domain or the N-terminus of two different heavy chains of the antibody. In certain embodiments, the knobs-into-hole mutations comprise 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 another polypeptide chain of the Fc domain or another heavy chain of the antibody (EU numbering). In certain embodiments, the knobs-into-hole mutations comprise 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).

[0016] In certain embodiments, the carrier moiety is an antibody Fc domain comprising two polypeptide chains comprising an amino acid sequence selected from SEQ ID NOs: 195-198 and an amino acid sequence selected from SEQ ID NOs: 132-137 and 139, respectively.

[0017] 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), and six transmembrane epithelial antigen of prostate 1 (six transmembrane epithelial antigen of prostate 1). 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), 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 EPCAM, or an antigen-binding fragment thereof.

[0018] In certain embodiments, the carrier moiety is an antibody comprising two heavy chains having the amino acid sequences SEQ ID NO: 209 and one of SEQ ID NOs: 210-215, respectively, and two light chains having the amino acid sequence SEQ ID NO: 216. In certain embodiments, the carrier moiety is an antibody comprising two heavy chains having the amino acid sequences SEQ ID NO: 191 and one of SEQ ID NOs: 192, 193, and 206-208, and two light chains having the amino acid sequence SEQ ID NO: 189.

[0019] In another aspect, the present disclosure provides an IL-2 mutein comprising a mutation at position A73, an IL-2 mutein comprising a K35N mutation, and an IL-2 mutein comprising an amino acid sequence selected from SEQ ID NOs: 23-33, 36, 37, and 39-41. The novel IL-2 muteins may exhibit significantly reduced binding to the trimeric IL-2 receptor.

[0020] In other aspects, the present disclosure provides a pharmaceutical composition comprising a prodrug or IL-2 mutein of the present disclosure and a pharmaceutically acceptable excipient; a polynucleotide encoding the prodrug or IL-2 mutein; an expression vector comprising the polynucleotide; and a host cell comprising the vector (the host cell may be a prokaryotic cell or a eukaryotic cell, such as a mammalian cell). In certain embodiments, the mammalian host cell has a gene knockout for uPA, MMP2, and / or MMP9 (e.g., comprises a null mutation in one or more of these genes). Thus, the present disclosure also provides a method for producing the prodrug or IL-2 mutein, comprising culturing the host cell under conditions that allow expression of the prodrug or IL-2 mutein (the host cell is a mammalian cell), and then isolating the prodrug or IL-2 mutein.

[0021] The present disclosure also provides a method for treating cancer or an infectious disease or activating the immune system in a patient (e.g., a human patient) in need thereof, comprising administering to the patient a therapeutically effective amount of a prodrug, IL-2 mutein, or pharmaceutical composition of the present disclosure. The patient may be suffering from, for example, a viral infection (e.g., HIV 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, colon cancer, and gastric cancer. Also provided herein are cytokine prodrugs or IL-2 muteins for use in treating cancer or an infectious disease or activating the immune system in the methods of the invention; use of a prodrug or IL-2 mutein for the manufacture of a medicament for treating cancer or an infectious disease or activating the immune system in the methods of the invention; and articles of manufacture (e.g., kits) containing one or more dosage units of a prodrug or IL-2 mutein of the invention.

[0022] Other features, objects, and advantages of the present invention will become apparent in the following detailed description. It should be understood, however, that the detailed description, while illustrating embodiments and aspects of the present invention, is given by way of example only and not by way of 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]

[0023] [Figure 1] FIG. 1 shows an SDS-PAGE analysis of mutant IL-2 polypeptides fused to a carrier protein. [Figure 2] Figures 2A-C show the results of a CTLL2-based biological activity assay of IL-2 muteins fused to carrier proteins. Figure 2A is a table listing the fusion proteins. Figure 2B shows the results for an IL-2 mutein / Fc fusion protein. Figure 2C shows the results for an IL-2 mutein / human serum albumin (HSA) fusion protein. [Figure 3]3 is a schematic diagram showing antibody-based IL-2 or IL-15 prodrugs. An IL-2 or IL-15 agonist polypeptide is fused to the C-terminus of one heavy chain of the carrier antibody, optionally via a non-cleavable peptide linker. An IL-2 or IL-15 antagonist polypeptide (a masking moiety, e.g., the IL-2Rβ extracellular domain) is fused to the C-terminus of the other heavy chain of the carrier antibody via a cleavable peptide linker. [Figure 4] Figure 4 shows SDS-PAGE analysis of 589A-IL-2 prodrug expressed in HEK293 cells. 589A is a humanized antibody against claudin 18.2 derived from a rabbit B cell clone. Prodrug samples were purified by protein A affinity chromatography. "Activated" samples were treated with proteases. The SDS-PAGE was run under non-reducing conditions. [Figure 5] FIG. 5 shows the SEC-HPLC analysis of antibody 589A and prodrug 589A-IL-2E. [Figure 6] Figure 6 shows activation of 589A-IL-2 prodrugs using a CTLL2-based activation assay. JR1.55.1: 589A-IL-2E. JR1.55.2: 589-IL-2F. MT: matriptase (protease). [Figure 7] Figures 7A and B show activation of the 589A-IL-2E (JR1.74.1) prodrug. Figure 7A shows binding of 589A-IL-2E and its activated form (+MT) to HEK293 cells (expressing IL-2Rαβγ or IL-2Rβγ) as measured by FACS analysis. MFI: mean fluorescence intensity. Figure 7B shows overall binding levels at 11 μg / ml of prodrug. [Figure 8] Figure 8 shows the activation of αPD-L1-IL-2B prodrugs using a CTLL2-based activation assay. MT: matriptase. [Figure 9]FIG. 9 shows an analysis of antibody-dependent cellular cytotoxicity (ADCC) function of antibody 589A, 589A with enhanced ADCC functionality, and 589A with enhanced ADCC functionality fused to IL-2. [Figure 10] Figure 10 shows the in vivo anti-cancer efficacy of the prodrug 589A-IL-2E, as measured by tumor volume in each mouse. Tecentriq®: Atezolizumab, an anti-PD-L1 antibody. [Figure 11] Figure 11 shows the in vivo anti-cancer efficacy of prodrug 589A-IL-2E, as measured by tumor volume in each mouse, which was identical to the experiment shown in Figure 10. [Figure 12] Figure 12 shows the in vivo anti-cancer efficacy of the prodrug 589A-IL-2E, as measured by survival rate, which was identical to the experiment shown in Figure 10. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description of the Invention As used in this specification and claims, the singular forms "a," "or," and "the" include plural referents unless otherwise clear from the context. References herein to "about" a value or parameter include (and describe) a degree of variation relative to the value or parameter itself. For example, reference to "about X" includes reference to "X." Furthermore, the use of "about" before a series of numbers includes "about" each of the numbers in that series. For example, a reference indicating "about X, Y, or Z" is intended to describe "about X, about Y, or about Z."

[0025] The term "antigen-binding portion" refers to a polypeptide or set of interacting polypeptides that specifically binds to an antigen, including, but not limited to, antibodies (e.g., monoclonal antibodies, polyclonal antibodies, multispecific antibodies, bispecific or diabodies, anti-idiotypic antibodies, or bifunctional hybrid antibodies) or antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single-domain antibodies (dAbs), or bispecific antibodies), single-chain antibodies, and Fc-containing polypeptides (e.g., immunoadhesins). In certain embodiments, the antibodies include 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 antibodies may be any light chain isotype (e.g., kappa or lambda). The antibody may be human, non-human (e.g., derived from a mouse, rat, rabbit, goat, or another non-human animal), chimeric (e.g., having a non-human variable region and a human constant region), or humanized (e.g., having non-human CDRs and a human framework and constant region). In certain embodiments, the antibody is a derivatized antibody.

[0026] 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. The analog may be, for example, a mutein (i.e., a polypeptide containing mutations) of the 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.

[0027] The term "cytokine antagonist" or "cytokine mask" refers to a moiety (e.g., a polypeptide) that binds to a cytokine, thereby preventing the cytokine from binding to its receptor on the surface of a target cell and / or from performing 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 the cytokine's natural receptor that contacts the cytokine.

[0028] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or composition sufficient to treat a particular disorder, illness, or disease, e.g., to relieve, alleviate, reduce, and / or delay one or more symptoms thereof. With respect to diseases such as cancer, an effective amount can be an amount sufficient to delay the onset or progression of cancer (e.g., reduce tumor growth rate and / or delay or prevent tumor angiogenesis, metastasis, or invasion of cancer cells into peripheral organs), reduce the number of epithelioid cells, cause regression of cancer (e.g., shrink or eradicate tumors), and / or prevent or delay the onset or recurrence of cancer. An effective amount can be administered in one or more administrations.

[0029] 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 the molecule of interest.

[0030] The terms "fused" or "fusion," in reference to two polypeptide sequences, refer to the linkage of the two polypeptide sequences by a backbone peptide bond. The two polypeptides may 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 coding sequences that include each of the coding sequences for two fusion partners, with or without a coding sequence for a peptide linker between them. In certain embodiments, fusion includes chemical conjugation.

[0031] The term "pharmaceutically acceptable excipient," when used to refer to an ingredient in a composition, means that the excipient is suitable for administration to a subject (including a human subject) to be treated without excessive adverse side effects to the subject and without affecting the biological activity of the active ingredient (API).

[0032] The term "subject" means a mammal, including, but not limited to, a human, a pet (e.g., a dog or cat), a livestock (e.g., a cow or horse), a rodent, or a primate.

[0033] As used herein, "treatment" or "treating" refers to an approach for obtaining a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, reducing the extent of the disease, ameliorating the disease state, stabilizing the disease (e.g., inhibiting or slowing the worsening or progression of the disease), inhibiting or slowing the spread of the disease (e.g., metastasis), inhibiting or slowing the recurrence of the disease, providing partial or total remission of the disease, reducing the dose of one or more other medications needed to treat the disease, improving the patient's quality of life, and / or increasing survival. The methods of the present disclosure include any one or more of these therapeutic aspects.

[0034] It should be 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 headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described below.

[0035] Cytokine Prodrugs The present disclosure provides cytokine prodrugs that are metabolized in vivo to become active cytokine therapeutics. The cytokine prodrugs have fewer side effects, a superior in vivo PK profile (e.g., a prolonged half-life), excellent target specificity, and higher efficacy compared to previous cytokine therapeutics. The prodrugs of the present invention include a cytokine agonist polypeptide (cytokine moiety) conjugated to a carrier moiety and masked (conjugated) by a cytokine antagonist (masking moiety). The cytokine antagonist may be, for example, the extracellular domain of a cytokine receptor, and is conjugated to the cytokine moiety or carrier moiety via a cleavable linker (e.g., a cleavable peptide linker). The mask inhibits the biological function of the cytokine moiety while binding to it. The prodrug can be activated at a target site in a patient (e.g., a tumor site or its surrounding environment) by cleavage of the linker, thereby releasing the cytokine mask from the prodrug and exposing the previously masked cytokine moiety, allowing the cytokine moiety to bind to its receptor on the target cell and perform its biological function in the target cell. In some embodiments, the prodrug carrier is an antigen-binding moiety, such as an antibody, that binds to an antigen at the target site.

