Novel IL-15 prodrugs and methods of use thereof

Abstract

Provided herein are IL-15 cytokine prodrugs and methods for their production and use.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 860,635, filed June 12, 2019; U.S. Provisional Application No. 62 / 888,444, filed August 17, 2019; U.S. Provisional Application No. 62 / 891,190, filed August 23, 2019; U.S. Provisional Application No. 62 / 959,973, filed January 11, 2020; and U.S. Provisional Application No. 63 / 029,473, filed May 23, 2020. The disclosures of the above priority applications are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application has been provided electronically in ASCII format and includes a Sequence Listing, which is hereby incorporated by reference in its entirety. The ASCII copy was created on June 8, 2020, is named 025471_WO004_SL.txt, and is 359,580 bytes in size. [Background technology]

[0003] 2. Background of the Invention Interleukin-15 (IL-15) is a cytokine with structural similarity to IL-2. IL-15 is secreted by mononuclear phagocytes and other immune cells after viral infection. IL-15 induces proliferation of natural killer (NK) and other cells of the immune system and participates in the killing of virus-infected and cancer cells. Like IL-2, IL-15 binds to its intermediate affinity receptor, the IL-2 receptor (IL-2R) β / γ complex, with a K of approximately 1 nM. D (Giri et al., EMBO J. (1994) 13:2822-30). IL-15 binds to the IL-15 receptor (IL-15R)α with much higher affinity (K D= approximately 0.05nM). IL-15Rα can combine with the IL-2Rβ / γ complex to form an IL-15-specific, functional high-affinity receptor (αβγ) (Minami et al., Annu Rev Immunol. (1993) 11:245-67; ​​Giri et al., J Leukoc Biol. (1995) 5745:763-6; and Lehours et al., Eur Cytokine Netw. (2000) 11:207-15).

[0004] The extracellular region of IL-15Rα contains a Sushi domain, a consensus motif for protein-protein interactions. It has been shown that an N-terminal fragment of IL-15Rα having the first 65 amino acids is partially active, whereas a fragment containing the first 85 amino acids is fully functional (Wei et al., J Immunol. (2001) 167(1):277-82).

[0005] Mutations of IL-15 have been carried out to test the interaction of IL-15 with its receptor. For example, D8 and Q108 have been shown to be involved in the binding of IL-15 to IL-2Rβ and γ subunits, respectively (Pettit et al., J Biol Chem. (1997) 272: 2312-18). Further mutations of IL-15 have been disclosed, including mutations of IL-15 residues L45, Q48, S51, L52, E64, N65, I68 and L69, which are involved in the binding of IL-15 to IL-15Rα or IL-2Rβ (US Pat. No. 7,858,081). IL-15 mutant proteins with mutations E64K, N65K, N65D, L66D, L66E, I67D, I67E or I68D have been shown to have reduced biological activity in cell-based assays (Zhu et al., J Immnol. (2009) 183(6):3598; and WO2005 / 085282A1). Mutations that target the interaction of IL-15 with IL-15Rα have also been reported. For example, E46, V49, L45, S51 and L52 have been shown to be involved in IL-15Rα binding (Bernard et al., J Biol Chem. (2004) 279:24313-22). E46 appears to be particularly important as substitution of the acidic side chain for a basic one (E46K) results in complete loss of IL-15 and IL-15Rα binding and bioactivity. Summary of the Invention [Problem to be solved by the invention]

[0006] Unfortunately, the adverse effects of current IL-15 drug candidates are significant, limiting the dosage of such drugs. Moreover, the activation of T cells, NK cells and other immune cells by these drug candidates is not site-specific. Furthermore, there are likely to be "PK sinkers" for IL-15 mutant proteins, even though their affinity for the IL-15 / 2 receptor is significantly reduced. There are also numerous challenges in producing IL-15-based protein therapeutics. All of the above highlights the need for improved IL-15-based therapeutics. [Means for solving the problem]

[0007] Summary of the Invention The present invention provides a prodrug comprising an IL-15 cytokine moiety (A), a masking moiety (M), a carrier moiety (C) and a Sushi domain (S), wherein the masking moiety binds to the IL-15 cytokine moiety and inhibits the biological activity of the IL-15 cytokine moiety, the masking moiety is fused to the carrier moiety, the Sushi domain is fused to the carrier moiety, and the IL-15 cytokine moiety is fused to the Sushi domain. In some embodiments, the masking moiety is fused to the carrier moiety via a first peptide linker, the Sushi domain is fused to the carrier moiety via a second peptide linker, and the IL-15 cytokine is fused to the Sushi domain via a third peptide linker, wherein at least one of the three peptide linkers (e.g., one, two or three) is cleavable. In some embodiments, at least one of the three peptide linkers (e.g., one, two or three) is not cleavable. In some embodiments, all of the peptide linkers are not cleavable. In specific embodiments, the third peptide linker is at least 15, 20, 25 or 30 amino acids in length (eg, 15-50 or 15-100 amino acids in length), optionally wherein the third peptide linker comprises SEQ ID NO:139 or 140.

[0008] The present invention also provides a prodrug comprising an IL-15 cytokine moiety (A), a masking moiety (M), a carrier moiety (C) and a Sushi domain (S), wherein the masking moiety binds to the IL-15 cytokine moiety and inhibits the biological activity of the IL-15 cytokine moiety, the IL-15 cytokine moiety is fused to the carrier moiety, the Sushi domain is fused to the carrier moiety, and the masking moiety is fused to the Sushi domain. In some embodiments, the IL-15 cytokine moiety is fused to the carrier moiety via a first peptide linker, the Sushi domain is fused to the carrier moiety via a second peptide linker, and the masking is fused to the Sushi domain via a third peptide linker, wherein at least one of the three peptide linkers (e.g., 1, 2 or 3) is cleavable. In some embodiments, at least one of the three peptide linkers (e.g., 1, 2 or 3) is not cleavable. In some embodiments, all three peptide linkers are not cleavable.

[0009] The present invention further provides a prodrug comprising an IL-15 cytokine moiety (A), a masking moiety (M), a carrier moiety (C) and a Sushi domain (S), wherein the masking moiety binds to the IL-15 cytokine moiety and inhibits the biological activity of the IL-15 cytokine moiety, the masking moiety is fused to the carrier moiety, the IL-15 moiety is fused to the carrier moiety, and the Sushi domain is fused to the IL-15 moiety. In some embodiments, the masking moiety is fused to the carrier moiety via a first peptide linker, the IL-15 moiety is fused to the carrier moiety via a second peptide linker, and the Sushi domain is fused to the IL-15 moiety via a third peptide linker, wherein at least one of the three peptide linkers (e.g., one, two or three) is cleavable. In some embodiments, at least one of the three peptide linkers (e.g., one, two or three) is not cleavable. In some embodiments, all of the peptide linkers are not cleavable. In specific embodiments, the third peptide linker is at least 15, 20, 25 or 30 amino acids in length (eg, 15-50 or 15-100 amino acids in length), optionally wherein the third peptide linker comprises SEQ ID NO:139 or 140.

[0010] The present invention also provides a prodrug comprising an IL-15 cytokine moiety (A), a masking moiety (M), a carrier moiety (C) and a Sushi domain (S), wherein the masking moiety binds to the IL-15 cytokine moiety and inhibits the biological activity of the IL-15 cytokine moiety, the IL-15 cytokine moiety is fused to the carrier moiety, the masking moiety is fused to the carrier moiety, and the Sushi domain is fused to the masking moiety. In some embodiments, the IL-15 cytokine moiety is fused to the carrier moiety via a first peptide linker, the masking moiety is fused to the carrier moiety via a second peptide linker, and the Sushi domain is fused to the masking moiety via a third peptide linker, wherein at least one of the three peptide linkers (e.g., one, two or three) is cleavable. In some embodiments, at least one of the three peptide linkers (e.g., one, two or three) is not cleavable. In some embodiments, all three peptide linkers are not cleavable.

[0011] The present invention also provides a prodrug comprising an IL-15 cytokine moiety (A), a masking moiety (M), a carrier moiety (C) and a Sushi domain (S), wherein the masking moiety binds to the IL-15 cytokine moiety and inhibits the biological activity of the IL-15 cytokine moiety, the IL-15 cytokine moiety is fused to the carrier moiety, the masking moiety is fused to the IL-15 moiety, and the Sushi domain is fused to the carrier moiety. In some embodiments, the IL-15 cytokine moiety is fused to the carrier moiety via a first peptide linker, the masking moiety is fused to the IL-15 moiety via a second peptide linker, and the Sushi domain is fused to the carrier moiety via a third peptide linker, wherein at least one of the three peptide linkers (e.g., 1, 2 or 3) is cleavable. In some embodiments, at least one of the three peptide linkers (e.g., 1, 2 or 3) is not cleavable. In some embodiments, all three peptide linkers are not cleavable.

[0012] The present invention also provides a prodrug comprising an IL-15 cytokine moiety (A), a masking moiety (M), a carrier moiety (C) and a Sushi domain (S), wherein the masking moiety binds to the IL-15 cytokine moiety and inhibits the biological activity of the IL-15 cytokine moiety, the masking moiety is fused to the carrier moiety, the IL-15 moiety is fused to the masking moiety, and the Sushi domain is fused to the carrier moiety. In some embodiments, the masking moiety is fused to the carrier moiety via a first peptide linker, the IL-15 moiety is fused to the masking moiety via a second peptide linker, and the Sushi domain is fused to the carrier moiety via a third peptide linker, wherein at least one of the three peptide linkers (e.g., 1, 2 or 3) is cleavable. In some embodiments, at least one of the three peptide linkers (e.g., 1, 2 or 3) is not cleavable. In some embodiments, all three peptide linkers are not cleavable.

[0013] In certain embodiments, the masking moiety comprises the extracellular domain (ECD) of the receptor of the IL-15 cytokine moiety. For example, the masking moiety comprises the ECD of human IL-2Rβ or a functional analog thereof and / or the ECD of human IL-2Rγ or a functional analog thereof. In a specific embodiment, 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 thereto. In other specific 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 thereto. In other embodiments, the masking moiety comprises an antibody fragment that binds to the IL-15 cytokine moiety.

[0014] The present invention further provides a prodrug comprising an IL-15 cytokine moiety (A), a masking moiety (M), a carrier moiety (C) and optionally a Sushi domain (S), where the masking moiety comprises an antibody fragment that binds to the IL-15 cytokine moiety and inhibits a biological activity of the IL-15 cytokine moiety, and where the masking moiety is fused to the carrier moiety, the IL-15 cytokine moiety or the Sushi domain, optionally via a peptide linker.

[0015] In certain embodiments, the antibody fragment in the prodrug is an ScFv or Fab comprising heavy chain CDR1-3 and light chain CDR1-3 of an anti-IL-15 antibody selected from 146B7, 146H5, 404E4 and 404A8. For example, the antibody fragment comprises a heavy chain CDR (HCDR)1 comprising SEQ ID NO: 100, an HCDR2 comprising SEQ ID NO: 101, an HCDR3 comprising SEQ ID NO: 102 or 106, a light chain CDR (LCDR)1 comprising SEQ ID NO: 103, an LCDR2 comprising SEQ ID NO: 104 and an LCDR3 comprising SEQ ID NO: 105. In a specific embodiment, the antibody fragment comprises (i) a heavy chain variable domain comprising SEQ ID NO: 107 or an amino acid sequence at least 95% identical thereto, and a light chain variable domain comprising SEQ ID NO: 108 or 123 or an amino acid sequence at least 95% identical thereto; (ii) SEQ ID NO: 109; (iii) SEQ ID NO: 110; or (iv) SEQ ID NO: 124. In certain embodiments, the Cys residue in the heavy chain CDR3 (SEQ ID NO: 102) is mutated to Ser, Thr, Met, Ala, Gly, Asn, or Gln.

[0016] In certain embodiments, the masking moiety does not interfere with or has a minimal effect on the binding of the IL-15 cytokine moiety to IL-15Rα.

[0017] In certain embodiments, the IL-15 cytokine portion is a human IL-15 polypeptide comprising SEQ ID NO:2 or a mutein thereof. In a specific embodiment, the human IL-15 polypeptide comprises any one of the following sequences: N1A, N1D, N4A, N4D, I6T, S7A, D8A, D8T, D8E, D8N, K10A, K10D, K11A, K11D, E46, V49, L45, S51, L52, D61A, D61N, T62L, T62A, E64A, E64L, E64K, E64Q, N65A, N65L, N65D, L66D, L66E, I67D, I67E, I68S, I68E, L69S, L69E, N72A, N72D, V63E, V63D ... , L66E, L66D, I67E, I67D, Q108E, N112A, N1D / D61N, N1D / E64Q, N4D / D61N, N4D / E64Q, D8N / D61N, D8N / E64Q, D61N / E64Q, E64Q / Q108E, N1D / N4D / D8N, D61N / E64Q / N65D, N1D / D61N / E64Q, N1D / Q108E, N1D / D61N / E64Q / Q108E, N4D / D61N / E64Q / Q108E and D30N / E64Q / N65D.

[0018] In certain embodiments, the carrier moiety is a PEG molecule, albumin, an albumin fragment, an antibody Fc domain or an antibody or an antigen-binding fragment thereof. In further embodiments, the carrier moiety is an antibody Fc domain or an antibody comprising the mutations L234A and L235A ("LALA") (EU numbering). In certain embodiments, the carrier moiety is an antibody Fc domain or an antibody comprising knob-into-hole mutations, wherein the IL-15 cytokine moiety and the masking moiety are fused to different polypeptide chains of the antibody Fc domain or different heavy chains of the antibody. In certain embodiments, the knob-into-hole mutation comprises a T366Y "knob" mutation in one polypeptide chain of the Fc domain or one heavy chain of the antibody and a Y407T "hole" mutation in the other polypeptide of the Fc domain or the other heavy chain of the antibody, or the knob-into-hole mutation comprises a Y349C and / or T366W mutation in the CH3 domain of the "knob chain" and an E356C, T366S, L368A and / or Y407V mutation in the CH3 domain of the "hole chain" (EU numbering). In certain embodiments, the carrier moiety is an IgG4 Fc domain, wherein the first polypeptide comprises an amino acid sequence at least 99% identical to that set forth in SEQ ID NO: 80, 81 or 87, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to that selected from SEQ ID NOs: 82-86.

[0019] In one embodiment, the carrier moiety is an anti-PD-1 antibody comprising a light chain having an amino acid sequence at least 99% identical to SEQ ID NO:55 or 56; a first heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO:54, 60 or 61; and a second heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO:52, 53, 58, 59, 62, 63 or 69. In a further embodiment, the carrier moiety is an anti-PD-1 antibody comprising a light chain having an amino acid sequence at least 99% identical to SEQ ID NO:55; a first heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO:66; and a second heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO:64, 65, 67 or 68.

