Masking polypeptides, novel activatable prodrugs, and methods of use thereof - Patent Application 20070122997

Abstract

The present invention provides masking polypeptides (MPs), cleavable moieties (CMs) and activatable prodrugs comprising the masking polypeptides and the cleavable moieties; isolated nucleic acid molecules encoding the masking polypeptides, the cleavable moieties or the activatable prodrugs; vectors comprising the nucleic acid molecules; host cells comprising the nucleic acid molecules or the vectors; pharmaceutical compositions comprising the activatable prodrugs, the isolated nucleic acid molecules, the vectors or the host cells; and methods of making and using the prodrugs or pharmaceutical compositions.

Description

[Technical Field]

[0001] This application relates to activatable prodrugs, such as cytokine or antibody prodrugs, comprising a masking polypeptide (MP), a cleavable moiety (CM), or the MP or / and CM, as well as methods for their preparation and use. The invention also relates to cleavage products of the activatable prodrugs and methods for their use.

[0002] (Submission of sequence listing) This application claims priority to U.S. Provisional Application No. 63 / 370,605, filed August 5, 2022, U.S. Provisional Application No. 63 / 370,606, filed August 5, 2022, U.S. Provisional Application No. 63 / 370,607, filed August 5, 2022, and U.S. Provisional Application No. 63 / 477,993, filed December 30, 2022, the contents of each of which are incorporated herein by reference in their entirety. The contents of the Electronic Sequence Listing (Masking Polypeptides, Novel Activatable Prodrugs and Methods of Use Thereof SEQ.xml, Size: 64KB, and Creation Date: 2022.08.05) are incorporated herein by reference in their entirety. [Background technology]

[0003] Although antibodies are considered ideal candidates for the treatment of cancer, autoimmune diseases, and chronic inflammatory diseases, antibody therapy can be limited by cross-reactivity with healthy tissues. Various methods have been described to improve tumor targeting by engineering antibodies to overcome these "off-target" effects, for example, by producing masking antibodies that are selectively activated in the tumor microenvironment (see WO2003 / 068934, WO2004 / 009638, WO2009 / 025846, WO2101 / 081173, and WO2014 / 103973).

[0004] Cytokines are considered very promising tumor therapeutic agents because they are potent immune agonists. For example, interleukin-15 (IL-15) is currently being investigated for its antitumor activity and is being used in human treatment. However, cytokines have a narrow therapeutic range and a short serum half-life. Therefore, therapeutic administration of cytokines can cause undesirable systemic effects and toxicity. Desired cytokine levels can only be achieved by administering large amounts of cytokines to the cytokine's intended site of action (e.g., tumor), which further exacerbates the above problems.

[0005] This application relates to conditionally activatable prodrugs (e.g., cytokine and antibody prodrugs) having a cleavable moiety linked to a masking polypeptide (MP) for the treatment of cancer or other diseases. The masking polypeptide (MP) may exert a steric hindrance effect on the biologically active moiety. The cleavable moiety can be designed to be cleaved by proteases in specific or pathological tissues, thereby allowing the prodrug to be preferentially activated at a desired site (e.g., tumor), thereby overcoming the dose-limiting effects of cytokines or the "off-target" effects of antibodies. Summary of the Invention

[0006] One aspect of the present application provides a synthetic masking polypeptide (MP) consisting of four or five amino acid residues selected from the group consisting of proline (P), alanine (A), serine (S), glycine (G), and glutamic acid (E).

[0007] In some embodiments, the masking polypeptide (MP) consists of five types of amino acids: G, S, P, E, and A; further, in the masking polypeptide, the percentage of amino acid residue G is about 15% to 30%, preferably 20%; in the masking polypeptide, the percentage of amino acid residue S is about 20% to 40%, preferably 40%; in the masking polypeptide, the percentage of amino acid residue P is about 15% to 40%, preferably 20%; in the masking polypeptide, the percentage of amino acid residue E is about 1% to 20%, preferably 10%; and in the masking polypeptide, the percentage of amino acid residue A is about 5% to 20%, preferably 10%; and if the number of amino acids is not an integer, it takes an integer value.

[0008] In some embodiments, the masking polypeptide (MP) consists of four types of amino acids: S, P, E, and G; further, in the masking polypeptide, the percentage of amino acid residues S is about 20% to 40%, preferably 23%; in the masking polypeptide, the percentage of amino acid residues P is about 15% to 40%, preferably 29%; in the masking polypeptide, the percentage of amino acid residues E is about 1% to 20%, preferably 18%; and in the masking polypeptide, the percentage of amino acid residues G is about 15% to 30%, preferably 30%; and if the number of amino acids is not an integer, it takes an integer value.

[0009] In some embodiments, the masking polypeptide (MP) comprises between about 40 and 720 amino acid residues. In some embodiments, the masking polypeptide (MP) comprises between 80 and 320 amino acid residues. In some embodiments, the masking polypeptide (MP) comprises between 80 and 240 amino acid residues.

[0010] In some embodiments, the masking polypeptide (MP) comprises: (i) Forms only random coils lacking secondary structure; (ii) detected by size exclusion chromatography, which indicates a molecule larger than the expected molecular size; (iii) does not induce immunogenicity in the host or bind nonspecifically to serum proteins; and / or (iv) It has the property of being stable in a buffer solution or plasma.

[0011] In some embodiments, the masking polypeptide (MP) comprises the amino acid sequence SEQ ID NO:6.

[0012] In some embodiments, the masking polypeptide (MP) comprises the amino acid sequence SEQ ID NO:1.

[0013] In some embodiments, the masking polypeptide (MP) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-5 or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-5.

[0014] Another aspect of the present application provides a cleavable moiety (CM) comprising the amino acid sequence MVX1X2AX3TX4SG (SEQ ID NO: 49), wherein X1 is selected from P, L, V, or A, X2 is selected from L or S, X3 is selected from L, V, P, or Y, and X4 is selected from A or V.

[0015] In some embodiments, the cleavable moiety (CM) comprises a substrate sequence for urokinase plasminogen activator (uPA), matrix metalloproteinase (MMP) 1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, fibroblast activation protein (FAP), matrix protease, cathepsin, caspase, thrombin, metalloproteinase, serine protease, cysteine protease, aspartic protease, legumain, kallikrein, cathepsin A, cathepsin B, chymase, a protease located at the tumor site or its surrounding environment, or any combination thereof.

[0016] In some embodiments, the cleavable moiety (CM) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 8-16 or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 8-16.

[0017] Another aspect of the present application provides prodrugs comprising (i) one or more biologically active moieties (B), (ii) one or more cleavable moieties (CM), and (iii) one or more masking polypeptides (MP). In some embodiments, the masking polypeptides (MP) reduce the activity of the biologically active moieties (B). In some embodiments, the cleavable moieties (CM) are susceptible to cleavage at or near a tumor, target cell, or the like.

[0018] In some embodiments, in the prodrugs described herein, the masking polypeptide (MP) and the biologically active moiety (B) are linked by the cleavable moiety (CM).

[0019] In some embodiments, the prodrug further comprises a non-cleavable linker (L). In some embodiments, the non-cleavable linker (L) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 17-21.

[0020] In some embodiments, in the prodrugs described herein, the masking polypeptide (MP) is selected from any of the polypeptides described above. In some embodiments, the cleavable moiety (CM) is selected from any of the polypeptides described above.

[0021] In some embodiments, the biologically active moiety (B) is selected from a cytokine, an antigen-binding fragment or antibody, or a small molecule drug that has cytotoxic or cytostatic activity against tumor cells.

[0022] In some embodiments, the cytokine is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IFNα, IFNβ, IFNγ, TNFα, TNFβ1, TNFβ2, TNFβ3, lymphotoxin, GM-CSF, CXCL10, CCL19, CCL20, CCL21, or a variant of the above cytokines.

[0023] In some examples, the antigen-binding fragments or antibodies described herein specifically bind a tumor-associated antigen, and optionally, the antigen-binding fragments or antibodies specifically bind one or more antigens, wherein the antigens are selected from the group consisting of TNFR2, CTLA4, PD1, PDL1, LAG3, TIM3, BCMA, HER2, CEA, EGFR, VEGFR1, and VEGFR2.

[0024] In some embodiments, the prodrug further comprises one or more half-life extending moieties (C).

[0025] In some embodiments, the half-life extending moiety (C) comprises a serum protein or a molecule that binds to a serum protein, optionally wherein the serum protein is selected from the group consisting of fibronectin, transferrin, and human serum albumin (HSA).

[0026] In some embodiments, the half-life extending moiety (C) comprises a biocompatible polymer, optionally, the polymer is selected from PEG or hydroxyethyl starch.

[0027] In some embodiments, the half-life extending moiety (C) comprises an Fc domain, or an antibody comprising an Fc domain, or a fragment that is involved in FcRn-mediated recycling.

[0028] In some embodiments, in the prodrugs described herein, the biologically active moiety (B) is linked to the half-life extending moiety (C).

[0029] In some embodiments, in the prodrugs described herein, the masking moiety (MP) and the half-life extending moiety (C) are linked by the cleavable moiety (CM).

[0030] In some embodiments, antibody prodrugs are provided that include (i) one or more antibodies or antigen-binding fragments, (ii) one or more cleavable moieties (CMs), and (iii) one or more masking polypeptides (MPs).

[0031] In some embodiments, in the antibody prodrugs described herein, the masking polypeptide (MP) is selected from any of the polypeptides described above, and in some embodiments, the cleavable moiety (CM) is selected from any of the polypeptides described above.

[0032] In some embodiments, in the antibody prodrugs described herein, the antibody or antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)'2, Fab'-SH, single-chain Fv (scFv), Fv fragment, dAb, Fd, VHH, or diabody.

[0033] In some embodiments, in the antibody prodrugs described herein, the masking polypeptide (MP) and V H Domain and / or V L The N-terminus and / or C-terminus of the domains are linked by said cleavable moiety (CM).

[0034] In some embodiments, in the antibody prodrugs described herein, the antibody or antigen-binding fragment specifically binds TNFR2. In some embodiments, in the antibody prodrugs described herein, the V H comprises an HC-CDR1 comprising the amino acid sequence SEQ ID NO: 50, an HC-CDR2 comprising the amino acid sequence SEQ ID NO: 51, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO: 52, or any of the above Vs comprising at most about five amino acid substitutions in the HC-CDRs. H and variants of V L comprises an LC-CDR1 comprising the amino acid sequence SEQ ID NO: 53, an LC-CDR2 comprising the amino acid sequence SEQ ID NO: 54, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO: 55, or any of the above V CDRs containing at most about five amino acid substitutions in the LC-CDRs. L Includes variants of.

[0035] In some embodiments, in the antibody prodrugs described herein, the V H comprises the amino acid sequence SEQ ID NO: 56 or a variant thereof, wherein the variant has at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO: 56, and wherein the V Lcomprises the amino acid sequence SEQ ID NO:57 or a variant thereof, wherein the variant has at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:57.

[0036] In some embodiments, the antibody prodrug comprises two heavy chains and two light chains, wherein the heavy chain comprises the amino acid sequence SEQ ID NO:58 or SEQ ID NO:60, or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO:58 or SEQ ID NO:60, and the light chain comprises the amino acid sequence SEQ ID NO:59 or SEQ ID NO:61, or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO:59 or SEQ ID NO:61.

[0037] Also provided are isolated nucleic acid molecules encoding any of the masking polypeptides (MPs), cleavable moieties (CMs), or prodrugs provided herein, vectors containing the nucleic acid molecules, host cells (e.g., CHO cells, HEK293 cells, Hela cells, or COS cells) containing the nucleic acid molecules or vectors, compositions (e.g., pharmaceutical compositions), kits, and articles of manufacture comprising any of the masking polypeptides, cleavable moieties, or prodrugs provided herein. Also provided are methods of using any of the prodrugs provided herein, or pharmaceutical compositions thereof, to treat a disease (e.g., a tumor) in an individual (e.g., a human). [Brief explanation of the drawings]

[0038] [Figure 1A]The sequence alignment of the amino acid sequences MP80 and MP100 is shown, and the identical amino acid sequences between MP80 and MP100 are marked in boxes (the amino acid sequence marked in the first box is SEQ ID NO: 6). [Figure 1B] The alignment of the amino acid sequences of MP80 and MP163, which are repeated twice, is shown, and the amino acid sequence marked in the first box is the sequence of MP80, i.e., MP163 contains the amino acid sequence of MP80. [Figure 1C] The alignment of the three repeated amino acid sequences of MP80 and MP240 is shown, and the amino acid sequence marked in the first box is the MP80 sequence, i.e., MP240 contains three copies of the MP80 amino acid sequence. [Figure 2A] Figure 1 shows an exemplary IL-15 prodrug with an Fc domain as a half-life extending moiety, in which the IL-15Rα_sushi domain is linked to the C-terminus of one Fc domain, optionally via a non-cleavable linker. IL-15 is linked to the C-terminus of another Fc domain, optionally via a non-cleavable linker. The masking polypeptide (MP) and IL-15 are linked by a cleavable moiety (CM). [Figure 2B] FIG. 1 is an exemplary schematic diagram showing activation of an IL-15 prodrug in target tissue (eg, tumors with high levels of MMPs) via release of a masking polypeptide (MP). [Figure 3] 1 shows non-reducing and reducing SDS-PAGE gel electropherograms used to analyze the purity of exemplary prodrugs SB1902-C2 and SB1902-C7, and drug SB1902-C1 (without masking polypeptide). [Figure 4] FIG. 1 shows the results of SEC-HPLC used to analyze the homogeneity of exemplary prodrug SB1902-C2 and drug SB1902-C1 (without masking polypeptide). [Figure 5A]1 shows an image of a polyacrylamide gel used to analyze an exemplary cleavable portion of the MMP2 enzyme-sensitive prodrug SB1902-C2. [Figure 5B] 1 shows an image of a polyacrylamide gel used to analyze an exemplary cleavable portion of the MMP9 enzyme-sensitive prodrug SB1902-C2. [Figure 5C] 1 shows an image of a polyacrylamide gel used to analyze SB1902-C4, which has a non-cleavable (G4S)2 linker instead of a cleavable linker that remains intact after digestion with the enzyme MMP2. [Figure 5D] 1 shows an image of a polyacrylamide gel used to analyze SB1902-C4, which has a non-cleavable (G4S)2 linker instead of a cleavable linker that remains intact after digestion with the enzyme MMP9. [Figure 5E] 1 shows an image of a polyacrylamide gel used to analyze an exemplary cleavable portion of the MMP9 enzyme-sensitive prodrug SB1902-C5. [Figure 5F] 1 shows an image of a polyacrylamide gel used to analyze an exemplary cleavable portion of the MMP2 enzyme-sensitive prodrug SB1902-C5. [Figure 6A] 1 shows that the drug SB1902-C1 (without a masking polypeptide), the prodrug SB1902-C2, the prodrug SB1902-C3, and the prodrug SB1902-C4 (without a cleavable moiety) do not bind nonspecifically to human serum proteins. [Figure 6B] 1 shows that the drug SB1902-C1 (without a masking polypeptide), the prodrug SB1902-C2, the prodrug SB1902-C3, and the prodrug SB1902-C4 (without a cleavable moiety) do not bind nonspecifically to cynomolgus monkey serum proteins. [Figure 6C] 1 shows that the drug SB1902-C1 (without a masking polypeptide), the prodrug SB1902-C2, the prodrug SB1902-C3, and the prodrug SB1902-C4 (without a cleavable moiety) do not bind nonspecifically to rat serum proteins. [Figure 6D]Shown are bands on WB membranes for prodrug SB1902-C2, prodrug SB1902-C3, and SB1902-C4 (without cleavable moiety) before incubation with plasma or PBS buffer. [Figure 6E] Figure 1 shows that prodrug SB1902-C2, prodrug SB1902-C3, and SB1902-C4 (without a cleavable moiety) show no detectable degradation bands on WB membranes after incubation with human plasma (simply referred to as plas in the figure) or PBS buffer. [Figure 7] 1 shows the binding affinity of an exemplary prodrug SB1902-C2 and an exemplary drug SB1902-C1 to the IL-2 / IL-15Rβγ receptor. [Figure 8] 1 shows the results of an immunogenicity detection test in Balbc mice, demonstrating that the MP80 masking polypeptide is not immunogenic. [Figure 9] 1 shows the results of a Mo7e cell proliferation assay, demonstrating that compared with the drug SB1902-C1, the prodrugs SB1902-C2, SB1902-C6, and SB1902-C7 significantly reduced the stimulatory function of IL-15 on Mo7e cell proliferation. [Figure 10A] 1 shows the results of exemplary drugs and prodrugs in a CD8+ T cell activation assay. [Figure 10B] 1 shows the results of exemplary drugs and prodrugs in a CD8+ T cell activation assay. [Figure 10C] 1 shows the results of exemplary drugs and prodrugs in a CD8+ T cell activation assay. [Figure 10D] 1 shows the results of exemplary drugs and prodrugs in a CD8+ T cell activation assay. [Figure 10E] 1 shows the results of exemplary drugs and prodrugs in a CD8+ T cell activation assay. [Figure 10F] 1 shows the results of exemplary drugs and prodrugs in a CD8+ T cell activation assay. [Figure 11]1 shows the results of an exemplary prodrug SB1902-C2 and MMP-digested SB1902-C2 (from which the masking polypeptide has been removed) in a CD8+ T cell activation assay. [Figure 12] 1 shows the results of an IFN-γ production study showing that mice treated with the prodrug SB1902-C2 produced less IFN-γ compared to the drug SB1902-C1. [Figure 13A] WEHI-164 tumor-bearing animals treated with 3 mg / kg of the IgG1 subtype control antibody MOPC-21 show that tumor growth in animals treated with SB1902-C2 was significantly inhibited compared to tumor growth in animals treated with the control antibody. [Figure 13B] WEHI-164 tumor-bearing animals treated with 3 mg / kg of the prodrug SB1902-C2 show that tumor growth in animals treated with SB1902-C2 was significantly inhibited compared to tumor growth in animals treated with the control antibody. [Figure 13C] This shows the antitumor activity of different molecules in WEHI-164 tumor-bearing animals. These animals were treated with the IgG1 subtype control antibody MOPC-21 on days 0, 4, 7, 10, and 14. All other drugs were administered at a dose of 1 mg / kg, except for the drug SB1902-C1 group, which was administered at a dose of 0.3 mg / kg. [Figure 13D] The antitumor activity of different molecules in WEHI-164 tumor-bearing animals was shown. These animals were treated with SB1902-C4 without a cleavable moiety on days 0, 4, 7, 10, and 14, and all other drugs were administered at a dose of 1 mg / kg, except for the drug SB1902-C1 group, which was administered at a dose of 0.3 mg / kg. [Figure 13E]This shows the antitumor activity of different molecules in WEHI-164 tumor-bearing animals, which were treated with the drug SB1902-C1 on days 0, 4, 7, 10, and 14, with all other drugs administered at a dose of 1 mg / kg, except for the drug SB1902-C1 group, which was administered at a dose of 0.3 mg / kg. [Figure 13F] This shows the antitumor activity of different molecules in WEHI-164 tumor-bearing animals. These animals were treated with the prodrug SB1902-C2 on days 0, 4, 7, 10, and 14, with all other drugs administered at a dose of 1 mg / kg, except for the drug SB1902-C1 group, which was administered at a dose of 0.3 mg / kg. [Figure 13G] This shows the antitumor activity of different molecules in WEHI-164 tumor-bearing animals. These animals were treated with the prodrug SB1902-C5 on days 0, 4, 7, 10, and 14, with all other drugs administered at a dose of 1 mg / kg, except for the drug SB1902-C1 group, which was administered at a dose of 0.3 mg / kg. [Figure 13H] This shows the antitumor activity of different molecules in WEHI-164 tumor-bearing animals. These animals were treated with the prodrug SB1902-C3 on days 0, 4, 7, 10, and 14, with all other drugs administered at a dose of 1 mg / kg, except for the drug SB1902-C1 group, which was administered at a dose of 0.3 mg / kg. [Figure 14A] FIG. 14A shows a structural diagram of an anti-TNFR2 antibody. [Figure 14B] Figure 14B shows a structural diagram of the masked antibody prodrug Pepbody-SB1901-H. [Figure 14C] Figure 14C shows a structural diagram of the masked antibody prodrug Pepbody-SB1901-L. [Figure 14D] FIG. 14D shows a structural diagram of the masked antibody prodrug Pepbody-SB1901-HL. [Figure 15A]1 shows antigen binding results for exemplary anti-TNFR2 antibody prodrugs and anti-TNFR2 antibodies. [Figure 15B] 1 shows the results of an exemplary anti-TNFR2 antibody prodrug, Pepbody-SB1901-H, compared to anti-TNFR2 antibody SB1901-72 in a human primary Treg cell proliferation assay. DETAILED DESCRIPTION OF THE INVENTION

[0039] Disclosed herein are activatable prodrugs comprising a masking polypeptide (MP), a cleavable moiety (CM), and the MP and / or CM. The prodrugs overcome toxicity issues that significantly limit the clinical application of biologically active moieties (e.g., cytokines and antibodies) to tumors. The activity of the cytokine, antibody, or other biologically active moiety of the prodrug is reduced. The cleavable moiety of the prodrug contains a protease cleavage site, and the masking polypeptide of the prodrug is cleaved by a protease associated with the desired site (e.g., within the tumor or tumor microenvironment), restoring the activity of the biologically active moiety (e.g., cytokine or antibody).

[0040] definition Unless the context clearly dictates otherwise, conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA technology within the skill of the art will be used to practice the present invention, and for purposes of illustration, various such methods are detailed below. Such techniques are fully described in the literature (e.g., Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., John Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatische et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vols. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984), and other similar references).

[0041] As used herein, "treatment" or "treating" refers to a method of obtaining beneficial or desired results, including clinical results. For purposes of this application, such beneficial or desired results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, reducing the severity of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease, preventing or slowing the recurrence of the disease, slowing or mitigating the progression of the disease, improving the state of the disease, alleviating the disease (partially or completely), reducing the dose of one or more other medications required to treat the disease, slowing the progression of the disease, improving or enhancing quality of life, and / or prolonging survival. "Treatment" also includes reducing the pathological consequences of the disease. The methods of this application contemplate any one or more of these aspects of treatment. For example, a patient is considered to be successfully "treated" if one or more symptoms associated with the disease are alleviated or eliminated, including, but not limited to, a reduction in symptoms caused by the disease, an improvement in the quality of life of a patient suffering from the disease, and a reduction in the dose of other medications required to treat the disease, and / or an extension of the individual's survival time.

[0042] The term "prevent" and similar words such as "prevented," "preventing," and the like refer to a method of preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition. It also refers to delaying the occurrence or recurrence of a disease or condition, or the occurrence or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the severity, impact, symptoms, and / or burden of a disease or condition before the disease or condition recurs.

[0043] As used herein, "delaying" disease progression means postponing, preventing, attenuating, slowing, stabilizing, and / or delaying the progression of the disease. The duration of the delay may vary depending on the disease history and / or the individual receiving the treatment. A method that "delays" disease progression is one that reduces the probability of disease progression within a given time range and / or reduces the severity of the disease within a given time range compared to the absence of the method. This comparison is usually based on clinical studies using a statistically significant number of individuals.