[0036] In one embodiment, the prodrug of the present invention is a pro-inflammatory cytokine prodrug that is metabolized to a pro-inflammatory cytokine at a target site in the body targeted by the carrier. In a further embodiment, the carrier in the prodrug is an antibody that targets a tumor antigen such that the prodrug is delivered to the tumor site in a patient and metabolized locally (e.g., within or near the tumor microenvironment) by cleavage of a linker connecting the cytokine mask to the carrier or cytokine moiety, making the pro-inflammatory cytokine moiety available to interact with its receptor on target cells and locally activating the target immune cells.

[0037] Although the description below exemplifies IL-2 and IL-15 prodrugs, prodrugs for other cytokines (especially cytokines that are potent immunomodulators and have strong side effects) are also encompassed by this disclosure. These other cytokine prodrugs can be made using the same principles as those exemplified below for IL-2 and IL-15 prodrugs.

[0038] A. Cytokine Moiety of the Prodrug In certain embodiments, the prodrugs of the invention include a pro-inflammatory cytokine agonist polypeptide, such as an IL-2 agonist polypeptide or an IL-15 agonist polypeptide.

[0039] 1. IL-2 agonist polypeptide An IL-2 prodrug may comprise an IL-2 agonist polypeptide (cytokine moiety), a carrier (carrier moiety), and an IL-2 antagonist (masking moiety), wherein the IL-2 agonist polypeptide is fused to the carrier directly or via a linker (e.g., a cleavable or non-cleavable peptide linker), and the IL-2 antagonist is linked to the IL-2 agonist polypeptide or the carrier via a cleavable peptide linker. In the IL-2 prodrug of the present invention, the IL-2 agonist polypeptide may be a wild-type IL-2 polypeptide, e.g., a wild-type human IL-2 polypeptide (SEQ ID NO: 1), or an IL-2 mutein, e.g., an IL-2 mutein derived from human IL-2. The IL-2 mutein may have significantly reduced affinity for CD25 or trimeric high-affinity IL-2R compared to wild-type IL-2. In certain embodiments, the IL-2 muteins have a binding affinity for the high-affinity IL-2R that is 100-fold, 300-fold, 500-fold, 1,000-fold, or 10,000-fold lower compared to wild-type IL-2. Unless otherwise indicated, all residue numbers in IL-2 and IL-2 muteins described herein are according to the numbering of SEQ ID NO: 1.

[0040] In one aspect, the present disclosure provides novel IL-2 muteins that can be used as IL-2 agonist polypeptides in IL-2 prodrugs. The novel IL-2 muteins include a mutation at A73 (e.g., to T or another amino acid residue) and / or a K35N mutation. A73 has not previously been identified as one of the amino acid residues that interact with CD25. Therefore, the inventors were surprised that introducing a mutation at this position (e.g., A73T) can result in a significantly reduced binding affinity of the IL-2 mutein to the trimeric IL-2 receptor, similar to the binding affinity of an IL-2 mutein with the mutations R38S / F42A / Y45A / E62A or F42A / Y45A / L72G (see Example 1 below). Without being bound by theory, the inventors believe that A73 and K35 are potential glycosylation sites in IL-2, and that mutations in these glycosylation sites modulate the affinity of IL-2 muteins for IL-2R. The novel muteins have a safer clinical profile and can be used in patients requiring IL-2 activity, e.g., patients requiring an activated immune system (e.g., cancer and AIDS patients). The novel IL-2 muteins can be used as free entities or in conjugates (e.g., fused to a carrier, e.g., in prodrugs of the invention).

[0041] In certain embodiments, the novel IL-2 muteins of the present disclosure may comprise a mutation at A73 (e.g., A73T) and one or more mutations at positions selected from T3, D20, K35, R38, F42, F44, Y45, E62, E68, L72, N88, N90, C125, and Q126. In certain embodiments, the novel IL-2 muteins comprise mutations at R38, F42, Y45, and A73.

[0042] In certain embodiments, the novel IL-2 muteins of the present invention may comprise a K35N mutation and one or more mutations at positions selected from T3, D20, R38, F42, F44, Y45, E62, E68, L72, A73, N88, N90, C125, and Q126. In certain embodiments, the novel IL-2 muteins comprise the mutation K35N and additional mutations at R38, F42, and Y45, with or without a mutation at A73.

[0043] In certain embodiments, the IL-2 agonist polypeptide of the IL-2 prodrug may contain one or more mutations at K35, R38, F42, F44, Y45, E62, E68, L72, and A73. In certain embodiments, the IL-2 agonist polypeptide further contains one or more mutations at D20, N88, N90, and Q126. Additional mutations at T3 and / or C125 may also be included. In certain embodiments, the IL-2 agonist polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 8-17, 19-33, 36, 37, and 39-46.

[0044] In certain embodiments, to ensure a baseline level of IL-2 agonist activity, the IL-2 prodrugs of the present invention may further comprise a second IL-2 agonist polypeptide containing mutations that result in significantly reduced affinity for the dimeric intermediate-affinity IL-2 receptor compared to wild-type IL-2. For example, an IL-2 mutein having the mutations T3A / R38S / F42A / Y45A / E62A / C125S / Q126W (SEQ ID NO: 30), with or without a linker (SEQ ID NO: 130) exhibits low but detectable levels of IL-2 activity (see Example 1 below).

[0045] 2. IL-15 agonist polypeptide In the IL-15 prodrugs of the present disclosure, the IL-15 agonist polypeptide may be a wild-type IL-15 polypeptide, e.g., a wild-type human IL-15 polypeptide (SEQ ID NO: 2), or an IL-15 mutein having reduced affinity for IL-15Rα or IL-2Rβ (CD122) compared to wild-type IL-15, e.g., an IL-15 mutein derived from human wild-type IL-15.

[0046] B. Masking moiety of the prodrug The cytokine antagonist, i.e., the masking moiety of the prodrug of the present invention, may comprise a peptide or an antibody or antibody fragment that binds to the cytokine moiety in the prodrug, masks the cytokine moiety and inhibits its biological function.

[0047] As an example, IL-2 and IL-15 antagonists can include peptides and antibodies that bind to IL-2 or IL-15 and prevent the binding of the IL-2 or IL-15 moiety to its receptor, resulting in a masked but reduced biological activity of the IL-2 or IL-15 moiety. In certain embodiments, the IL-2 antagonist comprises the IL-2Rβ or IL-2Rγ extracellular domain or a functional analog thereof (e.g., derived from human IL-2Rβ or IL-2Rγ (e.g., one of SEQ ID NOS: 3-6)). In certain embodiments, the IL-2 antagonist comprises a peptide identified from screening a peptide library. In certain embodiments, the IL-2 antagonist comprises an antibody or fragment thereof that inhibits the binding of IL-2 or an IL-2 mutein to the IL-2 receptor. In certain embodiments, the IL-2 antagonist comprises an scFv, Fab, or single-chain Fab having CDR sequences identical to an antibody selected from hybridoma clones 4E12B2D10, 4E12B2, and 4E12 disclosed in US Pat. No. 4,411,993.

[0048] Human IL-2 binds to IL-2Rβ (CD122) with relatively low affinity (K DIL-2Rβ binds to the IL-2 mutein agonist polypeptide with a binding affinity of 0.01 to 0.02 μM (approximately 3 μM), which is more than 1,000-fold weaker than the binding affinity of IL-2 to the intermediate affinity receptor IL-2Rβγ (Johnson et al., Eur Cytokine Netw. 5(1):23-34 (1994)). Thus, the present inventors were surprised to find that when the extracellular domain (ECD) of IL-2Rβ was fused to the same carrier molecule as the IL-2 mutein agonist polypeptide, the cell-based activity of the IL-2 mutein agonist polypeptide was significantly inhibited (see Example 4 below).

[0049] For IL-15 prodrugs, the masking moiety may be the extracellular domain of IL-2Rβ or IL-2Rγ or a functional analogue thereof (eg, one of SEQ ID NOs: 3-6).

[0050] C. Carrier Moieties of Prodrugs The carrier moiety of the prodrug of the present invention may be an antigen-binding moiety or a moiety that is not an antigen-binding moiety, and may improve the PK profile, such as serum half-life, of the cytokine agonist polypeptide and target the cytokine agonist polypeptide to a target site in the body (e.g., a tumor site).

[0051] 1. Antigen-binding carrier site The carrier moiety may be an antibody or antigen-binding fragment thereof, or an immunoadhesin. In certain embodiments, the antigen-binding 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 certain embodiments, the antigen-binding moiety is a bispecific antigen-binding moiety, capable of binding to two different antigens or two different epitopes on the same antigen. The antigen-binding moiety may provide the cytokine agonist polypeptide with a more potent synergistic therapeutic effect.

[0052] The cytokine (e.g., IL-2 or IL-15) 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. As an example, the cytokine (e.g., IL-2 or IL-15) agonist polypeptide and its mask can be fused to an antibody heavy chain or antigen-binding fragment thereof or an antibody light chain or antigen-binding fragment thereof. In one embodiment, the cytokine (e.g., IL-2 or IL-15) agonist polypeptide is fused to the C-terminus of one or both antibody heavy chains, and the cytokine mask is fused to the other end of the cytokine agonist polypeptide via a cleavable peptide linker. In one embodiment, the cytokine (e.g., IL-2 or IL-15) agonist polypeptide is fused to the C-terminus of one antibody heavy chain, and the cytokine mask is fused to the C-terminus of the other antibody heavy chain via a cleavable peptide linker, and the two heavy chains contain mutations that allow for specific pairing of two different heavy chains.

[0053] Techniques for forming heterodimers are well known (see, e.g., Spies et al., Mol Imm. 67(2)(A):95-106 (2015)). For example, the two heavy chain polypeptides in the prodrug may form stable heterodimers by "knobs-into-holes" mutations. "Knobs-into-holes" mutations are introduced to facilitate the formation of heterodimers in antibody heavy chains and are commonly used to create bispecific antibodies (see, e.g., U.S. Patent No. 8,642,745). For example, the Fc domain of the antibody may 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." An interchain disulfide bridge between additional 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 16:677-81 (1998)). 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 portion comprises 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, optionally with an additional Y349C mutation in one CH3 domain and an additional E356C or S354C mutation in the other CH3 domain, forming an interchain disulfide bridge (numbering always according to the EU index of Kabat; 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 techniques (e.g., those disclosed in EP1870459A1) can be used instead or in addition. Thus, another example of a knob-into-hole mutation on an antibody portion is to have 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).

[0054] In one embodiment, the antibody portion of the prodrug comprises L234A and L235A ("LALA") mutations in its Fc domain. The LALA mutations eliminate complement binding and fixation and Fcγ-dependent ADCC (see, e.g., Hezareh et al. J. Virol. 75(24):12161-8 (2001)). In a further embodiment, the LALA mutations are present in the antibody portion in addition to knob-into-hole mutations.

[0055] In certain embodiments, the antibody portion comprises M252Y / S254T / T256E ("YTE") mutations in the Fc domain. The YTE mutations allow for simultaneous modulation of the serum half-life, tissue distribution, and activity of IGG1 (see Dall'Acqua et al., J Biol Chem. 281: 23514-24 (2006); and Robbie et al., Antimicrob Agents Chemother. 57(12):6147-53 (2013)). In further embodiments, the YTE mutations are present in the antibody portion in addition to knob-into-hole mutations. In certain embodiments, the antibody portion has YTE, LALA, and knob-into-hole mutations, or any combination thereof.