[0020] In some embodiments, the carrier moiety is an anti-PD-L1 antibody comprising a light chain having an amino acid sequence at least 99% identical to SEQ ID NO: 50 or 51; a first heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO: 47, 48 or 49; and a second heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO: 45 or 46.

[0021] In certain embodiments, the carrier moiety is an antibody or antigen-binding fragment thereof that specifically binds to one or more antigens selected from PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, and TIGIT.

[0022] In certain embodiments, the carrier moiety is an antibody Fc domain or an antibody and the prodrug comprises the following polypeptide pair (N-terminus to C-terminus): C1-A and C2-SM, A-C1 and MS-C2, C1-SA and C2-M, C1-AS and C2-M, SA-C1 and M-C2 or AS-C1 and M-C2; and C1 and C2 are the first and second polypeptide chains of an Fc domain, respectively, or the first and second polypeptide chains of an antibody, respectively; and "-" is a direct peptidyl bond or a peptide linker.

[0023] In certain embodiments, the Sushi domain comprises SEQ ID NO: 7 or 9, or an amino acid sequence at least 90% identical thereto.

[0024] In certain embodiments, at least one of the first, second and third peptide linkers is a non-cleavable peptide linker, optionally selected from SEQ ID NOs: 11-16.

[0025] In certain embodiments, at least one of the first, second and third peptide linkers is a cleavable peptide linker comprising a substrate sequence for urokinase-type plasminogen activator (uPA), matriptase, matrix metallopeptidase (MMP)2 or MMP9. For example, the cleavable peptide linker comprises a substrate sequence for (i) both uPA and MMP2, (ii) both uPA and MMP9, (iii) uPA, MMP2 and MMP9 or (iv) MMP2 and matriptase. In a specific embodiment, the cleavable peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 17-36. The cleavable peptide linker is cleavable by one or more proteases located at the tumor site or its surrounding environment, and cleavage results in activation of the prodrug at the tumor site or its surrounding environment.

[0026] In another aspect, the present invention provides a pharmaceutical composition comprising the prodrug and a pharma- ceutically acceptable excipient; one or more polynucleotides encoding the prodrug; one or more expression vectors comprising the one or more polynucleotides; and a host cell comprising the vector. In one embodiment, the genes encoding uPA, matriptase, MMP-2 and / or MMP-9 are knocked out in the host cell.

[0027] Also provided is a method of producing the prodrug, comprising culturing a host cell, which is a mammalian cell, under conditions that permit expression of the prodrug, and isolating the prodrug.

[0028] In another embodiment, the present invention provides a method of treating cancer or infectious disease or stimulating the immune system in a patient in need of treatment, comprising administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of the prodrug. The patient may have, for example, 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, colorectal cancer and gastric cancer. Also provided are IL-15 prodrugs for use in such treatments and the use of IL-15 prodrugs in the manufacture of a medicament for such treatments.

[0029] Other characteristics, objects and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description, while describing embodiments and aspects of the present invention, is provided for the purpose of illustration only and is not intended to limit the present invention. 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 description of the drawings]

[0030] [Figure 1]1A-C are schematic diagrams of IL-15 prodrugs with an Fc domain as a carrier moiety. FIG. 1A shows an IL-15Rα Sushi domain polypeptide fused to the C-terminus of one Fc polypeptide, optionally via a non-cleavable peptide linker. The IL-15 polypeptide is fused to the C-terminus of the Sushi domain, optionally via a non-cleavable linker. A masking moiety is fused to the C-terminus of another Fc polypeptide, optionally via a cleavable linker. FIG. 1B shows an IL-15 polypeptide fused to the C-terminus of one Fc polypeptide, optionally via a non-cleavable peptide linker. The IL-15Rα Sushi domain is fused to the C-terminus of the IL-15 polypeptide, optionally via a non-cleavable linker. A masking moiety is fused to the C-terminus of another Fc polypeptide, optionally via a cleavable linker. FIG. 1C shows an IL-15 polypeptide fused to the C-terminus of one Fc polypeptide, optionally via a non-cleavable peptide linker. The IL-15Rα Sushi domain is fused to the C-terminus of another Fc polypeptide, optionally via a non-cleavable linker. The masking moiety is fused to the C-terminus of the Sushi domain via a cleavable linker. In all three configurations, the Fc domain contains a knob-into-hole mutation.

[0031] [Diagram 2]2A-C show schematic diagrams of IL-15 prodrugs with an Fc domain as a carrier moiety. FIG. 2A shows an IL-15Rα Sushi domain fused to the N-terminus of one Fc polypeptide, optionally via a non-cleavable linker. The IL-15 polypeptide is fused to the N-terminus of the Sushi domain, optionally via a non-cleavable peptide linker. A masking moiety is fused to the N-terminus of another Fc polypeptide, optionally via a cleavable linker. FIG. 2B shows an IL-15 polypeptide fused to the N-terminus of one Fc polypeptide, optionally via a non-cleavable linker. The IL-15Rα Sushi domain polypeptide is fused to the N-terminus of the IL-15 polypeptide, optionally via a non-cleavable peptide linker. A masking moiety is fused to the N-terminus of another Fc polypeptide, optionally via a cleavable linker. FIG. 2C shows an IL-15 polypeptide fused to the N-terminus of one Fc polypeptide, optionally via a non-cleavable peptide linker. The IL-15Rα Sushi domain is fused to the N-terminus of another Fc polypeptide, optionally via a non-cleavable linker. The masking moiety is fused to the N-terminus of the Sushi domain via a cleavable linker. In all three configurations, the Fc domain contains a knob-into-hole mutation.

[0032] [Diagram 3]3A-C show schematic diagrams of IL-15 prodrugs with an antibody (with two antigen binding sites) as a carrier moiety. FIG. 3A shows an IL-15 polypeptide fused to the C-terminus of one of the antibody's heavy chains, optionally via a non-cleavable peptide linker. An IL-15Rα Sushi domain is fused to the C-terminus of the IL-15 polypeptide, optionally via a non-cleavable linker. A masking moiety is fused to the C-terminus of the other antibody heavy chain, optionally via a cleavable linker. FIG. 3B shows an IL-15Rα Sushi domain polypeptide fused to the C-terminus of one of the antibody's heavy chains, optionally via a non-cleavable peptide linker. An IL-15 polypeptide is fused to the C-terminus of the Sushi domain, optionally via a non-cleavable linker. A masking moiety is fused to the C-terminus of the other antibody heavy chain, optionally via a cleavable linker. Figure 3C shows an IL-15 polypeptide fused to the C-terminus of one of the antibody heavy chains, optionally via a non-cleavable peptide linker. An IL-15Rα Sushi domain is optionally fused to the C-terminus of the other antibody heavy chain, optionally via a non-cleavable linker. A masking moiety is fused to the C-terminus of the Sushi domain via a cleavable linker. In all three figures, the antibody contains knob-into-hole mutations.

[0033] [Figure 4]Figures 4A and 4B are schematic diagrams of IL-15 prodrugs with an antibody as a carrier moiety. The antibody has a single antigen binding site. Figure 4A shows an IL-15 polypeptide fused to the C-terminus of one of the antibody's heavy chains, optionally via a non-cleavable peptide linker. The IL-15Rα Sushi domain is fused to the C-terminus of the IL-15 polypeptide, optionally via a non-cleavable linker. The masking moiety is fused to the C-terminus of the other antibody heavy chain, optionally via a cleavable linker. Figure 4B shows an IL-15Rα Sushi domain polypeptide fused to the C-terminus of one of the antibody's heavy chains, optionally via a non-cleavable peptide linker. The IL-15 polypeptide is fused to the C-terminus of the Sushi domain, optionally via a non-cleavable linker. The masking moiety is fused to the C-terminus of the other antibody heavy chain, optionally via a cleavable linker. In both configurations, the antibody contains a knob-into-hole mutation and the masking moiety is on the same polypeptide chain as the heavy chain variable region of the antibody.

[0034] [Diagram 5] Figures 5A and 5B are schematic diagrams of IL-15 prodrugs with an antibody as a carrier moiety. The antibody has a single antigen-binding moiety. Figure 5A shows an IL-15 polypeptide fused to the C-terminus of one of the antibody's heavy chains, optionally via a non-cleavable peptide linker. The IL-15Rα Sushi domain is fused to the C-terminus of the IL-15 polypeptide, optionally via a non-cleavable linker. The masking moiety is fused to the C-terminus of the other antibody heavy chain, optionally via a cleavable linker. Figure 5B shows an IL-15Rα Sushi domain polypeptide fused to the C-terminus of one of the antibody's heavy chains, optionally via a non-cleavable peptide linker. The IL-15 polypeptide is fused to the C-terminus of the Sushi domain, optionally via a non-cleavable linker. The masking moiety is fused to the C-terminus of the other antibody heavy chain, optionally via a cleavable linker. In both configurations, the antibody contains a knob-into-hole mutation, and the IL-15 polypeptide and the Sushi domain are on the same polypeptide chain as the heavy chain variable region of the antibody.

[0035] [Figure 6A] FIG. 6A shows sequence information for Fc-IL-15 prodrugs (JR3.68.1, JR3.68.2 and JR3.68.3) and control molecules (Fc-IL-15 fusion polypeptides, JR3.68.4 and JR3.68.5).

[0036] [Figure 6B] FIG. 6B shows the structure of the molecule of FIG. 6A. All molecules have an Fc domain as a carrier moiety. In JR3.68.1, the Sushi domain is fused to the C-terminus of one Fc polypeptide via a non-cleavable linker. The IL-15 polypeptide is fused to the C-terminus of the Sushi domain via a non-cleavable linker. The masking moiety is fused to the C-terminus of the other Fc polypeptide via a cleavable linker. In JR3.68.2, the IL-15 polypeptide is fused to the C-terminus of one Fc domain polypeptide via a non-cleavable linker. The Sushi domain is fused to the C-terminus of the IL-15 polypeptide via a non-cleavable linker. The masking moiety is fused to the C-terminus of the other Fc polypeptide via a cleavable linker. In JR3.68.3, the IL-15 polypeptide is fused to the C-terminus of one Fc polypeptide via a non-cleavable linker. The Sushi domain is fused to the C-terminus of the other Fc polypeptide via a non-cleavable linker. The masking moiety is fused to the C-terminus of the Sushi domain via a cleavable linker. JR3.68.4 and JR3.68.5 are the activated forms of JR3.68.1 and JR3.68.2, respectively (no masking moiety was designed into the construct).

[0037] [Figure 7] 7A and 7B are photographs of SDS-PAGE gels analyzing the activatable fusion polypeptides shown in FIG. 6B before and after activation.

[0038] [Figure 8]8A-C are graphs showing SEC-HPLC analysis of Fc-IL-15 / Sushi fusion protein samples JR3.68.1, JR3.68.2 and JR3.68.3, respectively, purified by Protein A column.

[0039] [Figure 9] Figures 9A-C show the cell-based activity of activatable Fc-IL-15 fusion polypeptides JR3.68.1, JR3.68.2 and JR3.68.3 before and after activation, respectively. In all three figures, IL-15 was used as a positive control.

[0040] [Figure 10A] FIG. 10A is a table showing sequence information for antibody-IL-15 fusion polypeptides JR3.74.1 and JR3.74.2 (unmasked) and activatable antibody-IL-15 fusion polypeptides JR3.73.2 and JR3.73.4.

[0041] [Figure 10B] FIG. 10B shows the structure of the molecule of FIG. 10A.

[0042] [Figure 11] 11A and 11B are graphs showing SEC-HPLC analysis of JR3.74.1, JR3.74.2, JR3.73.2 and JR3.73.4 samples purified by Protein A column.

[0043] [Figure 11C] FIG. 11C is a graph showing the results of a CTLL2 proliferation assay of prodrug samples before and after activation by protease treatment.

[0044] [Figure 12]Figures 12A and 12B show the results of NK92 proliferation assay of IL-15 prodrugs masked with scFv (scFv1 or scFv2) from anti-IL-15 antibody 146B7. Figure 12A shows the sequence information of activatable IL-15 fusion proteins. Figure 12B shows the results of NK92 proliferation assay. Control X1: XmAb® 24306, an IL-15 / IL-15-receptor alpha complex fused to XmAb Fc domain (IL-15 / IL-15Rα-Fc). Fc-IL-15*: activatable IL-15 fusion protein with IL-2Rβ extracellular domain (ECD) as masking moiety. Fc-IL-15: Fc-IL-15 fusion protein without masking moiety. RLU: relative light units.

[0045] [Figure 13] Figures 13A and 13B show the NK92 cell-based activity of activatable IL-15 fusion proteins before and after activation. Figure 13A shows the NK92 cell-based activity of an IL-15 fusion protein comprising wild type IL-15. Figure 13B shows the NK92 cell-based activity of an IL-15 fusion polypeptide comprising an IL-15 mutein with the N65D mutation. Control X1: XmAb® 24306, an IL-15 / IL-15-receptor alpha complex fused to the XmAb Fc domain (IL-15 / IL-15Rα-Fc). LUC: signal of fermentation unit. Act: activation.

[0046] [Figure 14A] FIG. 14A is a table showing sequence information for activatable IL-15 fusion proteins.

[0047] [Figure 14]Figures 14B-D show NK92 proliferation assay results of activatable IL-15 fusion proteins before and after activation. Figure 14B shows the results of wild type IL-15 masked with IL-2Rβ ECD and IL-2Rγ ECD. Figure 14C shows the results of IL-15 mutein Q108E masked with IL-2Rβ ECD and IL-2Rγ ECD. Figure 14D shows the results of activatable Fc-IL-15 fusion protein without Sushi domain (JR2.145.1) and activatable Fc-IL-15 fusion protein with long linker between Sushi domain and IL-15 polypeptide moiety (JR2.145.2). Control X1: XmAb® 24306, which is IL-15 / IL-15-receptor alpha complex fused to XmAb Fc domain (IL-15 / IL-15Rα-Fc). Control X2: is a PD-1 antibody-IL-15 mutein fusion protein lacking the Sushi domain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Detailed Description of the Invention As used in this specification and the appended claims, the singular terms "a," "an," and "the" include plurals unless the context clearly indicates otherwise.

[0049] The addition of "about" to a value or parameter herein includes (describes) the value or parameter itself as well as its variation. For example, a description of "about X" includes "X." Furthermore, the use of "about" before a series of numerical values ​​includes "about" each of the numerical values ​​in the series. For example, a description of "about X, Y, or Z" is intended to describe "about X, about Y, or about Z."