[0044] As used herein, the term "effective amount" refers to an amount of a drug or pharmaceutical composition sufficient to treat a particular disorder, condition, or disease, e.g., sufficient to ameliorate, alleviate, attenuate, and / or delay one or more symptoms. In some examples, an effective amount is an amount sufficient to slow the progression of the disease. In some examples, an effective amount is an amount sufficient to prevent or delay the onset or recurrence of the disease. An effective amount can be administered in one or more applications. Where the disease is, for example, cancer, an effective amount can be an amount that delays the onset or progression of the cancer (e.g., reduces the rate of tumor growth and / or delays or prevents tumor angiogenesis, metastasis, or cancer cell invasion into peripheral organs), reduces the number of epithelioid cells, causes cancer regression (e.g., tumor shrinkage or eradication), and / or prevents or delays the onset or recurrence of cancer. An effective amount can be administered in one or more applications.

[0045] As used herein, an "individual" or "subject" refers to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0046] The term "antibody" includes full-length antibodies and antigen-binding fragments thereof. Full-length antibodies contain two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of the two chains typically contain three hypervariable loops called complementarity-determining regions (CDRs) (light chain (LC) CDRs contain LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs contain HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein are defined or identified by the conventional method of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDR regions of a heavy or light chain are interposed between adjacent segments called framework regions (FRs), which are more conserved than the CDR regions and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified or typed based on the amino acid sequence of the heavy chain constant region. The five major antibody classes or isotypes are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some major antibody classes are further divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0047] As used herein, the term "antigen-binding fragment" includes antibody fragments, including, for example, diabodies, Fab, Fab', F(ab'), Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), VHHs, single-chain Fvs (scFv), scFv dimers (bivalent diabodies), multispecific antibodies composed of antibody fragments containing one or more CDRs, single-domain antibodies, and nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that can bind to an antigen but does not contain a complete antibody structure. Antigen-binding fragments also include fusion proteins containing the above antibody fragments. Antigen-binding fragments can bind to the same antigen as the parent antibody or parent antibody fragment (e.g., the parent scFv). In some examples, the antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted onto framework regions from one or more different human antibodies.

[0048] As used herein, the term "CDR" or "complementarity determining region" refers to the discontinuous antigen-binding sites found within the variable domains of heavy and light chain polypeptides. References: Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallumetal., J. Mo These special regions are described in J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc M. P. et al., Dev. Comp. Immunol., 27:55-77 (2003), and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001), where, when compared with each other, these definitions include any or a subset of amino acid residues. However, regardless of which definition system is used to refer to the CDRs of an antibody or grafted antibody or its variants, it is within the scope of the terms defined and used herein. For comparison, Table A lists the positions of the amino acid residues contained in the CDRs defined by each of the above-cited references. Algorithms and interfaces for CDR prediction are known in the art and are described, for example, in Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010), and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015).The contents of the references cited in this paragraph are incorporated by reference in their entirety for purposes of use in this application and any one or more claims contained herein.

[0049] [Table 1]

[0050] 1 The numbering of amino acid residues refers to the nomenclature in Kabat et al., supra.

[0051] 2 The numbering of amino acid residues refers to the nomenclature in Chothia et al., supra.

[0052] 3 The numbering of amino acid residues refers to the nomenclature in MacCallum et al., supra.

[0053] 4 The numbering of amino acid residues refers to the nomenclature in Lefranc et al., supra.

[0054] 5 The numbering of amino acid residues refers to the nomenclature in Honegger and Plückthun, supra.

[0055] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence than other portions of the immunoglobulin molecule, including the antigen-binding site variable domain. The constant domain is the C H 1. C H 2, and C H 3 domains (collectively referred to as C H (called C) and light chain L domains. HImmunoglobulins can be divided into various classes or subtypes based on the amino acid sequence of the constant domain of the immunoglobulin. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains of α, δ, ε, γ, and μ, respectively. γ and α are C H They are further divided into subclasses based on relatively minor differences in sequence and function; for example, humans express the IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2 subclasses.

[0056] As used herein, the terms "Fc," "Fc region," "fragment crystallizable region," "Fc domain," or "Fc moiety" are used to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as beginning at amino acid residue Cys226 or Pro230 and continuing to the carboxy-terminus thereof. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during protein production or purification, or by recombinant manipulation of the nucleic acid encoding the protein. Native-sequence Fc regions suitable for use in the constructs described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0057] As used herein, the term IgG "subtype" or "subclass" refers to any subclass of immunoglobulin defined by the chemical and antigenic properties of its constant domains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Several of these can be further divided into subclasses (subtypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different immunoglobulin classes are called α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known and are described in detail in Cellular and Molecular Immunology, 4th Edition, by Abbas et al. (WB Saunders, Co., 2000).

[0058] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region in an Fc-containing structure (e.g., an antibody or Fc fusion protein). A preferred FcR is a native human FcR sequence. Further, a preferred FcR is one that binds IgG antibodies (a type of gamma receptor), including the receptor subclasses FcγRI, FcγRII, and FcγRIII, as well as allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are described in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994), and de Haase et al., J. Lab. Clin. Med. 126:330-41 (1995). As used herein, the term "FcR" encompasses other FcRs, including those identified in the future.

[0059] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transport of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kimelthal, J. Immunol. 24:249 (1994)). Methods for measuring binding to FcRn are well known (see Ghetie and Ward, Immunol. Today 18:(12):592-8 (1997); Ghetie et al., Nature Biotechnology 15(7):637-40 (1997); Hintone et al., J. Biol. Chem. 279(8):6213-6 (2004); WO2004 / 92219 (Hintone et al.)). The half-life of human FcRn high-affinity binding polypeptides binding to FcRn in vivo and in serum can be measured, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with polypeptides having variant Fc regions. WO 2004 / 42072 (Presta) describes antibody variants with improved or reduced binding to FcRs in detail. See Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0060] "Antibody effector function" refers to a biological activity exerted by an Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) in an Fc-containing structure (e.g., an antibody or Fc fusion protein), and varies depending on the Fc subtype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. "Reducing or minimizing" an antibody effector function means a reduction of at least 50% (or 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) compared to a wild-type or unmodified Fc-containing structure (e.g., an antibody or Fc fusion protein). Measurement of antibody effector function can be readily determined and measured by one of ordinary skill in the art. In preferred embodiments, antibody effector functions of complement fixation, complement dependent cytotoxicity, and antibody dependent cellular cytotoxicity are affected. In some embodiments, effector function is abolished by mutations in the constant domain, e.g., by removing glycosylation through "effector function null mutations." In some embodiments, effector function null mutants are C H The N297A or DANA mutation (D265A + N297A) in the two regions is a mutation (Shields et al., J. Biol. Chem. 276(9):6591-6604(2001)). Other mutations that reduce or eliminate effector function include K322A and L234A / L235A (LALA). Effector function can also be reduced or eliminated through production techniques, such as expression in host cells that do not undergo glycosylation (e.g., E. coli) or host cells that result in altered glycosylation patterns that are ineffective or less effective in promoting effector function (e.g., Shinkawa et al., J. Biol. Chem. 278(5):3466-3473(2003)).

[0061] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a form of cytotoxicity in which secreted Ig (or ligand-Fc structures) binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer cells (NK), neutrophils, macrophages), enabling these cytotoxic effector cells to specifically bind to antigen-bearing (or ligand-receptor-bearing) target cells and subsequently kill them using cytotoxins. The antibody (or Fc-containing structure) "arms" the cytotoxic cell and is required for such killing. Of the major cell types mediating ADCC, NK cells express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 2 in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991), p. 464. To assess the ADCC activity of a target molecule, in vitro ADCC experiments can be performed, as described in U.S. Patent Nos. 5,500,362 or 5,821,337. Suitable effector cells for such experiments include peripheral blood mononuclear cells (PBMCs) and natural killer cells (NK). Alternatively, or in addition, the ADCC activity of a target molecule can be assessed in vivo, as described, for example, in the animal model disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

[0062] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an Fc-containing structure (of the appropriate subclass) that binds to its cognate receptor via a ligand fused to the Fc. To assess complement activation, CDC experiments can be performed as described in Gazzano-Santoroe et al., J. Immunol. Methods 202:163 (1996). Polypeptide variants with altered Fc region amino acid sequences and improved or reduced C1q binding ability are described in U.S. Pat. No. 6,194,551 B1 and WO 99 / 51642. The contents of these patent publications are expressly incorporated herein by reference. See also Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0063] As used herein, the terms "specific binding," "specific recognition," or "specifically used" refer to a measurable and reproducible interaction, such as binding between a ligand and a receptor, where the presence of the ligand can be determined in the presence of a heterogeneous population of molecules, including biomolecules. For example, a ligand that specifically binds to a receptor has greater affinity, avidity, greater ease, and / or longer duration of binding to the target receptor than to other receptors. In some examples, the binding of the ligand to an unrelated receptor is less than 10% of the binding of the ligand to the target receptor, as measured, for example, by radioimmunoassay (RIA). In some examples, a ligand that specifically binds to a target receptor has an equilibrium dissociation constant (Kd) of ≦10. -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, or ≤ 10 -12M. In some embodiments, the ligand specifically binds to a receptor that is conserved in different species. In some embodiments, specific binding may include exclusive binding, but this is not required. The binding specificity of a ligand can be determined experimentally using methods known in the art, including, but not limited to, Western blots, ELISA, RIA, ECL, IRMA, EIA, BIACORE™ tests, and peptide scans.

[0064] As used herein, when referring to a protease (e.g., a metalloprotease), the term "substrate" refers to any material or substance upon which the protease (e.g., a metalloprotease) acts. The material or substance may be, for example, a natural or non-natural organic compound or macromolecule, such as a polypeptide or peptidomimetic. In some examples, a metalloprotease substrate specifically interacts with and is cleaved by one or more metalloproteases. Using appropriate conditions, the metalloprotease cleaves at least one molecule of the substrate within an experimental time range. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the substrate may be cleaved by the metalloprotease.

[0065] The term "functional analog" refers to a molecule that has the same biological specificity (eg, binds to the same ligand) and / or activity (eg, activates or inhibits target cells) as a reference molecule.

[0066] The term "prodrug" refers to a therapeutic molecule that is inactive until activated in vivo.

[0067] The term "modulation" includes "increase," "enhancement," or "stimulation," as well as "decrease" or "reduction," usually by a statistically or physiologically significant amount or degree compared to a control group.

[0068] The term "variant" includes one or more substitutions, additions, deletions, and / or insertions compared to a reference polypeptide or polynucleotide. As discussed herein, a variant of a polypeptide or polynucleotide comprises an amino acid or nucleotide sequence that has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, similarity, or homology to the reference sequence and substantially retains the activity of the reference sequence. These include those that contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more amino acid or nucleotide additions, deletions, insertions, or substitutions, or that differ from a reference sequence while substantially retaining at least one activity of the reference sequence. In some embodiments, the additions or deletions include C-terminal and / or N-terminal additions and / or deletions.

[0069] The term "wild-type" refers to a gene or gene product (e.g., a polypeptide) that is most commonly observed in a population and is thus assumed to be the "normal" or "wild-type" form of the gene.

[0070] The term "linked" includes covalent and non-covalent linkages, meaning that a first moiety, such as a first amino acid sequence or nucleotide sequence, is covalently or non-covalently linked to a second moiety, such as a second amino acid sequence or nucleic acid sequence, respectively. The first moieties may be directly linked to the second moiety or adjacent to each other (hereinafter simply referred to as directly linked, e.g., in the case of polypeptides, directly linked by a peptide bond), or, optionally, the first moiety may be linked to the second moiety via an intervening moiety (e.g., a peptide linker) (hereinafter referred to as indirectly linked), which can be expressed as the first moiety being linked to the second moiety via an intervening moiety. With respect to polypeptides or proteins, the term "linked" includes not only linkage at the C-terminus and / or N-terminus of the first moiety (or second moiety), but also linkage of the entire first moiety (or second moiety) to any position within the second moiety (or first moiety) (e.g., a non-terminal amino acid residue). In one embodiment, the first moiety is linked to the second moiety by a peptide bond or a linker. In some examples, a first moiety can be linked to a second moiety by a phosphodiester bond or a linker. In some examples, the term "linker" refers to any chemical group, such as a molecule (including, but not limited to, unmodified or modified nucleic acids or amino acids), a group of molecules (e.g., two or more, e.g., 2, 3, 4, 10, 30, 50, 100 or more), or two polypeptides, that links two moieties.

[0071] As used herein, a "covalent bond" refers to a stable bond formed between two atoms through the sharing of one or more electrons. Examples of covalent bonds include, but are not limited to, peptide bonds and disulfide bonds. As used herein, a "peptide bond" refers to a covalent bond formed between the carboxyl of an amino acid and the amino of an adjacent amino acid. As used herein, a "disulfide bond" refers to a covalent bond formed between two sulfur atoms, such as two Fc fragments linked by one or more disulfide bonds. One or more disulfide bonds between two fragments may be formed by linking thiol groups within the two fragments. In some examples, one or more disulfide bonds may be formed between one or more cysteines of two Fc fragments. A disulfide bond may be formed when two thiol groups are oxidized. In some examples, the covalent linkage is a direct covalent linkage. In some examples, the covalent linkage is a direct peptide bond or disulfide bond.

[0072] When referring to two polypeptide sequences, the term "fused" or "fusion" refers to the linking of two polypeptide fragments by a backbone peptide bond. Two polypeptides can be fused directly or via a peptide linker containing one or more amino acids. A fusion protein is a polypeptide containing two or more regions derived from different or heterologous proteins or peptides. Fusion proteins are produced using conventional techniques of enzymatic digestion and ligation of fragments from the desired sequences. PCR technology to synthesize oligonucleotides can be used to produce and / or amplify the desired fragments. Overlapping synthetic oligonucleotides representing the desired sequences can also be used to produce DNA constructs encoding the fusion protein. Fusion proteins can contain multiple sequences, including leader (or signal peptide) sequences, linker sequences, leucine zipper sequences, or other oligomerization sequences, as well as sequences encoding highly antigenic moieties that allow for convenient purification or rapid detection of the fusion protein. Fusion proteins can be produced by recombinant techniques from coding sequences containing the coding sequences for both components of the fusion protein, with or without a peptide linker between them. In some examples, fusion involves chemical conjugation.

[0073] As provided herein, the term "IL-15 / IL-15Rα complex" refers to a complex in which the IL-15 cytokine and IL-15Rα or a functional fragment thereof are non-covalently linked to each other.

[0074] Half maximal inhibitory concentration (IC 50 IC ) is a measure of the effectiveness of a substance (e.g., a ligand) in inhibiting a particular biological or biochemical function. It indicates how much of a particular drug or other substance (inhibitor, e.g., ligand) is needed to inhibit a particular biological process by half. The value is usually expressed as a molar concentration. 50 is the "EC agonist" of an agonist drug or other substance (e.g., a ligand). 50 " EC 50Also represents the plasma concentration required to obtain 50% of the maximum effect in vivo. As stated herein, "IC 50 " is used to describe the effective concentration of a ligand required to neutralize 50% of the biological activity of a receptor in vitro. 50 or EC 50 can be measured by inhibition of ligand binding by FACS analysis (competitive binding assay), cell-based cytokine release assays, or bioassays such as amplified luminescent proximity homogeneous assay (AlphaLISA).

[0075] "Percentage (%) amino acid sequence identity" and "homology" of a peptide or polypeptide sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to a particular polypeptide or polypeptide sequence, taking into account sequence alignment and introducing gaps (if necessary) to achieve maximum percentage sequence identity, and conservative substitutions are not considered as part of sequence identity. Various alignment methods within the skill of the art can be used to determine percentage amino acid sequence identity, for example, BLAST, BLAST-2, ALIGN, MEGALIGN, etc. TM Publicly available computer software such as (DNASTAR) software can be used. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0076] As used herein, the "C-terminus" of a polypeptide refers to the last amino acid residue of the polypeptide that provides an amino group that forms a peptide bond with the carboxyl group of an adjacent amino acid residue. As used herein, the "N-terminus" of a polypeptide refers to the first amino acid of the polypeptide that provides a carboxyl group that forms a peptide bond with the amino acid of an adjacent amino acid residue.

[0077] As used herein, the term "moiety" refers to a portion of a molecule that has a distinct function within the molecule and that function can be performed by that portion in another molecule. A moiety may be a chemical entity with a specific function or a portion of a biological molecule with a specific function.

[0078] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also includes amino acid polymers modified by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation to a labeling component. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, including, but not limited to, glycine and D- or L-optical isomers, as well as amino acid analogs and peptidomimetics. Standard one-letter or three-letter abbreviations are used to represent amino acids.

[0079] An "isolated" polypeptide refers to a polypeptide that has been identified, isolated, and / or recovered from components (e.g., natural or recombinant) of the environment in which it is produced. Preferably, an isolated polypeptide is free from association with all other components of the environment in which it is produced. Contaminating components of the production environment, such as those produced by recombinantly transfected cells, often interfere with the study, diagnosis, or treatment of the peptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide is purified as follows: (1) to a polypeptide content greater than 95% by weight, and in some embodiments, greater than 99% by weight, as determined, for example, by the Lowry method; (2) sufficiently purified to obtain at least 15 N-terminal residues or internal amino acid sequence using a spin-cup sequencer; or (3) to homogeneity achieved by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. Isolated polypeptides include polypeptides in situ within recombinant cells, because at least one component of the polypeptide's natural environment is not present. However, an isolated peptide will usually undergo at least one purification step.

[0080] As used herein, the terms "polynucleotide," "nucleic acid," "nucleotide," and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, which can be deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications can be made to the nucleotide structure before or after assembly of the polymer. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component.

[0081] An "isolated" nucleic acid molecule encoding a construct (e.g., a masking polypeptide described herein) is one that has been identified and separated from nucleic acid molecules, including at least one contaminant nucleic acid molecule, that is typically associated with its production environment. Preferably, the isolated nucleic acid is free from association with all components of its production environment. An isolated nucleic acid molecule encoding a polypeptide described herein exists in a form or format that is different from that found in nature. Thus, an isolated nucleic acid molecule differs from a nucleic acid encoding a polypeptide described herein that is naturally present in a cell. Isolated nucleic acid includes a nucleic acid molecule contained within a cell that contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0082] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter and, optionally, an operator sequence and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0083] A nucleic acid is "operably linked" when it establishes a functional relationship with another nucleic acid sequence. For example, DNA for a leader sequence or secretory leader can be operably linked to DNA for a polypeptide if it is expressed as a proprotein that participates in the secretion of the polypeptide. A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence. Alternatively, a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote translation. Generally speaking, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader, not only contiguous but also in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at appropriate restriction sites. If no such sites exist, synthetic oligonucleotide aptamers or linkers are used in accordance with conventional practice.

[0084] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid molecule to which it is linked. This term includes vectors that are self-replicating nucleic acid structures and vectors that are introduced into the genome of a known host cell. Some vectors are capable of directing the expression of nucleic acids to which they are linked. Such vectors are referred to herein as "expression vectors."

[0085] As used herein, the terms "transfection" or "transformation" or "transduction" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. Such cells include the primary test cell and its progeny.

[0086] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid to the parent cell and may contain mutations. Included herein is screening or selection of mutant progeny that have the same function or biological activity as the originally transformed cell.

[0087] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a formulation of the active ingredient in a form effective for biological activity and containing no additional ingredients that would be unacceptably toxic to the subject to which it is administered. The formulation is sterile; a "sterile" formulation is sterile or free of any living microorganisms and their spores.

[0088] It should be understood that the embodiments of the present application described herein include embodiments that "consist of" and / or "consist essentially of."

[0089] References herein to "about" refer to a numerical value or parameter and include (and describe) variations of that value or parameter itself. For example, a statement relating to "about X" also includes a statement of "X."

[0090] As used herein, a reference to a value or parameter "not" generally means and describes the value or parameter as "other than." For example, "this method cannot be used to treat disease type X" means that this method is typically used to treat other types of disease besides disease type X.

[0091] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0092] As used herein and in the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0093] Masking Polypeptide (MP) A masking polypeptide (MP) provided herein refers to a moiety that can block the activity of a biologically active moiety (B). In some embodiments, the masking polypeptide (MP) inhibits the ability of a cytokine to bind to and / or activate its receptor. In some embodiments, the masking polypeptide (MP) inhibits the ability of an antibody or antigen-binding fragment to bind to its target.

[0094] In some embodiments, the masking polypeptide (MP) has a hydrodynamic radius greater than its actual molecular weight. In some embodiments, the masking polypeptide is capable of forming only a random coil, lacking secondary structure. In some embodiments, the masking polypeptide has a steric masking effect, i.e., due to its relative size, its proximity to a biologically active moiety generally inhibits or blocks the activity of the biologically active moiety.

[0095] In some embodiments, the masking polypeptide (MP) consists of four or five amino acid residues, and the amino acid residues are selected from the group consisting of proline (P), alanine (A), serine (S), glycine (G), and glutamic acid (E).

[0096] In some embodiments, the masking polypeptide (MP) consists of four types of amino acid residues, and the amino acid residues are selected from the group consisting of proline (P), alanine (A), serine (S), and glutamic acid (E).

[0097] In some embodiments, the masking polypeptide (MP) consists of four types of amino acid residues, and the amino acid residues are selected from the group consisting of proline (P), glycine (G), serine (S), and glutamic acid (E).

[0098] In some embodiments, the masking polypeptide (MP) consists of five amino acid residues, and the amino acid residues are selected from the group consisting of proline (P), alanine (A), serine (S), glycine (G) and glutamic acid (E).

[0099] In some embodiments, the net charge of the masking polypeptide (MP) is very important. Generally, negative charges are preferred over positive charges. A negative net charge on an MP can avoid potential interactions with tissues or cell membranes, whereas a positive net charge can cause it to "sink" before reaching its destination. However, excess negative charge can increase the likelihood of interactions with positively charged proteins. The percentage of net charge also depends on what the MP is fused to.

[0100] In some embodiments, the masking polypeptide (MP) consists of five types of amino acids: G, S, P, E, and A. Here, the percentage of amino acid residues G in the masking polypeptide is about 15% to 30%, the percentage of amino acid residues S in the masking polypeptide is about 20% to 40%, the percentage of amino acid residues P in the masking polypeptide is about 15% to 40%, the percentage of amino acid residues E in the masking polypeptide is about 1% to 20%, and the percentage of amino acid residues A in the masking polypeptide is about 5% to 20%, and if the number of amino acids is not an integer, it takes an integer value.

[0101] In some embodiments, the masking polypeptide (MP) consists of five amino acids: G, S, P, E, and A, wherein the percentage of amino acid residues G in the masking polypeptide is about 20%, the percentage of amino acid residues S in the masking polypeptide is about 40%, the percentage of amino acid residues P in the masking polypeptide is about 20%, the percentage of amino acid residues E in the masking polypeptide is about 10%, and the percentage of amino acid residues A in the masking polypeptide is about 10%, and when the number of amino acids is not an integer, it takes an integer value.

[0102] In some embodiments, the masking polypeptide (MP) consists of four types of amino acids: S, P, E, and G, wherein the percentage of amino acid residues S in the masking polypeptide is about 20% to 40%, the percentage of amino acid residues P in the masking polypeptide is about 15% to 40%, the percentage of amino acid residues E in the masking polypeptide is about 1% to 20%, and the percentage of amino acid residues G in the masking polypeptide is about 15% to 30%, and if the number of amino acids is not an integer, it takes an integer value.