[0056] The antigen-binding portion may bind to an antigen on the surface of a cell (e.g., an immune cell, e.g., a T cell, a NK cell, or a macrophage) or may bind to a cytokine. For example, the antigen-binding portion may bind to PD-1, LAG-3, TIM-3, TIGIT, CTLA-4, or TGF-beta, or may be an antibody. The antibody may have the ability to activate immune cells and enhance their anti-cancer activity.

[0057] The antigen-binding moiety can bind to an antigen on the surface of a tumor cell. For example, the antigen-binding moiety can bind to FAP alpha, 5T4, Trop-2, PD-L1, HER2, EGFR, claudin 18.2, DLL-3, GCP3, or carcinoembryonic antigen (CEA), or can be an antibody. The antibody may or may not have ADCC activity. The antibody may also be further conjugated to a cytotoxic drug.

[0058] In certain embodiments, the antigen-binding portion 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, carbonic anhydrase IX (CA9), endothelin B receptor (ETBR), and 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 some embodiments, the antigen-binding portion binds to the epidermal growth factor (EGF)-like domain of DLL3. In some embodiments, the antigen-binding portion binds to the delta / serrate / Lag2 (DSL)-like domain of DLL3. In certain embodiments, the antigen-binding portion binds to an epitope located after amino acid 374 of GPC3. In certain embodiments, the antigen-binding portion binds to a heparin sulfate glycan of GPC3. In certain embodiments, the antigen-binding portion binds to claudin 18.2 but not claudin 18.1.In certain embodiments, the antigen-binding portion binds to claudin 18.1 with a binding affinity that is at least 10-fold weaker than that of claudin 18.2.

[0059] Exemplary antigen-binding moieties include 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 some embodiments, the antigen-binding moiety has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity 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, 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 comprises an antibody light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity 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.

[0060] Many CDR delineations are known in the art and are included herein. One skilled in the art can easily determine the CDRs for a given delineation based on the sequence of the heavy or light chain variable region. "Kabat" CDRs are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). "Chothia" CDRs indicate the location of structural loops (Chothia & Lesk, Canonical structures for the hypervariable regions of immunoglobulins, J. Mol. Biol., vol. 196, pp. 901-917 (1987)). "AbM" CDRs represent an intermediate approach between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" CDRs are based on analysis of available complex crystal structures. Residues from each of these CDRs are set forth in Table 1 below with reference to common antibody numbering schemes. Unless otherwise specified herein, antibody amino acid numbering refers to the Kabat numbering scheme described in Kabat et al., supra, including when made with reference to the Kabat, Chothia, AbM, or contact schemes. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the framework regions (FRs) or CDRs of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52 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 can be determined for a given antibody by alignment of homology regions of the antibody sequence with the "standard" Kabat-numbered sequence. Table 1. CDR delineation by various schemes [Table 1]

[0061] In some embodiments, the CDRs are "extended CDRs" and include regions that begin or end according to different sequences. For example, extended CDRs can be: L24 to L36, L26 to L34, or L26 to L36 (VL-CDR1); L46 to L52, L46 to L56, or L50 to L55 (VL-CDR2); L91 to L97 (VL-CDR3); H47 to H55, H47 to H65, H50 to H55, H53 to H58, or H53 to H65 (VH-CDR2); and / or H93 to H102 (VH-CDR3).

[0062] In certain embodiments, the antigen-binding portion binds to HER2 and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 148, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 149, or a fragment thereof. In certain embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 148, and CDR1, CDR2, and CDR3 from SEQ ID NO: 149.

[0063] In certain embodiments, the antigen-binding portion binds to CD20 and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 150, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 151, or a fragment thereof. In certain embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 150, and CDR1, CDR2, and CDR3 from SEQ ID NO: 151.

[0064] In certain embodiments, the antigen-binding portion binds to CD30 and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 152, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 153, or a fragment thereof. In certain embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 152, and CDR1, CDR2, and CDR3 from SEQ ID NO: 153.

[0065] In certain embodiments, the antigen-binding portion binds to EGFR and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 154, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 155, or a fragment thereof. In certain embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 154, and CDR1, CDR2, and CDR3 from SEQ ID NO: 155.

[0066] In certain embodiments, the antigen-binding portion binds to EGFR and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 156, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 157, or a fragment thereof. In certain embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 156, and CDR1, CDR2, and CDR3 from SEQ ID NO: 157.

[0067] In certain embodiments, the antigen-binding portion binds to c-MET and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 158, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 159, or a fragment thereof. In certain embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 158, and CDR1, CDR2, and CDR3 from SEQ ID NO: 159.

[0068] In certain embodiments, the antigen-binding portion binds to GPC3 and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 160, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 161, or a fragment thereof. In certain embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 160, and CDR1, CDR2, and CDR3 from SEQ ID NO: 161.

[0069] In certain embodiments, the antigen-binding portion binds to claudin 18.2 and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 162, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 163, or a fragment thereof. In certain embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 162, and CDR1, CDR2, and CDR3 from SEQ ID NO: 163.

[0070] In certain embodiments, the antigen-binding portion binds to FAP alpha and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 180, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 182, or a fragment thereof. In certain embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 180 or 181, and CDR1, CDR2, and CDR3 from SEQ ID NO: 182. In certain embodiments, the antigen-binding portion binds to FAP alpha and comprises a light chain variable domain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 183, or a fragment thereof, and a heavy chain variable domain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 184, or a fragment thereof. In certain embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 183, and CDR1, CDR2, and CDR3 from SEQ ID NO: 184. In certain embodiments, the humanized FAP antibody comprises a light chain amino acid sequence set forth in SEQ ID NO: 180 or 181 and a heavy chain amino acid sequence set forth in SEQ ID NO: 182.

[0071] In some embodiments, the antigen-binding portion binds to carcinoembryonic antigen (CEA) and may be derived from antibody PR1A3 (U.S. Patent No. 8,642,742). The anti-CEA antibody comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 178, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 179, or a fragment thereof. In some embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 176 and CDR1, CDR2, and CDR3 from SEQ ID NO: 177. In one embodiment, the PR1A3 antibody is a humanized antibody comprising the light chain variable region amino acid sequence shown in SEQ ID NO: 178 and the heavy chain variable region amino acid sequence shown in SEQ ID NO: 179.

[0072] In certain embodiments, the antigen-binding portion binds to PDL1 and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 189, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 190, or a fragment thereof. In certain embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 189, and CDR1, CDR2, and CDR3 from SEQ ID NO: 190.

[0073] In certain embodiments, the antigen-binding portion binds to 5T4 and comprises a light chain variable domain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 187 or 188, or a fragment thereof, and a heavy chain variable domain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 185 or 186, or a fragment thereof. In certain embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 187 or 188, and CDR1, CDR2, and CDR3 from SEQ ID NO: 185 or 186.

[0074] In one embodiment, the antigen-binding portion binds to Trop-2 and comprises a light chain variable region comprising CDR1 comprising the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 164), CDR2 comprising the amino acid sequence of SASYRYT (SEQ ID NO: 165), and CDR3 comprising the amino acid sequence of QQHYITPLT (SEQ ID NO: 166); and a heavy chain variable region comprising CDR1 comprising the amino acid sequence of NYGMN (SEQ ID NO: 167), CDR2 comprising the amino acid sequence of WINTYTGEPTYTDDFKG (SEQ ID NO: 168), and CDR3 comprising the amino acid sequence of GGFGSSYWYFDV (SEQ ID NO: 169).

[0075] In one embodiment, the antigen-binding portion binds to mesothelin and comprises a light chain variable region comprising CDR1 comprising the amino acid sequence of SASSSVSYMH (SEQ ID NO: 170), CDR2 comprising the amino acid sequence of DTSKLAS (SEQ ID NO: 171), and CDR3 comprising the amino acid sequence of QQWSGYPLT (SEQ ID NO: 172); and a heavy chain variable region comprising CDR1 comprising the amino acid sequence of GYTMN (SEQ ID NO: 173), CDR2 comprising the amino acid sequence of LITPYNGASSYNQKFRG (SEQ ID NO: 174), and CDR3 comprising the amino acid sequence of GGYDGRGFDY (SEQ ID NO: 175).

[0076] 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 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.

[0077] 2. Other carrier parts Other non-antigen-binding carrier moieties may be used for the prodrugs of the present invention, such as antibody Fc domains (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc), polymers (e.g., PEG), albumin (e.g., human albumin), or fragments thereof, or nanoparticles.

[0078] For example, the cytokine (e.g., IL-2 or IL-15) agonist polypeptide and its antagonist may be fused to an antibody Fc domain to form an Fc fusion protein. In one embodiment, the cytokine (e.g., IL-2 or IL-15) agonist polypeptide is fused (directly or via a peptide linker) to the C-terminus or N-terminus of one of the Fc domain polypeptide chains, and the cytokine mask is fused to the C-terminus or N-terminus of the other Fc domain polypeptide chain via a cleavable peptide linker, and the two Fc domain polypeptide chains contain mutations that allow specific pairing of the two different Fc chains. In one embodiment, the Fc domain contains the hole-into-hole mutation described above. In a further embodiment, the Fc domain may also contain the YTE and / or LALA mutation described above.

[0079] The carrier moiety of the prodrug can include albumin (e.g., human serum albumin) or a fragment thereof. An exemplary sequence of albumin is set forth in SEQ ID NO: 124. In certain embodiments, the albumin or albumin fragment is about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, or about 99.8% or more identical to human serum albumin or a fragment thereof.

[0080] 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 one embodiment, the albumin fragment is about 10 to about 584 amino acids in length (e.g., about 10 to about 20, about 20 to about 40, about 40 to about 80, about 80 to about 160, about 160 to about 250, about 250 to about 350, about 350 to about 450, or about 450 to about 550 amino acids in length, etc.). In one embodiment, the albumin fragment comprises a Sudlow I domain or a fragment thereof, or a Sudlow II domain or a fragment thereof.

[0081] D. Prodrug Linker Configuration The cytokine (e.g., IL-2 or IL-15) agonist polypeptide may be fused to the carrier moiety with or without a peptide linker. The peptide linker may be non-cleavable. In certain embodiments, the peptide linker is selected from SEQ ID NOs: 47-51. In certain embodiments, the peptide linker comprises the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 49).

[0082] The cytokine (e.g., IL-2 or IL-15) mask may be fused to the cytokine moiety or the carrier via a cleavable linker. The cleavable linker may contain one or more (e.g., two or three) cleavable moieties (CMs). Each CM may be a substrate for an enzyme or protease selected from legumain, plasmin, TMPRSS-3 / 4, MMP2, MMP9, MT1-MMP, cathepsin, caspase, human neutrophil elastase, beta-secretase, uPA, and PSA. Examples of cleavable linkers include, but are not limited to, those comprising an amino acid sequence selected from SEQ ID NOs: 18, 34, 35, 38, 52-121, and 217.

[0083] Specific, non-limiting examples of cytokine agonist polypeptides, cytokine masks, carriers, peptide linkers, and prodrugs are shown in the sequence entries below. Furthermore, the prodrugs and novel IL-2 muteins of the present disclosure can be produced by well-known recombinant techniques. For example, one or more expression vectors containing coding sequences for the polypeptide chains of the prodrugs can be transfected into mammalian host cells (e.g., CHO cells), and the cells are cultured under conditions that allow expression of the coding sequences and assembly of the expressed polypeptides into prodrug conjugates. To ensure that the prodrugs remain inactive, host cells that do not express or barely express uPA, MMP2, and / or MMP9 can be used. In certain embodiments, the host cells can contain null mutations (knockouts) of the genes for these proteases.