[0050] 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, an antibody (e.g., a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a bispecific or bispecific antibody, an anti-idiotypic antibody, or a bifunctional hybrid antibody) or an antigen-binding fragment thereof (e.g., a Fab, a Fab', a F(ab')2, a Fv, a disulfide-linked Fv, a scFv, a single domain antibody (dAb) or a bispecific antibody), a single chain antibody, and an Fc-containing polypeptide such as an immunoadhesin. In certain embodiments, the antibody may be of any heavy chain isotype (e.g., IgG, IgA, IgM, IgE, or IgD) or subtype (e.g., IgG1, IgG2, IgG3, or IgG4). In certain embodiments, the antibody may be of any antibody light chain isotype (e.g., kappa or lambda). The antibody can be human, non-human (e.g., mouse, rat, rabbit, goat or other non-human animal), chimeric (e.g., having non-human variable regions and human constant regions) or humanized (e.g., having non-human CDRs and human framework and constant regions). In certain embodiments, the antibody is a derivatized antibody.

[0051] 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 (e.g., binds to the same receptor and activates the same target cells) as the wild-type cytokine, but the activity level of the analog may differ from that of the wild-type cytokine. An analog may be, for example, a mutant protein (i.e., mutant polypeptide) of a wild-type cytokine, and may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten mutations relative to the wild-type cytokine.

[0052] The term "cytokine antagonist" or "cytokine mask" refers to a moiety (e.g., a polypeptide) that binds to a cytokine, thereby inhibiting the cytokine from binding to its receptor on the surface of a target cell and / or exerting its biological function while bound to the antagonist or mask. Examples of cytokine antagonists or masks include, but are not limited to, polypeptides derived from the extracellular domain of the cytokine's natural receptor that contacts the cytokine.

[0053] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease, such as improving, alleviating, reducing, and / or delaying one or more of its symptoms. For diseases such as cancer, an effective amount can be an amount sufficient to delay the development or progression of cancer (e.g., reducing the rate of tumor growth and / or slowing or preventing tumor angiogenesis, metastasis, or cancer cell invasion into peripheral organs), reduce the number of epithelioid cells, induce cancer remission (e.g., tumor shrinkage or eradication), and / or prevent or delay cancer onset or recurrence. An effective amount can be administered in one or more administrations.

[0054] The term "functional analog" refers to a molecule that has the same biological specificity (eg, binding to the same ligand) and / or activity (eg, activating or inhibiting target cells) as a reference molecule.

[0055] The term "fused" or "fusion" with respect to two polypeptide sequences refers to the linkage of two polypeptide sequences via a backbone peptide bond. The two polypeptides can be fused directly or via a peptide linker of one or more amino acids in length. A fusion polypeptide can be produced by recombinant technology from a coding sequence that includes each coding sequence of two fusion partners, with or without a coding sequence for a peptide linker in between. In some embodiments, fusion includes chemical conjugation.

[0056] The term "pharmaceutical acceptable excipient," when used to refer to a component of a composition, means an excipient that is suitable for administration for treatment of a subject, including a human subject, without causing adverse side effects to the subject and without affecting the biological activity of the active pharmaceutical ingredient (API).

[0057] The term "subject" refers to a mammal, including, but not limited to, a human, a pet (eg, a dog or cat), a farm animal (eg, a cow or horse), a rodent, or a primate.

[0058] As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviating one or more symptoms caused by a disease, reducing the extent of the disease, alleviating the disease state, stabilizing the disease (e.g., preventing or slowing the worsening or progression of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, providing partial or complete remission of the disease, reducing the dosage of one or more other medications required to treat the disease, increasing the quality of life and / or prolonging the survival of a patient. The methods of the present invention contemplate any one or more of these aspects of treatment.

[0059] It is understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. The headings of the described paragraphs are for organizational purposes only and are not to be construed as limiting the subject matter described thereunder.

[0060] I. IL-15 Prodrugs The present invention relates to IL-15 prodrugs that are metabolized in vivo to become active IL-15 therapeutic agents. The IL-15 prodrugs have fewer side effects, better in vivo PK profiles (e.g., longer half-lives) and better target specificity, and are more effective than previous IL-15 therapeutic agents. The IL-15 prodrugs of the present invention have configurations that reduce aggregation levels and improve manufacturing efficiency, thereby overcoming common challenges in the manufacture of fusion and bispecific molecules.

[0061] The prodrug comprises an IL-15 polypeptide (A) (i.e., a cytokine agonist polypeptide or an IL-15 cytokine moiety), an optional IL-15Rα Sushi domain (S), a masking moiety (M) (i.e., a cytokine antagonist) and a carrier moiety (C). The components are operatively linked to each other via peptide linkers, one of which may be cleavable such that upon activation by a protease at the target site, the masking moiety and the IL-15 cytokine moiety are detached from each other. In certain embodiments, the masking moiety (IL-15 antagonist), which may be, for example, the extracellular domain of the receptor for IL-15 or a binding fragment of an antibody that binds to the cytokine, is linked to the cytokine moiety, the Sushi domain or the carrier moiety via a cleavable linker (e.g., a cleavable peptide linker). In other embodiments, the masking moiety is linked to the other moiety via a non-cleavable linker.

[0062] The mask inhibits the biological function of the IL-15 cytokine moiety while it is bound to it. In some embodiments, the masking moiety of the prodrug specifically binds to an epitope located in the IL-2Rβ and / or γ chain interacting domain of the IL-15 polypeptide. The inhibitory effect of the masking moiety is removed by protease digestion of the cleavable linker in the prodrug, allowing the masking moiety and the cytokine moiety to separate. In some embodiments, the masking moiety of the prodrug does not block or interfere with the binding of the IL-15 polypeptide (A) to IL-15Rα. The prodrug is activated at the target site (e.g., at the tumor site or the surrounding environment or at the site of infection) in the patient by cleavage of the linker, which then releases the remaining cytokine mask or IL-15 cytokine moiety of the prodrug, exposing the previously masked IL-15 cytokine moiety, allowing the IL-15 cytokine moiety to bind to its receptor on the target cell and exert its biological function on the target cell. In some embodiments, the carrier for the prodrug is an antigen-binding moiety, such as an antibody, that binds to the antigen at the target site.

[0063] In certain embodiments of the IL-15 prodrugs of the present invention, the Sushi domain is fused to the carrier, masking moiety and / or IL-15 cytokine moiety via a peptide linker (non-cleavable or cleavable). In certain embodiments, the IL-15 cytokine moiety is fused to the carrier moiety, masking moiety and / or Sushi domain via a peptide linker (non-cleavable or cleavable). In certain embodiments, the masking moiety is fused to the carrier moiety, cytokine moiety and / or Sushi domain via a peptide linker (non-cleavable or cleavable).

[0064] In some embodiments, the prodrug is metabolized to the active IL-15 cytokine, which is proinflammatory, at the target site targeted by the carrier in the body. In further embodiments, the carrier in the prodrug is an antibody that targets a tumor antigen such that the prodrug is delivered to the tumor site of the patient and metabolized locally (e.g., in or near the tumor microenvironment) by cleavage of the linker that connects the cytokine mask to the carrier or cytokine moiety, allowing the proinflammatory cytokine moiety to interact with its receptor on the target cell and stimulate the target immune cell locally.

[0065] A. IL-15 Moiety of the Prodrug In the present IL-15 prodrugs, the IL-15 cytokine moiety can be a wild-type IL-15 polypeptide, such as the wild-type human IL-15 polypeptide (SEQ ID NO:2), or an IL-15 mutein, such as an IL-15 mutein derived from human wild-type IL-15 that has reduced affinity for IL-2Rβ (CD122) compared to wild-type IL-15. The IL-15 mutein has significantly reduced affinity for CD122 or the dimeric IL-2R compared to wild-type IL-15.

[0066] In certain embodiments, the IL-15 portion, when masked, has a biological activity that is reduced by at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold; or an EC 50 The value is increased by at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold.

[0067] In certain embodiments, the IL-15 portion is an IL-15 mutein that includes at least one, two, three, four or five mutations at positions N1, N4, I6, S7, D8, K10, K11, E46, D61, T62, E64, N65, I68, L69, N72, V63, L66, I67, A70, N71, Q108, N112 of human IL-15. Examples of IL-15 muteins are those having one or more mutations selected from N1A, N1D, N4A, N4D, I6T, S7A, D8A, DAT, D8E, D8N, K10A, K10D, K11A, K11D, D61A, D61N, T62L, T62A, E64A, E64L, E64K, E64Q, N65A, N65L, N65D, L66D, L66E, I67D, I67E, I68S, I68E, L69S, L69E, N72A, N72D, V63E, V63D, L66E, L66D, I67E, I67D, Q108E and N112A. In certain embodiments, the IL-15 portion comprises a mutation or position selected from E46, V49, L45, S51, and L52. Unless otherwise specified, all residue numbers of IL-15 and IL-15 muteins described herein are according to the numbering of SEQ ID NO: 2. In other embodiments, the IL-15 portion comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2.

[0068] In specific embodiments, the IL-15 mutein comprises a mutation selected from N1D / D61N, N1D / E64Q, N4D / D61N, N4D / E64Q, D8N / D61N, D8N / E64Q, D30N / E64Q / N65D, D61N / E64Q, E64Q / Q108E, N1D / N4D / D8N, D61N / E64Q / N65D, N1D / D61N / E64Q, N1D / D61N / E64Q / Q108E and N4D / D61N / E64Q / Q108E.

[0069] B. IL-15 Receptor Alpha Sushi Domain In certain embodiments, the IL-15 prodrug comprises an IL-15Rα Sushi domain. The Sushi domain may be fused directly to the carrier or to the IL-15 cytokine moiety, optionally via a linker (e.g., a non-cleavable or cleavable peptide linker). The masking moiety may be fused to the Sushi domain or to the carrier via a cleavable or non-cleavable peptide linker. In certain embodiments, the Sushi domain is fused to the carrier and the cytokine is fused to the Sushi domain via a peptide linker. In the IL-15 prodrug, the Sushi domain may be a wild-type Sushi domain or a Sushi domain comprising the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9. In other embodiments, the Sushi domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7 or SEQ ID NO:9.

[0070] In certain embodiments, the human IL-15 receptor alpha (IL-15Rα) protein has the amino acid sequence set forth in SEQ ID NO:8. In certain examples, the coding sequence for human IL-15Rα is set forth in SEQ ID NO:137. The IL-15Rα protein of the prodrugs outlined herein comprises or consists of the Sushi domain of SEQ ID NO:8 (e.g., amino acids 31-95 or 31-105 of SEQ ID NO:8) or, in other words, the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:7. In certain embodiments, the IL-15Rα protein has the amino acid sequence of SEQ ID NO:7 and an amino acid insertion selected from the group consisting of D96, P97, A98, D96 / P97, D96 / C97, D96 / P97 / A98, D96 / P97 / C98 and D96 / C97 / A98, where the amino acid positions are relative to the full-length human IL-15Rα protein or SEQ ID NO:8. For example, amino acids such as D, P, A, DP, DC, DPA, DPC, or DCA can be added to the C-terminus of an IL-15Rα protein (e.g., SEQ ID NO: 9). In certain embodiments, the IL-15Rα protein has the amino acid sequence of SEQ ID NO: 9 and one or more amino acid substitutions selected from the group consisting of K34C, A37C, G38C, S40C, and L42C, where the amino acid positions are relative to SEQ ID NO: 9. In certain embodiments, the IL-15 analog and Sushi domain each have a set of amino acid substitutions or additions selected from the group consisting of E87C:D96 / P97 / C98; E87C:D96 / C97 / A98; V49C:S40C; L52C:S40C; E89C:K34C; Q48C:G38C; E53C:L42C; C42S:A37C; and L45C:A37C (mutations in IL-15 are shown before the colon; and mutations in the Sushi domain are shown after the colon).

[0071] C. Masking Moieties of Prodrugs The cytokine antagonist, i.e., masking moiety, of the prodrug 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. In one embodiment, the masking moiety comprises an antigen-binding portion or binding fragment of an antibody that binds to a human IL-15 polypeptide and inhibits the biological activity of the human IL-15 polypeptide.

[0072] As an example, the IL-15 antagonist may include peptides and antibodies that bind to IL-15 and prevent the binding of the IL-15 moiety to its receptor, reducing the biological activity of the IL-15 moiety when masked. In certain embodiments, the IL-15 antagonist includes an IL-2Rβ or IL-2Rγ extracellular domain or a functional analog thereof, such as one derived from human IL-2Rβ or IL-2Rγ (e.g., one of SEQ ID NOs: 3-6). In certain embodiments, the IL-15 antagonist includes a peptide identified by screening a peptide library. In certain embodiments, the IL-15 antagonist includes an antibody or fragment thereof that blocks the binding of IL-15 or an IL-15 mutein to the IL-15 receptor. In other embodiments, the antagonist inhibits the biological activity of an IL-15 polypeptide. In certain embodiments, the antagonist includes an scFv, Fab, or other type of antibody fragment known in the art. In a preferred embodiment, the antibody fragment is an scFv specific for IL-15. In another preferred embodiment, the antagonist specifically binds to an epitope located in the beta and / or gamma chain interaction domain of the IL-15 agonist polypeptide. In a specific embodiment, the masking moiety does not block or interfere with the binding of the IL-15 polypeptide to IL-15Rα. As an example, the IL-15 binding antibody can be selected from 146B7, 146H5, 404E4 and 404A8. In one embodiment, the scFv or Fab IL-15 antagonist comprises the CDR1, CDR2 and CDR3 domains of an anti-IL-15 antibody selected from 146B7, 146H5, 404E4 and 404A8; and the CDR1, CDR2 and CDR3 domains from the light chain of an anti-IL-15 antibody selected from 146B7, 146H5, 404E4 and 404A8, all of which are described in WO2003 / 017935A2.

[0073] In one embodiment, the IL-15 antagonist comprises a heavy chain CDR1, CDR2 and CDR3 domains having the amino acid sequences of SEQ ID NO: 100, 101 and 102, respectively; and a light chain CDR1, CDR2 and CDR3 domains having the amino acid sequences of SEQ ID NO: 103, 104 and 105, respectively. In one embodiment, the heavy chain CDR3 domain of SEQ ID NO: 102 comprises a substitution mutation at its Cys residue. The Cys residue in the CDR3 domain of SEQ ID NO: 102 can be mutated to Ser, Thr, Ala, Asn or Gln. In another embodiment, the CDR3 domain comprises the amino acid sequence of SEQ ID NO: 106. In one embodiment, the antagonist or masking moiety is an scFv or Fab comprising a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 107 or at least 95% identical to SEQ ID NO: 107, and a light chain variable domain having an amino acid sequence of SEQ ID NO: 108 or 123 or at least 95% identical to SEQ ID NO: 108 or 123. In certain portions, the masking portion comprises the amino acid sequence of SEQ ID NO:110 or 124.