[0103] In some embodiments, the masking polypeptide (MP) consists of four amino acids: S, P, E, and G, wherein the percentage of amino acid residues S in the masking polypeptide is about 23%, the percentage of amino acid residues P in the masking polypeptide is about 29%, the percentage of amino acid residues E in the masking polypeptide is about 18%, and the percentage of amino acid residues G in the masking polypeptide is about 30%, and when the number of amino acids is not an integer, it takes an integer value.

[0104] In some embodiments, the masking polypeptide (MP) can be tailored to meet certain requirements for certain subjects by altering its amino acid chain length and overall net charge.

[0105] In some embodiments, the masking polypeptide (MP) comprises between about 40 and 720 amino acid residues. In some embodiments, the masking polypeptide (MP) comprises between about 80 and 320 amino acid residues. In some embodiments, the masking polypeptide (MP) comprises between about 80 and 240 amino acid residues.

[0106] In some embodiments, the masking polypeptide (MP) comprises the amino acid sequence SEQ ID NO:6.

[0107] In some embodiments, the masking polypeptide (MP) comprises the amino acid sequence SEQ ID NO:1.

[0108] In some embodiments, the masking polypeptide (MP) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-5, or a variant thereof having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-5, or a variant thereof comprising one or more amino acid substitutions, additions, and / or deletions.

[0109] Crack potential portion (CM) The cleavable moiety (CM) is an enzyme or protease cleavage site or a polypeptide containing such a site. In some embodiments, the protease may be urokinase plasminogen activator (uPA); matrix metalloproteinases (e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, and / or MMP27); tobacco etch virus (TEV) protease; plasmin; thrombin; Thrombin; PSA; PSMA; ADAMS / ADAMTS (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM13, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, and / or ADAMTS5); caspases (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, caspase-15, caspase-16, caspase-17, caspase-18, caspase-19 ... caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13 and / or caspase-14; aspartic proteases (e.g., RACE and / or Renin); aspartic cathepsins (e.g., Cathepsin D and / or Cathepsin E); cysteine cathepsins (e.g., Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V / L2 and / or Cathepsin X / Z / P; cysteine proteases (e.g., Cruzipain, Legumain and / or Otubain-2); KLKs (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and / or KLK14); metalloproteinases (e.g., Meprin, Neprilysin, PSMA and / or BMP-1);Serine proteases (e.g., activated protein C, cathepsin A, cathepsin G, chymase, and / or coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FX1a, FXIIa)); elastase; granzyme B; guanidinobenzoic acid enzyme; HtrAl; human neutrophil elastase; lactoferrin; marapsin; NS3 / 4A; PACE4; tPA; tryptase; type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, TMPRSS2, TMPRSS3, and / or TMPRSS4);

[0110] In some embodiments, the cleavable moiety (CM) comprises a substrate sequence for at least one matrix metalloproteinase (MMP). Exemplary MMPs include MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27. In some embodiments, the CM comprises a substrate sequence for MMP2, MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. In some embodiments, the CM comprises a substrate sequence for MMP2. In some embodiments, the CM comprises a substrate sequence for MMP9. In some embodiments, the CM comprises substrate sequences for two or more MMPs. In some embodiments, the CM comprises substrate sequences for at least MMP2 and MMP9. In some embodiments, the CM comprises two or more substrates of the same MMP. In some embodiments, the CM comprises at least two or more MMP2 substrates. In some embodiments, the CM comprises at least two or more MMP9 substrates.

[0111] In some embodiments, the cleavable moiety (CM) comprises the amino acid sequence MVX1X2AX3TX4SG (SEQ ID NO: 49), wherein X1 is selected from P, L, V, or A; X2 is selected from L or S; X3 is selected from L, V, P, or Y; and X4 is selected from A or V.

[0112] The terminology used to describe the specificity of proteases, i.e., the cleavage of peptide bonds between amino acids at specific nearby positions, is based on terminology originally coined by Schechter & Berger (1967, 1968) to describe the specificity of papain. According to this model, amino acid residues in the cleaved substrate are designated P1, P2, P3, P4, etc., toward the N-terminus of the cleaved bond. Similarly, residues toward the C-terminus are designated P1', P2', P3', P4', etc.

[0113] In some embodiments, the CM comprises an amino acid sequence set forth in any one of SEQ ID NOs: 8-16 or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 8-16.

[0114] Biologically active moiety (B) In some embodiments, the biologically active moiety (B) may be a chemical entity. In some embodiments, the biologically active moiety (B) may be a therapeutic protein. In some embodiments, the biologically active moiety (B) is a cytokine. In some embodiments, the biologically active moiety (B) is an antibody or antigen-binding fragment that targets the prodrug to a site of action (e.g., a site of inflammation or tumor).

[0115] Cytokine: "Cytokine" is a term well known in the art and refers to any immunomodulatory protein (e.g., interleukin or interferon) secreted by cells, particularly cells of the immune system, and acting as a regulator of the immune system. In some embodiments, the cytokine includes a functional fragment, mutant, or variant of a cytokine. Examples of cytokines may include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Hereinafter, an IL-15 prodrug will be used as an example of a cytokine prodrug. However, prodrugs of other cytokines, particularly those that are potent immunomodulators and have potent side effects, are also contemplated within the scope of this application. Upon hydrolytic cleavage of the target protein at the cleavable moiety, the cytokine is converted to its activated form, allowing it to bind with higher affinity to its homologous receptor or protein. Other cytokine prodrugs can be prepared according to the same principles as those described below for the IL-15 prodrug.

[0116] In some embodiments, the cytokine is selected from the group consisting of IL-1α, IL-1β, IL-1 receptor antagonist (IL-1RA), IL-18, IL-33, IL-36α, IL-36β, IL-36γ, IL-36 receptor antagonist (IL-36RA), IL-37, and IL-38, or variants of the above cytokines.

[0117] In some embodiments, the cytokine is selected from the group consisting of IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-13, IL-15, IL-21, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), IL-6, IL-11, IL-12, growth hormone (GH), erythropoietin (EPO), prolactin (PRL), leukemia inhibitory factor (LIF), oncostatin (OSM), and thrombopoietin (TPO), or variants of the above cytokines.

[0118] In some embodiments, the cytokine is selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CCL1e, CCL2, CCL3, CCL3L1, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CX3CL1, XCL1 and XCL2, or variants of the above cytokines.

[0119] In some embodiments, the cytokine is selected from the group consisting of IFN-α (alpha), IFN-β (beta), IFN-γ (gamma), IFN-ε (epsilon), IFN-κ (kappa), IFN-(ω) (omega), IFN-τ (tau), IFN-ζ (zeta), IFN-δ (delta), and IFN-λ (lambda), or variants of the above cytokines.

[0120] In some examples, the cytokine is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL -20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A, IL-28B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, and IL-37.

[0121] In some embodiments, the cytokine is selected from the group consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), IFN-γ(gamma), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, and IL-12.

[0122] In some embodiments, the cytokine is selected from the group consisting of TNF-α (alpha), TNF-β (beta), TNF-γ (gamma), CD252, CD154, CD178, CD70, CD153, 4-1BB-L, TRAIL, RANKL, APO3L, CD256, CD257, CD258, TL1, AITRL, and EDA1.

[0123] In some examples, the cytokines disclosed herein are mutated or engineered to alter the properties of the native cytokine, including receptor binding affinity and specificity or serum half-life.

[0124] Although the following discussion of antibody prodrugs uses anti-TNFR2 antibody prodrugs as an example, the present application also contemplates prodrugs of other antibodies (especially antibodies with less than optimal selectivity for their intended targets), which are activated by tumor-associated proteases in the tumor microenvironment, thereby limiting their activity within the tumor microenvironment and minimizing "extratumor" toxicity. In some embodiments, the masking polypeptide (MP) reduces the binding ability of the antibody or antigen-binding fragment thereof to the target such that the dissociation constant (Kd) between the antibody or antigen-binding fragment bound to the masking polypeptide (MP) and the target is at least 10-fold, at least 100-fold, at least 1000-fold, or at least 10,000-fold higher than the Kd between the antibody or antigen-binding fragment not bound to the masking polypeptide (MP) and the target. The antibody or antigen-binding fragment is activated after proteolytic cleavage of the cleavable moiety (CM) at the target. Other antibody prodrugs can be produced according to the same principles as those described below for anti-TNFR2 antibody prodrugs.

[0125] In some examples, the antibody or antigen-binding fragment is specific for regulatory T cells (Tregs), e.g., targets CCR4 or CD39 receptors. In some examples, the specific antibody or antigen-binding fragment can bind to an antigen on the surface of immune cells such as T cells, NK cells, and macrophages, e.g., the specific antibody or antigen-binding fragment can bind to PD-1, LAG-3, TIM-3, TIGIT, CTLA-4, or TNF-α. In some examples, the specific antibody or antigen-binding fragment can activate immune cells and enhance their anti-cancer activity. In some examples, the specific antibody or antigen-binding fragment can bind to an antigen on the surface of diseased cells or tissues, e.g., tumor cells, where the tumor antigen is well known in the art. The specific antibody or antigen-binding fragment may bind to tumor antigens, including, but not limited to, fibroblast activation protein alpha (FAPα), trophoblast glycoprotein (5T4), tumor-associated calcium signal transducer 2 (Trop2), fibronectin EDB (EDB-FN), FOLR1, fibronectin EIIIB domain, CGS-2, EpCAM, EGFR, HER-2, HER-3, c-Met, and CEA.

[0126] In some embodiments, the specific antibody or antigen-binding fragment is specific for an immune checkpoint protein, including, but not limited to, CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, OX40, DNAM-1, PD-L1, PD-1, PD-L2, CTLA-4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDOL, IDO2, TDO, KIR, LAG-3, TIM-3, or VISTA.

[0127] In some embodiments, the specific antibody or antigen-binding fragment is specific for an immune response modifier, including, but not limited to, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), B7-1 (CD80), B7-2 (CD86), GITRL, CD3, or GITR.

[0128] In some embodiments, the specific antibody or antigen-binding fragment is specific for a cytokine receptor. Exemplary cytokine receptors include type I cytokine receptors such as GM-CSF receptor, G-CSF receptor, type I IL receptor, Epo receptor, LIF receptor, CNTF receptor, and TPO receptor; type II cytokine receptors such as IFN-alpha receptor (IFNAR1, IFNAR2), IFB-beta receptor, IFN-gamma receptor (IFNGR1, IFNGR2), and type II IL receptor; CC chemokine receptor, CXC chemokine receptor, CX3C chemokine receptor, and XC chemokine receptor. These include, but are not limited to, chemokine receptors; tumor necrosis receptor superfamily receptors such as TNFRSF5 / CD40, TNFRSF8 / CD30, TNFRSF7 / CD27, TNFRSFlA / TNFRl / CD120a, and TNFRSF1B / TNFR2 / CD120b; TGF-beta receptors such as TGF-beta receptor 1 and TGF-beta receptor 2; and Ig superfamily receptors such as IF-1 receptor, CSF-1R, PDGFR (PDGFRA, PDGFRB), or SCFR.

[0129] In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of CD47, CD3, CD19, CD20, CD22, CD30, CD33, CD34, CD40, CD44, CD52, CD70, CD79a, CD123, Her-2, EphA2, lymphocyte-associated antigen 1, VEGF or VEGFR, CTLA-4, LIV-1, nectin-4, CD74, SLTRK-6, EGFR, CD73, PD-L1, CD163, CCR4, CD147, EpCam, Trop-2, CD25, C5aR, Ly6D, alphavintegrin, and the like. The antibody binds to an antigen selected from the group consisting of glioma, B7H3, B7H4, Her-3, folate receptor, GD-2, CEACAM5, CEACAM6, c-MET, CD266, MUC1, CD10, MSLN, sialylTn, LewisY, CD63, CD81, CD98, CD166, tissue factor (CD142), CD55, CD59, CD46, CD164, TGFbeta receptor 1 (TGFpRl), TGFpR2, TGFpR3, FasL, MerTk, Axl, Clecl2A, CD352, FAP, CXCR3, and CD5.

[0130] Non-cleavable linker (L) In some embodiments, the linker is a non-cleavable linker. Exemplary non-cleavable linkers are stable under physiological conditions and at disease sites, such as tumor sites and inflammatory disease sites. In some embodiments, the non-cleavable linker is rich in the amino acid residues G and S. In some embodiments, the non-cleavable linker comprises a "G4S" repeat sequence. In some embodiments, the non-cleavable linker is a polypeptide chain comprising at least three residues. Portions of such linkers are likely flexible and hydrophilic, forming little or no secondary structure of their own (the linker portion or flexible linker portion). Linkers consisting of at least three amino acids can be used to connect adjacent domains and / or regions after assembly of the molecule. Longer linkers can also be used. In some examples, a linker can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 125, 150, 175, or 200 residues. When multiple linkers are used to connect various portions of the molecule, the linkers can be the same or different (e.g., same or different length and / or amino acid sequence).

[0131] In some embodiments, the non-cleavable linker comprises or consists of a Gly-Ser linker. As used herein, the term "Gly-Ser linker" refers to a peptide consisting of glycine and serine residues. In some embodiments, an exemplary Gly-Ser linker comprises the amino acid sequence GSG (SEQ ID NO: 17). In some embodiments, an exemplary Gly-Ser linker comprises the amino acid sequence (Gly4Ser). nwhere n is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, a preferred Gly-Ser linker is (Gly4Ser)1. In some embodiments, a preferred Gly-Ser linker is (Gly4Ser)2. In some embodiments, a preferred Gly-Ser linker is (Gly4Ser)3. In some embodiments, a preferred Gly-Ser linker is (Gly4Ser)4. In some embodiments, a preferred Gly-Ser linker is (Gly4Ser)5. In other embodiments, two or more Gly-Ser linkers are linked in tandem in the polypeptide linker.

[0132] In some embodiments, non-cleavable linkers are used in prodrugs described herein that comprise an immunoglobulin (Ig) / antibody hinge region. In one embodiment, the hinge region is obtained from an IgG1 antibody. In one embodiment, the term Ig "hinge" region refers to a polypeptide that comprises an amino acid sequence that shares sequence identity or similarity with a portion of a naturally occurring Ig hinge region sequence, which includes cysteine residues that form disulfide bonds to link the two heavy chains of an immunoglobulin.

[0133] In some examples, non-cleavable linkers are used to link any of the moieties in the prodrugs provided herein.

[0134] In some embodiments, the non-cleavable linker comprises an amino acid sequence set forth in any one of SEQ ID NOs: 17-21, or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 17-21.

[0135] Half-life extending moiety (C) Preferably, the prodrug comprises an in vivo half-life extending moiety (C). The term half-life extending moiety refers to a moiety that extends the half-life of a target component in serum. A long in vivo half-life is very important for therapeutic molecules; for example, cytokines administered to a subject typically have very short half-lives because they are rapidly removed from the subject by mechanisms such as renal clearance and endocytic degradation. For therapeutic molecules with naturally short half-lives, extending the in vivo half-life of these molecules may enable more acceptable and manageable dosing regimens without sacrificing efficacy. Therefore, in the prodrugs provided herein, it is preferred to link a half-life extending moiety to a biologically active moiety to achieve the goal of extending the in vivo half-life.

[0136] As described herein, a "half-life extending moiety" extends in vivo half-life and improves PK, for example, by altering its size (e.g., making it higher than the upper limit of renal filtration), shape, hydrodynamic radius, charge, or absorption, biodistribution, metabolism, and elimination parameters. One exemplary method for improving PK of a polypeptide is by expressing elements within the polypeptide chain that bind to receptors that can be recycled to the cell membrane without being degraded in lysosomes, such as the FcRn receptor and transferrin receptor on endothelial cells. Three proteins, such as human IgG, HSA (or fragments), and transferrin, persist in human serum much longer than would be predicted by their size, which is related to their ability to bind to receptors that are recycled in lysosomes rather than being degraded. These proteins or fragments thereof that retain FcRn-binding ability are often conjugated to other polypeptides to extend serum half-life.

[0137] In some embodiments, the half-life extending moiety (C) may be an antibody or antigen-binding fragment that binds to a protein with a long serum half-life, such as serum albumin, transferrin, etc. Examples of such antibodies or antigen-binding fragments include polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv); heavy chain variable domains (V); H ), light chain variable domain (V L ) and camel nanobodies (V HH ) and single domain antibodies; dAbs, etc.

[0138] In some embodiments, the half-life extending moiety (C) can also function as a linker, and optionally as a non-cleavable linker (L).

[0139] In some embodiments, the half-life extending moiety is an antibody Fc domain (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc) or a fragment thereof capable of participating in FcRn-mediated circulation, such as any heavy chain polypeptide or fragment thereof capable of participating in FcRn-mediated circulation. In some embodiments, the Fc domain is a monomer. In some embodiments, the Fc domain is a dimer comprising a first Fc domain and a second Fc domain.

[0140] FC Domain In some embodiments, the Fc domain is derived from any one of IgA, IgD, IgE, IgG, and IgM, and their subclasses. Of all immunoglobulins, IgG has the highest serum content and the longest half-life. Unlike other immunoglobulins, IgG can be efficiently recycled after binding to Fc receptors (FcRs). In some embodiments, the Fc domain is derived from an IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the Fc domain is derived from a human IgG. In some embodiments, the Fc domain comprises a CH2 domain and a CH3 domain. In some embodiments, the Fc domain further comprises all or a portion of a hinge region. In some embodiments, the Fc domain is derived from a human IgG1 or human IgG4. In some embodiments, two subunits of the Fc domain dimerize via one or more (e.g., 1, 2, 3, 4, or more) disulfide bonds. In some embodiments, each subunit of the Fc domain comprises a full-length Fc sequence. In some embodiments, each subunit of the Fc domain comprises an N-terminally truncated Fc sequence, e.g., a truncated Fc domain containing fewer N-terminal cysteines to reduce disulfide bond mismatches during the dimerization process, hi some embodiments, the Fc domain is truncated at the N-terminus, e.g., by deleting the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of an intact immunoglobulin Fc domain.

[0141] In some examples, the Fc domain comprises one or more mutations, such as an insertion, deletion, and / or substitution.

[0142] In some examples, the Fc domain comprises one or more amino acid mutations that alter effector function, and the Fc domain is engineered (e.g., comprises one or more amino acid mutations) to alter its binding to FcR, particularly binding to Fcγ receptors (responsible for ADCC), and / or alter effector function, e.g., antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). Preferably, such amino acid mutations do not reduce binding to the FcRn receptor (responsible for half-life).

[0143] An Fc domain (e.g., human IgG1 Fc) mutated to eliminate one or more effector functions, such as ADCC, ADCP, or CDC, is hereinafter referred to as a "null-effect" or "near-null-effect" Fc domain. For example, in some embodiments, the Fc domain is a null-effect human IgG1 Fc containing one or more of the following mutations (e.g., in each Fc subunit): L234A, L235E, G237A, A330S, and P331S. In some embodiments, the Fc domain of a prodrug contains L234A and L235A ("LALA") mutations. The combination of K322A, L234A, and L235A in IgG1 Fc is sufficient to completely eliminate FcγR and C1q binding (Hezarehe Tal., J Virol 75, 12161-12168, 2001). MedImmune found that a series of triple mutations, L234F / L235E / P331S, had very similar effects (Organotypica et al., Acta Crystallographica 64, 700-704, 2008). In some embodiments, the Fc domain includes a glycosylation modification on N297 of the IgG1 Fc domain, known to be required for optimal FcR interaction. The Fc domain modification may be any suitable IgG Fc engineering described by Wang et al. ("IgG Fc engineering to modulate antibody effector functions," Protein Cell. 2018 Jan;9(1):63-73), the contents of which are incorporated herein by reference in their entirety.

[0144] Glycosylation variants In some examples, the Fc domain is modified to increase or decrease the degree of glycosylation of the construct. One or more glycosylation sites can be created or removed by altering the amino acid sequence to add or remove glycosylation sites from the Fc domain.

[0145] Naturally occurring Fc-containing proteins produced by mammalian cells typically contain C-terminal Fc regions via N-linkages. H The Fc domain contains a monoantennary and a biantennary oligosaccharide attached to Asn297 of the Fc domain. See Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc on the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the Fc domain can be modified to produce certain improved properties.

[0146] In some embodiments, the Fc domains described herein have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc domain. For example, the fucose content in such Fc domains or IL-15 prodrugs or anti-TNFR2 antibody prodrugs may range from 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The fucose content was determined by MALDI-TOF mass spectrometry by measuring the average fucose content in the glycans linked to Asn297 relative to the sum of all glycans attached to Asn297 (e.g., complex, hybrid, and high-mannose structures), as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at position 297 of the Fc domain (Fc region residues are based on the EU numbering system); however, due to minor sequence variations in the Fc region, Asn297 may be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have enhanced ADCC function. See U.S. Patent Publication No. US2003 / 0157108 (Presta, L.) and U.S. Patent Publication No. US2004 / 0093621 (KyowaHakkoKogyoCo.,Ltd). Examples of publications relating to "afucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 01098 65; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing afucosylated Fc-containing proteins include Lec13 CHO cells, which lack the function of protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Pat. Appl. No. US2003 / 0157108A1, Presta, L; and WO2004 / 056312A1, Adams et al., especially Example 11), and gene knockout cell lines such as α-1,6-fucosyltransferase gene and FUT8 gene knockout CHO cells (see Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0147] Effector function variants In some embodiments, the present application contemplates Fc domains that possess some, but not all, Fc effector functions, making them ideal candidates for particular applications in which in vivo half-life is critical, but certain effector functions (CDC and ADCC) are unnecessary or deleterious. Cytotoxicity assays may be performed in vitro or in vivo to determine reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the Fc domain lacks FcγR binding capacity (and thus potentially lacks ADCC activity) but retains FcRn binding capacity. Natural killer cells (NK), the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcRs on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of target molecules are described in detail in U.S. Pat. Nos. 5,500,362 (Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and 5,821,337 (Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive detection methods can be used (see ACTI™ Non-Radiotoxicity Test for Flow Cytometry (CellTechnology, Inc. Mountain View, CA) and CytoTox96® Non-Radiotoxicity Test (Promega, Madison, WI)). Suitable effector cells for this detection include peripheral blood mononuclear cells (PBMCs) and NK cells.Furthermore, the ADCC activity of the target molecule can be evaluated in vivo in an animal model, for example, as disclosed in Clynesetal., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding test may be performed to determine that the Fc domain does not bind to C1q and therefore lacks CDC activity. See the C1q and C3c binding enzyme-linked immunosorbent assays in WO 2006 / 029879 and WO 2005 / 100402. A CDC test may be performed to assess complement activity (see Gazzano-Santoroeta l., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). Measurements of FcRn binding and in vivo clearance / half-life can be performed using methods known in the art (see Petkova, S.B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0148] Fc domains with reduced effector function include substitutions of one or more residues at positions 238, 265, 269, 270, 297, 327, and 329 of the Fc region (U.S. Pat. No. 6,737,056). Such Fc mutants contain substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581). Certain antibody variants with enhanced or reduced binding to FcRs have been described in detail (see U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)). In some examples, the Fc region is modified to alter (i.e., increase or decrease) C1q binding and / or CDC, as described, for example, in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0149] In some embodiments, the Fc domain contains one or more amino acid substitutions that increase half-life and / or enhance binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-life and enhanced binding to neonatal FcRn are involved in the transport of maternal IgG to the fetus, as detailed in (Guyer et al., J. Immunol. 117:587 (1976) and Kimelthall, J. Immunol. 24:249 (1994)) and U.S. Patent No. 2005 / 0014934 A1 (Hinton et al.). Thus, antibodies comprising an Fc domain with one or more substitutions enhance binding of the Fc region to FcRn. Such Fc variants include variants with one or more Fc region residue substitutions, e.g., substitutions at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0150] For other examples of Fc domain variants, see Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0151] Cysteine engineered variants In some examples, it may be desirable to create a cysteine-engineered Fc domain in which one or more residues of the Fc domain are substituted with cysteine residues. In some examples, the substituted residues occur at accessible sites on the Fc domain. By substituting these residues with cysteine, reactive thiol groups are positioned at accessible sites on the Fc domain, which can be used to attach the molecule to other moieties, such as drug moieties or linker-drug moieties, thereby producing long-acting drug or prodrug conjugates. In some examples, any one or more of the following residues can be substituted with cysteine: heavy chain A118 (according to the EU numbering system) and heavy chain Fc domain S400 (according to the EU numbering system). Cysteine-engineered molecules can be generated as described in U.S. Patent No. 7,521,541.