[0084] Pharmaceutical Composition Pharmaceutical compositions comprising the prodrugs and muteins (i.e., active pharmaceutical ingredients or APIs) of the present disclosure can be prepared by mixing the API having the desired degree of purification with one or more optional pharmaceutically acceptable excipients (see, e.g., Remington's Pharmaceutical Sciences, 16th Edition., Osol, A. Ed. (1980)) in the form of a lyophilized formulation or aqueous solution. Pharmaceutically acceptable excipients (or carriers) are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers containing, for example, phosphate, citric acid, succinic acid, 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; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol. azoles); low molecular weight (about 10 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 sugars (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).

[0085] Buffers are used to adjust the pH to a range that optimizes therapeutic efficacy, particularly when stability is pH-dependent. Buffers are preferably present at concentrations 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 contain histidine and trimethylamine salts, such as Tris.

[0086] Preservatives are added to prevent microbial growth and are typically 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.

[0087] Tonicity agents (also known as "stabilizers") are present to adjust or maintain the osmotic pressure of the liquid in the composition. When used with large, charged biomolecules (e.g., proteins and antibodies), they are sometimes called "stabilizers" because they can interact with the charged groups on amino acid side chains, thereby reducing the potential for intra- and intermolecular interactions. Tonicity agents can be present in amounts ranging from 0.1% to 25% by weight, or more preferably, from 1% to 5% by weight, taking into account the relative amounts with other components. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0088] The non-ionic surfactant or detergent (also known as a "wetting agent") is present to help solubilize the therapeutic agent, prevent aggregation of the therapeutic protein caused by agitation, and allow the formulation to be exposed to shear surface stresses without denaturing the active therapeutic protein or antibody. The non-ionic surfactant is present in the 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.

[0089] Suitable nonionic surfactants include polysorbates (e.g., 20, 40, 60, 65, 80), poloxamers (e.g., 184, 188), PLURONIC® polyol, TRITON®, polyoxyethylene sorbitan monoether (e.g., TWEEN®-20, TWEEN®-80), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0090] The choice of pharmaceutical carrier, excipient, or diluent can be selected with regard to the intended route of administration and standard medical practice. The pharmaceutical composition may further comprise any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), or solubilizing agent(s).

[0091] There may be different composition / formulation requirements for different delivery systems. As an example, pharmaceutical compositions useful in the present invention may be formulated for administration using a minipump, or by a mucosal route, e.g., as a nasal spray or aerosol of an inhalable or ingestible solution, or parenterally, where the composition is formulated in an injectable form, e.g., for delivery by intravenous, intramuscular, or subcutaneous routes.

[0092] In certain embodiments, the pharmaceutical composition of the present disclosure is a lyophilized protein formulation, hi other embodiments, the pharmaceutical composition may be an aqueous liquid formulation.

[0093] Treatment method The cytokine (e.g., IL-2 or IL-15) prodrug can be used to treat diseases caused by the antigen bound by the antigen-binding domain. In some embodiments, the IL-2 or IL-15 prodrug is used to treat cancer. In some embodiments, the IL-2 or IL-15 prodrug is used to treat infectious diseases, for example, when the drug molecule is an antibacterial or antiviral drug.

[0094] In certain embodiments, a method for treating a disease (e.g., cancer, a viral infection, or a bacterial infection) in a subject comprises administering to said subject an effective amount of an IL-2 or IL-15 prodrug.

[0095] In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a blood cancer or a solid tumor. Exemplary cancers 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, colon cancer, gastric cancer, squamous cell carcinoma, prostate cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer), cholangiocarcinoma, breast cancer, and ovarian cancer.

[0096] In some embodiments, the cytokine (e.g., IL-2 or IL-15) prodrug is used to treat a bacterial infection (e.g., sepsis). In some embodiments, the bacteria causing the bacterial infection are drug-resistant bacteria. In some embodiments, the antigen-binding portion binds to a bacterial antigen.

[0097] In some embodiments, the cytokine (e.g., IL-2 or IL-15) prodrug is used to treat viral infection. In some embodiments, the virus causing the 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 portion binds to a viral antigen.

[0098] Generally, the dosage and administration route of the pharmaceutical composition of the present invention will be determined according to the size and condition of the subject according to standard medical practice.In some embodiments, the pharmaceutical composition will be administered to the subject by any route, including oral, transdermal, inhalation, intravenous, intraarterial, intramuscular, direct application to wound site, application to surgical site, intraperitoneal, suppository, subcutaneous, intradermal, transdermal, spray, intrapleural, intraventricular, intraarticular, intraocular, intracranial or intraspinal.In some embodiments, the composition will be administered to the subject intravenously.

[0099] In some embodiments, the dosage of the pharmaceutical composition is a single dose or repeated doses.In some embodiments, the dosage is given to the subject once a day, twice a day, three times a day, or four or more times a day.In some embodiments, about one or more doses (for example, about 2, 3, 4, 5, 6, or 7 or more) are given per week.In some embodiments, the pharmaceutical composition is administered once a week, once every two weeks, once every three weeks, once every four weeks, once a week for two weeks out of three weeks, or once a week for three weeks out of four weeks.In some embodiments, multiple doses are given over multiple days, weeks, months, or years.In some embodiments, a course of treatment is about one or more doses (for example, about 2, 3, 4, 5, 7, 10, 15, or 20 or more doses).

[0100] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure have the 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 can also be used in the practice or testing of this disclosure. In the case of conflicts, the present specification, including definitions, will control. Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, drug and medicinal chemistry, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly practiced in the art or as described herein. Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular. Throughout this specification and the embodiments, the terms "have" and "comprise," or variations such as "has," "having," "comprises," or "comprising," are understood to refer to the inclusion of the stated integer or group of integers but not to the exclusion of any other integer or group of integers. It is understood that aspects and variations of the invention described herein include "consisting of" and / or "consisting essentially of" aspects and variations. All publications and other references mentioned herein are incorporated herein by reference in their entirety. Although a number of documents have been cited herein, this citation is not an admission that any of these documents form part of the common general knowledge.