[0074] D. Carrier Moieties of Prodrugs The carrier moiety of the prodrug may be an antigen-binding moiety or a non-antigen-binding moiety. The carrier moiety may improve the PK profile, such as serum half-life, of the cytokine agonist polypeptide and may direct the cytokine agonist polypeptide to a target site in the body, such as a tumor site.

[0075] In certain embodiments, the carrier moiety (C) is an Fc domain composed of a first and a second polypeptide chain (i.e., two different heavy chains), wherein the polypeptide chains comprise a molecular formula (N-terminus to C-terminus) selected from one of the following pairs: a) F1-PL1-A-PL2-S, F2-CL-M (Figure 1A); b) F1-PL1-S-PL2-A, F2-CL-M (Figure 1B); and c) F1-PL1-S-PL2-A, F2-CL-M (Figure 1C); where F1 and F2 are subunits of a carrier moiety (e.g., an Fc domain) that form a heterodimer; PL1 and PL2 are peptide linkers; CL is a cleavable peptide linker; S is a Sushi domain; and A is an IL-15 polypeptide.

[0076] In certain embodiments, the carrier moiety (C) is an Fc domain composed of a first and a second polypeptide chain (i.e., two different heavy chains), wherein the polypeptide chains comprise a molecular formula (N-terminus to C-terminus) selected from one of the following pairs: a) A-PL1-S-F1, M-CL-F2 (Figure 2A); b) S-PL1-A-F1, M-CL-F2 (Figure 2B); and c) A-PL1-F1, M-CL-S-F2 (Figure 2C); where F1 and F2 are subunits of a carrier moiety (e.g., an Fc domain) that form a heterodimer; PL1 and PL2 are peptide linkers; CL is a cleavable peptide linker; S is a Sushi domain; and A is an IL-15 polypeptide.

[0077] In certain embodiments, the carrier moiety (C) is an antibody comprising two antibody fragments, a first antibody heavy chain and a second antibody heavy chain, a) the first heavy chain comprises the molecular formula (N-terminus to C-terminus) C1-CL-M; and b) the first duplex comprises the molecular formula (N-terminus to C-terminus) C2-PL1-S-PL2-A; wherein C1 and C2 are antibody heavy chains; PL1 and PL2 are peptide linkers; CL is a cleavable peptide linker; S is a Sushi domain; and A is an IL-15 polypeptide. In other embodiments, the order of the first and second heavy chains is reversed (Figures 3A and 3B).

[0078] In certain embodiments, the carrier moiety (C) is an antibody comprising two antibody fragments, a first antibody heavy chain and a second antibody heavy chain, a) the first heavy chain comprises the molecular formula (N-terminus to C-terminus) C1-A; and b) the first double-stranded polypeptide chain comprises the molecular formula (N-terminus to C-terminus) C2-PL1-S-CL-M; where C1 and C2 are antibody heavy chains; PL1 and PL2 are peptide linkers; CL is a cleavable peptide linker; S is a Sushi domain; and A is an IL-15 polypeptide (FIG. 3C).

[0079] In one embodiment, the prodrug of the invention comprises three polypeptide chains - one antibody light chain and two heavy chains - where the first polypeptide chain is an antibody light chain variable region, the first heavy chain comprises the heavy chain variable and constant regions of an antibody, and the second chain comprises CH2 and CH3 domains, where the first and second chains comprise a molecular formula (N-terminus to C-terminus) selected from one of the following pairs: a) F-PL1-A-PL2-S, HC-CL-M (Figure 4A); b) F-PL1-S-PL2-A, HC-CL-M (Figure 4B); c) HC-PL1-A-PL2-S, F-CL-M (Figure 5A); and d) HC-PL1-S-PL2-A, F-CL-M (Figure 5B). where F is a subunit of the Fc domain (including the CH2 and CH3 domains); HC is an antibody heavy chain that forms the antigen-binding portion with the light chain; PL1 and PL2 are peptide linkers; CL is a cleavable peptide linker; S is a Sushi domain; and A is an IL-15 polypeptide.

[0080] 1. Antigen-binding carrier moiety The carrier moiety can be an antibody or an antigen-binding fragment thereof or an immunoadhesin. In some embodiments, the antigen-binding moiety is a full-length antibody of two heavy chains and two light chains, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a disulfide-linked Fv fragment, a single domain antibody, a nanobody, or a single-chain variable fragment (scFv). In some embodiments, the antigen-binding moiety is a bispecific antigen-binding moiety and can bind to two different antigens or two different epitopes of the same antigen. The antigen-binding moiety can provide additional and possibly synergistic therapeutic efficacy to the cytokine agonist polypeptide.

[0081] The cytokine (IL-15) polypeptide and its mask may be fused to the N-terminus or C-terminus of the light and / or heavy chain of the antigen-binding portion. As an example, the cytokine (e.g., IL-15) polypeptide and its mask may be fused to an antibody heavy chain or an antigen-binding fragment thereof or an antibody light chain or an antigen-binding fragment thereof. In certain embodiments, the cytokine (IL-15) polypeptide is fused to the C-terminus of one or both heavy chains of the antibody, and the cytokine mask is fused to the other end of the heavy chain or to the C-terminus of the cytokine agonist polypeptide via a cleavable or non-cleavable peptide linker. In certain embodiments, the cytokine (IL-15) polypeptide is fused to the C-terminus of one of the heavy chains of the antibody, and the cytokine mask is fused to the C-terminus of the other heavy chain of the antibody via a cleavable peptide linker, where the two heavy chains optionally contain a mutation that allows for specific pairing of the two different heavy chains.

[0082] Strategies for forming heterodimers of Fc fusion polypeptides or bispecific antibodies are well known (see, e.g., Spies et al., Mol Imm. (2015) 67(2)(A):95-106). For example, two heavy chain polypeptides of a prodrug can form stable heterodimers via "knob-into-hole" mutations. "Knob-into-hole" mutations are made to promote the formation of heterodimers of antibody heavy chains and are commonly used in the production of bispecific antibodies (see, e.g., U.S. Patent No. 8,642,745). For example, the Fc domain of an antibody can contain a T366W mutation in the CH3 domain of the "knob chain" and a T366S, L368A and / or Y407V mutation in the CH3 domain of the "hole chain". Additional interchain disulfide bridges between the CH3 domains can also be used, for example by introducing a Y349C mutation in the CH3 domain of the "knob chain" and an E356C or S354C mutation in the CH3 domain of the "hole chain" (see, e.g., Merchant et al., Nature Biotech (1998) 16:677-81). In other embodiments, the antibody moiety can include 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 certain 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, 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 et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).Other knobs-into-hole technologies such as those described in EP1870459A1 may be used alternatively or in addition. Thus, other examples of knobs-into-hole mutations in antibody moieties include the R409D / K370E mutations in the CH3 domain of the "knob chain" and the D399K / E357K mutations in the CH3 domain of the "hole chain" (EU numbering).

[0083] In some embodiments, the antibody portion of the prodrug contains L234A and L235A ("LALA") mutations in the Fc domain. The LALA mutations eliminate complement binding and fixation and Fcγ-dependent ADCC (see, e.g., Hezareh et al. J. Virol. (2001) 75(24):12161-8). In further embodiments, the LALA mutations are present in the antibody portion in addition to the knob-into-hole mutations.

[0084] In certain embodiments, the antibody portion comprises M252Y / S254T / T256E ("YTE") mutations in the Fc domain. The YTE mutations allow for simultaneous modulation of serum half-life, tissue distribution and IgG1 activity (Dall'Acqua et al., J Biol Chem. (2006) 281: 23514-24; and Robbie et al., Antimicrob Agents Chemother. (2013) 57(12):6147-53). In further embodiments, the YTE mutations are present in the antibody portion in addition to knob-into-hole mutations. In specific embodiments, the antibody portion has YTE, LALA and knob-into-hole mutations or any combination thereof.

[0085] The antigen-binding portion binds to an antigen on the surface of a cell, such as an immune cell, for example, a T cell, a NK cell, and a macrophage, or binds to a cytokine. For example, the antigen-binding portion can bind to PD-1, LAG-3, TIM-3, TIGIT, CTLA-4, or TGF-beta, and can be an antibody. The antibody has the ability to activate the immune cell and enhance its anti-cancer activity.

[0086] 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, HER-2, EGFR, claudin 18.2, DLL-3, GCP3 or carcinoembryonic antigen (CEA) and can be an antibody.The antibody may or may not have ADCC activity.The antibody can also be further conjugated to a cytotoxic drug.

[0087] In some embodiments, the antigen binding moiety is selected from the group consisting of guanylate cyclase C (GCC), carbohydrate antigen 19-9 (CA19-9), glycoprotein A33 (gpA33), mucin 1 (MUC1), insulin-like growth factor 1 receptor (IGF1-R), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), delta-like protein 3 (DLL3), delta-like protein 4 (DLL4), epidermal growth factor receptor (EGFR), glypican-3 (GPC3), c-MET, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), nectin-4, Liv-1, glycoprotein NMB (GPNMB), prostate specific membrane antigen (PSMA), Trop-2, carbonate Binds to anhydrase IX (CA9), endothelin B receptor (ETBR), six transmembrane epithelial antigen of the prostate 1 (STEAP1), folate receptor alpha (FR-α), SLIT and NTRK-like protein 6 (SLITRK6), carbonic anhydrase VI (CA6), ectonucleotide pyrophosphatase / phosphodiesterase family member 3 (ENPP3), mesothelin, trophoblast glycoprotein (TPBG), CD19, CD20, CD22, CD33, CD40, CD56, CD66e, CD70, CD74, CD79b, CD98, CD123, CD138, CD352, CD47, signal regulatory protein alpha (SIRPα), claudin 18.2, claudin 6, BCMA, or EPCAM. In certain embodiments, the antigen binding moiety binds to the epidermal growth factor (EGF)-like domain of DLL3. In some embodiments, the antigen-binding moiety binds to the delta / serrate / Lag2 (DSL)-like domain of DLL3. In some embodiments, the antigen-binding moiety binds to an epitope located after amino acid 374 of GPC3. In some embodiments, the antigen-binding moiety binds to heparin sulfate glycans of GPC3. In some embodiments, the antigen-binding moiety binds to claudin 18.2 and does not bind to claudin 18.1. In some embodiments, the antigen-binding moiety binds to claudin 18.1 with at least 10-fold weaker binding affinity than to claudin 18.2.

[0088] In one embodiment, the antigen-binding portion (carrier portion) comprises an antibody or fragment thereof that binds to PD-1 and interferes with the interaction of PD-1 with its ligand (PD-L1) so as to stimulate an anti-tumor immune response, as known in the art. In one embodiment, the antibody or antigen-binding portion thereof specifically binds to PD-1. For example, antibodies that target PD-1 and may be useful in the present invention include nivolumab (BMS-936558, Bristol-Myers Squibb), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck), humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.), pidilizumab (anti-PD-1 mAb CT-011, Medivation), anti-PD-1 monoclonal antibody BGB-A317 (BeiGene) and / or anti-PD-1 antibody SHR-1210 (ShangHai HengRui), human monoclonal antibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb) and / or humanized anti-PD-1 Anti-PD-1 antibodies include, but are not limited to, the IgG4 antibody PDR001 (Novartis). In one embodiment, the PD-1 antibody is from clone: ​​RMP1-14 (rat IgG) - BioXcell cat# BP0146. Other suitable anti-PD-1 antibodies include those disclosed in US Patent 8,008,449. In one embodiment, the antibody or antigen-binding portion thereof specifically binds to PD-L1 and disrupts the interaction with PD-1, thereby enhancing immune activity. Any antibody known in the art that binds to PD-L1, disrupts the interaction of PD-1 with PD-L1, and stimulates an anti-tumor immune response is suitable for use in the combination treatment methods described herein. By way of example, PD-L1-targeting antibodies include BMS-936559 (Bristol-Myers Squibb) and MPDL3280A (Genetech; currently in human clinical trials). Other suitable PD-L1-targeting antibodies are disclosed in US Patent 7,943,743.One of skill in the art will appreciate that any antibody that binds to PD-1 or PD-L1, disrupts the PD-1 / PD-L1 interaction, and stimulates an anti-tumor immune response is suitable for use in the combination treatment methods disclosed herein.

[0089] In certain embodiments, wherein the carrier is an antibody selected from ASKB1296, avelumab, atezolizumab, and durvalumab.

[0090] In some embodiments, the carrier is an antibody that binds to the antigen expressed in cancer cell.In some embodiments, the carrier antibody has ADCC activity.In some embodiments, the carrier antibody binds to the antigen selected from HER2, HER3, EGFR, CMET, Trop-2, GPC3, claudin 18.2, claudin 6, 5T4, BCMA, CD38, CD20, CD30, CD47 and VEGFR2.

[0091] In one embodiment, the carrier is a bispecific antibody that binds to two antigens selected from PD-1, PD-L1, CTLA-4, LAG-4, TIM-3, CD47, and TIGIT.

[0092] In one embodiment, the carrier antibody binds to human PD-1, where the PD-1 antibody comprises the same heavy chain CDR1, CDR2, and CDR3 domains and light chain CDR1, CDR2, and CDR3 domains from the heavy and light chains of nivolumab, pembrolizumab, toripalimab, sintilimab, or tislelizumab.

[0093] In one embodiment, the carrier antibody binds to human PD-1, wherein the light chain comprises an amino acid sequence at least 99% identical to one selected from SEQ ID NOs: 55 and 56; wherein the first heavy chain polypeptide chain comprises an amino acid sequence at least 99% identical to one selected from SEQ ID NOs: 54, 60, or 61; and wherein the second heavy chain polypeptide chain comprises an amino acid sequence at least 99% identical to one selected from SEQ ID NOs: 52, 53, 58, 59, 62, 63, and 69.

[0094] In one embodiment, the antibody binds to human PD-1, wherein the light chain comprises an amino acid sequence at least 99% identical to SEQ ID NO:55; wherein the first heavy chain polypeptide chain comprises an amino acid sequence at least 99% identical to SEQ ID NO:66; and wherein the second heavy chain polypeptide chain comprises an amino acid sequence at least 99% identical to one selected from SEQ ID NOs:64, 65, 67, and 68.