[0152] In some embodiments, the Fc domain is derived from IgG1 Fc. In some embodiments, the Fc domain is derived from human IgG1 Fc. In some embodiments, the Fc domain is derived from wild-type IgG1 Fc (IGHG1*05). In some embodiments, the Fc domain is a naturally occurring variant of IgG1 (e.g., IGHG1*03, which comprises a D239E and L241M double mutation compared to IGHG1*05). In some embodiments, the Fc domain does not comprise the hinge region of IgG1 Fc. In some embodiments, the Fc domain comprises a truncation of up to five amino acids from the N-terminus of IgG1 Fc, for example, a truncation of the first 1, first 2, first 3, first 4, or first 5 amino acids from the N-terminus of IgG1 Fc. In some embodiments, the Fc domain comprises one or more null-effect mutations and / or deglycosylation mutations.

[0153] In some embodiments, the Fc domain is derived from IgG4Fc. In some embodiments, the Fc domain is derived from human IgG4Fc. In some embodiments, the Fc domain is wild-type IgG4Fc. In some embodiments, the Fc domain is a naturally occurring variant of IgG4. In some embodiments, the Fc domain does not comprise the hinge region of IgG4Fc. In some embodiments, the Fc portion comprises a truncation of up to 5 amino acids from the N-terminus of IgG4Fc, for example, a truncation of the first 1, first 2, first 3, first 4, or first 5 amino acids from the N-terminus of IgG4Fc. In some embodiments, the Fc domain comprises one or more null-effect mutations and / or deglycosylation mutations.

[0154] Strategies for forming Fc fusion protein polypeptides or bispecific antibodies are well known (see Spiesetal., MolImm. (2015) 67(2)(A):95-106). For example, in some embodiments, the first and / or second polypeptide chain of the Fc domain comprises one or more modifications that promote heterodimerization of the first and second Fc domains, respectively. Thus, one or more amino acid modifications to the first Fc domain and one or more amino acid alterations to the second Fc region can be made using any strategy available in the art (including any of the strategies described in Kleinettal. (2012), MAbs, 4(6):653-663). An exemplary strategy and modification is the "knob-holes" approach. In some embodiments, the first Fc domain comprising the CH3 domain is a heavy chain polypeptide or a fragment thereof. The CH3 domains of both Fc domains can be modified using the "knob-into-holes" technique (Fc knobs and Fc holes), as described in detail in WO 1996 / 027011; Ridgway, JB et al. Protein Eng (1996) 9(7):617-621; Merchant, AM, et al. Nat. Biotechnoi. (1998) 16(7):677-681, but also in Kleine et al. (2012), MAbs, 4(6):653-663. Using the knob-into-holes method, the interaction surfaces of the two CH3 domains are altered, thereby increasing heterodimerization of two moieties containing two modified CH3 structures. This is achieved by introducing a bulky residue into the CH3 domain of one of the Fc domains, which acts as a "knob." To accommodate the bulky residue, a "hole" is then created in the other Fc domain that can accommodate the knob. One of the engineered CH3 domains can be the "knob" and the other can be the "hole."The introduction of disulfide bonds further stabilizes heterodimers (Merchant, AM, et al., Nat. Biotechnol. (1998) 16(7); Atwell, S., et al., J. Mol. Biol. (1997) 270(1):26-35), improving yield. It is known that heterodimerization can be achieved by introducing T366W and / or S354C mutations into the heavy chain to create a "knob" and by introducing T366S, L368A, Y407V, and / or Y349C mutations into the heavy chain to create a "hole" (residues numbered according to the Kabat EU numbering system). Carter et al. (2001), J. Immunol. Methods, 248:7-15; Kleine et al. (2012), MAbs, 4(6):653-663.

[0155] In some embodiments, the Fc domain or fragment thereof comprises T366S, L368A, and Y407V mutations, forming a "hole." In some embodiments, the Fc domain or fragment thereof comprises T366W mutations, forming a "knob." In some embodiments, the Fc domain or fragment thereof comprises Y349C, T366S, L368A, and Y407V mutations, forming a "hole." In some embodiments, the Fc domain or fragment thereof comprises S354C and T366W mutations, forming a "knob." In some embodiments, the first Fc domain or fragment thereof comprises a hole mutation and the second Fc domain or fragment thereof comprises a knob mutation. In some embodiments, the first Fc domain or fragment thereof comprises a knob mutation and the second Fc domain or fragment thereof comprises a hole mutation, and residue numbering is according to the EU numbering system.

[0156] In some examples, in addition to the LALA (L234A and L235A) mutations, the knob-into-holes mutations are also present in the Fc domain.

[0157] In some examples, the first Fc domain comprises the amino acid sequence SEQ ID NO:27 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:27, and the second Fc domain comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:28.

[0158] In some examples, the first Fc domain comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:28, and the second Fc domain comprises the amino acid sequence SEQ ID NO:27 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:27.

[0159] In some examples, the first Fc domain comprises the amino acid sequence SEQ ID NO:29 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:29, and the second Fc domain comprises the amino acid sequence SEQ ID NO:30 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:30.

[0160] In some examples, the first Fc domain comprises the amino acid sequence SEQ ID NO: 30 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO: 30, and the second Fc domain comprises the amino acid sequence SEQ ID NO: 29 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO: 29.

[0161] Prodrug One aspect of the present application provides activatable prodrugs that are metabolized in vivo to active therapeutic agents with fewer side effects and better target specificity. In some embodiments, the prodrugs include one or more biologically active moieties (B), one or more cleavable moieties (CM), and one or more masking polypeptides (MP). In some embodiments, the masking polypeptides (MP) of the prodrug inhibit the biological function of the biologically active moiety (B). Digestion of the cleavable moiety (CM) of the prodrug by a protease at the target site in a patient (e.g., a tumor site or the surrounding environment) releases the masking polypeptide (MP) from the prodrug, exposing the previously masked biologically active moiety (B), allowing the biologically active moiety (B) to exert its biological function on the target cell, thereby activating the prodrug.

[0162] In some embodiments, the prodrug further comprises one or more non-cleavable linkers (L).

[0163] In some embodiments, in the prodrugs according to the present invention, the masking polypeptide (MP) and the biologically active moiety (B) are linked by the cleavable moiety (CM).

[0164] In some embodiments, in the prodrugs according to the present invention, the cleavable moiety (CM) and the masking polypeptide (MP) are linked, preferably by a non-cleavable linker (L).

[0165] In some embodiments, in the prodrugs herein, the cleavable moiety (CM) and the biologically active moiety (B) are linked, preferably by a non-cleavable linker (L).

[0166] In some embodiments, the prodrug further comprises one or more half-life extending moieties (C).

[0167] In some embodiments, in the prodrugs herein, the biologically active moiety (B) and the half-life extending moiety (C) are linked.

[0168] In some embodiments, in the prodrugs herein, the biologically active moiety (B) and the half-life extending moiety (C) are linked, preferably by a non-cleavable linker (L).

[0169] In some embodiments, in the prodrugs herein, the masking polypeptide (MP) and the half-life extending moiety (C) are linked by the cleavable moiety (CM).

[0170] In some embodiments, in the prodrugs herein, the cleavable moiety (CM) and the half-life extending moiety (C) are linked, preferably by a non-cleavable linker (L).

[0171] In some embodiments, in the prodrugs according to the present invention, the cleavable moiety (CM) and the masking polypeptide (MP) are linked, preferably by a non-cleavable linker (L).

[0172] In some examples, in the prodrugs according to the present disclosure, the prodrug comprises a construct in which the N to C-terminus or C to N-terminus is BC-CM-MP, where "-" represents a covalent bond with or without a non-cleavable linker (L).

[0173] In some examples, in the prodrugs according to the present disclosure, the prodrug comprises a construct in which the N to C-terminus or C to N-terminus is CB-CM-MP, where "-" represents a covalent bond with or without a non-cleavable linker (L).

[0174] In some embodiments, the prodrugs provided herein are monomeric. In some embodiments, the prodrugs provided herein are dimeric. In some embodiments, the dimers are monovalent. In some embodiments, the dimers are bivalent. In some embodiments, the dimers are homodimers. In some embodiments, the dimers are heterodimers.

[0175] In some examples, the prodrugs herein are dimeric, with one of the monomers comprising a construct N to C-terminal or C to N-terminally CB, and the other of the monomers comprising a construct N to C-terminal or C to N-terminally C-CM-MP, where "-" represents a covalent bond with or without a non-cleavable linker (L).

[0176] Exemplary Antibody Prodrugs The present application provides antibody prodrugs having a masking polypeptide (MP) and a cleavable moiety (CM), which are metabolized in vivo to an active antibody, with fewer side effects associated with the masked antibody. In some embodiments, the masking polypeptide (MP) and VCM are H The N- or C-termini of the domains are linked by a cleavable moiety (CM). In some embodiments, the masking polypeptide (MP) and V LThe N- or C-termini of the domains are linked by a cleavable moiety (CM). In some embodiments, the masking polypeptide (MP) and V H N-terminal and V domains L The N-termini of the domains are linked by a cleavable moiety (CM).

[0177] In some embodiments, the antibody prodrug is a TNFR2 antibody prodrug. Schematic structures of the anti-TNFR2 antibody or antibody prodrug are shown in Figures 14A-14D.

[0178] In some examples, an anti-TNFR2 antibody prodrug according to the present specification comprises (i) an antibody or antigen-binding fragment thereof that specifically binds to human TNFR2, (ii) one or more masking polypeptides (MPs) described herein, and (iii) one or more cleavable moieties (CMs) described herein.

[0179] In some embodiments, the masking polypeptide (MP) reduces the binding affinity of the anti-TNFR2 antibody or antigen-binding fragment to human TNFR2 compared to the anti-TNFR2 antibody or antigen-binding fragment without the masking polypeptide (MP). In some embodiments, the binding affinity is reduced by at least about 10-fold compared to the anti-TNFR2 antibody or antigen-binding fragment without the masking polypeptide (MP). In some embodiments, the binding affinity is reduced by at least about 100-fold compared to the anti-TNFR2 antibody or antigen-binding fragment without the masking polypeptide (MP). In some embodiments, the binding affinity is reduced by at least about 200-1500-fold compared to the anti-TNFR2 antibody or antigen-binding fragment without the masking polypeptide (MP). In some embodiments, the masking polypeptide (MP) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-5 or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-5.

[0180] In some embodiments, in the anti-TNFR2 antibody prodrugs provided herein, the masking polypeptide (MP) and V H The N- and / or C-termini of the domains are linked by a cleavable moiety (CM).

[0181] In some embodiments, in the anti-TNFR2 antibody prodrugs provided herein, the masking polypeptide (MP) and V L The N- and / or C-termini of the domains are linked by a cleavable moiety (CM).

[0182] In some embodiments, in the anti-TNFR2 antibody prodrugs provided herein, the masking polypeptide (MP) and V H The N-terminus and / or C-terminus of the domain are linked by a cleavable moiety (CM), and the masking polypeptide (MP) and VL The N- and / or C-termini of the domains are linked by a cleavable moiety (CM).

[0183] In some embodiments, the anti-TNFR2 antibody in the anti-TNFR2 antibody prodrug is a full-length antibody. In some embodiments, the isolated anti-TNFR2 antibody is a full-length IgG1, IgG2, IgG3, or IgG4.

[0184] In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG1 is human wild-type IgG1. In some embodiments, the IgG1 comprises one or more mutations compared to human wild-type IgG1.

[0185] In some embodiments, the IgG2 is human IgG2. In some embodiments, the IgG2 is human wild-type IgG2. In some embodiments, the IgG2 comprises one or more mutations compared to human wild-type IgG2.

[0186] In some embodiments, the IgG3 is a human IgG3. In some embodiments, the IgG3 is a human wild-type IgG3. In some embodiments, the IgG3 comprises one or more mutations compared to human wild-type IgG3.

[0187] In some embodiments, the IgG4 is human IgG4. In some embodiments, the IgG4 is human wild-type IgG4. In some embodiments, the IgG4 comprises one or more mutations compared to human wild-type IgG4.

[0188] In some embodiments, the anti-TNFR2 antibody prodrug comprises two heavy chains and two light chains, and the masking polypeptide (MP) and the N-terminus of one or two heavy chains are linked by a cleavable moiety (CM).

[0189] In some embodiments, the anti-TNFR2 antibody prodrug comprises two heavy chains and two light chains, and the N-terminus of the masking polypeptide (MP) and one or two light chains are linked by a cleavable moiety (CM).

[0190] In some embodiments, the anti-TNFR2 antibody prodrug comprises two heavy chains and two light chains, wherein the masking polypeptide (MP) and the N-terminus of the two heavy chains are linked by a cleavable moiety (CM), and the masking polypeptide (MP) and the N-terminus of the two light chains are linked by a cleavable moiety (CM).

[0191] In some embodiments, the cleavable moiety (CM) and the masking polypeptide (MP) are linked, preferably by a non-cleavable linker (L).

[0192] In some embodiments, the cleavable moiety (CM) and V H Domain and / or V L The domains are linked, preferably by a non-cleavable linker (L).

[0193] In some embodiments, the cleavable moiety (CM) described herein comprises a cleavable site that can be cleaved under certain circumstances to separate the N-terminal fragment from the C-terminal fragment, such that the cleavable site between the masking polypeptide and the heavy or light chain of the antibody allows the masking polypeptide to be removed at the cleavable site under certain circumstances, thereby releasing the fully functional anti-TNFR2 antibody.

[0194] In some embodiments, screening for a suitable cleavable moiety (CM) depends on the desired site of action of the anti-TNFR2 antibody. For example, if the desired site of action is a tumor site, the cleavage site of a tumor-specific protease is used to construct a masked anti-TNFR2 antibody designed to act at the tumor site. A tumor-specific protease refers to any protease that has higher levels and / or activity at tumor sites compared to normal tissues.

[0195] In some embodiments, the protease cleavage site may be a matrix metalloproteinase (MMP) cleavage site. In some embodiments, the protease cleavage site may be a cleavage site for MMP2. In some embodiments, the protease cleavage site may be a cleavage site for MMP9. In some embodiments, the protease cleavage site may be a cleavage site for MMP2 and MMP9. Other tumor-specific proteases and their corresponding cleavage sites are known in the art, such as those disclosed in Vasiljeva et al., Scientific Reports, 10:5894, 2020, the relevant disclosure of which is incorporated herein by reference and consistent with the subject matter and purpose of this specification. In some embodiments, the cleavable moiety (CM) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 8-16, or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 8-16.

[0196] In some embodiments, in the anti-TNFR2 antibody prodrugs provided herein, the anti-TNFR2 antibody is H And, V L and V H comprises an HC-CDR1 comprising the amino acid sequence SEQ ID NO: 50, an HC-CDR2 comprising the amino acid sequence SEQ ID NO: 51, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO: 52, or a V comprising at most about five amino acid substitutions in said HC-CDRs. H and the V L comprises an LC-CDR1 comprising the amino acid sequence SEQ ID NO: 53, an LC-CDR2 comprising the amino acid sequence SEQ ID NO: 54, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO: 55, or any of the above V CDRs containing at most about five amino acid substitutions in the LC-CDRs. L Includes variants of.

[0197] In some embodiments, in the anti-TNFR2 antibody prodrugs provided herein, the anti-TNFR2 antibody is H And, V L and V H comprises the amino acid sequence SEQ ID NO: 56 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO: 56, L comprises the amino acid sequence SEQ ID NO:57 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:57.

[0198] In some embodiments, the anti-TNFR2 antibody prodrug comprises a heavy chain, wherein the heavy chain comprises a construct N- to C-terminally comprising MP-CM-V H -C H 1-hinge-C H 2-C H 3, where "-" represents a covalent bond with or without a non-cleavable linker (L). In some examples, the heavy chain of the anti-TNFR2 antibody prodrug comprises the amino acid sequence SEQ ID NO: 58 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 58.

[0199] In some embodiments, the anti-TNFR2 antibody prodrug comprises a light chain, and the light chain comprises a construct N- to C-terminally comprising MP-CM-V L -C Lwhere "-" represents a covalent bond with or without a non-cleavable linker (L). In some examples, the anti-TNFR2 antibody prodrug light chain comprises the amino acid sequence SEQ ID NO:61 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO:61.

[0200] In some examples, the anti-TNFR2 antibody prodrug comprises two heavy chains and two light chains, each of the heavy chains comprising the amino acid sequence SEQ ID NO: 58 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 58, and each of the light chains comprising the amino acid sequence SEQ ID NO: 59 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 59.

[0201] In some embodiments, the anti-TNFR2 antibody prodrug comprises two heavy chains and two light chains, each of the heavy chains comprising the amino acid sequence SEQ ID NO: 60 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 60, and each of the light chains comprising the amino acid sequence SEQ ID NO: 61 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 61.

[0202] In some examples, the anti-TNFR2 antibody prodrug comprises two heavy chains and two light chains, each of the heavy chains comprising the amino acid sequence SEQ ID NO: 58 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 58, and each of the light chains comprising the amino acid sequence SEQ ID NO: 61 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 61.

[0203] Exemplary Cytokine Prodrugs The present application provides, by way of example, IL-15 prodrugs that are metabolized in vivo to active IL-15 cytokines. The IL-15 prodrugs have fewer side effects. In some embodiments, the IL-15 prodrugs have a better PK spectrum in vivo (e.g., a longer half-life).

[0204] A structural schematic of an exemplary IL-15 cytokine prodrug is shown in Figure 2A.

[0205] In some examples, an IL-15 prodrug according to the present specification comprises (i) an IL-15 cytokine (I), (ii) one or more masking polypeptides (MPs) described herein, and (iii) one or more cleavable moieties (CMs) described herein.

[0206] In some embodiments, in the IL-15 prodrug according to the present invention, the IL-15 cytokine (I) and the masking polypeptide (MP) are linked by a cleavable moiety (CM).

[0207] In some embodiments, in the IL-15 prodrugs according to the present invention, the cleavable moiety (CM) and the IL-15 cytokine (I) are linked, preferably by a non-cleavable linker (L).

[0208] In some embodiments, in the IL-15 prodrugs according to the present invention, the cleavable moiety (CM) and the masking polypeptide (MP) are linked, preferably by a non-cleavable linker (L).

[0209] In some examples, IL-15 prodrugs according to the present disclosure include constructs in which the N to C-terminus or C to N-terminus is I-CM-MP, where "-" represents a covalent bond with or without a non-cleavable linker (L).

[0210] In some examples, the IL-15 prodrugs according to the present invention further comprise one or more half-life extending moieties (C).

[0211] In some embodiments, in the IL-15 prodrugs herein, the IL-15 cytokine (I) and the half-life extending moiety (C) are linked.

[0212] In some embodiments, in the IL-15 prodrugs herein, the IL-15 cytokine (I) and the half-life extending moiety (C) are linked, preferably by a non-cleavable linker (L).

[0213] In some embodiments, in the IL-15 prodrugs herein, the masking polypeptide (MP) and the half-life extending moiety (C) are linked by a cleavable moiety (CM).

[0214] In some embodiments, in the IL-15 prodrugs herein, the cleavable moiety (CM) and the masking polypeptide (MP) are linked by a non-cleavable linker (L).

[0215] In some embodiments, in the IL-15 prodrugs herein, the cleavable moiety (CM) and the half-life extending moiety (C) are linked, preferably by a non-cleavable linker (L).

[0216] In some examples, IL-15 prodrugs according to the present disclosure include constructs that are N to C-terminal or C to N-terminal CI-CM-MP, where "-" represents a covalent bond with or without a non-cleavable linker (L).

[0217] In some examples, IL-15 prodrugs according to the present disclosure include constructs that are N to C-terminal or C to N-terminal IC-CM-MP, where "-" represents a covalent bond with or without a non-cleavable linker (L).

[0218] In some examples, IL-15 prodrugs according to the present disclosure include constructs that are N to C-terminal or C to N-terminally C-CM-MP-I, where "-" represents a covalent bond with or without a non-cleavable linker (L).

[0219] In some examples, an IL-15 prodrug according to the present disclosure is a dimer, wherein one of the monomers comprises a construct whose N to C-terminus or C to N-terminus is CI, and the other of the monomers comprises a construct whose N to C-terminus or C to N-terminus is C-CM-MP, where "-" represents a covalent bond with or without a non-cleavable linker (L).

[0220] In some examples, an IL-15 prodrug according to the present specification comprises (i) an IL-15 cytokine (I), (ii) an IL-15Rα or a functional fragment thereof (S), (iii) one or more masking polypeptides (MPs) described herein, and (iv) one or more cleavable moieties (CMs) described herein.

[0221] In some embodiments, in the IL-15 prodrug according to the present invention, the IL-15 cytokine (I) and the masking polypeptide (MP) are linked by a cleavable moiety (CM).

[0222] In some embodiments, in the IL-15 prodrugs according to the present invention, the cleavable moiety (CM) and the IL-15 cytokine (I) are linked, preferably by a non-cleavable linker (L).

[0223] In some embodiments, in the IL-15 prodrugs according to the present invention, the cleavable moiety (CM) and the masking polypeptide (MP) are linked, preferably by a non-cleavable linker (L).

[0224] In some embodiments, in the IL-15 prodrug according to the present invention, the IL-15Rα or functional fragment thereof (S) and the IL-15 cytokine (I) are linked together.

[0225] In some embodiments, in the IL-15 prodrug according to the present invention, the IL-15Rα or functional fragment thereof (S) and the masking polypeptide (MP) are linked by a cleavable moiety (CM).

[0226] In some embodiments, in the IL-15 prodrugs herein, the cleavable moiety (CM) and the IL-15Rα or functional fragment thereof (S) are linked, preferably by a non-cleavable linker (L).

[0227] In some embodiments, in the IL-15 prodrugs according to the present invention, the cleavable moiety (CM) and the masking polypeptide (MP) are linked, preferably by a non-cleavable linker (L).

[0228] In some embodiments, in the IL-15 prodrug herein, the IL-15Rα or functional fragment thereof (S) and the IL-15 cytokine (I) are covalently linked, hi some embodiments, the IL-15Rα or functional fragment thereof (S) and the IL-15 cytokine (I) are linked by a non-cleavable linker (L).

[0229] In some embodiments, in the IL-15 prodrug according to the present specification, the IL-15Rα or a functional fragment thereof (S) and the IL-15 cytokine (I) are non-covalently linked to form an IL-15 / IL-15Rα complex.