[0101] Exemplary Embodiments Further specific embodiments of the present disclosure are described below. These embodiments are intended to be illustrative of the compositions and methods described in this disclosure and are not intended to limit the scope of the disclosure. 1. An isolated mutant interleukin-2 (IL-2) polypeptide comprising the amino acid sequence of SEQ ID NO: 1 with seven or fewer amino acid substitutions, one of said mutations being at position 73. 2. An isolated mutant interleukin-2 (IL-2) polypeptide, comprising the amino acid sequence of SEQ ID NO: 1 with seven or fewer amino acid substitutions, one of said mutations being K35N. 3. An isolated mutant interleukin-2 (IL-2) polypeptide, comprising the amino acid sequence of SEQ ID NO: 1 with seven or fewer amino acid substitutions, one of said mutations being A73T. 4. A mutant interleukin-2 polypeptide according to any one of embodiments 1 to 3, further comprising additional amino acid mutations, said mutations being at positions corresponding to residues 42 and 45 of human IL-2. 5. A mutant interleukin-2 polypeptide according to any one of embodiments 1 to 3, further comprising additional amino acid mutations, said mutations being at positions corresponding to residues 38, 42, and 45 of human IL-2. 6. The mutant interleukin-2 polypeptide of any one of embodiments 1 to 3, further comprising additional amino acid mutations, said mutations being at positions corresponding to residues 38, 42, 45, and 62 of human IL-2. 7. The mutant interleukin-2 polypeptide according to any one of embodiments 4 to 6, wherein the mutation at position 42 is selected from the group consisting of F42A, F42G, F42I, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, and F42K. 8. A mutant interleukin-2 polypeptide according to any one of embodiments 4 to 6, wherein the mutation at position 45 is selected from the group consisting of Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R and Y45K. 9. A mutant interleukin-2 polypeptide according to any one of embodiments 5 and 6, wherein the mutation at position 38 is selected from the group of R38A, R38K and R38S. 10. A mutant interleukin-2 polypeptide according to embodiment 6, wherein the mutation at position 62 is selected from the group of E62L, E62A and E62I. 11. A mutant interleukin-2 polypeptide according to any one of embodiments 1 to 10, further comprising the mutations T3A and C125S. 12. A chimeric molecule comprising a mutant interleukin-2 polypeptide according to any one of embodiments 1 to 11 and a carrier, wherein the mutant IL-2 polypeptide is optionally conjugated to the carrier, and the carrier is selected from a PEG molecule, an albumin molecule, an albumin fragment, an IgG Fc, and an antigen-binding molecule. 13. The chimeric molecule of embodiment 12, wherein the carrier is an antigen-binding molecule, and the antigen-binding molecule is an antibody or antibody fragment. 14. The chimeric molecule of embodiment 12, wherein the carrier is an antigen-binding molecule, and the antigen-binding molecule is a bispecific antibody. 15. The chimeric molecule of any of embodiments 13 and 14, wherein the antigen is selected from the group consisting of PD-L1, PD-1, fibroblast activation protein alpha (FAP alpha), CEA, BCMA, CD20, Trop-2, HER2, 5T4, melanoma-associated chondroitin sulfate proteoglycan (MCSP), PSMA, EGFR, and claudin 18.2. 16. A prodrug of a cytokine (e.g., IL-2 or IL-15) (e.g., a prodrug of IL-2) comprising a cytokine (e.g., IL-2 or IL-15) mutein, a masking moiety or antagonist of the cytokine (e.g., IL-2 or IL-15), and a cleavable peptide linker, the prodrug comprising an IL-2 agonist polypeptide (A), a masking moiety (MM), and at least one cleavable peptide linker; the masking moiety comprises the IL-2 receptor beta subunit extracellular domain or a functional analog thereof. 17. The prodrug of embodiment 16, wherein the IL-2 antagonist or masking moiety (MM) comprises the extracellular domain of the IL-2 receptor beta subunit, comprising an amino acid sequence at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:3. 18. The prodrug of embodiment 16, wherein the IL-2 antagonist or masking moiety comprises the extracellular domain of the IL-2 receptor beta subunit, which comprises the amino acid sequence of SEQ ID NO:3. 19. The prodrug of any of embodiments 16-18, wherein the IL-2 agonist polypeptide (A) comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1, or the IL-15 agonist polypeptide (A) comprises an amino acid sequence at least 90% identical to SEQ ID NO: 2. 20. The prodrug of any of embodiments 16 to 18, wherein the IL-2 agonist polypeptide (A) comprises an analog of human IL-2 comprising one or more mutations at positions selected from T3, K35, R38, F42, Y45, E62, E68, L72, A73, and C125; and wherein the mutations are as shown according to the numbering of human IL-2 having the amino acid sequence of SEQ ID NO: 1. 21. The prodrug according to any one of embodiments 16 to 18, wherein said IL-2 agonist polypeptide (A) is a mutant IL-2 selected from any one of embodiments 1 to 11. 22. The prodrug of any of embodiments 16-18, wherein the IL-2 agonist polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 8-17, 19-33, 36, 37, and 39-46. 23. The prodrug of any of embodiments 16-22, further comprising a carrier (C), wherein said carrier is selected from a PEG molecule, albumin, an albumin fragment, Fc, and an antigen-binding molecule. 24. A prodrug of IL-2 (or IL-15) that activates IL-2 (or IL-15) activity at or around a tumor site, comprising: an IL-2 (or IL-15) agonist polypeptide suitably fused or conjugated to a carrier (an IL-2 (or IL-15) antagonist is one that inhibits or affects the binding of said IL-2 (or IL-15) agonist polypeptide to its receptor); and a cleavable peptide linker connecting the IL-2 (or IL-15) antagonist to said IL-2 (or IL-15) agonist polypeptide or its carrier; said cleavable peptide linker is cleavable by one or more proteases found within or in the tumor's surrounding environment; and said carrier is selected from a protein, an antibody, or a polyethylene glycol (PEG) polymer. 25. The prodrug of embodiment 24, wherein the IL-2 or IL-15 antagonist comprises the extracellular domain of the IL-2 receptor beta subunit or a functional analogue thereof. 26. The prodrug of any of embodiments 24 and 25, wherein the IL-2 or IL-15 antagonist or masking moiety (MM) comprises the extracellular domain of the IL-2 receptor beta subunit comprising an amino acid sequence at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:3. 27. The prodrug of any of embodiments 24 and 25, wherein the IL-2 or IL-15 antagonist or masking moiety is the extracellular domain of the IL-2 receptor beta subunit, comprising the amino acid sequence of SEQ ID NO:3. 28. The prodrug of any of embodiments 24-27, wherein the IL-2 or IL-15 antagonist or masking moiety further comprises an IL-2 receptor gamma subunit or a functional equivalent thereof. 29. The prodrug of any of embodiments 24-27, wherein the IL-2 or IL-15 antagonist or masking moiety further comprises a second IL-2 receptor beta subunit or a functional equivalent thereof. 30. The prodrug of any of embodiments 24-29, wherein said IL-2 agonist polypeptide (A) has an amino acid sequence that is at least 90% identical to SEQ ID NO:1. 31. The prodrug of any of embodiments 24 to 29, wherein the IL-2 agonist polypeptide (A) is an analog of human IL-2 comprising one or more mutations at positions selected from T3, K35, R38, F42, Y45, E62, E68, L72, A73, and C125 (e.g., a mutation at A73 and a K35N mutation); and the mutations are as shown according to the numbering of human IL-2 having the amino acid sequence of SEQ ID NO: 1. 32. The prodrug according to any of embodiments 24 to 29, wherein said IL-2 agonist polypeptide (A) is a mutant IL-2 selected from any one of embodiments 1 to 11. 33. The prodrug of any of embodiments 24-29, wherein the IL-2 agonist polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 8-17, 19-33, 36, 37, and 39-46. 34. A prodrug of IL-15, comprising an IL-15 agonist polypeptide (A), a masking moiety (MM), a carrier (C), and at least one cleavable peptide linker; wherein the IL-15 agonist polypeptide (A) comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:2; the masking moiety (MM) is selected from the group consisting of an IL-2 receptor beta subunit extracellular domain, a functional analog of the IL-2 receptor beta subunit extracellular domain, an IL-2 receptor beta subunit extracellular domain fused to an IL-2 receptor gamma subunit extracellular domain via a peptide linker, and a dimer of IL-2 receptor beta subunit extracellular domains linked to each other via a cleavable peptide linker; and the carrier is selected from albumin, an albumin fragment, Fc, and an antigen-binding molecule. 35. The prodrug of embodiment 34, wherein the prodrug of IL-15 comprises the sushi domain of the IL-15 receptor alpha subunit; and the sushi domain comprises an amino acid sequence at least 95% or 100% identical to SEQ ID NO:7. 36. The prodrug of any one of embodiments 34 and 35, wherein the IL-2 receptor beta subunit extracellular domain comprises an amino acid sequence at least 95% or 100% identical to SEQ ID NO:3. 37. The prodrug of any one of embodiments 28 and 34, wherein the gamma subunit extracellular domain comprises an amino acid sequence at least 95% or 100% identical to SEQ ID NO:6. 38. The prodrug according to any one of embodiments 23 to 37, wherein the carrier (C) is an antigen-binding molecule; and the antigen-binding molecule is an antibody comprising two heavy chains and two light chains. 39. The prodrug of embodiment 38, wherein the cytokine (e.g., IL-2 or IL-15) agonist polypeptide is fused to the C-terminus of one of the antibody heavy chains, optionally via a peptide linker, and the cytokine (e.g., IL-2 or IL-15) antagonist or masking moiety (MM) is fused to the C-terminus of a second heavy chain via a cleavable peptide linker; and the two heavy chain fusion proteins form a heterodimer by a "knob-into-hole" mutation. 40. The prodrug of embodiment 38, wherein the cytokine (e.g., IL-2 or IL-15) agonist polypeptide is fused to the N-terminus of one of the antibody heavy chains, optionally via a peptide linker, and the cytokine (e.g., IL-2 or IL-15) antagonist or masking moiety (MM) is fused to the N-terminus of a second heavy chain via a cleavable peptide linker; and the two heavy chain fusion proteins form a heterodimer by a "knob-into-hole" mutation. 41. The prodrug of embodiment 38, wherein the cytokine (e.g., IL-2 or IL-15) agonist polypeptide is fused or conjugated, directly or via a peptide linker, to the N-terminus of one or both of the heavy chains of the antibody or antibody fragment, and the cytokine (e.g., IL-2 or IL-15) antagonist or masking moiety (MM) is fused via a cleavable peptide linker to the N-terminus of the light chain, forming a heavy chain fusion polypeptide and a light chain fusion polypeptide. 42. The carrier is an antigen-binding molecule, and the antigen-binding molecule is selected from the group consisting of guanyl 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), and delta-like protein 1 (DL1). 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), prostate six-transmembrane epithelial antigen 1 (six 42. The prodrug of any of embodiments 23-41, which binds to one or more antigens selected from 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), PD1, claudin 18.2, claudin 6, FAP-alpha, 5T4, BCMA, PD-L1, PD-1, and EPCAM. 43. The prodrug according to any one of embodiments 23 to 42, further comprising another effector polypeptide. 44. The prodrug according to any one of embodiments 23 to 42, further comprising another effector polypeptide, said effector polypeptide being another IL-2 mutein comprising an amino acid mutation at position 126. 45. The prodrug of any one of embodiments 23-42, further comprising another effector polypeptide, wherein said effector polypeptide is a CCL19 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 123. 46. ​​The prodrug of any one of embodiments 16-45, wherein the cleavable peptide linker is cleavable by one or more proteases found at the tumor site or its surrounding environment. 47. The prodrug of any one of embodiments 16-45, wherein the prodrug is activated at the tumor site. 48. The prodrug of any one of embodiments 16-45, wherein the cleavable peptide linker comprises a substrate for uPA. 49. The prodrug of any one of embodiments 16-45, wherein the cleavable peptide linker comprises a substrate for MMP2 and / or MMP9. 50. The prodrug of any one of embodiments 16-45, wherein the cleavable peptide linker comprises a substrate for both uPA and MMP2, both uPA and MMP9, or uPA, MMP2, and MMP9. 51. The prodrug of any one of embodiments 16-45, wherein the cleavable peptide linker comprises an enzyme substrate of an amino acid sequence selected from LSGRSDNH (SEQ ID NO: 52), ISSGLLSS (SEQ ID NO: 53), and GPLGVR (SEQ ID NO: 54). 52. The prodrug of any one of embodiments 16-45, wherein the cleavable peptide linker comprises enzyme substrates for both amino acid sequences LSGRSDNH (SEQ ID NO: 52) and ISSGLLSS (SEQ ID NO: 53). 53. The prodrug of any one of embodiments 16-45, wherein the cleavable peptide linker comprises enzyme substrates of both the amino acid sequences LSGRSDNH (SEQ ID NO: 52) and GPLGVR (SEQ ID NO: 54); or both ISSGLLSS (SEQ ID NO: 53) and GPLGVR (SEQ ID NO: 54). 54. The prodrug of any one of embodiments 16-45, wherein the cleavable peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 55-78. 55. A polynucleotide encoding a mutant IL-2 according to any one of embodiments 1 to 11. 56. A polynucleotide encoding a chimeric molecule according to any one of embodiments 12 to 15, or a prodrug according to any one of embodiments 16 to 54. 57. An expression vector comprising a polynucleotide according to embodiment 55 or 56. 58. A host cell transfected with a vector according to embodiment 57. 59. The host cell of embodiment 58, wherein the host cell has a gene encoding uPA, MMP2, and / or MMP9 knocked out. 60. A method for producing a mutant IL-2 according to any one of embodiments 1 to 11, a chimeric molecule according to any one of embodiments 12 to 15, or a prodrug according to any one of embodiments 16 to 54, the method comprising culturing a host cell according to embodiment 58 or 59. 61. A pharmaceutical composition comprising as an active ingredient a mutant IL-2 according to any one of embodiments 1 to 11 or a prodrug according to any one of embodiments 16 to 54. 62. A pharmaceutical composition comprising, as an active ingredient, a chimeric molecule according to any one of embodiments 12 to 15. 63. A method for treating breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colon cancer, rectal cancer or gastric cancer, or an infectious disease in a human subject in need thereof, comprising administering to said human subject a pharmaceutical composition according to embodiment 61 or 62.

[0102] 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]

[0103] The materials and methods used in the experiments described in Examples 1-6 are described below.

[0104] Transient transfection of HEK293 cells The expression plasmid was diluted to 3x10 with PEI (polyethyleneimine). 6 The IL-2 mutant protein fusion polypeptide and the Fc-masking moiety fusion polypeptide were co-transfected into FreeStyle HEK293 cells at 2.5-3 μg / ml. For Fc-based IL-2 prodrugs, the ratio of Fc-IL-2 mutein fusion polypeptide to Fc-masking moiety fusion polypeptide was 1:2. For antibody-based IL-2 prodrugs, the ratio of knob heavy chain (containing the IL-2 agonist polypeptide), hole heavy chain (containing the masking moiety), and light chain DNA was 2:1:2 molar ratio. The cell cultures were harvested 6 days post-transfection by centrifugation at 9,000 rpm for 45 minutes followed by 0.22 μM filtration.

[0105] Protein purification Protein purification of the antibody-based IL-2 prodrug was performed using three chromatographic steps, including 1) Protein A affinity chromatography; 2) Q Sepharose Fast Flow; and 3) Capto MMC ImpRes. Q Sepharose Fast Flow was equilibrated with a buffer containing 25 mM Tris and 100 mM NaCl (pH 7.5). Capto MMC ImpRes was equilibrated with buffer A (20 mM phosphate, 30 mM NaCl, pH 6.2) and eluted with a 10 CV linear gradient of buffer B (20 mM phosphate, 0.5 M arginine, pH 6.2).

[0106] SEC-HPLC analysis SEC-HPLC was performed using an Agilent 1100 series HPLC system equipped with a TSKgel G3000SWXL column (7.8 mm IDX 30 cm, 5 μm particle size) ordered from Tosoh Bioscience. Up to 100 μl of sample was loaded. The column was flushed with buffer (containing 200 mM KPO, 250 mM KCl, pH 6.5). The flow rate was 0.5 ml / min. The column was run at room temperature. Protein elution was monitored at both 220 nm and 280 nm.