[0095] In one embodiment, the carrier antibody binds to PD-1, wherein the light chain comprises an amino acid sequence at least 99% identical to one selected from SEQ ID NOs: 55 and 56; wherein the first heavy chain comprises an amino acid sequence at least 99% identical to one selected from SEQ ID NOs: 80, 81, or 87; and wherein the second heavy chain comprises an amino acid sequence at least 99% identical to one selected from SEQ ID NOs: 52, 53, 58, 59, 62, 63, and 69.

[0096] In one embodiment, the carrier antibody binds to PD-1, wherein the light chain comprises an amino acid sequence at least 99% identical to one selected from SEQ ID NOs: 55 and 56; wherein the first heavy chain comprises an amino acid sequence at least 99% identical to one selected from SEQ ID NOs: 54, 60, or 61; and wherein the second heavy chain comprises an amino acid sequence at least 99% identical to one selected from SEQ ID NOs: 82, 83, 84, 85, and 86.

[0097] In one embodiment, the carrier antibody binds to PD-L1, wherein the light chain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 50 or 51; wherein the first heavy chain polypeptide chain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 47, 48 or 49; and wherein the second heavy chain polypeptide chain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 45 or 46.

[0098] In certain embodiments, the carrier antibody is a bispecific antibody that binds to two antigens selected from HER2, HER3, EGFR, CMET, Trop-2, GPC3, claudin 18.2, claudin 6, 5T4, BCMA, CD38, CD20, CD30, and VEGFR2. In certain embodiments, the carrier is a bispecific antibody that binds to cMet and EGFR; wherein the EGFR binding domain comprises a light chain CDR1, CDR2, and CDR3 from SEQ ID NO: 88 or 90 and a heavy chain CDR1, CDR2, and CDR3 from SEQ ID NO: 89 or 91.

[0099] In one embodiment, the carrier moiety is an IgG1 Fc domain, wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 37, 70-72, and 73, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 38, 39, 75-78, and 79.

[0100] In certain embodiments, the carrier moiety is an IgG4 Fc domain, wherein the first polypeptide comprises an amino acid sequence at least 99% identical to that set forth in SEQ ID NO: 80, 81 or 87, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to that selected from SEQ ID NOs: 82-85 and 86.

[0101] In some embodiments, the antigen-binding portion comprises an antibody or fragment thereof known in the art that binds to CTLA-4 and disrupts its interaction with CD80 and CD86. Examples of antibodies that target CTLA-4 include FDA-approved ipilimumab (MDX-010, MDX-101, Bristol-Myers Squibb) and tremelimumab (ticilimumab, CP-675, 206, Pfizer), which is currently in human clinical trials. Other suitable antibodies that target CTLA-4 are disclosed in WO2012 / 120125, US Patents 6,984,720, 6,682,7368, and US Patent Applications 2002 / 0039581, 2002 / 0086014, and 2005 / 0201994. One of skill in the art will appreciate that any antibody that binds to CTLA-4, interferes with its interaction with CD80 and CD86, and stimulates an anti-tumor immune response is suitable for use in the combination treatment methods disclosed herein.

[0102] In some embodiments, the combination therapy includes an antibody known in the art that binds to LAG-3 and blocks its interaction with MHC class II molecules. An example of an antibody that targets LAG-3 is IMP321 (Immutep), which is currently in human clinical trials. Other suitable antibodies that target LAG-3 are disclosed in US patent application 2011 / 0150892. Those skilled in the art will understand that any antibody that binds to LAG-3, blocks its interaction with MHC class II molecules, and stimulates anti-tumor immune responses is suitable for use in the combination treatment methods disclosed herein.

[0103] In certain embodiments, the antigen binding portion comprises an antibody or fragment thereof known in the art that binds to TIM-3 and disrupts its interaction with Galectin 9. Suitable antibodies that target TIM-3 are disclosed in US Patent Application 2013 / 0022623. One of skill in the art will appreciate that any antibody that binds to TIM-3, disrupts its interaction with Galectin 9, and stimulates an anti-tumor immune response is suitable for use in the combination treatment methods disclosed herein.

[0104] In certain embodiments, the antigen binding portion comprises an antibody or fragment thereof known in the art that binds 4-1BB / CD137 and disrupts its interaction with CD137L. One of skill in the art will appreciate that any antibody that binds 4-1BB / CD137, disrupts its interaction with CD137L or other ligands, and stimulates an anti-tumor immune response or an immunostimulatory response that results in overall anti-tumor activity is suitable for use in the combination treatment methods disclosed herein.

[0105] In some embodiments, the antigen-binding portion comprises an antibody or fragment thereof known in the art that binds to GITR and blocks its interaction with its ligand. Those skilled in the art will understand that any antibody that binds to GITR, blocks its interaction with GITRL or other ligands, and stimulates an immune stimulatory response that results in an anti-tumor immune response or overall anti-tumor activity is suitable for use in the combination treatment methods disclosed herein.

[0106] In some embodiments, the antigen-binding portion comprises an antibody or fragment thereof known in the art that binds to OX40 and disrupts its interaction with its ligands. Those skilled in the art will understand that any antibody that binds to OX40, disrupts its interaction with OX40L or other ligands, and stimulates an anti-tumor immune response or an immune stimulatory response that results in overall anti-tumor activity is suitable for use in the combination treatment methods disclosed herein.

[0107] In certain embodiments, the antigen-binding portion comprises an antibody or fragment thereof known in the art that binds to CD40 and disrupts its interaction with its ligands. One of skill in the art will appreciate that any antibody that binds to CD40, disrupts its interaction with its ligands, and stimulates an anti-tumor immune response or an immune stimulatory response that results in overall anti-tumor activity is suitable for use in the combination treatment methods disclosed herein.

[0108] In one embodiment, the antigen-binding portion comprises an antibody or fragment thereof known in the art that binds to ICOS and blocks its interaction with its ligand. Those skilled in the art will appreciate that any antibody that binds to ICOS, blocks its interaction with its ligand, and stimulates an anti-tumor immune response or an immune stimulatory response that results in overall anti-tumor activity is suitable for use in the combination treatment methods disclosed herein.

[0109] In certain embodiments, the antigen-binding portion comprises an antibody or fragment thereof known in the art that binds to CD28 and disrupts its interaction with its ligand. One of skill in the art will appreciate that any antibody that binds to CD28, disrupts its interaction with its ligand, and stimulates an anti-tumor immune response or an immune stimulatory response that results in overall anti-tumor activity is suitable for use in the combination treatment methods disclosed herein.

[0110] Further examples of antigen-binding moieties (carrier moieties) are trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereof) and anti-EGFR antibody mAb806 (or a humanized version thereof). In certain embodiments, the antigen-binding moiety has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with trastuzumab, rituximab, brentuximab, cetuximab or panitumumab, GC33 (or a humanized version thereof) or anti-EGFR antibody mAb806 (or a humanized version thereof). In some embodiments, the antigen-binding portion has an antibody heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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) or a fragment thereof. In some embodiments, the antigen-binding portion has an antibody light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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) or a fragment thereof. The antigen-binding portion is fused to an IL-15 polypeptide. In some embodiments, the antigen-binding portion comprises the six complementarity determining regions (CDRs) of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33, or the anti-EGFR antibody mAb806.

[0111] Several CDR delineations are known in the art and are encompassed herein. One skilled in the art can easily determine certain delineations of CDRs based on the sequences of the heavy or light chain variable regions. "Kabat" CDRs are based on sequence variability and are most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). "Chothia" CDRs refer to 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, which represent a compromise between Kabat CDRs and Chothia structural loops, are used in 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 a general antibody numbering scheme. Unless otherwise noted herein, antibody amino acid numbers refer to the Kabat numbering scheme as described in Kabat et al., supra, including when the CDR delineations were made with reference to Kabat, Chothia, AbM or contact schemes. Using this numbering system, the actual linear amino acid sequence may contain fewer or more amino acids corresponding to a shortening or insertion of a framework region (FR) or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and an inserted residue after heavy chain FR residue 82 (e.g., residues 82a, 82b and 82c, etc. according to Kabat). The Kabat numbering of residues may be determined for a given antibody by aligning the regions of homology of the antibody's sequence with the "standard" Kabat numbering sequence. [Table 1]

[0112] In some embodiments, the CDR is an "extended CDR" and encompasses regions beginning or ending according to different schemes. For example, the extended CDR can be: L24-L36, L26-L34 or L26-L36 (VL-CDR1); L46-L52, L46-L56 or L50-L55 (VL-CDR2); L91-L97 (VL-CDR3); H47-H55, H47-H65, H50-H55, H53-H58 or H53-H65 (VH-CDR2); and / or H93-H102 (VH-CDR3).

[0113] In certain embodiments, the antigen binding portion binds to EGFR and comprises a light chain or fragment thereof having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 88 and a heavy chain or fragment thereof having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 89. In certain embodiments, the antigen binding domain comprises CDR1, CDR2 and CDR3 from SEQ ID NO:88 and CDR1, CDR2 and CDR3 from SEQ ID NO:89.

[0114] In certain embodiments, the antigen binding portion binds to EGFR and comprises a light chain or fragment thereof having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 90 and a heavy chain or fragment thereof having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 91. In certain embodiments, the antigen binding domain comprises CDR1, CDR2 and CDR3 from SEQ ID NO: 90 and CDR1, CDR2 and CDR3 from SEQ ID NO:91.

[0115] In certain embodiments, the antigen binding portion binds to c-MET and comprises a light chain or fragment thereof having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 92 and a heavy chain or fragment thereof having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 93. In certain embodiments, the antigen binding domain comprises CDR1, CDR2 and CDR3 from SEQ ID NO: 92 and CDR1, CDR2 and CDR3 from SEQ ID NO:93.

[0116] In one embodiment, the antigen binding portion binds to GPC3 and comprises a light chain or fragment thereof having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 94 and a heavy chain or fragment thereof having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 95. In one embodiment, the antigen binding domain comprises CDR1, CDR2 and CDR3 from SEQ ID NO: 94 and CDR1, CDR2 and CDR3 from SEQ ID NO:95.

[0117] In certain embodiments, the antigen binding portion binds to 5T4 and comprises a light chain variable domain having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 98 or 99 and a heavy chain variable domain having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 96 or 97, or a fragment thereof. In certain embodiments, the antigen binding domain comprises CDR1, CDR2 and CDR3 from SEQ ID NO: 98 or 99 and CDR1, CDR2 and CDR3 from SEQ ID NO: 96 or 97.

[0118] In one embodiment, the antigen binding portion binds to Trop-2 and comprises a light chain variable region comprising a CDR1 comprising the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 125), a CDR2 comprising the amino acid sequence of SASYRYT (SEQ ID NO: 126), and a CDR3 comprising the amino acid sequence of QQHYITPLT (SEQ ID NO: 127); and a heavy chain variable region comprising a CDR1 comprising the amino acid sequence of NYGMN (SEQ ID NO: 128), a CDR2 comprising the amino acid sequence of WINTYTGEPTYTDDFKG (SEQ ID NO: 129), and a CDR3 comprising the amino acid sequence of GGFGSSYWYFDV (SEQ ID NO: 130).

[0119] 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: 131), a CDR2 comprising the amino acid sequence of DTSKLAS (SEQ ID NO: 132), and a CDR3 comprising the amino acid sequence of QQWSGYPLT (SEQ ID NO: 133); and a heavy chain variable region comprising CDR1 comprising the amino acid sequence of GYTMN (SEQ ID NO: 134), a CDR2 comprising the amino acid sequence of LITPYNGASSYNQKFRG (SEQ ID NO: 135), and a CDR3 comprising the amino acid sequence of GGYDGRGFDY (SEQ ID NO: 136).

[0120] In some embodiments, the antigen-binding portion comprises one, two or three antigen-binding domains.For example, the antigen-binding portion can be bispecific and bind 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 portion can bind to two different epitopes of the same antigen.For example, the bispecific antibody can bind to two different epitopes of HER2.

[0121] 2. Other carrier parts Other non-antigen-binding carrier moieties may be used in the prodrug, for example, 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.

[0122] As an example, an IL-15 polypeptide and a Sushi domain and an IL-15 antagonist can be fused to an antibody Fc domain to form an Fc fusion protein. In one embodiment, a Sushi domain is fused to the C-terminus or N-terminus of one of the heavy chains of the Fc domain as desired, an IL-15 polypeptide is fused to the C-terminus or N-terminus of the Sushi domain via a non-cleavable linker, and a masking moiety is fused to the C-terminus or N-terminus of the other heavy chain of the Fc domain via a cleavable peptide or a non-cleavable linker. In one embodiment, each of the heavy chains of the Fc domain contains a mutation that allows them to pair. In one embodiment, the mutation can be a knob-into-hole, YTE and / or LALA mutation.

[0123] The carrier moiety of the prodrug may comprise albumin (e.g., human serum albumin) or a fragment thereof. In some 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.

[0124] In some embodiments, the carrier portion comprises an albumin fragment (e.g., a human serum albumin fragment) that is about 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 120 or more, 140 or more, 160 or more, 180 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, or 550 or more amino acids in length. In some embodiments, the albumin fragment is about 10 amino acids 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). In certain embodiments, the albumin fragment comprises a Sudlow I domain or a fragment thereof or a Sudlow II domain or a fragment thereof.

[0125] D. Prodrug Linker Moieties The IL-15 polypeptide and the Sushi domain 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: 11-16. In a specific embodiment, the peptide linker comprises the amino acid sequence GGGSGGGGSGGGGS (SEQ ID NO: 13). In certain embodiments, the IL-15 polypeptide (A) is fused to the Sushi domain (S) via a peptide linker. The peptide linker can be at least 25, 30 or 35 amino acids in length. In certain embodiments, the peptide linker can be 25-45 amino acids. In other embodiments, the peptide linker has 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acids. In certain embodiments, the linker comprises the amino acid sequence GSAGSAAGSGEF (SEQ ID NO: 138). In certain embodiments, the linker comprises the amino acid sequence (GGGGS) n1 GSAGSAAGSGEF(GGGGS) n2(SEQ ID NO: 139) (wherein n1=1, 2 or 3 and n2=1, 2 or 3). In one embodiment, the linker comprises the amino acid sequence (GGGGS) n1 AA(GGGGS) n2 (SEQ ID NO: 140) (wherein n1=2 or 3 and n2=2 or 3).

[0126] The masking moiety may be fused to the carrier via a cleavable linker. The cleavable linker may include 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, MMP-2, MMP-9, 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: 17-35 and 36.

[0127] In certain embodiments, the IL-15 prodrug of the invention comprises an IL-15 receptor alpha Sushi domain (S) fused to an IL-15 polypeptide via a peptide linker. In certain embodiments, the peptide linker comprises at least 20 amino acids, 25 amino acids, at least 30 amino acids, at least 35 amino acids, or at least 40 amino acids; or 27 amino acids, 32 amino acids, 37 amino acids, 42 amino acids, or 47 amino acids.