[0230] In some examples, in an IL-15 prodrug according to the present invention, IL-15Rα or a functional fragment thereof and IL-15 cytokine are transfected, respectively, to form an IL-15 / IL-15Rα complex.

[0231] In some examples, the IL-15 prodrugs according to the present invention further comprise one or more half-life extending moieties (C).

[0232] In some embodiments, in the IL-15 prodrugs herein, the IL-15 cytokine (I) and the half-life extending moiety (C) are linked.

[0233] In some embodiments, in the IL-15 prodrugs herein, the IL-15 cytokine (I) and the half-life extending moiety (C) are linked, preferably by a non-cleavable linker (L).

[0234] In some embodiments, in the IL-15 prodrugs herein, the IL-15Rα or functional fragment thereof (S) and the half-life extending moiety (C) are linked.

[0235] In some embodiments, in the IL-15 prodrugs herein, the IL-15Rα or functional fragment thereof (S) and the half-life extending moiety (C) are linked, preferably by a non-cleavable linker (L).

[0236] In some embodiments, in the IL-15 prodrugs herein, the masking polypeptide (MP) and the half-life extending moiety (C) are linked by a cleavable moiety (CM).

[0237] In some embodiments, in the IL-15 prodrugs according to the present invention, the cleavable moiety (CM) and the masking polypeptide (MP) are linked, preferably by a non-cleavable linker (L).

[0238] In some embodiments, in the IL-15 prodrugs herein, the cleavable moiety (CM) and the half-life extending moiety (C) are linked, preferably by a non-cleavable linker (L).

[0239] In some embodiments, in the IL-15 prodrug herein, the IL-15 cytokine (I) and the half-life extending moiety (C) are linked, the IL-15Rα or a functional fragment thereof (S) and the IL-15 cytokine (I) are linked, and the masking polypeptide (MP) and the IL-15Rα or a functional fragment thereof (S) are linked by a cleavable moiety (CM).

[0240] In some embodiments, in the IL-15 prodrugs herein, the IL-15 cytokine (I) and the half-life extending moiety (C) are linked, the IL-15Rα or a functional fragment thereof (S) and the half-life extending moiety (C) are linked, and the masking polypeptide (MP) and the IL-15 cytokine (I) are linked by a cleavable moiety (CM).

[0241] In some embodiments, in the IL-15 prodrugs herein, the IL-15Rα or functional fragment thereof (S) and the half-life extending moiety (C) are linked, the IL-15 cytokine (I) and the IL-15Rα or functional fragment thereof (S) are linked, and the masking polypeptide (MP) and the IL-15 cytokine (I) are linked by a cleavable moiety (CM).

[0242] In some embodiments, in the IL-15 prodrugs herein, the IL-15 cytokine (I) and the half-life extending moiety (C) are linked, the IL-15Rα or functional fragment thereof (S) and the half-life extending moiety (C) are linked, and the masking polypeptide (MP) and the IL-15Rα or functional fragment thereof (S) are linked by a cleavable moiety (CM).

[0243] In some embodiments, in the IL-15 prodrugs herein, the masking polypeptide (MP) and the half-life extending moiety (C) are linked by a cleavable moiety (CM), the IL-15Rα or functional fragment thereof (S) and the half-life extending moiety (C) are linked, and the IL-15 cytokine (I) and the IL-15Rα or functional fragment thereof (S) are linked.

[0244] In some embodiments, in the IL-15 prodrugs herein, the masking polypeptide (MP) and the half-life extending moiety (C) are linked by a cleavable moiety (CM), the IL-15 cytokine (I) and the half-life extending moiety (C) are linked, and the IL-15Rα or functional fragment thereof (S) and the IL-15 cytokine (I) are linked.

[0245] In some embodiments, in the IL-15 prodrug herein, the IL-15Rα or functional fragment thereof (S) and the IL-15 cytokine (I) are covalently linked, hi some embodiments, the IL-15Rα or functional fragment thereof (S) and the IL-15 cytokine (I) are linked by a non-cleavable linker (L).

[0246] In some embodiments, in the IL-15 prodrug according to the present specification, the IL-15Rα or a functional fragment thereof (S) and the IL-15 cytokine (I) are non-covalently linked to form an IL-15 / IL-15Rα complex.

[0247] In some examples, IL-15 prodrugs according to the present disclosure include constructs that are N to C-terminal or C to N-terminal CIS-CM-MP, where "-" represents a covalent bond with or without a non-cleavable linker (L).

[0248] In some examples, IL-15 prodrugs according to the present disclosure include constructs in which the N to C-terminus or C to N-terminus is CSI-CM-MP, where "-" represents a covalent bond with or without a non-cleavable linker (L).

[0249] In some examples, IL-15 prodrugs according to the present disclosure include constructs that are N to C-terminal or C to N-terminal ICS-CM-MP, where "-" represents a covalent bond with or without a non-cleavable linker (L).

[0250] In some examples, IL-15 prodrugs according to the present disclosure include constructs that are N to C-terminal or C to N-terminal SCI-CM-MP, where "-" represents a covalent bond with or without a non-cleavable linker (L).

[0251] In some examples, IL-15 prodrugs according to the present disclosure include constructs that are N to C-terminal or C to N-terminal SIC-CM-MP, where "-" represents a covalent bond with or without a non-cleavable linker (L).

[0252] In some examples, IL-15 prodrugs according to the present disclosure include constructs that are N to C-terminal or C to N-terminally ISC-CM-MP, where "-" represents a covalent bond with or without a non-cleavable linker (L).

[0253] In some embodiments, the IL-15 prodrugs herein are monomeric.

[0254] In some embodiments, the IL-15 prodrug provided herein is a dimer. In some embodiments, the dimer is monovalent. In some embodiments, the dimer is bivalent. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer is a heterodimer.

[0255] In some embodiments, the IL-15 prodrugs provided herein are dimeric, one monomer comprising a first half-life extending moiety (C), said IL-15Rα or a functional fragment thereof (S), and said IL-15 cytokine (I), and the other monomer comprising a second half-life extending moiety (C), said masking polypeptide (MP), and said cleavable moiety (CM), wherein said masking polypeptide (MP) and said half-life extending moiety (C) are linked by the cleavable moiety (CM).

[0256] In some embodiments, the IL-15 prodrugs provided herein are dimeric, one monomer comprising a first half-life extending moiety (C), said IL-15Rα or a functional fragment thereof (S), said masking polypeptide (MP), and said cleavable moiety (CM), and the other monomer comprising a second half-life extending moiety (C) and said IL-15 cytokine (I).

[0257] In some embodiments, the IL-15 prodrugs provided herein are dimeric, one monomer comprising a first half-life extending moiety (C), said IL-15 cytokine (I), said masking polypeptide (MP), and said cleavable moiety (CM), and the other monomer comprising a second half-life extending moiety (C) and said IL-15Rα or functional fragment thereof (S).

[0258] In some embodiments, the IL-15 prodrugs herein are dimeric, both monomers comprising a half-life extending moiety (C), said IL-15 cytokine (I), said IL-15Rα or a functional fragment thereof (S), and said masking polypeptide (MP).

[0259] In some embodiments, the IL-15 prodrugs herein are dimeric, in which in one of the monomers, the IL-15 cytokine (I) and a first half-life extending moiety (C) are linked, and the IL-15Rα or functional fragment thereof (S) and a first half-life extending moiety (C) are linked, and in the other monomer, the masking polypeptide (MP) and a second half-life extending moiety (C) are linked by the cleavable moiety (CM).

[0260] In some embodiments, the IL-15 prodrugs herein are dimeric, in which in one of the monomers, the IL-15 cytokine (I) and a first half-life extending moiety (C) are linked and the IL-15Rα or functional fragment thereof (S) and the IL-15 cytokine (I) are linked, and in the other monomer, the masking polypeptide (MP) and a second half-life extending moiety (C) are linked by the cleavable moiety (CM).

[0261] In some embodiments, the IL-15 prodrugs provided herein are dimeric, in one of the monomers, the IL-15Rα or functional fragment thereof (S) and a first half-life extending moiety (C) are linked, the masking polypeptide (MP) and the first half-life extending moiety (C) are linked by the cleavable moiety (CM), and the other of the monomers, the IL-15 cytokine (I) and a second half-life extending moiety (C) are linked.

[0262] In some embodiments, the IL-15 prodrugs provided herein are dimeric, in which in one of the monomers, the IL-15Rα or functional fragment thereof (S) and a first half-life extending moiety (C) are linked, and the masking polypeptide (MP) and IL-15Rα or functional fragment thereof (S) are linked by the cleavable moiety (CM), and in the other monomer, the IL-15 cytokine (I) and a second half-life extending moiety (C) are linked.

[0263] In some embodiments, the IL-15 prodrugs provided herein are dimeric, in one of the monomers, the IL-15 cytokine (I) and a first half-life extending moiety (C) are linked, and the masking polypeptide (MP) and the first half-life extending moiety (C) are linked by the cleavable moiety (CM), and in the other monomer, the IL-15Rα or functional fragment thereof (S) and a second half-life extending moiety (C) are linked.

[0264] In some embodiments, the IL-15 prodrugs provided herein are dimeric, in one of the monomers, the IL-15 cytokine (I) and a first half-life extending moiety (C) are linked, and the masking polypeptide (MP) and the IL-15 cytokine (I) are linked by the cleavable moiety (CM), and in the other monomer, the IL-15Rα or functional fragment thereof (S) and a second half-life extending moiety (C) are linked.

[0265] In some embodiments, in the IL-15 prodrug herein, the IL-15Rα or functional fragment thereof (S) and the IL-15 cytokine (I) are covalently linked, hi some embodiments, the IL-15Rα or functional fragment thereof (S) and the IL-15 cytokine (I) are linked by a non-cleavable linker (L).

[0266] In some embodiments, in the IL-15 prodrug according to the present specification, the IL-15Rα or a functional fragment thereof (S) and the IL-15 cytokine (I) are non-covalently linked to form an IL-15 / IL-15Rα complex.

[0267] IL-15: Interleukin-15 (IL-15), a member of the four α-helical bundle family, has a molecular weight of 14-15 kDa and 114 amino acids (Fehniger TA, Caligiuri MA. Interleukin 15: biology and relevance to human disease. Blood. 2001;97:14-32). It is produced by mononuclear phagocytes and other immune system cells. IL-15 is crucial for the development and function of natural killer (NK) cells, natural killer T cells (NKT), and memory CD8+ T cells.

[0268] IL-15 is a cytokine similar to IL-2, originally called T-cell growth factor. Both cytokines exert their cell signaling functions by binding to receptors composed of a trimeric complex consisting of two shared receptor chains (i.e., the co-γ chain (γc; CD132) and the IL-2 receptor β chain (IL-2Rβ; CD122)) and a cytokine-specific α chain receptor (IL-2 receptor α (IL-2Rα; CD25) or IL-15 receptor α (IL-15Rα; CD215)).

[0269] IL-15 shares receptor components with IL-2. The α chain of the IL-2 receptor (IL-2R) is not essential, but the β and co-γ chains are required for IL-15-mediated biological activities (Giri JG, et al. IL-15, a novel cell growth factor that shares activities and receptor components with IL-2. J Leukoc Biol. 1995 May;57(5):763-6.). IL-15R is composed of three subunits: IL-15Rα, IL-2 / IL-15Rβ, and the γ chain. IL-15Rα is required for high-affinity binding but does not transduce IL-15 signals. IL-15 exerts its effects primarily through trans-presentation (TP), in which IL-15-expressing APCs bind to IL-15Rα and present the ligand to βγ receptor heterodimers on surrounding T / NK cells (Kenesei A, Volko J, et al. IL-15 Trans-Presentation Isan Autonomous, Antigen-Independent Process. J Immunol. 2021 Nov 15;207(10):2489-2500).

[0270] In some embodiments, the IL-15 cytokine comprises an IL-15 variant. In eukaryotic cells, IL-15 is initially synthesized as a 162 amino acid precursor polypeptide, which is subsequently processed to mature IL-15 by removing amino acid residues 1-48. The mature form of IL-15 consists of 114 amino acids (amino acid residues 49-162) and is secreted in its active mature form.

[0271] In some examples, the IL-15 cytokine comprises the amino acid sequence SEQ ID NO:22 or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:22.

[0272] In some examples, the IL-15 cytokine comprises the amino acid sequence SEQ ID NO:23 or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:23.

[0273] In some embodiments, the IL-15 cytokine comprises an IL-15 variant or functional fragment thereof. In some embodiments, the IL-15 cytokine is a naturally occurring interleukin-15 (IL-15) protein. In some embodiments, the IL-15 cytokine is a modified variant thereof that binds to or otherwise exhibits affinity for the interleukin-15 receptor (IL-15R) or a component thereof (e.g., the IL-15Rα, IL-2 / IL-15Rβ, and / or γ chains).

[0274] In some embodiments, the IL-15 cytokine comprises an amino acid sequence obtained by modifying at least one amino acid in the amino acid sequence SEQ ID NO: 22. The at least one amino acid modification may be any amino acid modification, such as a substitution, insertion, or deletion, respectively. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, as long as the final construct possesses the desired properties, such as retained / improved ligand-receptor binding, retained / enhanced biological activity, etc. In some embodiments, the IL-15 cytokine comprises an amino acid sequence obtained by making 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 amino acid substitutions in the amino acid sequence SEQ ID NO: 22.

[0275] IL-15Rα or a functional fragment thereof The IL-15Rα or functional fragment thereof according to the present application may be any species of IL-15Rα or functional fragment thereof.

[0276] In some embodiments, the IL-15Rα or functional fragment thereof is selected from the extracellular region of human IL-15Rα or the sushi domain or functional analog.

[0277] Extracellular region of IL-15Rα: The extracellular region of IL-15Rα is generally defined as the sequence extending from the first N-terminal amino acid of the IL-15Rα sequence to the last amino acid of the tail region (or region rich in glycosylation sites). The tail region of the IL-15Rα sequence can be determined by those skilled in the art, for example, using auxiliary software.

[0278] IL-15Rα_sushi domain: The extracellular region of the IL-15Rα contains a domain known as the sushi domain (Weiet al. 2001, J. Immunol. 167:277-282). The IL-15Rα_sushi domain contains a β-sheet.

[0279] The IL-15Rα_sushi domain has the highest binding affinity for IL-15 and enhances the binding and biological effects of IL-15 (proliferation and protection from apoptosis) via the IL-15Rβγ heterodimer without affecting IL-15 binding and function (Mortier E, et al. J Biol Chem. 2006 Jan 20;281(3):1612-9), making it an effective IL-15 antagonist.

[0280] It is encoded by exon 2 of IL-15Rα (Anderson DM, et al. Functional characterization of the human interleukin-15 receptor alpha chain and close linkage of IL15RA and IL2RA genes. J Biol Chem. 1995 Dec 15;270(50):29862-9). It begins with the first cysteine residue (C1) encoded by exon 2 and ends with the fourth cysteine residue (C4) encoded by exon 2. From the IL-15Rα protein sequence (canonical N- to C-terminal orientation), the sushi domain of IL-15Rα is defined as beginning with the first cysteine residue (C1) after the signal peptide and ending with the fourth cysteine residue (C4) after the signal peptide. Both residues C1 and C4 are included in the sushi sequence. The IL-15Rα sushi domain can also be determined by analyzing the amino acid sequence of IL-15Rα using appropriate analytical software, such as those described in Prosite (http: / / us.expasy.org / prosite / ), (http: / / www.ebi.ac.uk / lnterProScan / ), and SMART (http: / / elm.eu.org / ).

[0281] In some examples, the IL-15Rα_sushi domain comprises the amino acid sequence SEQ ID NO:24 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:24.

[0282] In some examples, the IL-15Rα_sushi domain comprises the amino acid sequence SEQ ID NO:25 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:25.

[0283] In some examples, the IL-15Rα_sushi domain comprises the amino acid sequence SEQ ID NO:26 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:26.

[0284] In some examples, in the IL-15 prodrug, the IL-15 cytokine or the IL-15Rα or functional fragment thereof (eg, IL-15Rα_sushi domain) has one or more conservative amino acid substitutions.

[0285] "Conservative substitution" refers to a substitution of another amino acid with the same net charge and approximately the same size and shape as the amino acid being substituted. Amino acids with aliphatic or substituted aliphatic amino acid side chains will be approximately the same size if the total number of carbon and heteroatoms in their side chains differs by no more than four. Amino acids will be approximately the same shape if the number of branches in their side chains differs by no more than one. Amino acids with phenyl or substituted phenyl in their side chains can be considered to be approximately the same size and shape. Unless otherwise specified, it is preferable to use natural amino acids in conservative substitutions.

[0286] As used herein, the term "amino acid" is used in its broadest sense and includes both naturally occurring amino acids and non-naturally occurring amino acids, including amino acid analogs and derivatives. The latter includes molecules containing an amino acid moiety. Based on this broad definition, those skilled in the art will recognize that the amino acids described herein include, for example, naturally occurring proteinogenic L-amino acids; D-amino acids; chemically modified amino acids, such as amino acid analogs and derivatives; naturally occurring non-proteinogenic amino acids, such as norleucine, β-alanine, ornithine, and GABA; and chemically synthesized compounds having amino acid characteristics known in the art. As used herein, the term "proteinogenic" refers to amino acids from which cellular peptides, polypeptides, or proteins can be synthesized through metabolic pathways.

[0287] Inserting synthetic unnatural amino acids, substituted amino acids, or unnatural amino acids, including one or more D-amino acids, into a polypeptide (e.g., an IL-15 cytokine in an IL-15 cytokine prodrug described herein) can offer numerous advantages. Compared to peptides containing L-amino acids, peptides containing D-amino acids, etc., exhibit greater stability in vitro and in vivo. Therefore, constructing polypeptides, for example, by adding D-amino acids, is particularly useful when improved intracellular stability is required. In particular, D-peptides and their analogs can withstand the activity of endogenous peptidases and proteases, thereby improving the bioavailability and extending the molecule's lifespan in the body, as needed. Furthermore, D-peptides and their analogs are not processed effectively due to limited presentation by class II major histocompatibility complex (MHC) molecules to helper T cells, making them less likely to induce a humoral immune response in a subject.

[0288] Table B shows conservative substitutions. More substantial substitutions are provided in Table B under the heading "Exemplary Substitutions," as explained in more detail in the Amino Acid Side Chain Categories section below. Amino acids can be classified according to general side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile. (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln. (3) Acidic: Asp, Glu. (4) Alkaline: His, Lys, Arg. (5) Residues that influence chain direction: Gly, Pro. (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve replacing a member of one of these classes with a member of another class. Amino acid substitutions can be introduced into protein constructs and screened for products that meet the desired activity described above.

[0289] [Table 2]

[0290] In some embodiments, in an IL-15 prodrug provided herein, the masking polypeptide (MP) reduces the binding affinity of IL-15 or a functional fragment thereof to the IL-2 / IL-15Rβγ chain compared to IL-15 or a functional fragment thereof without the MP. In some embodiments, the binding affinity is reduced by at least about 10-fold compared to IL-15 or a functional fragment thereof without the MP. In some embodiments, the binding affinity is reduced by at least about 100-fold compared to IL-15 or a functional fragment thereof without the MP. In some embodiments, the binding affinity is reduced by at least about 200-1500-fold compared to IL-15 or a functional fragment thereof without the MP. In some embodiments, the EC 50The value increases by at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold. In some embodiments, the masking polypeptide (MP) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-5, or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-5.

[0291] In some embodiments, the cleavable moiety (CM) comprising a protease cleavable site is located between the masking polypeptide (MP) and the IL-15 cytokine. The cleavable moiety (CM) described herein can be cleaved under certain circumstances to separate the N-terminal fragment from the C-terminal fragment. By introducing the cleavable moiety (CM), under certain circumstances, the masking polypeptide (MP) is removed at the cleavable site, thereby releasing the intact functional IL-15 cytokine.

[0292] In some embodiments, screening for a suitable cleavable moiety (CM) depends on the desired site of action of the IL-15 cytokine. For example, if a tumor site is the desired site of action, the cleavage site of a tumor-specific protease is used to construct an IL-15 prodrug designed to act at the tumor site. A tumor-specific protease refers to any protease that has elevated levels and / or activity at tumor sites compared to normal tissues.

[0293] In some embodiments, the protease cleavage site may be a cleavage site for a matrix metalloproteinase (MMP). In some embodiments, the protease cleavage site may be a cleavage site for MMP2. In some embodiments, the protease cleavage site may be a cleavage site for MMP9. In some embodiments, the protease cleavage site may be a cleavage site for MMP2 and MMP9. Other tumor-specific proteases and corresponding cleavage sites are known in the art, such as those disclosed in Vasiljeva et al., Scientific Reports, 10:5894, 2020, the relevant disclosure of which is incorporated herein by reference and consistent with the subject matter and purpose of this specification. In some embodiments, the cleavable moiety (CM) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 8-16, or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 8-16.

[0294] In some embodiments, in the IL-15 prodrug according to the present specification, said half-life extending moiety (C) comprises an Fc domain, preferably said Fc domain is selected from the group consisting of a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, an IgA Fc domain, an IgD Fc domain, an IgE Fc domain and an IgM Fc domain, more preferably said Fc domain is a human IgG1 Fc domain.

[0295] In some examples, in the IL-15 prodrugs herein, the half-life extending moiety (C) comprises a human IgG1 Fc domain comprising L234A and L235A (LALA) mutations, and the mutation sites are numbered according to the EU numbering system.

[0296] In some embodiments, in the IL-15 prodrugs according to the present specification, the half-life extending moiety (C) further comprises knobs-into-holes mutations (Fc knobs (Fcknob) and Fc holes (Fchole)).

[0297] In some embodiments, in an IL-15 prodrug according to the present specification, the Fcknob comprises a T366W mutation in the Fc domain, and the Fchole comprises T366S, L368A, and Y407V mutations in the Fc domain, and the mutation sites are numbered according to the EU numbering system.

[0298] In some embodiments, in the IL-15 prodrug according to the present specification, the Fcknob further comprises a S354C mutation in the Fc domain, and the Fchole further comprises a Y349C mutation in the Fc domain, and the mutation sites are numbered according to the EU numbering system.

[0299] In some examples, in the IL-15 prodrugs provided herein, the Fcknob comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:28.

[0300] In some examples, in an IL-15 prodrug according to the present specification, the Fchole comprises the amino acid sequence SEQ ID NO:27 or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:27.

[0301] In some embodiments, in the IL-15 prodrug according to the present invention, the Fcknob and Fc hole further comprise a LALA mutation.

[0302] In some examples, in the IL-15 prodrugs described herein, the FcknobLALA comprises the amino acid sequence SEQ ID NO: 30 or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO: 30.

[0303] In some examples, in the IL-15 prodrugs provided herein, the FchoeLALA comprises the amino acid sequence SEQ ID NO:29 or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:29.

[0304] In some embodiments, the IL-15 prodrug comprises two monomers, in one of which the IL-15Rα or a functional fragment thereof and the Fc domain are linked, and in the other monomer, the IL-15 cytokine (I) and the Fc domain are linked, and the masking polypeptide (MP) and the IL-15 cytokine (I) are linked by the cleavable moiety (CM).

[0305] In some examples, an IL-15 prodrug according to the present invention comprises a construct shown in Table 2.