[0107] SDS-PAGE analysis Ten μl of culture supernatant or 20 μg of purified protein sample was mixed with Bolt® LDS sample buffer (Novex) with or without a reducing agent. The sample was heated at 70°C for 3 minutes and then loaded onto a NuPAGE® 4-12% BisTris gel (Invitrogen). The gel was run in NuPAGE® MOPS SDS running buffer (Invitrogen) at 200 volts for 40 minutes and stained with Coomassie.

[0108] Proteolytic processing The proteases, human u-plasminogen activator (uPA) / urokinase (R&D systems) or human matriptase / ST14 (R&D systems), were added to the precursor molecules at 81 nM and 250 nM, respectively, and incubated overnight at 37°C.

[0109] CTLL2 assay CTLL2 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. CTLL2 cells were grown at 5 × 10 in medium containing 100 ng / ml IL-2. 4 ~1x10 6Cells were maintained without attachment at 1000 cells / ml. Generally, cells were split twice a week. For bioassays, it was best to use cells at least 48 hours after passage.

[0110] Samples were diluted to 2x concentration in 50 μl / well in a 96-well plate. The IL-2 standard was titrated in 12 wells from 20 ng / ml (2x concentration) to 3x serial dilutions. Samples were titrated as needed. CTLL2 cells were washed five times to remove IL-2, plated at 5000 cells / well in 50 μl, and cultured with the samples overnight or for at least 18 hours. Subsequently, 100 μl / well of Cell Titer Glo reagent (Promega) was added, and luminescence was measured.

[0111] Enzyme-linked immunosorbent assay (ELISA) 10 μg / ml IL-2 protein in PBS was seeded at 100 μl per well onto a 96-well plate and coated overnight at 4°C. The wells were washed three times with PBS and blocked with 100 μl of 2% milk / PBS for 1 hour. The wells were then washed three times with PBS, and 100 μl of 3-fold serially diluted protein samples were added to the wells and incubated at room temperature for 1 hour. After washing three times with PBS, 100 μl of HRP-conjugated anti-IgG antibody was added and incubated at room temperature for 1 hour. The wells were then washed three times with PBS, and detection reagent was added, and OD450nM was measured.

[0112] FACS analysis Stable HEK293 cell lines expressing IL-2Rαβγ or IL-2Rβγ were cultured. The cells were detached with a non-enzymatic cell dissociation solution. The cells were counted and the cell density was adjusted to approximately 3,000,000 cells / ml with FACS wash buffer (containing 3% FBS in PBS). 50 μl of cells (150,000 cells) were added to each well of a 96-well plate. Primary antibodies or supernatants expressing the antibodies of interest were added to the cells at the indicated concentrations. The plate was incubated on ice for 1 hour. The plate was washed three times with FACS wash buffer. Fluorescently conjugated secondary antibodies were added to the cells (at the concentration specified by the manufacturer's instructions). The plate was incubated on ice for 1 hour. The plate was washed again. PI staining solution was added at 0.1 μg / ml, and the plate was incubated on ice for 10 minutes. The fluorescence of the cells was measured using a flow cytometer.

[0113] Antibody-dependent cellular cytotoxicity (ADCC) Samples of claudin 18.2 antibody, claudin 18.2 antibody with high ADCC, and claudin 18.2 antibody-IL-2 were analyzed for their ability to induce ADCC against HEK293 cells stably expressing human CLD18.2 or human CLD18.1.

[0114] To enrich for human peripheral blood mononuclear cells, human blood from healthy donors was diluted two-fold with phosphate-buffered saline (PBS), and blood cells were layered on Ficoll (Lymphocyte Separation Medium 1077 μg / ml, PAA Laboratories, Cat. No. J15-004). Peripheral blood mononuclear cells (MNCs) were collected from the interphase, washed, and resuspended in RPMI 1640 culture medium (supplemented with 10% heat-inactivated fetal bovine serum and 2 mM L-glutamine).

[0115] To initiate the ADCC assay, target cells were labeled with a fluorescently enhanced ligand (BADTA, DELFIA EuTDA Cytotoxicity Reagent from the Perkin Elmer Cytotoxicity Assay Kit, Catalog No. AD0116) for 30 min. After spreading and washing in RPMI-10 (supplemented with 10 mM probenecid (Sigma, Catalog No. P8761), 10-20 mM HEPES, and 10% heat-inactivated fetal bovine serum), the cells were diluted to 1 × 10 5 The concentration of each antibody was adjusted to 100 cells / ml. Labeled target cells, effector cells (MNCs), and supernatants (containing monoclonal antibodies adjusted to a concentration of 10 μg / ml) were added to round-bottom microtiter plates. For isolated effector cells, an effector-to-target (E:T) ratio of 100:1 was used (data for 50:1 and 25:1 are not shown). After 2 hours of incubation at 37°C, the assay was stopped by centrifugation, and duplicate fluorescent ligand release was measured as europium counts in a time-resolved fluorometer. Percent cytotoxic activity was calculated using the following formula: % specific lysis (experimental release count - spontaneous release count) / (maximum release count - spontaneous release count) × 100. Maximum fluorescent ligand release was determined by adding Triton X-100 (0.25% final concentration) to target cells, and spontaneous release was measured in the absence of antibody and effector cells.

[0116] In vivo efficacy testing using syngeneic tumor models Six-week-old Balb / c mice (Taconic Biosciences) were inoculated with 1x10 6 CT26 / 18.2 cells were injected subcutaneously. Seven days later, tumors were measured using digital calipers, and tumor volume was calculated (V = (ab2)p / 6, where b is the shorter of the two measured lengths). Mice were then separated into groups until all groups had approximately the same mean tumor size (127.6 mm). 3 Mice were then randomized into treatment groups as indicated. Mice were then treated with placebo or test substance at 10 mg / Kg in 100 μl by intraperitoneal injection. Dosing occurred on days 7, 9, 11, 13, 15, and 18. Tumors were measured every 2-3 days and were monitored until tumors reached 2000 mm 3Mice were sacrificed when

[0117] Example 1: Expression and testing of mutant IL-2 agonist polypeptides Human IL-2 (SEQ ID NO: 1) is a 133 amino acid polypeptide. A number of mutant human IL-2 agonist polypeptides were expressed as part of fusion molecules and tested for their biological activity (Table 2). Where appropriate, counterpart polypeptides are also shown. Table 2. Selected mutant IL-2 agonist polypeptide fusions [Table 2] SEQ: SEQ ID NO. HSA: Human serum albumin. N-HSA: Carrier HSA is located at the N-terminus of the IL-2 polypeptide; N-Fc: Carrier Fc is located at the N-terminus of the IL-2 polypeptide. C-Fc: Carrier Fc is located at the C-terminus of the IL-2 polypeptide.

[0118] The expressed IL-2 polypeptides were examined by SDS-PAGE (FIG. 1). Their biological activities were tested using the CTLL2 cell-based activation assay described above. As shown in FIG. 2B, Fc fusion proteins with the IL-2 muteins T3A / C125S / R38S / F42A / Y45A / A73T (SEQ ID NO: 135) and T3A / C125S / R38S / F42A / Y45A / E62A (SEQ ID NO: 132) exhibited an EC of approximately 60 nM. 50 While the Fc fusion protein with the IL-2 mutein T3A / C125S / K35N / R38S / F42A / Y45A / A73T (SEQ ID NO: 136) exhibited similar activity in a cell-based assay with an EC 50 The IL-2 mutein T3A / C125S / R38S / F42R / Y45K / E62A (SEQ ID NO: 133) showed low activity of approximately EC 50The Fc fusion protein with the IL-2 mutein T3A / C125S / R38S / F42A / Y45A / E62A (SEQ ID NO: 137), which does not have a peptide linker between the IL-2 mutein and the Fc, showed an EC of approximately 72 nM (data not shown). 50 Figure 2C shows that human albumin fusion proteins with the IL-2 muteins T3A / C125S / F42A / Y45A / L72G (SEQ ID NO: 126) and T3A / C125S / R38S / F42A / Y45A / E62A (SEQ ID NO: 127) had EC values ​​of approximately 77 nM and 76 nM, respectively. 50 It has been shown to have similar cell-based activity as

[0119] These results indicate that the introduction of the additional mutation A73T into the IL-2 mutein had a similar effect on IL-2 activity as the E62A mutation. In general, the IL-2 muteins represented by SEQ ID NOs: 135, 132, 126, and 127 exhibited similar cell-based activities, which were significantly lower than those of wild-type IL-2. This was presumably due to a significant reduction or loss of binding of the muteins to IL-2Rα. The IL-2 mutein with the mutations T3A / C125S / R38S / F42A / Y45A / E62A (SEQ ID NO: 132) exhibited a significantly reduced binding affinity to IL-2Rα, as shown in Figure 8 (see also below). Furthermore, the introduction of two additional mutations, A73T and K35N, further reduced the activity of this IL-2 mutein.

[0120] The inventors found that further mutations at position 126 resulted in even more significantly reduced levels of cell-based activity, although still possessing some IL-2 activity, as shown by the IL-2 muteins T3A / C125S / R38S / F42A / Y45A / E62A / Q126W (SEQ ID NO: 130) and T3A / C125S / R38S / F42A / Y45A / E62A / Q126W (no linker) (SEQ ID NO: 129) (Figure 2C).

[0121] Example 2: Design of IL-2 antagonists or masking moieties To build a prodrug platform for IL-2, we designed various IL-2 antagonists (masks) using the human IL-2 receptor beta and gamma subunits. Exemplary mask designs are listed in Table 3 and include: 1) A cleavable peptide linker (GGGGSGGGGSGGGGS) containing one protease substrate peptide at the C-terminus of the Fc fragment LSGRSDNH a single copy of the IL-2Rβ subunit extracellular domain (SEQ ID NO: 195) fused via a nucleotide sequence (SEQ ID NO: 18) of the IL-2Rβ subunit extracellular domain (SEQ ID NO: 195); 2) At the C-terminus of Fc, a cleavable peptide linker (GGGGSGGGGSGGGGS) containing two protease cleavage sites ISSGLLSS GGSGGS LSGRSDNH one copy of the IL-2R beta subunit extracellular domain fused to one copy of the IL-2R gamma subunit extracellular domain via a fusion protein (SEQ ID NO: 196) containing the IL-2R beta subunit extracellular domain fused ... 3) a single copy of the IL-2R β subunit extracellular domain (SEQ ID NO: 197) fused to the C-terminus of the Fc with the cleavable linker SEQ ID NO: 38; 4) Two copies of the IL-2R β subunit extracellular domain linked together (SEQ ID NO: 198) fused to the C-terminus of Fc via a cleavable linker SEQ ID NO: 38 The underlined sequence indicates the protease substrate sequence. Table 3. IL-2 antagonist or mask design [Table 3] SEQ: Sequence number.

[0122] The Fc fragment used in these masked polypeptides contained the "hole" mutation Y407T. The IL-2 muteins were fused to an Fc fragment that contained the "knob" mutation T366Y.