[0128] II. Examples of IL-15 Prodrugs In certain embodiments, the activatable IL-15 prodrug has a molecular structure shown in any of Figures 1A-1C and 2A-2C. In certain embodiments, the IL-15 prodrug has a molecular structure shown in any of Figures 1B or 2B. In certain embodiments, the IL-15 prodrug comprises a structure shown in any of Figures 3A-3C. In certain embodiments, the IL-15 prodrug comprises a structure shown in Figure 3B. In certain embodiments, the carrier moiety is an antibody comprising one antigen-binding moiety as shown in Figures 4A, 4B, 5A or 5B. In preferred embodiments, the IL-15 prodrug comprises a structure selected from Figures 4B and 5B.

[0129] The IL-15 prodrug may not contain any of the Sushi domains or functional analogs thereof. In certain embodiments, the IL-15 prodrug comprises an IL-15 polypeptide comprising one or more mutations at one or more positions selected from E46, V49, L45, S51 and L52 (numbered according to SEQ ID NO:2). In certain embodiments, the IL-15 polypeptide comprises the mutation E46K (numbered according to SEQ ID NO:2). In other embodiments, the IL-15 polypeptide comprises the mutations E46K / N65D (numbered according to SEQ ID NO:2). In yet other embodiments, the IL-15 polypeptide comprises the mutations E46K / Q108E (numbered according to SEQ ID NO:2).

[0130] In certain embodiments, the IL-15 prodrug of the present invention comprises an IgG1 Fc domain as a carrier moiety. For example, the IL-15 prodrug can be selected from Table 2. In other embodiments, the IL-15 prodrug of the present invention comprises an IgG4 Fc domain. For example, the IL-15 prodrug can be selected from Table 3. In certain embodiments, the IL-15 prodrug of the present invention comprises an antibody that binds to human PD-L1 as a carrier moiety. For example, the IL-15 prodrug can be selected from Table 4. In certain embodiments, the IL-15 prodrug of the present invention comprises an antibody that binds to human PD-1 as a carrier moiety. For example, the IL-15 prodrug can be selected from Table 5. [Table 2] [Table 3] [Table 4] [Table 5]

[0131] Specific non-limiting examples of IL-15 polypeptides, Sushi domains, cytokine antagonists / masks, carriers, peptide linkers and prodrugs are shown in the sequence section below. Furthermore, the prodrugs of the present invention can be produced by well-known recombinant technology. As an example, a further expression vector containing the coding sequence of the polypeptide chain of the prodrug can be transfected into a mammalian host cell (e.g., CHO cell), and the cell can be cultured under conditions that allow the expression of the coding sequence and the assembly of the expressed polypeptide into a prodrug complex. Host cells that do not express or only slightly express uPA, MMP-2 and / or MMP-9 can be used so that the prodrug remains inactive. In some embodiments, the host cell can contain null mutations (knockouts) of the genes of these proteases.

[0132] III. Pharmaceutical Compositions Pharmaceutical compositions containing the prodrugs and muteins (i.e., drug substances or APIs) of the invention can be prepared by mixing the API of the desired purity in the form of a lyophilized formulation or an aqueous solution with one or more optional pharma- ceutically acceptable additives (see, e.g., Remington's Pharmaceutical Sciences, 16th Edition., Osol, A. Ed. (1980)). Pharmaceutically acceptable additives (or carriers) are non-toxic to recipients at commonly used dosages and concentrations, and include, for example, buffers including phosphate, citric acid, 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, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight ( Examples of suitable surfactants include, but are not limited to, polypeptides (fewer than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including sucrose, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0133] Buffers are used to control pH within a range that optimizes therapeutic efficacy, especially when stability is pH dependent. Buffers are preferably present at a concentration ranging from about 50 mM to about 250 mM. Suitable buffers for use in the present invention include both organic and inorganic acids and their salts, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, and acetate. Additionally, buffers may include trimethylamine salts, such as histidine and Tris.

[0134] Preservatives are added to retard microbial growth and are generally present in the range of 0.2% to 1.0% (w / v). Suitable preservatives for use in the present invention include octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0135] Tonicity agents, sometimes known as "stabilizers", are present to adjust or maintain the tonicity of the liquid in the composition. When used with large charged biological materials such as proteins and antibodies, they are often referred to as "stabilizers" because they may interact with the charged groups of the amino acid side chains, thereby reducing the possibility of inter- and intra-molecular interactions. Tonicity agents may be present in amounts anywhere from 0.1% to 25% by weight or more, preferably 1% to 5% by weight, taking into account the relative amounts of other components. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0136] The non-ionic surfactant or detergent (also known as a "wetting agent") is present to aid in solubilization of the therapeutic agent, to protect the therapeutic protein against agitation-induced aggregation, and also to allow the formulation to be exposed to shear surface stresses without causing denaturation of 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.

[0137] Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), and pluronics. (登録商標) Polyol, Triton (登録商標) , polyoxyethylene sorbitan monoether (Tween (登録商標) -20, Tween (登録商標) -80, 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.

[0138] The pharmaceutical carrier, excipient, or diluent of choice can be selected according to the intended route of administration and standard pharmaceutical techniques. The pharmaceutical composition can further comprise any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), or solubilizing agent(s).

[0139] Different delivery systems may have different composition / formulation conditions.As an example, the pharmaceutical compositions useful in the present invention may be formulated to be administered by mucosal route, for example, by nasal spray or aerosol for inhalation or ingestible solution, or parenterally, where the composition is formulated in an injectable form, for example, for delivery by intravenous, intramuscular or subcutaneous route.

[0140] In some embodiments, the pharmaceutical composition of the present invention is a lyophilized protein formulation. In other embodiments, the pharmaceutical composition may be an aqueous liquid formulation.

[0141] IV. Treatment IL-15 prodrugs can be used to treat diseases depending on the antigen that is bound by the antigen-binding domain.In some embodiments, IL-15 prodrugs are used to treat cancer.In some embodiments, IL-15 prodrugs are used to treat infections, for example, when the drug molecule is an antibacterial or antiviral agent.

[0142] In some embodiments, the method of treating a disease (e.g., cancer, viral infection, or bacterial infection) in a subject comprises administering to the subject an effective amount of an IL-15 prodrug. In other embodiments, the method of treatment further comprises administering an additional therapeutic agent in combination with (before, after, or simultaneously with) the IL-15 prodrug. The additional agent can be an antibody or fragment thereof, a small molecule drug, or other type of therapeutic drug, some of which are disclosed herein.

[0143] In some embodiments, cancer is solid cancer.In some embodiments, cancer is blood cancer or solid tumor.The examples of cancer that can be treated include but are not limited to leukemia, lymphoma, kidney cancer, bladder cancer, urinary tract cancer, cervical cancer, brain cancer, head and neck cancer, skin cancer, uterine cancer, testicular cancer, esophageal cancer, liver cancer, colorectal cancer, gastric cancer, squamous cell carcinoma, prostate cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer, cholangiocarcinoma, breast cancer and ovarian cancer.

[0144] In some embodiments, IL-15 prodrugs are used to treat bacterial infections, such as sepsis. In some embodiments, the bacteria causing the bacterial infection are drug-resistant bacteria. In some embodiments, the antigen-binding moiety binds to a bacterial antigen.

[0145] In some embodiments, IL-15 prodrugs are used to treat viral infection.In some embodiments, the virus causing viral infection is Hepatitis C (HCV), Hepatitis B (HBV), Human Immunodeficiency Virus (HIV), Human Papilloma Virus (HPV).In some embodiments, the antigen-binding portion binds to viral antigen.

[0146] Generally, the dosage and route of administration of the pharmaceutical composition is determined according to the physique and condition of the subject according to standard pharmaceutical practice.In some embodiments, the pharmaceutical composition is administered to the subject via any route, including oral, transdermal, inhalation, intravenous, intraarterial, intramuscular, wound site specific application, surgical site application, intraperitoneal, suppository, subcutaneous, intradermal, transdermal, aerosol, intrapleural, intraventricular, intraarticular, intraocular, intracranial or intrathecal.In some embodiments, the composition is administered to the subject intravenously.

[0147] In some embodiments, the dosage of the pharmaceutical composition is a single dose or multiple doses.In some embodiments, the dosage is administered to the subject once a day, twice a day, three times a day, or four times a day or more.In some embodiments, about one or more doses (for example, about 2, 3, 4, 5, 6 or 7 or more) are administered per week.In some embodiments, the pharmaceutical composition is administered every week, once every two weeks, once every three weeks, once every four weeks, every two weeks out of three weeks, or every three weeks out of four weeks.In some embodiments, multiple doses are administered over several days, weeks, months, or years.In some embodiments, the course of treatment is about one or more doses (for example, about 2, 3, 4, 5, 7, 10, 15, or 20 or more doses).

[0148] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall 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 may also be used in the practice or testing of the present invention. In case of conflict, the present specification, including definitions, shall control. In general, the terms used in connection with and in the techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly practiced in the art or as described herein. Furthermore, singular terms include plurals and plural terms include the singular, unless the context requires otherwise. Throughout the specification and embodiments, the terms "having" and variations thereof, such as "including" or "having", "containing", "including" or "comprising", are understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. The embodiments and variations of the invention described herein are understood to include embodiments and variations that "consist of" and / or "consist essentially of". All publications and other references cited herein are incorporated herein by reference in their entirety. Although a number of references are cited herein, this citation is not an admission that any of these references form part of the common general knowledge in the art. EXAMPLES

[0149] Transient transfection of HEK293 cells Expression plasmids were diluted to 3 × 10 at 2.5–3 μg / ml using polyethyleneimine (PEI). 6cells / ml freestyle HEK293 cells were co-transfected. For Fc-based IL-15 prodrugs, the Fc-IL-15 mutein fusion polypeptide and the Fc-masking moiety fusion polypeptide were in a 1:2 ratio. For antibody-based IL-15 prodrugs, the knob heavy chain (containing the IL-15 polypeptide), hole heavy chain (containing the masking moiety) and light chain DNA were in a 2:1:2 molar ratio. Cell cultures were harvested 6 days after transfection by centrifugation at 9,000 rpm for 45 minutes followed by 0.22 μM filtration.

[0150] Protein purification Fc and antibody-based IL-15 fusion polypeptides are generally purified by Protein A affinity chromatography followed by ion exchange, hydrophobic interaction and / or size exclusion chromatography. In one example, the protein purification of an antibody-based IL-15 prodrug was performed using 1) Protein A affinity chromatography; 2) Capto chromatograph operated in flow-through mode. TM Adhere;3) Capto TM The analysis was carried out using four chromatography steps including MMC ImpRes and 4) Q Sepharose® HP operated in flow-through mode. TM The Adhere was equilibrated with a buffer containing 50 mM acetic acid, 30 mM NaCl (pH 5.5). TM MMC ImpRes was equilibrated using Buffer A (50 mM acetate, 30 mM NaCl, pH 5.5) and eluted using a 30 CV linear gradient of Buffer B (50 mM acetate, 0.5 M arginine, pH 5.5). Q Sepharose® HP was equilibrated with 40 mM Bis Tris, pH 6.5.

[0151] SEC-HPLC analysis SEC-HPLC was performed using an Agilent 1100 Series HPLC system equipped with a TSKgel® G3000SWXL column (7.8 mm ID x 30 cm, 5 μm particle size) from Tosoh Bioscience. Up to 100 μl of sample was loaded. The column was run with a buffer containing 200 mM K3PO4, 250 mM KCl, pH 6.5. The flow rate was 0.5 ml / min. The column was run at room temperature. Protein elution was monitored at both 220 nm and 280 nm. SDS-PAGE analysis

[0152] 10 μl of culture supernatant or 20 μg of purified protein sample was incubated with or without reducing agent in Bolt TM The samples were mixed with LDS sample buffer (Novex). The samples were heated at 70°C for 3 min and then NuPAGE TM The gels were loaded onto 4-12% BisTris Gels (Invitrogen). TM The sections were run in MOPS SDS running buffer (Invitrogen) at 200 volts for 40 minutes and then stained with Coomassie.

[0153] Proteolytic processing 1 μg of protease, human MMP-2 (R&D systems), human MMP-9 (R&D systems), mouse MMP-2 (R&D systems) or mouse MMP-9 (R&D systems), was added to 50 μg of precursor protein and incubated overnight at 37°C.

[0154] 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 are non-adherent and were grown at 5 × 10 4 ~1×10 6Cells were maintained at 1000 cells / ml. Generally, cells were split twice a week. For bioassays, cells were best used 48 hours or later after passaging.

[0155] Samples were diluted to 2x concentration in 50μl / well in 96-well plates. IL-15 standards were titrated in 12 wells in 3x serial dilutions starting at 20ng / ml (2x concentration). Samples were titrated accordingly. CTLL2 cells were washed 5 times to remove IL-15, dispensed at 5000 cells / well in 50μl, and incubated with samples overnight or for at least 18 hours. 100μl / well Cell Titer Glo reagent (Promega) was then added and luminescence was measured.

[0156] NK92 proliferation assay The NK92 cell proliferation assay was performed according to the following protocol.

[0157] The NK92 cell line is a factor-dependent cell line that requires IL-2 for growth and survival. Prior to the assay, cells are washed to remove IL-2 and cultured overnight in the absence of growth factors. Cells are harvested and washed again to remove residual growth factors. Cells (20,000 / well) are then added to a 96-well plate containing serial dilutions of test articles and controls. Plates are incubated overnight, Cell Titer Glo (Promega) is added, and luminescence is measured. This provides a measurement of ATP levels as an indicator of cell viability.

[0158] Assays were performed using several IL-2Rβ extracellular domains (ECDs), IL-2Rβ ECD and IL-2Rγ ECD and an IL-15 prodrug masked with an scFv molecule derived from the IL-15 antibody 146B7.

[0159] pSTAT5 analysis NK92 / pSTAT5 stable cell lines were starved overnight in RPMI 1640 medium supplemented with 0.1% FBS. 5 × 10 5The cells were seeded with 100 μl of Pierce IL-15 fusion polypeptide and incubated overnight at 37° C. and 5% CO2. The cells were then added with IL-15 fusion polypeptide and incubated in the incubator for 5-6 hours. TM Firefly Luc One-Step Glow assay solution was added and bioluminescence was measured in a luminometer.