[0306] In some examples, an IL-15 prodrug according to the present disclosure comprises two monomers, one of which comprises the amino acid sequence SEQ ID NO: 31 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 31, and the other of which comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.

[0307] In some examples, an IL-15 prodrug provided herein comprises two monomers, one of which comprises the amino acid sequence SEQ ID NO: 33 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 33, and the other of which comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.

[0308] In some examples, an IL-15 prodrug according to the present disclosure comprises two monomers, one of which comprises the amino acid sequence SEQ ID NO: 34 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 34, and the other of which comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.

[0309] In some examples, an IL-15 prodrug according to the present disclosure comprises two monomers, one of which comprises the amino acid sequence SEQ ID NO: 36 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 36, and the other of which comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.

[0310] In some examples, an IL-15 prodrug according to the present disclosure comprises two monomers, one of which comprises the amino acid sequence SEQ ID NO: 37 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 37, and the other of which comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.

[0311] In some examples, an IL-15 prodrug according to the present disclosure comprises two monomers, one of which comprises the amino acid sequence SEQ ID NO: 38 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 38, and the other of which comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.

[0312] In some examples, an IL-15 prodrug according to the present disclosure comprises two monomers, one of which comprises the amino acid sequence SEQ ID NO: 39 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 39, and the other of which comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.

[0313] In some examples, an IL-15 prodrug according to the present disclosure comprises two monomers, one of which comprises the amino acid sequence SEQ ID NO: 31 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 31, and the other of which comprises the amino acid sequence SEQ ID NO: 40 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 40.

[0314] In some examples, an IL-15 prodrug according to the present disclosure comprises two monomers, one of which comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 41, and the other of which comprises the amino acid sequence SEQ ID NO: 42 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 42.

[0315] In some examples, an IL-15 prodrug according to the present disclosure comprises two monomers, one of which comprises the amino acid sequence SEQ ID NO: 43 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 43, and the other of which comprises the amino acid sequence SEQ ID NO: 44 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 44.

[0316] In some examples, an IL-15 prodrug according to the present disclosure comprises two monomers, one of which comprises the amino acid sequence SEQ ID NO:45 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO:45, and the other of which comprises the amino acid sequence SEQ ID NO:46 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO:46.

[0317] In some examples, an IL-15 prodrug according to the present disclosure comprises two monomers, one of which comprises the amino acid sequence SEQ ID NO:47 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO:47, and the other of which comprises the amino acid sequence SEQ ID NO:40 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO:40.

[0318] binding affinity The binding affinity of a molecule (e.g., IL-15 or a functional fragment thereof) to its binding partner (e.g., IL-2 / IL-15Rβγ) can be determined by any suitable ligand-binding test or antibody / antigen-binding test known in the art, such as Western blot, enzyme-linked immunosorbent assay (ELISA), MesoScaleDiscovery (MSD) electrochemiluminescence, bead-based multiplex immunoassay (MIA), RIA, surface plasmon resonance (SPR), ECL, IRMA, EIA, Biacore test, Octet analysis, peptide scan, etc. For example, the assay can be easily performed using IL-15, its functional fragments, its receptor (e.g., IL-2 / IL-15Rβγ), or its subunits labeled with various labeling reagents, or using an OTC assay kit or a similar kit, BiacoreX (Amersham Biosciences), which can be operated according to the user manual and experimental procedures provided with the kit.

[0319] In some embodiments, protein microarrays are used for large-scale analysis of the interaction, function, and activity of IL-15 or functional fragments thereof and its receptor as described herein. Protein microarrays have a support surface to which a series of capture proteins (e.g., IL-15 receptor or its subunits) are bound. Fluorescently labeled probe molecules (e.g., IL-15 or functional fragments thereof as described herein) are then added to the array, where they interact with the bound capture proteins, emitting a fluorescent signal that is read by a laser scanner.

[0320] Binding affinity can also be measured using SPR (Biacore T-200). For example, anti-human IgG antibodies were bound to a CM-5 sensor chip surface using EDC / NHS chemistry. Human IL-2 / IL-15Rβγ-Fc fusion protein was then used as a capture ligand for this surface. By combining a dilution series of the IL-15 prodrug or drug described herein with the capture ligand, the binding and dissociation of the IL-15 prodrug or drug from IL-2 / IL-15Rβγ can be monitored in real time. The dissociation constant (K d ) and dissociation rate constants can be determined by kinetic analysis using BIA evaluation software.

[0321] Pharmacokinetics (PK) Pharmacokinetics (PK) refers to the absorption, distribution, metabolism, and excretion of a drug (e.g., an IL-15 cytokine or IL-15 prodrug described herein) following administration to a subject. Pharmacokinetic parameters that can be used to determine clinical utility include serum / plasma concentration, serum / plasma concentration over time, and maximum serum / plasma concentration (C max ), time to maximum concentration (T max ), half-life (t 1 / 2 ), area under the concentration-time curve within the dosing interval (AUC τ ), but are not limited to these.

[0322] Techniques for obtaining PK curves for drugs, such as the IL-15 cytokines or IL-15 prodrugs described herein, are known in the art. See Heller et al., AnnuRevAnalChem, 11, 2018; and GhandforoushSattariet al., JA MinoAcids, Article ID 346237, Volume 2010. In some examples, the PK curve for an IL-15 cytokine or IL-15 prodrug described herein is measured in an individual's blood, plasma, or serum sample. In some examples, the PK curve for an IL-15 cytokine or IL-15 prodrug described herein is measured in an individual using mass spectrometry techniques (e.g., LC-MS / MS or ELISA). PK analysis can be performed on PK curves by any method known in the art, for example, non-compartmental analysis using PKSolver V2 software (Zhang Y. et al., "PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel," Comput Methods Programs Biomed. 2010;99(3):306-1).

[0323] "C" represents the concentration of a drug or prodrug (e.g., an IL-15 cytokine or an IL-15 prodrug) in a subject's plasma, serum, or any suitable bodily fluid or tissue, typically expressed as mass per unit volume, e.g., nanograms per milliliter. For convenience, the drug concentration in serum or plasma is referred to herein as the "serum concentration" or "plasma concentration." The serum / plasma concentration at any time after administration (e.g., an IL-15 cytokine or an IL-15 prodrug, intravenous injection, intraperitoneal injection, subcutaneous injection, etc.) is expressed as the C time or C t The maximum serum / plasma drug concentration during administration is called C max It is called C min refers to the minimum serum / plasma drug concentration at the end of the dosing interval, and C averefers to the average concentration during the dosing interval.

[0324] The term "bioavailability" refers to the extent or rate at which a drug or prodrug (eg, an IL-15 cytokine or an IL-15 prodrug) passes through the systemic circulation and reaches its site of action.

[0325] "AUC" is the area under the serum / plasma concentration-time curve, and is the area under the concentration-time curve over the dosing interval (AUC τ ), “total exposure” or “total drug exposure over a period of time” (AUC 0-last or AUC 0-inf ), the area under the concentration-time curve within t hours after administration (AUC 0-t ) is considered to be the most reliable measure of bioavailability.

[0326] The time to peak serum / plasma concentration (Tmax) is the time to peak serum / plasma concentration (C) after administration of a drug or prodrug (e.g., IL-15 cytokine or IL-15 prodrug). max ) is the time it takes for the signal to reach its peak.

[0327] Half-life (t 1 / 2Elimination half-life (IOL) refers to the time required for the concentration of a drug or prodrug (e.g., IL-15 cytokine or IL-15 prodrug) measured in plasma or serum (or other biological matrix) to decrease to half of the concentration or amount at a particular time point. For example, after intravenous administration, the drug concentration in plasma or serum decreases due to distribution and elimination. In the curve of the change in plasma or serum drug concentration over time after intravenous administration, the initial or rapid decline phase is thought to be primarily due to distribution, while the subsequent declines are usually slower and primarily due to elimination, although both processes occur in both phases. After a sufficient amount of time has passed, drug distribution is complete. Generally, the elimination half-life is determined by the terminal or elimination (dominant) phase of the plasma / serum concentration-time curve. See "Chapter 3 - Pharmacokinetics and Toxicokinetics" in Michael Schrag and Kelly Regal, "Comprehensive Guide to Toxicology for Preclinical Drug Development," 2013.

[0328] stability In some examples, the masking polypeptides (MPs) and prodrugs (e.g., IL-15 prodrugs, anti-TNFR2 antibody prodrugs) described herein have excellent stability, such as physical stability, chemical stability, and / or biological stability. In some examples, the IL-15 prodrugs and anti-TNFR2 antibody prodrugs described herein have excellent stability under accelerated stress (e.g., elevated temperatures), such as little or no fragmentation, aggregate formation, and / or increase in aggregates.

[0329] Protein stability, particularly susceptibility to aggregation, primarily depends on the conformational and colloidal stability of the protein molecule. It is generally believed that the first step in non-native protein aggregation, the most common form of aggregation, is a slight disruption of molecular structure, such as partial unfolding of the protein, i.e., a conformational change. This is determined by the protein's conformational stability. In the second step, some unfolded molecules gather together by diffusion and random Brownian motion to form aggregates. This second step is primarily determined by the colloidal stability of the molecule (see Chet. al., Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony-stimulating factor. Protein Science, 2003 May;12(5):903-913). As used herein, the term "stability" generally refers to maintaining the integrity of a biologically active substance (e.g., a protein) or minimizing its degradation, denaturation, aggregation, or unfolding. As used herein, "improved stability" generally means that a target protein (e.g., an IL-15 prodrug described herein) maintains better stability than a control protein (e.g., another IL-15 prodrug) under conditions known to cause degradation, denaturation, aggregation, or unfolding.

[0330] Differential scanning calorimetry (DSC) and differential scanning fluorescence (DSF) are well-known techniques in the art for predicting the stability of protein formulations. Specifically, these techniques can be used to estimate the unfolding temperature (T) of a protein in a given formulation. m ) can be determined. m Correlating measurements with reliable and stable protein formulations that have long-term shelf-life is standard practice in the art.

[0331] A "stable" masking polypeptide (MP) or prodrug (or formulation), e.g., an IL-15 prodrug or anti-TNFR2 antibody prodrug described herein, substantially maintains its physical stability and / or chemical stability and / or biological activity during the manufacturing process and / or storage. The art has a variety of analytical techniques for measuring protein stability, as summarized in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991), and Jones, A. (1993) Adv. Drug Delivery Rev. 10:29-90. For example, in one embodiment, protein stability is determined based on the percentage of monomeric protein in solution and a relatively low percentage of degraded (e.g., fragmented) and / or aggregated protein. Preferably, the protein (or formulation) is stable for at least one month at room temperature (about 30°C) or 40°C, and / or for at least six months, or at least one year, or at least two years at about 2-8°C. Furthermore, the protein (or formulation) is preferably stable upon freezing (e.g., at -70°C) and thawing (hereinafter referred to as a "freeze / thaw cycle").

[0332] For a prodrug, e.g., an IL-15 prodrug or anti-TNFR2 antibody prodrug described herein, the protein "maintains its physical stability" in a formulation if there is substantially no evidence of instability, such as aggregation, precipitation, and / or denaturation, as measured by visual inspection of color and / or clarity or by UV light scattering or size exclusion chromatography. Aggregation is the process by which individual protein molecules or complexes associate covalently or noncovalently to form aggregates, which may proceed until a visible precipitate forms.

[0333] A prodrug, e.g., an IL-15 prodrug or anti-TNFR2 antibody prodrug described herein, "maintains its chemical stability" in a formulation if its chemical stability over a given period of time is such that the protein still retains its biological activity (see, e.g., the "Biological Activity" section above). Chemical stability can be assessed, for example, by detecting and quantifying various chemical changes in the protein. Chemical changes can include dimensional changes (e.g., shearing), which can be assessed using size-exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOFMS). Other types of chemical changes include charge changes (e.g., deamidation or oxidative changes), which can be assessed, for example, by ion-exchange chromatography.

[0334] For a prodrug, e.g., an IL-15 prodrug or an anti-TNFR2 antibody prodrug described herein, a protein "retains its biological activity" in a pharmaceutical formulation if the protein in the pharmaceutical formulation is biologically active for its intended purpose. For example, a protein retains its biological activity if the biological activity of the protein in the formulation is within 30%, 20%, or 10% (within analytical error) of the biological activity exhibited at the time the formulation was manufactured.

[0335] Those skilled in the art know that the stability of a prodrug (e.g., an IL-15 prodrug or an anti-TNFR2 antibody prodrug described herein) depends on other characteristics in addition to the composition of the formulation. For example, stability may be affected by temperature, pressure, humidity, pH, and external radiation. The stability of a protein (e.g., an IL-15 prodrug or an anti-TNFR2 antibody prodrug described herein) in a protein formulation can be determined by various methods. In some examples, protein stability is determined by size exclusion chromatography (SEC). SEC separates analytes (e.g., macromolecules such as proteins) based on their hydrodynamic size, diffusion coefficient, and surface properties. Thus, for example, SEC can separate an IL-15 prodrug or an anti-TNFR2 antibody prodrug described herein in its native three-dimensional conformation from proteins in various denatured states and / or degraded proteins. In SEC, the stationary phase is typically composed of inert particles in a dense three-dimensional matrix packed into a glass or steel column. The mobile phase can be pure water, an aqueous buffer, an organic solvent, a mixture thereof, or other solvents. Stationary phase particles have small pores or channels that only allow entry of substances smaller than a certain size. Thus, larger particles are excluded from these pores or channels, while smaller particles are transported from the mobile phase. The time a particle remains immobilized within the stationary pores depends in part on the depth to which the particle can penetrate the pores. Once particles are transported from the mobile phase stream, they take longer to elute from the column, thus separating the particles based on size differences.

[0336] In some examples, SEC is combined with an identification technique to identify or characterize a protein (e.g., an IL-15 prodrug or an anti-TNFR2 antibody prodrug described herein) or a fragment thereof. Protein identification and characterization can be performed by a variety of techniques, including, but not limited to, chromatographic techniques such as high-performance liquid chromatography (HPLC), sodium dodecyl sulfate capillary electrophoresis (CE-SDS), immunoassays, electrophoresis, UV / visible / infrared spectroscopy, Raman spectroscopy, surface-enhanced Raman spectroscopy, mass spectrometry, gas chromatography, static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism spectroscopy (CD), urea-induced protein unfolding techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and / or ANS protein binding.

[0337] In some examples, optionally, a sample formulation (e.g., comprising an IL-15 prodrug or anti-TNFR2 antibody prodrug described herein) and a control formulation are measured prior to a treatment step to determine monomer content, aggregate and / or fragmented protein (and / or % increase in fragments, % increase in aggregates, etc.). Each protein formulation then undergoes a treatment step. For example, each protein formulation can be stored at a particular temperature (e.g., 40°C, 25°C, or 5°C) for an extended period of time (e.g., 3 months, 6 months, 12 months, or longer). In some examples, a protein formulation is subjected to a physical stress test, such as an agitation stress test. In some examples, a protein formulation is subjected to an accelerated stability test, such as treatment under accelerated stresses including high temperature (e.g., 40°C), high humidity, and / or low pH. In some examples, a protein formulation is subjected to freeze-thaw cycles. In some examples, samples of the same protein formulation are subjected to different treatments, e.g., stored at different temperatures for a period of time. After the treatment step, the protein preparation is measured to determine the protein monomer, aggregate and / or fragment content (and / or % increase in fragments, % increase in aggregates, etc.).

[0338] "Large amounts of protein aggregation" means that the level of protein aggregation in a protein formulation is significantly higher than the level of protein aggregation in a control protein formulation, which can be the same protein formulation before a period of storage or before processing (e.g., before exposure to unstable conditions such as high temperature, humidity, pH, and / or long-term storage).

[0339] "Substantially free of protein aggregation" means that the level or percentage of protein aggregation of a protein (or formulation) of the invention is not significantly higher than that of a control formulation. In some embodiments, the stability is measured by SEC. In some embodiments, the stability is measured by CE-SDS.

[0340] In some examples, stability refers to reduced fragmentation of an IL-15 prodrug or anti-TNFR2 antibody prodrug described herein. As used herein, the term "low to undetectable levels of fragmentation" refers to a sample containing 80%, 85%, 90%, 95%, 98%, or 99% or more of the total protein, e.g., a single peak when measured by HPSEC, or multiple peaks (e.g., as many peaks as there are subunits) when measured by reduced capillary gel electrophoresis (rCGE), representing undegraded protein or undegraded fragments, and excluding other single peaks that account for more than 5%, more than 4%, more than 3%, more than 2%, more than 1%, or more than 0.5% of the total protein peak. As used herein, the term "reduced capillary gel electrophoresis" refers to capillary gel electrophoresis under reducing conditions sufficient to reduce disulfide bonds in an Fc-containing protein, such as an IL-15 prodrug or anti-TNFR2 antibody prodrug described herein.

[0341] vector The present invention also relates to isolated nucleic acids encoding any of the masking polypeptides (MPs), any cleavable moieties (CMs), any non-cleavable linkers (Ls), or any prodrugs (e.g., anti-TNFR2 antibody prodrugs or IL-15 prodrugs) described herein, including vectors encoding the nucleic acids described herein. The present invention also relates to isolated host cells (e.g., CHO cells, HEK293 cells, HeLa cells, or COS cells) containing nucleic acids or vectors encoding the nucleic acids or vectors described herein. Suitable nucleic acid constructs include, but are not limited to, constructs capable of expression in eukaryotic or prokaryotic cells. The selection of an expression construct will typically be compatible with the host cell used. In some examples, the vector encodes a masking polypeptide (e.g., MP80, MP96new, MP100, MP163, or MP240). In some examples, the vector encodes a cleavable moiety (e.g., CM1, CM2, or CM4). In some embodiments, the vector encodes a non-cleavable linker (e.g., lk, lk1, lk2, lk3, or lk5). In some embodiments, the vector encodes a protein or prodrug (e.g., a masking polypeptide or an IL-15 prodrug).

[0342] In some embodiments, a vector encoding a prodrug or any prodrug moiety described herein is suitable for replication and integration in eukaryotic cells, such as mammalian cells (e.g., CHO cells, HEK293 cells, HeLa cells, COS cells). In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector, such as pTT5.

[0343] Many virus-based systems have been developed for gene transfer into mammalian cells. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, herpes simplex viral vectors, and their derivatives. Viral vector technology is well known in the art and is described in detail, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology handbooks. Retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to engineered mammalian cells under in vitro or ex vivo conditions. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, a self-inactivating lentiviral vector carrying the construct protein coding sequence can be packaged using laboratory methods known in the art. The resulting lentiviral vector can be used to transduce mammalian cells using methods known in the art. Retrovirus (e.g., lentivirus)-derived vectors are suitable tools for long-term gene transfer because they allow long-term stable integration of the transgene and propagation in progeny cells; lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.

[0344] In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a pTT5 vector. In some embodiments, the vector is a transposon, such as the Sleeping Beauty (SB) transposon system or the PiggyBac transposon system. In some embodiments, the vector is a polymer-based non-viral vector, including, for example, poly(lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA), poly(ethyleneimine) (PEI), and dendrimers. In some embodiments, the vector is a cationic lipid-based non-viral vector, such as cationic liposomes, lipid nanoemulsions, and solid lipid nanoparticles (SLNs). In some embodiments, the vector is a peptide-based non-viral gene vector, such as poly-L-lysine. Any known non-viral vector suitable for genome editing can be used to introduce a nucleic acid encoding an IL-15 prodrug or an anti-TNFR2 antibody prodrug into a host cell. See Yin H. et al., Nature Rev. Genetics (2014) 15:521-555; Aronovich E. et al., "The Sleeping Beauty transposon system: anon-viral vector for gene therapy." Hum. Mol. Genet. (2011) R1:R14-20, and Zhao S. et al., "PiggyBac transposon vectors: the tools for the human gene editing." Transl. Lung Cancer Res. (2016) 5(1):120-125, which are incorporated herein by reference. In some examples, any one or more nucleic acids or vectors encoding prodrugs described herein are introduced into host cells (e.g., CHO, HEK293, Hela, or COS) by physical methods, including, but not limited to, electroporation, sonoporation, photoporation, magnetofection, and hydroporation.

[0345] In some examples, the vector comprises a selectable marker gene or reporter gene for selecting cells expressing a prodrug described herein from a population of host cells transfected with the vector (e.g., a lentiviral vector, a pTT5 vector). Both the selectable marker and the reporter gene may be surrounded by appropriate regulatory sequences to enable expression in the host cell. For example, the vector may comprise transcription and translation terminators, initiation sequences, and a promoter for regulating expression of the nucleic acid sequence.

[0346] Any molecular cloning method known in the art can be used, including, for example, cloning a nucleic acid into a vector using a restriction endonuclease site and one or more selectable markers. In some examples, the nucleic acid is operably linked to a promoter. A variety of promoters have been developed for gene expression in prokaryotic or eukaryotic cells (e.g., mammalian cells), and any promoter known in the art can be used in the present invention. Promoters can be broadly classified as constitutive promoters or regulatable promoters, such as inducible promoters.

[0347] In some embodiments, the nucleic acid encoding the prodrug described herein is operably linked to a constitutive promoter. Constitutive promoters allow for constitutive expression of a heterologous gene (also referred to as a transgene) in a host cell. Examples of promoters contemplated herein include, but are not limited to, the CMV promoter (CMV), human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerate kinase promoter (PGK), simian virus 40 early promoter (SV40), chicken β-actin promoter / CMV early enhancer (CAGG) complex, Rous sarcoma virus (RSV) promoter, polyoma virus enhancer / herpes simplex thymidine kinase (MC1) promoter, β-actin (β-ACT) promoter, and the "myeloproliferative sarcoma virus enhancer, negative control region deleted, d1587rev primer binding site substitution (MND)" promoter. The efficiency of these constitutive promoters in driving transgene expression has been extensively compared in numerous studies. In some examples, the nucleic acid encoding a prodrug described herein is operably linked to a CMV promoter.

[0348] In some examples, the nucleic acid encoding the prodrug described herein is operably linked to an inducible promoter. Inducible promoters belong to the category of regulated promoters. Inducible promoters can be induced by one or more conditions, such as a physical condition, a host cell microenvironment or a physiological state of the host cell, an inducing agent (i.e., an inducing drug), or a combination thereof. In some examples, the inducing condition does not induce expression of an endogenous gene in the host cell. In some examples, the inducing condition is selected from an inducing agent, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox conditions, and an activation state of the host cell. In some examples, the inducible promoter can be an NFAT promoter, a TETON® promoter, or an NFκB promoter.

[0349] Manufacturing method Also provided are methods of producing any masking polypeptide (MP), any cleavable moiety (CM), any non-cleavable linker (L), or any prodrug described herein. Accordingly, in some embodiments, methods of producing the masking polypeptide (MP), the cleavable moiety (CM), the non-cleavable linker (L), or the prodrug are provided, the methods comprising: (a) culturing a host cell (e.g., a CHO cell, a HEK293 cell, a Hela cell, or a COS cell) containing a nucleic acid or vector encoding the masking polypeptide (MP), the cleavable moiety (CM), the non-cleavable linker (L) or the prodrug described herein under conditions effective to express the encoded prodrug; and (b) obtaining the expressed masking polypeptide (MP), the cleavable moiety (CM), the non-cleavable linker (L), or the prodrug from the host cell. In some examples, the method of step (a) further comprises producing a host cell comprising a nucleic acid or vector encoding said masking polypeptide (MP), said cleavable moiety (CM), said non-cleavable linker (L), or said prodrug herein. The masking polypeptide (MP), said cleavable moiety (CM), said non-cleavable linker (L), or said prodrug herein can be produced using any method known in the art or described herein.