[0123] The IL-2 prodrug designs are shown in Table 4. Each of the prodrugs consisted of an IL-2 agonist polypeptide (SEQ ID NO: 132, 133, or 136) fused to the C-terminus of Fc and co-expressed with one of the Fc-masked fusion polypeptides (SEQ ID NO: 195, 196, 197, or 198). Table 4. IL-2 prodrug designs [Table 4]

[0124] The prodrugs were treated with the proteases human u-plasminogen activator (uPA) / urokinase or human matriptase / ST14. The data show that protease treatment resulted in 0.5- to 22-fold activation of IL-2 function in the CTLL2 assay (Table 4). These results demonstrate that both the IL-2Rβ extracellular domain and the IL-2Rβ extracellular domain dimer functioned as masks for the IL-2 agonist polypeptide. Cleavable peptide linkers with one or two cleavable sites were both functional. We found that masks containing both the IL-2Rβ and γ subunit extracellular domains were poorly expressed.

[0125] Example 3: Optimization of the masking part To search for improved IL-2 antagonists or masks that would be activated by protease cleavage, numerous mutations in the IL-2Rβ extracellular domain were constructed. These constructs were expressed as homodimers in HEK293 cells, and their binding affinities with IL-2 were measured by the ELISA method described above and are shown in Table 5. IL-2Rβ extracellular domains with the single mutations R15Y (SEQ ID NO: 199), V75Q (SEQ ID NO: 202), or V75F (SEQ ID NO: 203) completely lost binding affinity for IL-2 in the ELISA assay. IL-2Rβ extracellular domains with the single mutations S69H (SEQ ID NO: 201) or E136Q (SEQ ID NO: 204) lost binding affinity for IL-2 at pH 7.4 but showed a two-fold higher binding affinity for IL-2 at pH 6.4 (Table 5). The IL-2Rβ extracellular domain with the double mutation E136Q / H138R (SEQ ID NO: 205) showed similar binding affinity to IL-2 as the wild type at pH 7.4, but its binding affinity to IL-2 at pH 6.4 was up to two-fold higher (Table 5). The IL-2Rβ extracellular domain with the mutation D68E (SEQ ID NO: 200) showed two-fold higher binding affinity to IL-2 at both pH 7.4 and pH 6.4 (Table 5). Table 5. Design of IL-2Rβ mutations and their binding affinity to IL-2 [Table 5] "-": None or minimal binding

[0126] Example 4: IL-2 prodrug with antibody molecule as carrier Fusing cytokine polypeptides to antibodies allows for targeted delivery of cytokines to disease sites. However, when high-affinity cytokine receptors are present on immune cells, which may be abundant in immune organs, cytokines compete significantly for binding to the cytokine receptor. In this experiment, an IL-2 mutein with significantly reduced binding affinity to IL-2Rα was fused to an antibody carrier. This type of antibody-IL-2 prodrug can be activated at the disease site targeted by the antibody and can exhibit significantly improved PK profiles and extremely high disease site specificity.

[0127] An antibody against claudin 18.2 (589A sequence) and an antibody against PD-L1 (atezolizumab) were used as examples to demonstrate the feasibility of the novel IL-2 prodrug platform. The structures of the antibody-based prodrugs are shown in Figure 3. The different combinatorial designs of the 589A-IL-2-mask fusion molecules are listed in Table 6. Table 6. 589A-IL-2 Prodrug Design [Table 6] HC: heavy chain. LC: light chain. SEQ: sequence number.

[0128] More than 80% of the 589A-IL-2A molecule was cleaved without protease treatment, likely due to the presence of proteases in the cells or secreted from the cells during cell culture (Figure 4). 589A-IL-2B (with a non-cleavable linker [(GGGGS)3] (SEQ ID NO: 49)) demonstrated stable construction of the heterotetrameric antibody and showed no stimulatory activity in the CTLL2 assay (Figure 4, Table 7). This data demonstrates the effectiveness of the masking moiety as an IL-2 antagonist. The 589A-IL-2C molecule was properly constructed and showed 38-fold inhibition of IL-2 mutein activity (Figure 4 and Table 7). The 589A-IL-2E and 589A-IL-2F molecules were more stably constructed and showed more than 4,000-fold inhibition of IL-2 mutein activity (Figure 4 and Table 7). Possibly due to the higher affinity of the masked mutant protein D68E, 589A-IL-2E exhibited higher prodrug stability than 589A-IL2-F upon preparation (FIG. 4). Table 7. CTLL2 activity of 589A-IL-2 prodrugs [Table 7]

[0129] In a separate experiment, 589A-IL-2E showed approximately a 10- to 20-fold increase in binding to the HEK293-IL-2Rαβγ and HEK293-IL-2Rβγ cell lines after protease treatment (Figure 6). Furthermore, 589A-IL-2E showed similar binding to HEK293-IL-2Rαβγ and HEK293-IL-2Rβγ, indicating that the α subunit does not contribute significantly to IL-2 mutein binding and that IL-2 muteins with the mutations T3A / C125S / R38S / F42A / Y45A / E62A significantly reduced binding affinity for IL-2Rα (Figures 7A and B).

[0130] The anti-PD-L1-IL-2 prodrug designs are listed in Table 8. Table 8. Anti-PD-L1-IL-2 prodrug designs [Table 8]

[0131] The anti-PD-L1-IL-2A molecule has two cleavage sites in its cleavable peptide linker and exhibited band cleavage during expression in HEK293 cells, likely due to the presence of proteases in the cell culture medium or cells (data not shown). Anti-PD-L1-IL-2B demonstrated proper assembly of the heterotetrameric molecule, and purified samples showed significant activity after protease cleavage (Figure 8). Anti-PD-L1-IL-2C, anti-PD-L1-IL-2D, and anti-PD-L1-IL-2E did not assemble properly, forming HC-IL-2 homodimers (data not shown), and showed no inhibition of IL-2 activity in the CTLL2 assay. These data suggest that shorter cleavage linkers may prevent proper formation of heterotetrameric molecules.

[0132] Example 5: ADCC activity of 589A-IL-2 mutein fusion molecules The anti-claudin 18.2 antibody 589A, the nonfucosylated form of 589A (af-589A), and a fusion of IL-2 mutein with af-589A were tested for their in vitro activity in an ADCC assay as described above. Af-589A has little or no fucose in its N-glycans and showed enhanced ADCC function. The IL-2 mutein contained the mutations T3A / C125S / R38S / F42A / Y45A / E62A (SEQ ID NO: 10). The data show that further addition of the IL-2 mutein to the 589A antibody further enhanced its ADCC activity (Figure 9).

[0133] Example 6: In vivo efficacy of 589A-IL-2 prodrugs In vivo anti-cancer efficacy studies were performed by combining 589A-IL-2E with an anti-PD-L1 antibody. Both the prodrug and the PD-L1 antibody were administered subcutaneously at 10 mg / kg every other day. CT26 mouse tumor cells transfected with human claudin 18.2 were implanted into Balb / c mice. Tumors approximately 100 mm in size were observed. 3 Once tumor size reached 100 mg / kg, the mice were randomized into three groups based on their tumor size. Mice received subcutaneous buffer placebo (Group 1) (Figure 10, upper left panel), 10 mg / kg anti-PD-L1 antibody (Group 2) (Figure 10, upper right panel), or 10 mg / kg anti-PD-L1 antibody plus 10 mg / kg 589A-IL-2E prodrug (Group 3) (Figure 10, lower panel). Dosing occurred on days 7, 9, 11, 13, 15, and 18. Tumor size and body weight were monitored throughout the experiment.

[0134] As shown in Figure 10, the group treated with both the prodrug and antibody showed more uniform tumor size compared to the placebo and PD-L1 antibody groups. As shown in Figure 11, the group treated with both the prodrug and PD-L1 antibody showed the most delayed tumor growth until approximately 35 days, and its survival curves did not cross until day 42 (Figure 12). Treatment was terminated on day 18. Without being bound by theory, the inventors believe that one possible factor for crossover with the PD-L1 antibody group may be that the mice were wild-type and there may have been antibodies produced against 589A-IL-2E. 589A was a humanized antibody derived from rabbit B cell cloning.