[0160] Enzyme-linked immunosorbent assay (ELISA) IL-15 fusion protein at 10 μg / ml in PBS was seeded at 100 μl / well in 96-well plates and coated overnight at 4°C. Wells were washed 3 times with PBS and blocked with 100 μl 2% milk / PBS for 1 h. Wells were then washed 3 times with PBS and 100 μl protein samples in 3-fold serial dilutions were added with 1 h incubation at room temperature (RT). After washing 3 times with PBS, 100 μl of HRP-conjugated anti-IgG antibody was added and incubated for 1 h at RT. Wells were then washed again 3 times with PBS followed by addition of detection reagent and optical density (OD) was measured at 450 nm.

[0161] Example 1: Expression and testing of IL-15 prodrugs A number of prodrugs were constructed and recombinantly expressed in HEK293 cells (see Figures 6A and 10A). In the IL-15 prodrugs, the IL-15 polypeptide was expressed as part of a fusion polypeptide and tested for its biological activity. Some of the sequences of the expressed IL-15 fusion polypeptides are listed in Figures 6A, 10A, and 12A.

[0162] The expressed IL-15 fusion polypeptide was examined by SDS-PAGE before and after activation (FIG. 7A; non-reduced; and FIG. 7B; reduced). The data showed that the masking moiety in JR3.68.1, JR3.68.2 and JR3.68.3 samples was successfully cleaved by protease treatment.

[0163] Example 2: Purification of activatable IL-15 prodrug moieties The activatable IL-15 prodrugs JR3.68.1, JR3.68.2 and JR3.68.3 were purified on a Protein A column and analyzed using SEC-HPLC. JR3.68.1 (Figure 1A) has a Sushi domain fused via a peptide linker to the C-terminus of one of the heavy chains of the Fc domain, an IL-15 polypeptide is fused via a peptide linker to the C-terminus of the Sushi domain, and a masking moiety (IL2Rβ ECD) is fused to the C-terminus of the other heavy chain of the Fc domain. JR3.68.2 is shown in Figure 1B and JR3.68.3 is shown in Figure 1C.

[0164] It was surprising that the format, configuration, relative position or conformation of several components of the prodrug molecule significantly affected the level of drug aggregation when purified on a Protein A affinity column. It was clear that Fc-Sushi-IL-15 (containing two polypeptide chains SEQ ID NO:37 and SEQ ID NO:38) (JR3.68.1) had significantly higher purity (evident by the higher main peak; FIG. 8A) and lower levels of aggregation when compared to the format of Fc-IL-15-Sushi (JR3.68.2, having two polypeptide chains SEQ ID NO:37 and SEQ ID NO:40) (FIG. 8B). At the same time, the formats in which the Sushi domain and cytokine are on different heavy chains of the Fc domain had better SEC-HPLC main peak purity than JR3.68.2 (JR3.68.3, FIG. 8C), but lower than JR3.68.1 (FIG. 8A). The trends were essentially the same when the carrier was an antibody (e.g., nivolumab, an antibody against human PD-1; FIG. 11B, JR3.73.2 vs. JR3.73.4).

[0165] It was also unexpectedly observed that the addition of the masking moiety significantly improved the purity of the fusion polypeptide. JR3.73.2 IL-15 prodrug with an antibody as the carrier moiety was observed to have a higher monomer purity by SEC-HPLC than the activated version JR3.74.1 (FIGS. 11A and 11B). The monomer peak of JR3.74.1 was also observed to have a significant shoulder (FIG. 11A), which may indicate a potential challenge for further purification.

[0166] Example 3: Cell-Based Activity of IL-15 Prodrugs CTLL2 assay The CTLL2 cell-based activity of IL-15 prodrugs JR3.68.1, JR3.68.2 and JR3.68.3 was determined before and after activation, as shown in Figures 9A-9C. The results show that JR3.68.1 was significantly activated after protease treatment. The cell-based activity of IL-15 prodrugs with antibodies as carrier moieties is shown in Figure 11C. The results show that IL-15 prodrug JR3.73.2 was activatable.

[0167] NK92 assay NK92 cell proliferation assays were also performed with several IL-15 prodrugs masked with scFv molecules (derived from IL-15 antibody 146B7), IL-2Rβ ECD, or IL-2Rβ ECD and IL-2Rγ ECD. NK92 proliferation assay results for IL-15 prodrugs masked with scFv1 or scFv2 of IL-15 antibody 146B7 show that both scFv2 and scFv1 significantly masked the activity of IL-15 WT and IL-15 mutein with the N65D mutation (Figure 12B).

[0168] The NK92 cell-based activity of the activatable IL-15 fusion polypeptides before and after activation was determined using the pSTAT5 method. Figure 13A shows that both scFv2 and scFv1 masked wild-type IL-15 to the same extent, and the fusion polypeptides were activatable by protease treatment. Figure 13B shows that scFv2 significantly masked the activity of the IL-15 mutein N65D. The results also show that scFv1 efficiently masked the IL-15 mutein. It was unexpected that both IL-15 prodrugs were activatable in vitro by protease treatment without further purification to remove cleaved scFv molecules. It was also surprising that scFv2 had a significantly stronger masking effect on the IL-15 mutein N65D than scFv1.

[0169] The NK92 cell-based activity of additional activatable IL-15 fusion polypeptides masked with IL-2Rβ ECD or IL-2Rβ ECD and IL-2Rγ ECD was determined. In these fusion polypeptides, wild-type IL-15 was masked with IL-2Rβ ECD and IL-2Rγ ECD. The results show that IL-2Rβ ECD in combination with IL-2Rγ ECD formed an effective mask against wild-type IL-15, and the IL-15 prodrug was activatable by protease treatment (Figure 14A). The activity of the IL-15 mutein Q108E (which was activatable by protease treatment) was also masked by IL-2Rβ ECD and IL-2Rγ ECD (Figure 14C).

[0170] The results of NK92 cell-based assays of an activatable Fc-IL-15 fusion polypeptide lacking a Sushi domain (JR2.145.1) and an activatable Fc-IL-15 fusion protein with a long linker between the Sushi domain and the IL-15 polypeptide moiety (JR2.145.2) were also determined. The data showed significant masking of the IL-15 mutein N65D in both. The results showed that the scFv2 mask was effective in masking the IL-15 polypeptide in the absence of a Sushi domain. The masking domain also worked well when the linker between the Sushi domain and the IL-15 polypeptide was long (32 amino acids).

[0171] The above non-limiting examples are provided for illustrative purposes only to facilitate a more complete understanding of the subject matter of the present disclosure, and should not be construed as limiting any of the embodiments described herein, including those relating to the antibodies, pharmaceutical compositions or methods and uses for the treatment of cancer, neurodegenerative or infectious diseases. Furthermore, the present invention includes the following aspects. 1. A prodrug comprising an IL-15 cytokine moiety (A), a masking moiety (M), a carrier moiety (C) and a Sushi domain (S), wherein: the masking moiety binds to the IL-15 cytokine moiety and inhibits the biological activity of the IL-15 cytokine moiety; The masking portion is fused to the carrier portion, The Sushi domain is fused to a carrier moiety, and The IL-15 cytokine is fused to the Sushi domain. Prodrug. 2. a masking moiety is fused to a carrier moiety via a first peptide linker; the Sushi domain is fused to a carrier moiety via a second peptide linker; and the IL-15 cytokine is fused to the Sushi domain via a third peptide linker, wherein at least one of the three peptide linkers is cleavable; A prodrug of item 1. 3. The prodrug of paragraph 2, wherein the third peptide linker is at least 15, 20, 25 or 30 amino acids in length, optionally wherein the third peptide linker comprises SEQ ID NO: 139 or 140. 4. A prodrug comprising an IL-15 cytokine moiety (A), a masking moiety (M), a carrier moiety (C) and a Sushi domain (S), wherein: the masking moiety binds to the IL-15 cytokine moiety and inhibits the biological activity of the IL-15 cytokine moiety; The IL-15 cytokine moiety is fused to a carrier moiety, The Sushi domain is fused to a carrier moiety, and The masking moiety is fused to the Sushi domain. Prodrug. 5. The IL-15 cytokine moiety is fused to a carrier moiety via a first peptide linker; the Sushi domain is fused to a carrier moiety via a second peptide linker; and the masking moiety is fused to the Sushi domain via a third peptide linker, optionally wherein at least one of the three peptide linkers is cleavable; A prodrug of item 4. 6. The prodrug of any of paragraphs 1 to 5, wherein the masking moiety comprises the extracellular domain (ECD) of the receptor for the IL-15 cytokine moiety. 7. The prodrug of paragraph 6, wherein the masking moiety comprises the ECD of human IL-2Rβ or a functional analogue thereof and / or the ECD of human IL-2Rγ or a functional analogue thereof. 8. The prodrug of paragraph 7, wherein 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 thereto. 9. The prodrug of paragraph 7, 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 at least 90% identical thereto. 10. The prodrug of any of paragraphs 1 to 5, wherein the masking moiety comprises an antibody fragment that binds to the IL-15 cytokine moiety. 11. A prodrug comprising an IL-15 cytokine moiety (A), a masking moiety (M), a carrier moiety (C) and optionally a Sushi domain (S), wherein: the masking moiety comprises an antibody fragment that binds to the IL-15 cytokine moiety and inhibits a biological activity of the IL-15 cytokine moiety; and the masking moiety is fused to the carrier moiety, the IL-15 cytokine moiety or the Sushi domain via a peptide linker; Prodrug. 12. The prodrug of item 10 or 11, wherein the antibody fragment is an ScFv or Fab comprising heavy chain CDR1-3 and light chain CDR1-3 of an anti-IL-15 antibody selected from 146B7, 146H5, 404E4 and 404A8. 13. The prodrug of paragraph 10 or 11, wherein the antibody fragment comprises a heavy chain CDR (HCDR)1 comprising SEQ ID NO: 100, an HCDR2 comprising SEQ ID NO: 101, an HCDR3 comprising SEQ ID NO: 102 or 106, a light chain CDR (LCDR)1 comprising SEQ ID NO: 103, an LCDR2 comprising SEQ ID NO: 104 and an LCDR3 comprising SEQ ID NO: 105. 14. The prodrug of clause 10 or 11, wherein the antibody fragment comprises (i) a heavy chain variable domain comprising SEQ ID NO: 107, or an amino acid sequence that is at least 95% identical thereto, and a light chain variable domain comprising SEQ ID NO: 108 or 123, or an amino acid sequence that is at least 95% identical thereto; (ii) SEQ ID NO: 109; (iii) SEQ ID NO: 110; or (iv) SEQ ID NO: 124. 15. The prodrug of paragraph 13 or 14, wherein the Cys residue in the heavy chain CDR3 is mutated to Ser, Thr, Met, Ala, Gly, Asn, or Gln. 16. The prodrug of any of paragraphs 1 to 15, wherein the masking moiety does not interfere with or has a minimal effect on binding of the IL-15 cytokine moiety to IL-15Rα. 17. The prodrug of any of items 1 to 16, wherein the IL-15 cytokine moiety is a human IL-15 polypeptide comprising SEQ ID NO: 2 or a mutated protein thereof. 18. The human IL-15 polypeptide is selected from the group consisting of N1A, N1D, N4A, N4D, I6T, S7A, D8A, D8T, D8E, D8N, K10A, K10D, K11A, K11D, E46, V49, L45, S51, L52, D61A, D61N, T62L, T62A, E64A, E64L, E64K, E64Q, N65A, N65L, N65D, L66D, L66E, I67D, I67E, I68S, I68E, L69S, L69E, N72A, N72D, V63E, V63D, L66E, L66D, I 18. The prodrug of claim 17, comprising one or more mutations selected from 67E, I67D, Q108E, N112A, N1D / D61N, N1D / E64Q, N4D / D61N, N4D / E64Q, D8N / D61N, D8N / E64Q, D61N / E64Q, E64Q / Q108E, N1D / N4D / D8N, D61N / E64Q / N65D, N1D / D61N / E64Q, N1D / Q108E, N1D / D61N / E64Q / Q108E, N4D / D61N / E64Q / Q108E and D30N / E64Q / N65D. 19. The prodrug of any of paragraphs 1 to 18, wherein the carrier moiety is a PEG molecule, albumin, an albumin fragment, an antibody Fc domain, or an antibody or an antigen-binding fragment thereof. 20. The prodrug of paragraph 19, wherein the carrier moiety is an antibody Fc domain or an antibody comprising the mutations L234A and L235A ("LALA") (EU numbering). 21. The prodrug of paragraph 19 or 20, wherein the carrier moiety is an antibody Fc domain or an antibody containing a knob-into-hole mutation, wherein the IL-15 cytokine moiety and the masking moiety are fused to different polypeptide chains of the antibody Fc domain or different heavy chains of the antibody. 22. A knob-into-hole mutation comprises a T366Y "knob" mutation in one polypeptide chain of an Fc domain or one heavy chain of an antibody and a Y407T "hole" mutation in the other polypeptide chain of an Fc domain or the other heavy chain of an antibody, or Knob-into-hole mutations include 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); Item 21. A prodrug according to item 21. 23. The carrier moiety is IgG4 20. The prodrug of item 19, which is an Fc domain, wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to that set forth in SEQ ID NO: 80, 81 or 87, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to that selected from SEQ ID NOs: 82-86. 24. The prodrug of paragraph 19 or 20, wherein the carrier moiety is an anti-PD-1 antibody, comprising: a light chain having an amino acid sequence at least 99% identical to SEQ ID NO: 55 or 56; a first heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO: 54, 60 or 61; and a second heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO: 52, 53, 58, 59, 62, 63 or 69. 25. The prodrug of paragraph 19 or 20, wherein the carrier moiety is an anti-PD-1 antibody, comprising: a light chain having an amino acid sequence at least 99% identical to SEQ ID NO:55; a first heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO:66; and a second heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO:64, 65, 67, or 68. 26. The prodrug of clause 19 or 20, wherein the carrier moiety is an anti-PD-L1 antibody, comprising: a light chain having an amino acid sequence at least 99% identical to SEQ ID NO: 50 or 51; a first heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO: 47, 48 or 49; and a second heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO: 45 or 46. 27. The prodrug of paragraph 19 or 20, wherein the carrier moiety is an antibody or antigen-binding fragment thereof that specifically binds to one or more antigens selected from PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, CD47 and TIGIT. 28. The carrier moiety is an antibody Fc domain or an antibody and the prodrug is the following polypeptide pair (N-terminus to C-terminus): a) C1-A and C2-SM, b) A-C1 and MS-C2, c) C1-SA and C2-M, d) C1-AS and C2-M, e) SA-C1 and M-C2 or f) AS-C1 and M-C2 Including, 28. The prodrug of any of paragraphs 19 to 27, wherein C1 and C2 are the first and second polypeptide chains of an Fc domain, respectively, or the first and second polypeptide chains of an antibody, respectively; and "-" is a direct peptidyl bond or a peptide linker. 29. The prodrug of any of paragraphs 1 to 28, wherein the Sushi domain comprises an amino acid sequence set forth in SEQ ID NO: 7 or 9 or at least 90% identical thereto. 30. The prodrug of any of paragraphs 1 to 29, wherein at least one of the first, second and third peptide linkers is a non-cleavable peptide linker, optionally selected from SEQ ID NOs: 11 to 16. 31. The prodrug of any of paragraphs 1 to 30, wherein at least one of the first, second and third peptide linkers is a cleavable peptide linker containing a substrate sequence for urokinase-type plasminogen activator (uPA), matriptase, matrix metallopeptidase (MMP) 2 or MMP9. 32. The prodrug of paragraph 31, wherein the cleavable peptide linker comprises substrate sequences for (i) both uPA and MMP2, (ii) both uPA and MMP9, (iii) uPA, MMP2 and MMP9, or (iv) MMP2 and matriptase. 33. The prodrug of item 31, wherein the cleavable peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 17 to 36. 34. The prodrug of any of paragraphs 1 to 33, wherein the cleavable peptide linker is cleavable by one or more proteases located at the tumor site or in its surrounding environment, and cleavage results in activation of the prodrug at the tumor site or in its surrounding environment. 35. A pharmaceutical composition comprising the prodrug of any one of items 1 to 34 and a pharma- ceutically acceptable excipient. 36. A polynucleotide or polynucleotides encoding the prodrug of any one of items 1 to 34. 37. An expression vector or vectors comprising one or more polynucleotides of paragraph 36. 38. A host cell comprising the vector of paragraph 37. 39. The host cell of paragraph 38, wherein genes encoding uPA, matriptase, MMP-2 and / or MMP-9 are knocked out in the host cell. 40. A method for producing a prodrug according to any one of items 1 to 34, comprising the steps of: Cultivating the host cell of claim 38 or 39, which is a mammalian cell, under conditions that allow expression of the prodrug; and and isolating the prodrug. method. 41. A method for treating cancer or an infectious disease or stimulating the immune system in a patient in need of treatment, comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of paragraph 35. 42. An IL-15 prodrug for use in a method for treating cancer or an infectious disease or stimulating the immune system according to paragraph 41. 43. Use of an IL-15 prodrug for the manufacture of a medicament for use in a method for treating cancer or an infectious disease or stimulating the immune system according to item 41. 44. The method of paragraph 41, the prodrug for use of paragraph 42 or the use of paragraph 43, wherein the patient has a viral infection or a cancer selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer and gastric cancer.