[0350] In some embodiments, the masking polypeptide (MP), the cleavable moiety (CM), the non-cleavable linker (L), or the prodrug herein is expressed in a eukaryotic cell, such as a mammalian cell. In some embodiments, the masking polypeptide (MP), the cleavable moiety (CM), the non-cleavable linker (L), or the prodrug herein is expressed in a prokaryotic cell.

[0351] 1. Recombinant products of prokaryotic cells a) Vector construction Polynucleic acid sequences encoding the protein constructs described herein can be obtained using standard recombinant techniques. Polynucleotides can be synthesized using a nucleotide synthesizer or PCR technology. Once the polypeptide-encoding sequence is obtained, it is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors known and available in the art can be used in the present invention. The selection of an appropriate vector depends primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains various components depending on the function of the vector (amplification and / or expression of the heterologous polynucleotide) and the compatibility of the vector with the specific host cell in which it will be used. Vector components typically include, but are not limited to, an origin of replication site, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert, and a transcription termination sequence.

[0352] Plasmid vectors generally contain replicon and control sequences derived from species compatible with the host cells they are used with. Vectors usually contain a replication site and marker sequences that provide phenotypic selection in transformed cells. For example, Escherichia coli is commonly transformed using pBR322, a plasmid derived from E. coli. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, providing a simple method for identifying transformed cells. pBR322, its derivatives, or other bacterial plasmids or phages may contain, or be modified to contain, promoters that can be used by the microorganism to express endogenous proteins. U.S. Patent No. 5,648,237 to Carter et al. details examples of pBR322 derivatives used to express specific antibodies.

[0353] Additionally, phage vectors containing replicon and control sequences compatible with the host microorganism can be used as transforming vectors with these host cells. For example, phages such as GEM™-11 can be used to produce recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.

[0354] A promoter is a non-translated regulatory sequence located upstream (5') of a cistron that controls downstream gene expression. Prokaryotic promoters are generally divided into two categories: inducible and constitutive promoters. Inducible promoters are promoters that initiate and increase the level of transcription of a cistron in response to changes in culture conditions (e.g., the presence or absence of nutrients or a change in temperature).

[0355] Many promoters recognized by potential host cells are known. The promoter is removed from the source DNA with restriction enzymes, and the isolated promoter sequence is inserted into the vector of the present application, where the selected promoter is operably linked to the cistron DNA encoding the polypeptide. Both the native promoter sequence and many heterologous promoters can be used to direct amplification and / or expression of the target gene. In some embodiments, a heterologous promoter is utilized because heterologous promoters generally allow for greater transcription and higher target gene expression yields compared to the native target polypeptide promoter.

[0356] Promoters suitable for prokaryotic hosts include the PhoA promoter, the galactosidase / lactose promoter system, the tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter or the trc promoter. However, other promoters that function in bacteria (such as other known bacterial promoters or phage promoters) can also be used. Their nucleic acid sequences are disclosed, allowing those skilled in the art to provide any necessary restriction sites using linkers or adapters and ligate them to the cistrons encoding the light and heavy chains of interest (Siebenlist et al., (1980) Cell 20:269).

[0357] In some embodiments, each cistron in a recombinant vector contains a secretory signal sequence component that directly guides transmembrane translocation of the expressed polypeptide. Generally speaking, the signal sequence may be a component of the vector, or it may be part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the present invention should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that are unable to recognize and process signal sequences native to heterologous polypeptides, the signal sequence may be substituted with a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamP, PhoE, PelB, OmpA, and MBP.

[0358] In some embodiments, production of the protein constructs of the present application can occur in the cytoplasm of the host cell, eliminating the need for a secretory signal sequence in each cistron. In some embodiments, polypeptide components are expressed, folded, and assembled to form protein constructs in the cytoplasm. Certain host strains (e.g., E. coli trxB- strains) provide cytoplasmic conditions favorable for disulfide bond formation, thereby allowing the expressed protein subunits to fold and assemble properly. See Proba and Pluckthun, Gene, 159:203 (1995).

[0359] b) Prokaryotic host cells Prokaryotic host cells suitable for expression of the proteins of the present application include archaebacteria and eubacteria, such as gram-negative or gram-positive bacteria. Examples of useful bacteria include Escherichia coli (e.g., E. coli), Bacillus (e.g., Bacillus subtilis), Enterobacteriaceae, Pseudomonas (e.g., Pseudomonas aeruginosa), Salmonella typhimurium, Serratia marcescens, Klebsiella, Proteus, Shigella, Rhizobium, Vitreoscilla, or Paracoccus. In some embodiments, gram-negative cells are used. In some embodiments, E. coli cells serve as hosts in the present invention. Examples of E. coli strains include the W3110 strain (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) and its derivatives, which have the genotype W3110AfhuA (AtonA) ptr3lacIqlacL8AompTA (nmpcfepE) degP41kan. RExamples of suitable bacterial strains include strain 33D3 (U.S. Pat. No. 5,639,635) having the genotype E. coli 294 (ATCC 31446), E. coli B, E. coli 1776 (ATCC 31537), and E. coli RV308 (ATCC 31608), and their derivatives. These examples are illustrative rather than limiting. Methods for constructing bacterial derivatives of any of the above known genotypes are known in the art and are described in detail, for example, in Bassetal., Proteins, 8:309-314 (1990). It is often necessary to select an appropriate bacterium based on the replicability of the replicon in bacterial cells. For example, when a well-known plasmid, such as pBR322, pBR325, pACYC177, or pKN410, is used to provide the replicon, E. coli, Serratia, or Salmonella are suitable hosts.

[0360] Typically, the host cells should secrete minimal amounts of proteolytic enzymes and the cell culture should be appropriately supplemented with additional protease inhibitors.

[0361] c) Protein production Host cells are transformed with the expression vector and cultured in conventional nutrient media, modified as appropriate, to induce promoters, select transformants, or amplify the gene encoding the desired sequence. Transformation refers to the introduction of DNA into a prokaryotic host so that the DNA can replicate either as an extrachromosomal element or through chromosomal integration. Depending on the host cell used, transformation is performed using standard techniques appropriate for such cells. Calcium treatment with calcium chloride is typically used for bacterial cells, which contain many cell wall barriers. Another transformation method uses polyethylene glycol / dimethyl sulfoxide. Another technique is electroporation.

[0362] Prokaryotic cells used to produce the protein constructs of the present application are known in the art and are suitable for growth in media for culturing selected host cells. Suitable media include luria broth (LB) and necessary nutritional supplements. In some embodiments, the media further contains a selection agent selected based on the structure of the expression vector to selectively allow growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the media for growth of cells expressing an ampicillin resistance gene.

[0363] In addition to the carbon source, nitrogen source, and inorganic phosphate source, any necessary supplements can be added at appropriate concentrations, either alone or in mixtures with other supplements or media (e.g., complex nitrogen sources). Optionally, the medium may contain one or more reducing agents selected from glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiothreitol. Prokaryotic host cells are cultured at an appropriate temperature. For example, for growth of E. coli, the preferred temperature range is 20°C to 39°C, more preferably 25°C to 37°C, and even more preferably 30°C. The pH value of the medium will be anywhere between 5 and 9, depending primarily on the host organism. For E. coli, a pH value of 6.8 to 7.4 is preferred, with 7.0 being more preferred.

[0364] When an inducible promoter is used in the expression vector of the present application, protein expression is induced under conditions suitable for promoter activation. In one embodiment of the present application, the PhoA promoter is used to control the transcription of the polypeptide. Therefore, the transformed host cell is cultured in a phosphate-limited medium for induction. Preferably, the phosphate-limited medium is CRAP medium (see Simons et al., J. Immunol. Methods (2002), 263:133-147). Depending on the vector structure used, various other inducers known in the art can be used, which are known in the art.

[0365] The protein constructs expressed in this application are secreted into the periplasm of the host cells and recovered therefrom. Protein recovery typically involves disrupting the microorganisms by osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. For example, the protein can be further purified by affinity resin chromatography. Alternatively, the protein can be transported into the culture medium and isolated there. The cells can be removed from the culture medium, and the culture supernatant can be filtered and concentrated to further purify the produced protein. The expressed polypeptides can be further isolated and identified by common methods such as polyacrylamide gel electrophoresis (PAGE) or Western blot assays.

[0366] Alternatively, proteins can be produced on a large scale through a fermentation process. Various large-scale fed-batch fermentation procedures are available for the production of recombinant proteins. Large-scale fermentations have a volume of at least 1,000 liters, preferably 1,000 to 100,000 liters. These fermentation tanks use impellers to distribute oxygen gas and nutrients, especially glucose (which is the primary carbon / energy source). Small-scale fermentation typically refers to fermentation in fermentation tanks with volumes ranging from 1 liter to 100 liters, but not exceeding 100 liters.

[0367] During the fermentation process, protein expression typically begins after cells have grown under appropriate conditions to a desired density, e.g., when the cells are in early stationary phase, at OD 550 The vector is induced when the β-glucanase activity is approximately 180-220 bp. Depending on the vector construct used, various inducers known in the art and described above can be used. Cells can be grown for a short period of time before induction. Cells are typically induced for approximately 12-50 hours, although longer or shorter induction times can be used.

[0368] Various fermentation conditions can be modified to improve the yield and quality of the protein constructs of the present application. For example, to improve the correct assembly and folding of secreted polypeptides, prokaryotic host cells can be co-transformed with an additional vector overexpressing a chaperone protein, such as a Dsb protein (DsbA, DsbB, DsbC, DsbD, or DsbG) or FkpA (a peptide prolyl cis-trans isomerase with chaperone activity). Chaperone proteins have been shown to be useful in promoting the correct folding and solubility of heterologous proteins produced in bacterial host cells. Chenetal.,(1999)JBioChem274:19601-19605;Georgiouetal.,USPat.No.6,083,715;Georgiouetal.,USPat.No.6,027,888;Bothmann and Pluckthu n(2000) J.Biol.Chem.275:17100-17105;Ramm and Pluckthun(2000)J.Biol.Chem.275:17106-17113;Arieetal.,(2001)Mol.Microbiol.39:199-210.

[0369] To minimize hydrolysis of expressed heterologous proteins (especially proteolytically sensitive proteins), certain host strains lacking proteolytic enzymes can be used in the present invention. For example, host cell strains can be modified to genetically mutate genes encoding known bacterial proteases, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Several E. coli protease-deficient strains are available, which are described in detail in Joly et al., (1998), supra; Georgiouet al., US Pat. No. 5,264,365; Georgiouet al., US Pat. No. 5,508,192; Haraet al., Microbial Drug Resistance, 2:63-72 (1996).

[0370] E. coli strains that are deficient in proteolytic enzymes and transformed with plasmids that overexpress one or more chaperone proteins can serve as host cells in expression systems encoding the protein constructs described herein.

[0371] d) Protein purification The protein constructs produced herein can be further purified to obtain substantially homogeneous preparations for further analysis and use. Standard protein purification methods known in the art can be used. Processes such as fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse-phase liquid chromatography (HPLC), silica or cation exchange resin (e.g., DEAE) chromatography, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration (e.g., Sephadex G-75) are examples of suitable purification processes.

[0372] In some embodiments, Protein A immobilized on a solid phase is used for immunoaffinity purification of protein constructs containing the Fc region described herein. Protein A is a 42 kDa surface protein from Staphylococcus aureus that has high binding affinity for Fc-containing constructs, such as the IL-15 prodrug or anti-TNFR2 antibody prodrug described herein (Lindmarket et al., (1983) J. Immunol. Meth. 62:1-13). The solid phase for immobilizing Protein A is preferably a column containing a glass or silica surface, more preferably a glass column or silicic acid column with controllable pore size. In some applications, the column is coated with a reagent such as glycerol to prevent nonspecific adhesion of contaminants. The solid phase is then washed to remove contaminants nonspecifically bound to the solid phase. Finally, the target protein construct is recovered from the solid phase by elution.

[0373] 2. Recombinant products of eukaryotic cells For eukaryotic expression, vector components usually include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0374] a) Signal Sequence Element Vectors for use in eukaryotic hosts can also have inserts encoding signal sequences or other polypeptides with specific cleavage sites at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences and viral secretory leaders, such as the herpes simplex gD signal, may also be obtained and are both useful. The DNA for this precursor region is ligated in an open reading frame to the DNA encoding the protein construct of the present application.

[0375] b) Copy start point Generally, mammalian expression vectors do not need the origin of replication element (the SV40 origin is commonly used only because it contains the early promoter).

[0376] c) Selectable Genetic Elements Expression and cloning vectors can contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that: (a) confer resistance to antibiotics or other toxins (such as ampicillin, neomycin, methotrexate, or tetracycline); (b) complement auxotrophic proteins; or (c) provide important nutrients that cannot be provided by complex media, such as Bacillus D-alanine racemase.

[0377] An example of a dominant selection strategy is the use of drugs to prevent the growth of host cells. Cells successfully transformed with a heterologous gene produce drug resistance proteins and thus survive the dominant selection strategy. Examples of this type of dominant selection include the use of drugs such as neomycin, mycophenolic acid, and hygromycin.

[0378] Further examples of suitable selectable markers for mammalian cells are those that allow for the identification of cells capable of harboring nucleic acids encoding the protein constructs described herein, such as DHFR, thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

[0379] For example, in the case of cells transformed with a DHFR selection gene, all transformants are first identified by culturing them in medium containing methotrexate (Mtx), a competitive antagonist of DHFR. When wild-type DHFR is used, a suitable host cell is a Chinese hamster ovary (CHO) cell line lacking DHFR activity (e.g., ATCC CRL-9096).

[0380] Alternatively, host cells transformed or co-transformed with a DNA sequence encoding the polypeptide, a wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) (particularly wild-type hosts containing endogenous DHFR) can be selected by growing the cells in medium containing the selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.

[0381] d) promoter elements Expression and cloning vectors usually contain a promoter that is recognized by the host and is operably linked to a nucleic acid encoding the desired polypeptide sequence. Nearly all eukaryotic genes have an AT-rich region located approximately 25–30 bases upstream of the transcription start site. Another sequence found 70–80 bases upstream of the transcription start site of many genes is a CNCAAT region, where N can be any nucleotide. Most eukaryotic genes have an AATAAA sequence at the 3' end, which may be a signal for adding a poly(A) tail to the 3' end of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors. See the "Vector" section above.

[0382] Transcription of the polypeptide in the mammalian host cell vector is controlled by a promoter, for example, a promoter obtained from a viral genome such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably simian virus 40 (SV40), a promoter from a heterologous mammalian source, for example, the actin promoter or immunoglobulin promoter from a heat shock promoter, provided that such a promoter is compatible with the host cell system.

[0383] The early and late promoters of SV40 virus are readily available as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate-early promoter of human cytomegalovirus is readily available as a HindIII E restriction fragment. U.S. Pat. No. 4,419,446 discloses a system for expressing DNA in mammalian hosts using bovine papilloma virus as a vector. Improvements to this system are detailed in U.S. Pat. No. 4,601,978. For expression of human interferon cDNA in mouse cells under the control of the herpes simplex virus thymidine kinase promoter, see Reye et al., Nature 297:598-601 (1982). Alternatively, the Rous sarcoma virus long terminal repeat can be used as a promoter.

[0384] e) enhancer elements Transcription of DNA encoding the protein constructs of the present application by higher eukaryotes is typically increased by inserting an enhancer sequence into the vector. Many enhancer sequences are found in mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, enhancers from eukaryotic cell viruses are often used. Examples include the SV40 enhancer located distal to the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma virus enhancer located distal to the replication origin, and adenovirus enhancers. For enhancer elements that activate eukaryotic promoters, see Yaniv, Nature 297:17-18 (1982). The enhancer can be spliced into the vector at a position 5' or 3' from the polypeptide-coding sequence, but is preferably located 5' from the promoter.

[0385] f) transcription termination element Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells of other multicellular organisms) also contain sequences necessary for the termination of transcription and stabilization of mRNA. These sequences are usually obtained from the 5'-untranslated region, and occasionally the 3'-end, of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the polypeptide. A suitable transcription termination element is the bovine growth hormone polyadenylation region; see WO 94 / 11026 and the expression vector disclosed therein.

[0386] g) Host Cell Selection and Transformation Suitable host cells for cloning or expressing the DNA in the vectors described herein include higher eukaryotic cells, including the vertebrate host cells described herein. The cultivation and propagation of vertebrate cells (tissue culture) is a routine procedure. Examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7, ATC CRL 1651); a COS fibroblast-like cell line derived from monkey kidney tissue; a human embryonic kidney line (293 or 293 cell subclones for growth in suspension culture, Graham et al., J. GenVirol. 36:59 (1977)); milk hamster kidney cells (BHK, ATC CCL10); Chinese hamster ovary cells / -DHFR (CHO, Urlaubet al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)). ); monkey kidney cells (CV1 ATCCCCL70); African green monkey kidney cells (VERO-76, ATCCCRL-1587); human cervical carcinoma cells (HELA, ATCC-CCL2); dog kidney cells (MDCK, ATCC-CCL34); buffalo-rat hepatocytes (BRL3A, ATCCCRL1442); human lung cells (W138, ATCCCCL75); human hepatocytes (HepG2, HB8065); mouse mammary tumor (MMT060562, ATCCCCL51); TR1 cells (Matheretal., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells and a human hepatoma cell line (HepG2).

[0387] Host cells are transformed with the above-described expression or cloning vectors to produce the protein constructs and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants, or amplify the genes encoding the desired sequences.

[0388] h) Cultivation of host cells The host cells used to produce the protein constructs of the present application can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), minimal essential medium ((MEM) Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's medium ((DMEM) Sigma) are suitable for culturing the host cells. In addition, any of the media described in Hammetal., Meth. Enz. 58:44 (1979), Barnesetal., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469, WO90 / 03430, WO87 / 00195, or U.S. Pat. No. 30,985 can also be used as a culture medium for the host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug gentamicin™), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or energy equivalents. Other necessary supplements may also be added at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature and pH, have been previously used for host cell expression and will be apparent to those skilled in the art.

[0389] i) Protein purification When using recombinant techniques, the protein constructs of the present invention can be produced intracellularly, intraperiplasmically, or directly secreted into the culture medium. If the protein construct is produced intracellularly, the first step is to remove particulate debris (i.e., host cells or cleavage fragments) by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) details a process for isolating antibodies secreted into the periplasm of E. coli. Briefly, cell bodies are thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethanesulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation. If the protein construct is secreted into the culture medium, the supernatant from such expression systems is usually first concentrated using a commercially available protein concentration filter, e.g., an Amicon or MilliporePellicon ultrafiltration device. Protease inhibitors, such as PMSF, can be included in any of the above steps to inhibit protein hydrolysis, and antibiotics can also be included to prevent the growth of adventitious contaminants.

[0390] Protein compositions produced from cells can be purified using methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the type and subtype of any immunoglobulin Fc domain present in the Fc protein-containing construct. Protein A can be used to purify Fc-containing proteins based on human immunoglobulins containing one, two, or four heavy chains (Lindmarket et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G has been recommended for use with all mouse subtypes and human type 3 (Gusset et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is typically agarose, although other matrices can also be used. Mechanically stable matrices, such as controllable pore size glass or poly(styrene-divinyl)benzene, allow for faster flow rates and shorter processing times compared to agarose. Bakerbond ABX™ resin is C H It can be used to purify protein constructs containing the three domains (JT Baker, Phillipsburg, NJ). Other protein purification techniques, such as ion exchange column fractionation, ethanol precipitation, reverse phase liquid chromatography (HPLC), silica gel chromatography, heparin SEPHAROSE™ chromatography, anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation (depending on the protein construct being recovered), are also suitable.

[0391] After any preliminary purification steps, the mixture containing the protein construct of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer with a pH of about 2.5-4.5, preferably with a low salt concentration (e.g., 0-0.25 M salt).

[0392] Pharmaceutical Composition Additionally, pharmaceutical compositions are provided that include a prodrug described herein (e.g., an IL-15 prodrug or an anti-TNFR2 antibody prodrug) and any pharmaceutically acceptable carrier. The pharmaceutical composition can be prepared in the form of a lyophilized formulation or aqueous solution by mixing the prodrug of the desired purity described herein with any pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0393] Recombinant formulations can be prepared by dissolving the lyophilized prodrug in a diluent to uniformly distribute the protein. Examples of pharmaceutically acceptable (safe and non-toxic for human administration) diluents suitable for use in this application include, but are not limited to, sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate-buffered saline), sterile saline, Ringer's solution or glucose solution, or aqueous solutions of salts and / or buffers.

[0394] In some embodiments, the pharmaceutical composition comprises a homogeneous population of prodrugs (e.g., IL-15 prodrugs) described herein. A homogeneous population means that the prodrugs are completely identical to one another, e.g., the same IL-15 prodrug structure, the same IL-15 cytokine, the same IL-15Rα sushi domain, the same masking polypeptide, the same cleavable moiety, the same non-cleavable linker (if present), and the same Fc domain. In some embodiments, at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of the IL-15 prodrugs in the pharmaceutical composition are homogeneous.

[0395] Pharmaceutical compositions are preferably stable, and the proteins contained therein substantially maintain their physical and chemical stability and integrity during storage. Various analytical techniques for measuring protein stability are available in the art and are summarized in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pub. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature and for a selected period of time. For rapid screening, formulations can be stored at 40°C for two weeks to one month, and stability measured during that time. For example, the degree of aggregation during storage can be used as an indicator of protein stability.

[0396] In some examples, the pharmaceutical composition has a shelf life of at least 15 days, e.g., at least 20 days, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years, or more, e.g., at 2-25°C (e.g., 2-8°C). As used herein, "shelf life" refers to a shelf life during which degradation of an active ingredient in a pharmaceutical formulation, such as a therapeutic protein (e.g., an IL-15 prodrug or an anti-TNFR2 antibody prodrug described herein), is minimized (e.g., 5% or less degradation, e.g., 4%, 3%, or 2% or less degradation occurs) when the pharmaceutical formulation is stored under specified storage conditions, e.g., at 2-8°C. Exemplary techniques for assessing protein or formulation stability include size exclusion chromatography (SEC)-HPLC, e.g., to detect aggregation; reversed-phase liquid chromatography (RP)-HPLC, e.g., to detect protein fragments; ion-exchange HPLC, e.g., to detect charge changes in proteins; and mass spectrometry, fluorescence spectroscopy, circular dichroism (CD) spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and Raman spectroscopy to detect conformational changes in proteins. All of these techniques can be used individually or in combination to assess protein degradation in pharmaceutical formulations and to determine the shelf life of the formulations.

[0397] Acceptable carriers, excipients, or stabilizers are non-toxic to subjects at the dosages and concentrations employed, including buffers; antioxidants, including ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives, isotonicity agents (e.g., sodium chloride), stabilizers, metal complexes (e.g., zinc protein complexes); chelating agents, such as EDTA and / or non-ionic surfactants.

[0398] Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight (less than 10 residues) peptides; serum albumin, gelatin, or immunoglobulins. hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts such as sodium; metal complexes (e.g., zinc-protein complexes); and / or non-ionic surfactants, such as Tween™, polyethylene glycol (PEG), and PLURONICS™.