[0135] The above non-limiting examples are provided for illustrative purposes only to facilitate a fuller understanding of the disclosed subject matter, and should not be construed as limiting any of the embodiments described herein, including those relating to antibodies, pharmaceutical compositions, or methods and uses for treating cancer, neurodegenerative disorders, or infectious diseases. The present invention also includes the following aspects. 1. A prodrug comprising a cytokine moiety, a masking moiety, and a carrier moiety, the masking moiety binds to the cytokine moiety and inhibits the biological activity of the cytokine moiety; the cytokine moiety is fused to the carrier moiety; The prodrug, wherein the masking moiety is fused to the cytokine moiety or the carrier moiety via a cleavable peptide linker. 2. The prodrug of paragraph 1, wherein the masking moiety comprises the extracellular domain (ECD) of a receptor for the cytokine moiety. 3. The prodrug according to item 1 or 2, wherein the cytokine moiety is a wild-type human cytokine or a mutant protein thereof. 4. The prodrug of paragraph 3, wherein the cytokine moiety is a human IL-2 agonist polypeptide. 5. The prodrug of paragraph 4, wherein the human IL-2 agonist polypeptide comprises SEQ ID NO:1 or an amino acid sequence that is at least 90% identical to SEQ ID NO:1. 6. The prodrug of paragraph 5, wherein the human IL-2 agonist polypeptide comprises one or more mutations at positions selected from T3, K35, R38, F42, Y45, E62, E68, L72, A73, N88, C125, and Q126 (numbering according to SEQ ID NO: 1). 7. The prodrug according to Item 6, wherein the human IL-2 agonist polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 8 to 17, 19 to 33, 36, 37, and 39 to 46. 8. The prodrug according to any one of items 4 to 7, wherein the masking moiety comprises the ECD of human IL-2Rβ or a functional analog thereof. 9. The prodrug of paragraph 8, wherein the masking moiety comprises (i) two copies of the ECD of human IL-2Rβ or a functional analog thereof fused together via a peptide linker, or (ii) the ECD of human IL-2Rβ or a functional analog thereof fused via a peptide linker to the ECD of human IL-2Rγ or a functional analog thereof. 10. The prodrug of paragraph 9, wherein the ECD of human IL-2Rγ or a functional analog thereof comprises SEQ ID NO:6 or an amino acid sequence that is at least 90% identical to SEQ ID NO:6. 11. The prodrug according to any one of items 8 to 10, wherein the ECD of human IL-2Rβ or a functional analog thereof comprises an amino acid sequence of SEQ ID NO: 3, 4, or 5, or an amino acid sequence which is at least 90% similar to SEQ ID NO: 3, 4, or 5. 12. The prodrug of paragraph 3, wherein the cytokine moiety is a human IL-15 agonist polypeptide. 13. The prodrug of paragraph 12, wherein the human IL-15 agonist polypeptide comprises SEQ ID NO:2 or an amino acid sequence that is at least 90% identical to SEQ ID NO:2. 14. The prodrug of paragraph 12 or 13, wherein the IL-15 agonist polypeptide comprises (i) an IL-15Rα sushi domain comprising SEQ ID NO:7, or (ii) an amino acid sequence that is at least 90% identical to SEQ ID NO:7. 15. The prodrug according to any one of items 12 to 14, wherein the masking domain comprises the ECD of human IL-2Rβ or a functional analogue thereof, or IL-2Rγ or a functional analogue thereof. 16. The prodrug of paragraph 15, wherein the masking domain comprises SEQ ID NO: 3, 4, 5 or 6, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, 4, 5 or 6. 17. The prodrug according to any one of items 1 to 16, further comprising a second effector polypeptide. 18. The prodrug of paragraph 17, wherein the second effector polypeptide is (i) a human IL-2 agonist polypeptide comprising a mutation at position 126 (numbering according to SEQ ID NO: 1), or (ii) a CCL19 polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 123. 19. The prodrug of any one of paragraphs 1 to 18, wherein the cytokine moiety is fused to the carrier moiety via a non-cleavable peptide linker. 20. The prodrug according to item 19, wherein the non-cleavable peptide linker is selected from SEQ ID NOs: 47 to 51. 21. The prodrug according to any one of items 1 to 19, wherein the cleavable peptide linker comprises a substrate sequence for urokinase-type plasminogen activator (uPA), matrix metallopeptidase (MMP) 2, or MMP9. 22. The prodrug of paragraph 21, wherein the cleavable peptide linker comprises substrate sequences for (i) both uPA and MMP2, (ii) both uPA and MMP9, or (iii) uPA, MMP2, and MMP9. 23. The prodrug according to item 21, wherein the cleavable peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 18, 34, 35, 38, 52-121, and 217. 24. The prodrug according to any one of paragraphs 1 to 23, wherein the cleavable peptide linker is cleavable by one or more proteases localized at the tumor site or its surrounding environment, and the cleavage results in activation of the prodrug at the tumor site or its surrounding environment. 25. The prodrug according to any one of items 1 to 24, wherein the carrier moiety is a PEG molecule, albumin, an albumin fragment, an antibody Fc domain, or an antibody or antigen-binding fragment thereof. 26. The prodrug of paragraph 25, wherein the carrier moiety is an antibody Fc domain or antibody comprising the mutations L234A and L235A ("LALA") (EU numbering). 27. The prodrug of paragraph 25 or 26, wherein the masking moiety is fused to the cytokine moiety via a cleavable peptide linker. 28. The prodrug of paragraph 25 or 26, wherein the carrier moiety is an antibody Fc domain or an antibody comprising a knobs-into-holes mutation, and the cytokine moiety and the masking moiety are fused to different polypeptide chains of the antibody Fc domain or to different heavy chains of the antibody. 29. The prodrug of paragraph 28, wherein the cytokine moiety and the masking moiety are fused to the C-terminus of two different polypeptide chains of the Fc domain or to the C-terminus of two heavy chains of the antibody. 30. The prodrug of paragraph 28, wherein the cytokine moiety and the masking moiety are fused to the N-terminus of two different polypeptide chains of the Fc domain or to the N-terminus of two different heavy chains of the antibody. 31. The prodrug of any one of clauses 28 to 30, wherein the knob-into-hole mutations comprise a T366Y "knob" mutation in a polypeptide chain of the Fc domain or in the heavy chain of the antibody, and a Y407T "hole" mutation (EU numbering) in the other polypeptide of the Fc domain or in the other heavy chain of the antibody. 32. The prodrug of any one of clauses 28 to 31, wherein the knob-into-hole mutations comprise Y349C and / or T366W mutations in the CH3 domain of the "knob strand," and E356C, T366S, L368A, and / or Y407V mutations (EU numbering) in the CH3 domain of the "hole strand." 33. The prodrug of paragraph 25, wherein the carrier moiety is an antibody Fc domain comprising two polypeptide chains comprising an amino acid sequence selected from SEQ ID NOs: 195-198, and an amino acid sequence selected from SEQ ID NOs: 132-137 and 139, respectively. 34. 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), and prostate six transmembrane epithelial antigen 1 (STEP 1). 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, CD 34. The prodrug according to any one of items 25 to 33, which is an antibody or an antigen-binding fragment thereof that specifically binds to one or more antigens selected from 138, 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 EPCAM. 35. The prodrug of paragraph 25, wherein the carrier moiety is an antibody comprising two heavy chains whose amino acid sequences comprise SEQ ID NO: 209 and one of SEQ ID NOs: 210-215, respectively, and two light chains whose amino acid sequence comprises SEQ ID NO: 216. 36. The prodrug of paragraph 25, wherein the carrier moiety is an antibody comprising two heavy chains whose amino acid sequences comprise SEQ ID NO: 191 and one of SEQ ID NOs: 192, 193, and 206-208, respectively, and two light chains whose amino acid sequence comprises SEQ ID NO: 189. 37. The prodrug of paragraph 25, wherein the carrier moiety is human serum albumin (HSA). 38. An IL-2 mutein comprising a mutation at position A73. 39. IL-2 mutant protein containing the K35N mutation. 40. An IL-2 mutein comprising one of SEQ ID NOs: 23-33, 36, 37 and 39-41. 41. A pharmaceutical composition comprising the prodrug according to any one of items 1 to 37 or the IL-2 mutein according to any one of items 38 to 40, and a pharmaceutically acceptable excipient. 42. A polynucleotide encoding the prodrug according to any one of items 1 to 37 or the IL-2 mutein according to any one of items 38 to 40. 43. An expression vector comprising the polynucleotide according to item 42. 44. A host cell comprising the vector of paragraph 43. 45. The host cell of paragraph 44, wherein the genes encoding uPA, MMP2, and / or MMP9 are knocked out in the host cell. 46. ​​A method for producing the prodrug according to any one of items 1 to 37 or the IL-2 mutant protein according to any one of items 38 to 40, comprising: Culturing the host cell of paragraph 44 or 45 under conditions that allow expression of the prodrug or IL-2 mutein, wherein the host cell is a mammalian cell, and isolating said prodrug or IL-2 mutein. A method comprising: 47. A method for treating cancer or an infectious disease or activating the immune system in a patient in need of such treatment or activation, characterized in that a therapeutically effective amount of the pharmaceutical composition described in paragraph 41 is administered to the patient. 48. A cytokine prodrug or IL-2 mutant protein for use in the treatment of cancer or an infectious disease or for activating the immune system in a method according to paragraph 47. 49. Use of a prodrug or an IL-2 mutein for the manufacture of a medicament for the treatment of cancer or an infectious disease or for activating the immune system in a method according to paragraph 47. 50. The method of clause 47, the prodrug or IL-2 mutein for use of clause 48, or the use of clause 49, wherein the patient is suffering from HIV 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, colon cancer, and gastric cancer.

[0136] array In the sequences below, boxed residues indicate mutations, and the underlined portion of the cleavable linker indicates the protease substrate sequence. [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16] [Table 17] Table 18 Table 19 Table 20 Table 21 Table 22 Table 23 Table 24 Table 25 Table 26 Table 27 Table 28 Table 29 Table 30 Table 31 Table 32 Table 33

Claims

1. A prodrug comprising a cytokine moiety, a masking moiety, and a carrier moiety, the masking moiety binds to the cytokine moiety and inhibits the biological activity of the cytokine moiety; the cytokine moiety is fused to the carrier moiety, the cytokine moiety comprising an IL-15 agonist polypeptide; the masking moiety is fused to the cytokine moiety via a cleavable peptide linker; the masking moiety comprises the extracellular domain (ECD) of a receptor for the cytokine moiety; the carrier moiety is an antibody Fc domain or an antibody comprising a knob-into-hole mutation; The prodrug.

2. The prodrug of claim 1 , wherein the IL-15 agonist polypeptide comprises an IL-15Rα sushi domain.

3. 3. The method of claim 2, wherein the IL-15 agonist polypeptide comprises SEQ ID NO:2 or an amino acid sequence that is at least 90% identical to SEQ ID NO:

2.

4. 3. The method of claim 2, wherein the IL-15Rα sushi domain (i) comprises SEQ ID NO:7, or (ii) comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

7.

5. the masking portion comprises an ECD of human IL-2Rβ or an ECD of human IL-2Rγ; the IL-2Rβ comprises SEQ ID NO: 3, 4, or 5, or an amino acid sequence at least 90% identical to SEQ ID NO: 3, 4, or 5; The prodrug of any one of claims 1 to 4, wherein the IL-2Rγ comprises SEQ ID NO:6 or an amino acid sequence that is at least 90% identical to SEQ ID NO:

6.

6. The prodrug of any one of claims 1 to 5, wherein the cytokine moiety is fused to the carrier moiety via a non-cleavable peptide linker.

7. The prodrug of claim 6, wherein the non-cleavable peptide linker is selected from SEQ ID NOs: 47-51.

8. The prodrug of any one of claims 1 to 5, wherein the cleavable peptide linker comprises a substrate sequence for urokinase-type plasminogen activator (uPA), matrix metallopeptidase (MMP) 2, or MMP 9.

9. The prodrug of claim 8 , wherein the cleavable peptide linker comprises substrate sequences for (i) both uPA and MMP2, (ii) both uPA and MMP9, or (iii) uPA, MMP2, and MMP9.

10. 9. The prodrug of claim 8, wherein the cleavable peptide linker comprises any one of SEQ ID NOs: 18, 34, 35, 38, 52-121, and 217.

11. 11. The prodrug of any one of claims 1 to 10, wherein the cleavable peptide linker is cleavable by one or more proteases localized at a tumor site or its surrounding environment, and wherein the cleavage results in activation of the prodrug at the tumor site or its surrounding environment.

12. 12. The prodrug of any one of claims 1 to 11, wherein the carrier moiety is an antibody Fc domain or antibody comprising the mutations L234A and L235A ("LALA") (EU numbering).

13. The prodrug of claim 1, wherein the masking moiety is fused to the C-terminus of the cytokine moiety and the carrier moiety is fused to the N-terminus of the cytokine moiety.

14. The prodrug of claim 1, wherein the masking moiety is fused to the N-terminus of the cytokine moiety and the carrier moiety is fused to the C-terminus of the cytokine moiety.

15. The prodrug of any one of claims 1 to 14, wherein the knob-into-hole mutations comprise a T366Y "knob" mutation in one polypeptide chain of the Fc domain or in the heavy chain of the antibody, and a Y407T "hole" mutation (EU numbering) in the other polypeptide chain of the Fc domain or in the other heavy chain of the antibody.

16. 16. The prodrug of any one of claims 1 to 15, wherein the knob-into-hole mutations comprise Y349C and / or T366W mutations in the CH3 domain of the "knob strand," and E356C, T366S, L368A, and / or Y407V mutations in the CH3 domain of the "hole strand" (EU numbering).

17. The carrier moiety may be 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), and six transmembrane prostate epithelial antigen 1 (six transmembrane prostate antigen 1 (STP-1)). 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, CD 17. The prodrug of any one of claims 1 to 16, which is an antibody or antigen-binding fragment thereof that specifically binds to one or more antigens selected from 138, 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 EPCAM.

18. A pharmaceutical composition comprising the prodrug of any one of claims 1 to 17 and a pharmaceutically acceptable excipient.

19. A polynucleotide encoding the prodrug of any one of claims 1 to 17.

20. 20. An expression vector comprising the polynucleotide of claim 19.

21. A host cell comprising the vector of claim 20.

22. 1. A method for producing a cytokine prodrug, comprising:

22. Culturing the host cell of claim 21 under conditions that allow expression of the prodrug, wherein the host cell is a mammalian cell, and Isolating the prodrug. A method comprising:

23. 19. The pharmaceutical composition of claim 18 for treating cancer, or treating an infectious disease, or activating the immune system in a patient in need of such treatment or activation.

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