[0172] array In the sequence below, boxed residues indicate mutations and the underlined portion in the cleavable linker indicates the protease substrate sequence. [Table 6]

Table 7

[0173]

Table 8

Table 9

[0174]

Table 10

Table 11

[0175]

Table 12

Table 13

[0176]

Table 14

Table 15

[0177]

Table 16

Table 17

[0178]

Table 18

Table 19

[0179]

Table 20

Table 21

[0180]

Table 22

Table 23

[0181]

Table 24

Table 25

[0182]

Table 26

Table 27

Claims

1. A prodrug comprising an IL-15 cytokine moiety (A), a masking moiety (M), a carrier moiety (C) and a Sushi domain (S), wherein: the masking moiety comprises an anti-IL-15 scFv or the extracellular domain (ECD) of IL-2Rβ or IL-2Rγ; the masking moiety binds to the IL-15 cytokine moiety and inhibits the biological activity of the IL-15 cytokine moiety; Each of the masking moiety and the Sushi domain is fused to a carrier moiety; and The IL-15 cytokine portion is fused to a Sushi domain; Prodrug.

2. the masking moiety is fused to the carrier moiety via a first peptide linker; the Sushi domain is fused to a carrier moiety via a second peptide linker; and The IL-15 cytokine is fused to the Sushi domain via a third peptide linker, where at least one of the three peptide linkers is cleavable. The prodrug of claim 1.

3. The prodrug of claim 2, wherein the third peptide linker is at least 15, 20, 25 or 30 amino acids in length and contains one or more GGGGS (SEQ ID NO:11).

4. A prodrug comprising an IL-15 cytokine moiety (A), a masking moiety (M), a carrier moiety (C) and a Sushi domain (S), wherein: the masking moiety comprises an anti-IL-15 scFv or the extracellular domain (ECD) of IL-2Rβ or IL-2Rγ; the masking moiety binds to the IL-15 cytokine moiety and inhibits the biological activity of the IL-15 cytokine moiety; each of the IL-15 cytokine portion and the Sushi domain is fused to a carrier portion; and The masking portion is fused to the Sushi domain. Prodrug.

5. the IL-15 cytokine moiety is fused to a carrier moiety via a first peptide linker; the Sushi domain is fused to a carrier moiety via a second peptide linker; and the masking moiety is fused to the Sushi domain via a third peptide linker, where at least one of the three peptide linkers is cleavable; The prodrug of claim 4.

6. A prodrug comprising an IL-15 cytokine portion (A), a masking portion (M), a carrier portion (C) and a Sushi domain (S), wherein: the masking moiety comprises an anti-IL-15 scFv or the extracellular domain (ECD) of IL-2Rβ or IL-2Rγ; the masking moiety binds to the IL-15 cytokine moiety and inhibits the biological activity of the IL-15 cytokine moiety; and The masking moiety is fused to the carrier moiety, the IL-15 cytokine moiety or the Sushi domain via a peptide linker; Prodrug.

7. The prodrug of any one of claims 1 to 6, wherein the masking moiety comprises the ECD of human IL-2Rβ and / or the ECD of human IL-2Rγ.

8. 8. The prodrug of claim 7, wherein the ECD of human IL-2Rγ comprises SEQ ID NO:6 or an amino acid sequence at least 90% identical thereto.

9. 8. The prodrug of claim 7, wherein the ECD of human IL-2Rβ comprises SEQ ID NO: 3, 4 or 5, or an amino acid sequence at least 90% identical thereto.

10. A prodrug of any of claims 1 to 6, wherein the anti-IL-15 scFv comprises a heavy chain CDR (HCDR) 1 comprising SEQ ID NO: 100, a HCDR2 comprising SEQ ID NO: 101, a HCDR3 comprising SEQ ID NO: 102 or 106, a light chain CDR (LCDR) 1 comprising SEQ ID NO: 103, a LCDR2 comprising SEQ ID NO: 104, and a LCDR3 comprising SEQ ID NO:

105.

11. A prodrug of any of claims 1 to 6 or 10, wherein the anti-IL-15 scFv comprises (i) a heavy chain variable domain comprising SEQ ID NO: 107 or an amino acid sequence at least 95% identical thereto, and a light chain variable domain comprising SEQ ID NO: 108 or 123 or an amino acid sequence at least 95% identical thereto; (ii) SEQ ID NO: 109; (iii) SEQ ID NO: 110; or (iv) SEQ ID NO:

124.

12. 12. The prodrug of claim 10 or 11, wherein the Cys residue in the heavy chain CDR3 is mutated to Ser, Thr, Met, Ala, Gly, Asn or Gln.

13. The prodrug of any of claims 1 to 12, wherein the masking moiety does not interfere with binding of the IL-15 cytokine moiety to IL-15Rα.

14. A prodrug of any of claims 1 to 13, wherein the masking moiety comprises an scFv that binds to IL-15 and does not interfere with the interaction of IL-15 with IL-2Rγ.

15. 17. The prodrug of any of claims 1-16, wherein the IL-15 cytokine moiety is a human IL-15 polypeptide or a human IL-15 polypeptide mutein comprising SEQ ID NO:

2.

16. The prodrug of claim 15, wherein the human IL-15 polypeptide mutein contains one or more mutations that reduce the interaction of IL-15 with IL-2Rβ.

17. Human IL-15 polypeptide muteins are selected from the group consisting of N1A, N1D, N4A, N4D, I6T, S7A, D8A, D8T, D8E, D8N, K10A, K10D, K11A, K11D, E46, V49, L45, S51, L52, D61A, D61N, T62L, T62A, E64A, E64L, E64K, E64Q, N65A, N65L, N65D, L66D, L66E, I67D, I67E, I68S, I68E, L69S, L69E, N72A, N72D, V63E, V63D, L66E, L66D 17. The prodrug of claim 16, comprising one or more mutations selected from: I67E, I67D, Q108E, N112A, N1D / D61N, N1D / E64Q, N4D / D61N, N4D / E64Q, D8N / D61N, D8N / E64Q, D61N / E64Q, E64Q / Q108E, N1D / N4D / D8N, D61N / E64Q / N65D, N1D / D61N / E64Q, N1D / Q108E, N1D / D61N / E64Q / Q108E, N4D / D61N / E64Q / Q108E and D30N / E64Q / N65D.

18. 18. The prodrug of any of claims 1 to 17, wherein the carrier moiety is a PEG molecule, albumin, an albumin fragment, an antibody Fc domain or an antibody or an antigen-binding fragment thereof.

19. 20. The prodrug of claim 18, wherein the carrier moiety is an antibody Fc domain or an antibody comprising the mutations L234A and L235A ("LALA") (Eu numbering).

20. 20. The prodrug of claim 18 or 19, wherein the carrier moiety is an antibody Fc domain or an antibody containing a knob-into-hole mutation, wherein the IL-15 cytokine moiety and the masking moiety are fused to different polypeptide chains of the antibody Fc domain or different heavy chains of the antibody.

21. the knob-into-hole mutation comprises a T366Y "knob" mutation in one polypeptide chain of the Fc domain or one heavy chain of the antibody and a Y407T "hole" mutation in the other polypeptide chain of the Fc domain or the other heavy chain of the antibody; or The knob-into-hole mutation comprises a Y349C and / or T366W mutation in the CH3 domain of the "knob strand" and an E356C, T366S, L368A and / or Y407V mutation in the CH3 domain of the "hole strand" (Eu numbering); 21. The prodrug of claim 20.

22. An IgG comprising a first polypeptide chain comprising an amino acid sequence at least 99% identical to that set forth in SEQ ID NO:80, 81 or 87, and a second polypeptide chain comprising an amino acid sequence at least 99% identical to that set forth in SEQ ID NO:82-86. ４ 20. The prodrug of claim 18 which is an Fc domain.

23. 20. The prodrug of claim 18 or 19, wherein the carrier moiety is an anti-PD-1 antibody comprising a light chain having an amino acid sequence at least 99% identical to SEQ ID NO:55 or 56; a first heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO:54, 60 or 61; and a second heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO:52, 53, 58, 59 or 69.

24. 20. The prodrug of claim 18 or 19, wherein the carrier moiety is an anti-PD-1 antibody comprising a light chain having an amino acid sequence at least 99% identical to SEQ ID NO:55; a first heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO:66; and a second heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO:67 or 68.

25. 20. The prodrug of claim 18 or 19, wherein the carrier moiety is an anti-PD-L1 antibody comprising a light chain having an amino acid sequence at least 99% identical to SEQ ID NO:50 or 51; a first heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO:47, 48 or 49; and a second heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO:45 or 46.

26. 20. The prodrug of claim 18 or 19, wherein the carrier moiety is an antibody or antigen-binding fragment thereof that specifically binds to one or more antigens selected from PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, CD47, and TIGIT.

27. The carrier moiety is an antibody Fc domain or an antibody, and the prodrug is the following polypeptide pair (N-terminus to C-terminus): a) C1-A and C2-S-M; b) A-C1 and M-S-C2; c) C1-S-A and C2-M; d) C1-A-S and C2-M; e) S-A-C1 and M-C2, or f) A-S-C1 and M-C2 Including, 27. The prodrug of any of claims 18-26, wherein C1 and C2 are the first and second polypeptide chains of an Fc domain, respectively, or the first and second chains of an antibody, respectively; and "-" is a direct peptidyl bond or a peptide linker.

28. 28. The prodrug of any of claims 1 to 27, wherein the Sushi domain comprises an amino acid sequence of SEQ ID NO: 7 or 9 or at least 90% identical thereto.

29. 6. The prodrug of any of claims 2, 3 or 5, wherein at least one of the first, second and third peptide linkers is a non-cleavable peptide linker comprising an amino acid sequence selected from SEQ ID NOs: 11-16.

30. The prodrug of any of claims 2, 3 or 5, wherein at least one of the first, second and third peptide linkers is a cleavable peptide linker comprising a substrate sequence for urokinase-type plasminogen activator (uPA), matriptase, matrix metallopeptidase (MMP) 2 or MMP9.

31. The prodrug of claim 6, wherein the peptide linker is a cleavable peptide linker.

32. 32. The prodrug of claim 30 or 31, wherein the cleavable peptide linker comprises substrate sequences for (i) both uPA and MMP2, (ii) both uPA and MMP9, (iii) uPA, MMP2 and MMP9, or (iv) MMP2 and matriptase.

33. The prodrug of claim 32, wherein the cleavable peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 17-36.

34. 32. The prodrug of claim 30 or 31, wherein the cleavable peptide linker is cleavable by one or more proteases located at the tumor site or its surrounding environment, and cleavage results in activation of the prodrug at the tumor site or its surrounding environment.

35. The prodrug of claim 1, comprising a first polypeptide having SEQ ID NO:122 without a signal peptide and a second polypeptide having SEQ ID NO:119 without a signal peptide.

36. The prodrug of claim 1, comprising a first polypeptide having SEQ ID NO:79 without a signal peptide and a second polypeptide having SEQ ID NO:112 without a signal peptide.

37. The prodrug of claim 1, comprising a light chain having the amino acid sequence of SEQ ID NO:55; a first heavy chain having the amino acid sequence of SEQ ID NO:66; and a second heavy chain having an amino acid sequence that is at least 99% identical to sequence number 68.

38. A pharmaceutical composition comprising the prodrug of any one of claims 1 to 37 and a pharma- ceutically acceptable excipient.

39. One or more polynucleotides encoding the prodrug of any of claims 1 to 37.

40. 40. An expression vector or vectors comprising one or more polynucleotides of claim 39.

41. A host cell comprising the vector of claim 40.

42. 42. The host cell of claim 41, wherein the genes encoding uPA, matriptase, MMP-2 and / or MMP-9 are knocked out in the host cell.

43. A method for producing a prodrug according to any one of claims 1 to 37, comprising the steps of: Cultivating the host cell of claim 41 or 42, which is a mammalian cell, under conditions that allow expression of the prodrug; and and isolating the prodrug. method.

44. 39. The pharmaceutical composition of claim 38 for treating cancer or an infectious disease or for stimulating the immune system.

45. 45. The pharmaceutical composition of claim 44, wherein the infection is a viral infection.

46. 45. The pharmaceutical composition of claim 44, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer and gastric cancer.

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