[0399] In particular, when stability is dependent on pH, buffers are used to control the pH within a range that optimizes therapeutic efficacy. Suitable buffers for this application include organic acids, inorganic acids, and their salts, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, and acetate. Additionally, buffers may include histidine and trimethylamine salts such as Tris.

[0400] Preservatives are added to prevent microbial growth. For example, adding a preservative facilitates the production of multi-use (multi-dose) formulations. Preservatives suitable for use in the present application include stearyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butanol, or benzyl alcohol; alkylparabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol.

[0401] Isotonicity agents, also known as "stabilizers," are used to adjust or maintain the tonicity of the liquid in a composition. When used with large, charged biomolecules (e.g., proteins), they are typically called "stabilizers" because they interact with the charged groups on the amino acid side chains, thereby reducing the likelihood of inter- and intramolecular interactions. Isotonicity agents may be present in any amount between 0.1% and 25% by weight, preferably between 1% and 5% by weight, taking into account the relative amounts of other components. Preferred isotonicity agents include polysaccharide alcohols, preferably trihydric or higher sugar alcohols such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0402] Other excipients include one or more of (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to container walls. Such excipients include polyhydric sugar alcohols (as listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myonisitose, myo-inisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), and polyethylene glycol. alcohols; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0403] The presence of a non-ionic surfactant or detergent (also called a "wetting agent") aids in solubilizing the protein and protects it from aggregation due to agitation. This also allows the formulation to be exposed to shear surface stresses without causing denaturation of the active protein.

[0404] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (e.g., 184, 188), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (e.g., Tween®-20, Tween®-80, etc.), lauromacrogol 400, polyoxyethylene 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glyceryl monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic surfactants that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0405] For pharmaceutical compositions to be useful for in vivo administration, they must be sterile. Pharmaceutical compositions can be sterilized by filtration through sterile membranes. The pharmaceutical compositions are usually placed into a container with a sterile access port, for example, an intravenous solution bag or vial equipped with a stopper that can be pierced by a hypodermic injection needle.

[0406] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), L-glutamic acid and L-ethyl glutamate copolymers, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprorelin acetate)), and poly-D-(-)-3-hydroxybutyric acid.

[0407] The pharmaceutical compositions described herein may also contain multiple active compounds as necessary for the particular condition being treated, preferably compounds with complementary activities that do not adversely affect each other, such molecules being combined in amounts effective to achieve their intended purpose.

[0408] The active ingredient can also be encapsulated in microcapsules prepared, for example, by gelation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and polymethylmethacrylate microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or crude emulsions. Remington's Pharmaceutical Sciences (18th Edition) discloses these techniques.

[0409] In some embodiments, the pharmaceutical composition is packaged in a single-use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical composition is provided in a reusable vial. In some embodiments, the pharmaceutical composition is packaged loose in a container. In some embodiments, the pharmaceutical composition is stored frozen.

[0410] Treatment of Disease Also provided are methods for treating a subject suffering from or at risk of a disease, such as a proliferative disease, a neoplastic disease, an inflammatory disease, an immune disease, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, or graft-versus-host disease. The methods disclosed herein involve administering to a subject in need thereof an effective amount of an activatable prodrug, typically in a pharmaceutical composition, where the prodrug is activated upon enzymatic cleavage. In some embodiments, the method further comprises selecting a subject suffering from or at risk of suffering from such a disease or condition. In some embodiments, the prodrug is activated in the tumor microenvironment. The prodrug has therapeutic activity upon cleavage from the masking polypeptide. Thus, in some embodiments, the activator is a cleavage product. In some embodiments, the prodrug may rely on binding of an antigen to the antigen-binding domain to treat the disease.

[0411] In some examples, methods are provided for treating a disease (e.g., a tumor, a viral infection, or a bacterial infection) in an individual (e.g., a human), the methods comprising administering to the individual an effective amount of any of the prodrugs described herein (e.g., an IL-15 prodrug or an anti-TNFR2 antibody prodrug) or pharmaceutical compositions thereof. In some examples, the prodrug (or pharmaceutical composition thereof) is administered by intravenous, intramuscular, or subcutaneous injection. In some examples, the treatment method further comprises administering an additional therapeutic agent in combination with (before, after, or simultaneously with) the prodrug. The additional drug may be an antibody or antigen-binding fragment thereof, a small molecule drug, or other types of therapeutic agents.

[0412] In some embodiments, the IL-15 prodrug or anti-TNFR2 antibody prodrug is used to treat cancer or tumor in a subject, comprising administering a therapeutically effective amount of the IL-15 prodrug or anti-TNFR2 antibody prodrug to the subject. As described herein, in some embodiments, the term "tumor or cancer" refers to any type of cancer, tumor, or malignant tumor in a mammal, including leukemia, lymphoma, melanoma, neuroendocrine tumor, carcinoma, and sarcoma. Examples of cancers that may be treated with the masked cytokines, pharmaceutical compositions, or methods provided herein include lymphoma, sarcoma, bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g., triple negative, ER positive, ER negative, chemotherapy resistant, Herceptin resistant, HER2 positive, doxorubicin resistant, tamoxifen resistant, glandular cancer, leukemia ... cancer, including ductal carcinoma, lobular carcinoma, primary, and metastatic), ovarian cancer, pancreatic cancer, liver cancer (including hepatocellular carcinoma), lung cancer (including non-small cell lung cancer, squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, carcinoid, and sarcoma), glioblastoma multiforme, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple-negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (such as of the head and neck or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma.Other examples include thyroid cancer, endocrine cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, gastric cancer, uterine or medulloblastoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, essential thrombocythemia, primary macroglobulinemia, primary brain tumors, cancer, malignant islet tumor, malignant carcinoid, bladder cancer, and precancer. These include: cutaneous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, pancreatic endocrine or exocrine tumors, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, Paget's disease of the nipple, phyllodes tumors, lobular carcinoma, ductal carcinoma, pancreatic stellate cell carcinoma, hepatic stellate cell carcinoma, or prostate cancer.

[0413] In some embodiments, the IL-15 prodrug is used to treat a bacterial infection, such as sepsis. In some embodiments, the bacteria causing the bacterial infection is a drug-resistant bacteria. In some embodiments, the antigen-binding moiety binds to a bacterial antigen.

[0414] In some embodiments, the IL-15 prodrug is used to treat a viral infection. In some embodiments, the virus causing the viral infection is hepatitis C (HCV), hepatitis B (HBV), human immunodeficiency virus (HIV), or human papillomavirus (HPV). In some embodiments, the antigen-binding moiety binds to a viral antigen.

[0415] Administration of the prodrugs or pharmaceutical compositions thereof described herein can be by any convenient method, including injection or infusion. The route of administration follows known and accepted methods, such as single or multiple bolus injections or prolonged infusion in an appropriate manner. The prodrugs or pharmaceutical compositions thereof can be administered orally, subcutaneously, intravenously, intracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, intravaginally, rectally, intraocularly, topically, intraarterially, intradermally, intranodally, intracavity, or intramedullary, intrathecal, intraventricular, intracerebral, intraspinal, intrathecal, intralesional, or intraocularly. In some embodiments, the prodrugs or pharmaceutical compositions thereof are administered systemically. In some embodiments, the prodrugs or pharmaceutical compositions thereof are administered to an individual by infusion (e.g., intravenous infusion). Infusion techniques used in immunotherapy are known in the art (see Rosenberg et al., New Eng. J. of Med. 319:1676 (1988)). In some embodiments, the prodrug or pharmaceutical composition thereof is administered to an individual by intradermal or subcutaneous (i.e., under the skin) injection. For subcutaneous injection, a syringe can be used to inject the prodrug or pharmaceutical composition thereof. However, there are other devices for administering the prodrug or pharmaceutical composition thereof, such as injection devices, injection pens, auto-injection devices, needle-free devices, and subcutaneous patch delivery systems. In some embodiments, the prodrug or pharmaceutical composition thereof is administered by intravenous injection. In some embodiments, the prodrug or pharmaceutical composition thereof is injected directly into the brain or spine. In some embodiments, the prodrug or pharmaceutical composition thereof is administered via sustained-release or extended-release technology.

[0416] The dosage and desired drug concentration of the pharmaceutical composition of the present invention may vary depending on the specific intended use. Determining the appropriate dosage or route of administration is entirely within the skill of those skilled in the art. Animal experiments provide reliable guidance for determining effective dosages for human treatment. Interspecies analogies of effective dosages can be made according to the principles set forth in Mordenti, J. and Chappell, W., "The Use of Interspecies Scaling in Toxicological Kinetics," in Toxicological Kinetics and New Drug Development, Yacobietal., Eds., Pergamon Press, New York, 1989, pp. 42-46.

[0417] When the prodrug or pharmaceutical composition thereof is administered in vivo, the dosage will vary depending on the route of administration and the type of mammal. Within the scope of this application, different formulations may be effective for different treatments and different diseases, and the administration method intended for the treatment of a particular organ or tissue may differ from that intended for another organ or tissue. Furthermore, the dosage may be administered in one or more separate administrations or by continuous infusion. In the case of repeated administration over several days or longer, depending on the condition and severity, treatment may be continued until the desired degree of inhibition of disease symptoms is achieved. However, other dosage regimens may also be useful. The progress of this treatment can be easily monitored through routine techniques and assays.

[0418] In some embodiments, the prodrug or pharmaceutical composition thereof is administered in a single dose (e.g., a bolus injection). In some embodiments, the prodrug or pharmaceutical composition thereof is administered multiple times (e.g., 2, 3, 4, 5, 6, or more times). When multiple doses are administered, they may be administered by the same route or different routes, and at the same site or at other sites. The prodrug or pharmaceutical composition thereof may be administered from once daily to once yearly. The interval between two doses may be any time between 24 hours and one year, and the intervals may also be irregular (e.g., depending on tumor progression). In some embodiments, there is no break in the dosing regimen. The optimal dosage and treatment regimen for a particular patient can be determined by one skilled in the medical field by monitoring the patient's symptoms of disease and adjusting accordingly.

[0419] In some embodiments, the prodrug or pharmaceutical composition thereof is administered in divided doses, such as two, three, four, five, or more doses. In some embodiments, the divided doses are administered over a period of more than one week, one month, two months, three months, or longer. In some embodiments, the dose is divided evenly. In some embodiments, the divided doses are 20%, 30%, and 50% of the total dose. In some embodiments, the interval between successive divided doses is one day, two days, three days, one week, two weeks, three weeks, one month, three months, six months, or longer. In the case of repeated administration over several days or more depending on the condition, treatment is continued until the desired degree of inhibition of disease symptoms is achieved, depending on severity. However, other dosing regimens may also be useful. The progress of this treatment can be easily monitored using routine techniques and assays.

[0420] Products and Kits Kits, unit doses, and articles of manufacture comprising any of the prodrugs described herein are further provided. In some examples, kits are provided that comprise any of the prodrug pharmaceutical compositions described herein, and preferably, instructions for their use, such as for treating a disease (e.g., a tumor) described herein.

[0421] The kits of the present invention include one or more containers containing a prodrug described herein, e.g., for treating a disease. For example, instructions describing administration of the prodrug to treat a disease (e.g., a tumor) are included. The kits may further include instructions for selecting an appropriate individual (e.g., a human) for treatment based on identifying whether the individual has a disease and the stage of the disease. Instructions related to the use of the prodrug typically include information regarding the dosage, dosing regimen, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages), or subunit doses. The instructions provided in the kits of the present invention are typically written instructions in the form of a label or pharmaceutical instructions (e.g., a paper sheet included with the kit), although machine-readable instructions (e.g., instructions stored on a magnetic or optical disk) are also acceptable. The kits of the present application employ suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, cans, soft packaging (e.g., sealed Mylar or plastic bags), and the like. Packaging for use in combination with specific devices, such as infusion devices such as micropumps, is also contemplated. The kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). At least one activating agent in the composition is a prodrug described herein. The container may further comprise a second pharmaceutical activating agent. The kit may optionally provide additional components, such as buffers or interpretive information. Generally, the kit includes a container and a label or pharmaceutical insert on or associated with the container.

[0422] Accordingly, the present application also provides articles such as vials (e.g., sealed vials), bottles, cans, soft packaging, and the like. The article includes a container and a label or pharmaceutical instruction attached to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be made of various materials, such as glass or plastic. Generally, the composition contained in the container can effectively treat a disease or condition (e.g., a tumor) described herein, and the container may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic syringe). The label or package insert indicates that the composition is used to treat a particular condition in an individual. The label or pharmaceutical instruction further includes instructions for administering the composition to an individual. The label may include instructions for reconstitution and / or use. The container holding the pharmaceutical composition may be a multi-use vial that allows for repeated administration (e.g., 2-6 administrations) of the reconstituted formulation. Pharmaceutical leaflet means the leaflet typically included in the commercial packaging of a therapeutic product, including information regarding the indications, usage, dosage, administration, contraindications, and / or warnings for such a therapeutic product. Additionally, the product may further include a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. From a commercial and user perspective, other necessary materials may further be included, such as other buffers, diluents, filters, needles, syringes, etc.

[0423] The kit or article of manufacture includes a plurality of unit doses of the pharmaceutical composition and instructions for use, packaged in an amount sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies.

[0424] (Example) The following examples are intended to be merely illustrative of the present invention and therefore should not be construed as limiting the present invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0425] Example 1: Production of masking polypeptide (MP) Composition of the masking polypeptide: Considering the selection of amino acids for a chemically stable and largely unstructured masking polypeptide, the masking polypeptide contains at least 40 consecutive amino acids and is essentially unable to nonspecifically bind to serum proteins. The amino acids ultimately selected for the masking polypeptide consist of four or five amino acid residues, selected from the group consisting of proline (P), alanine (A), serine (S), glutamic acid (E), and glycine (G). The masking polypeptide sequence is arranged such that, except for serine (S), no single amino acid is repeated three times, and the percentage of each amino acid in the masking polypeptide corresponds to a specific ratio. The percentage of amino acid residue A in the masking polypeptide is about 5% to 20%, the percentage of amino acid residue E in the masking polypeptide is about 1% to 20%, and the percentage of amino acid residue G in the masking polypeptide is about 15% to 30%. The percentage of amino acid residues P in the masking polypeptide is about 15% to 40%, and the percentage of amino acid residues S in the masking polypeptide is about 20% to 40%. The masking polypeptide contains at least 40 amino acids, and the length of the sequence can be extended with a non-repetitive, unstructured polypeptide. An exemplary masking polypeptide, MP80, was initially designed as the sequence of SEQ ID NO: 1, and masking polypeptides MP163 and MP240 contain the amino acid sequence of MP80. Following the above rules, masking polypeptides can be designed as, but not limited to, the masking polypeptides shown in Table 1 and tested for masking activity. Figures 1A-1C show sequence alignments of MP100 and MP80, MP163 and two-repeat MP80, and MP240 and three-repeat MP80, respectively. MP80 and MP100 contain the amino acid sequence of SEQ ID NO: 6, and both MP163 and MP240 contain the amino acid sequence of MP80.

[0426] All sequences were analyzed using in silico T-cell epitope prediction tests, B-cell immunogenicity tests, and secondary structure predictions, and the masking peptides contained no detectable T- or B-cell epitopes and no detectable protease cleavage sites.

[0427] [Table 3]

[0428] Example 2: Expression and characterization of IL-15 prodrugs To reduce the toxicity of IL-15-related therapeutics, IL-15 prodrugs containing a masking polypeptide were constructed and recombinantly expressed in HEK293 cells. The constructs of the IL-15 prodrugs are shown in Table 2. Figure 2A is a schematic diagram of an exemplary structure of an IL-15 prodrug containing a masking polypeptide. Figure 2B shows an exemplary schematic diagram illustrating the release of a masking polypeptide (MP) from an IL-15 prodrug in target tissues (e.g., tumors with high levels of MMP) to activate the prodrug. The activity of the prodrug is very low until the masking polypeptide is cleaved by proteases in the target tissue. The drug SB1902-C1, which does not contain a masking polypeptide, and an inactivatable IL-15 cytokine construct that does not contain a cleavable moiety, such as SB1902-C4, served as controls in the experiments described below.

[0429] [Table 4]

[0430] The prodrug SB1902-C2 (hIgGFc(hole)-lk1-IL15-lk2-CM1-lk2-MP80 / hIgGFc(knob)-lk5-IL15Rα_sushi) is used as an example of plasmid construction. Similar methods are used for other prodrugs and drugs. The prodrug expression vectors are cloned using standard molecular techniques. Gene fragments for the masking polypeptide, human IL-15, and human IL-15Rα_sushi were commercially synthesized (Genscripts USA) and digested with restriction enzymes accordingly. Human IgG1Fc(hole) or Fc(knob) was amplified by PCR and digested with restriction enzymes. The cleavable portion of CM1 was synthesized by annealing at 50 °C using forward and reverse single-stranded nucleotides with corresponding restriction endonuclease sites at the 5' and 3' ends. All synthesized gene fragments and PCR fragments were purified and cloned into the plasmid pcDNA3.1 (Invitrogen). After transformation and plating, colonies were picked and grown overnight at 37°C in LB medium containing carbenicillin. Recombinant plasmids were extracted using a commercially available kit (Qiagen, catalog no. 27104) and sequenced using the T7 forward and BGH reverse primers. The entire coding sequence was verified by DNA sequencing. Exemplary sequences of the cleavable portions (CM1-CM10) are listed in Table 3, the non-cleavable linkers (lk, lk1, lk2, lk3, and lk5) are listed in Table 4, exemplary sequences of human IL-15 (mature or precursor) and IL-15Rα_sushi (long or short) are listed in Table 5, the sequences of human IgG1 Fc(hole), Fc(knob), Fc(knob-LALA), and Fc(hole-LALA) are listed in Table 6, and exemplary sequences of IL-15 prodrug constructs are listed in Table 7. In Table 7, the human IL-15 or IL-15Rα_sushi domain is marked in italics, the non-cleavable linker is marked in bold, the cleavable portion is marked with a single underline, the masking polypeptide is marked with a double underline, and the introduced restriction endonuclease recognition site is marked with a dashed line.

[0431] For characterization and evaluation, prodrug and drug expression constructs with N-terminal 8xHis tags were cloned and validated accordingly.

[0432] Exemplary cleavable portions of the activatable cytokines used in this application (eg, CM1, CM2, or CM4) can be cleaved by matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9).

[0433] [Table 5]

[0434] [Table 6]

[0435] [Table 7]

[0436] [Table 8]

[0437] [Table 9] TIFF2025527215000010.tif249164TIFF2025527215000011.tif250164TIFF2025527215000012.tif248164TIFF2025527215000013.tif248164 TIFF2025527215000014.tif248164TIFF2025527215000015.tif248164TIFF2025527215000016.tif248163TIFF2025527215000017.tif133164

[0438] Production: Vectors encoding IL-15 prodrugs or inactivatable IL-15 cytokines were transiently transfected into Expi293 cells (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer's recommendations, and the proteins were expressed. The culture supernatant medium was clarified by centrifugation and filtered through a 0.2 μm membrane. Exemplary IL-15 prodrugs (SB1902-C2, SB1902-C3, SB1902-C5, SB1902-C6, SB1902-C7, SB1902-C9, SB1902-C9-variant4, and SB1902-C10-variant1), exemplary IL-15 drugs without masking polypeptide (SB1902-C, SB1902-C1-variant1, SB1902-C1-variant2, and SB1902-C1-variant3), and the non-activatable IL-15 cytokine SB1902-C4 (without a cleavable moiety) were purified by a two-step purification process, including a pre-packed MabSelect SuRepcc column (Cytivalifescience, catalog number 17549112) and size exclusion chromatography (Superdex200, Cytiva, USA), according to the manufacturer's recommended methods. MBP-MP80 is a fusion protein of maltose-binding protein and masking polypeptide MP80 (SEQ ID NO: 1). It was purified using an amylose resin pre-packed column (NEB, Cat. No. E8021L, USA) according to the manufacturer's standard procedure.

[0439] SDS-PAGE analysis: Exemplary purified prodrugs SB1902-C2, SB1902-C7, and SB1902-C1 were analyzed for purity using 4-20% polyacrylamide SDS-PAGE under reducing or non-reducing conditions and staining the gel with SimplyBlueSafeStain (Figure 3).

[0440] SEC-HPLC Analysis: Analytical SEC-HPLC was used to analyze the prodrug SB1902-C2 and drug SB1902-C1 for homogeneity assessment (Figure 4). HPLC analysis was performed using a TSKgel G3000SWxl column according to the manufacturer's recommended method. All purified drugs and prodrugs were analyzed in the same manner using SDS-PAGE and SEC-HPLC.

[0441] The SDS-PAGE and HPLC results are shown in Figures 3 and 4. This exemplary data demonstrates that the constructs described herein were successfully produced and that the purified prodrugs SB1902-C2 and SB1902-C7, and drug SB1902-C1, have good purity and homogeneity.

[0442] Example 3: Protease cleavage of prodrugs by MMP2 and MMP9 The experiments involved the discovery of peptide substrates that are sensitive to MMP2 and MMP9, and the efficiency of substrate cleavage can be measured in various ways.

[0443] To obtain suitable polypeptide substrates with appropriate sensitivity to both MMP2 and MMP9, mutagenesis was performed using a known substrate sequence (i.e., phage clone A3 with the AKPRALTA sequence from U.S. Patent Publication No. US2009 / 0253896A1) as a template. Through mutagenesis and screening, a series of MMP2 and MMP9 substrate sequences (cleavable moieties) with appropriate sensitivity were obtained and named CM1 to CM10. The sequences and in vitro cleavage efficiency results are shown in Table 8.

[0444] To test the in vitro cleavage efficiency, an in vitro enzyme assay was performed. Briefly, recombinant human MMP2 (rhMMP2) (R&D Systems, catalog number 902-MP-010) and recombinant human MMP9 (rhMMP9) (R&D Systems, catalog number 911-MP-010) were diluted to 100 μg / mL in detection buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w / v) Brij 35, pH 7.5), and rhMMP2 and rhMMP9 were activated with 1 mM APMA (4-aminophenylmercuric acetate, Sigma-Aldrich, USA). Briefly, 1 μg each of the diluted MMP2 and MMP9 was transferred to an Eppendorf tube, and 4-aminophenylmercuric acetate (APMA) (Sigma, catalog number A-9563) was added to a final concentration of 1 mM. To activate both rhMMP2 and rhMMP9, the mixture was incubated at 37°C for 2 hours for rhMMP2 and overnight for rhMMP9. Activated rhMMP-2 or rhMMP9 was diluted to 1 ng / µL in detection buffer. 16 µL of activated rhMMP2 and rhMMP9 were each placed in the first tube, and 4 µL of detection buffer was added. A two-fold serial dilution was performed starting from tube 1 to tube 7, resulting in final enzyme concentrations ranging from 400 ng / mL to 6.25 ng / mL. Each Eppendorf tube was preloaded with activated rhMMP2 or rhMMP9 at a different enzyme concentration. For protease digestion, 1.5 µg of SB1902-C2, SB1902-C5, or SB1902-C4 was added, respectively, and the mixture was digested at 37°C for 5 hours. After the 5-hour incubation, the prodrug or drug was analyzed before and after enzyme digestion using a 4-20% polyacrylamide gel.

[0445] [Table 10]

[0446] Figures 5A-5B show the bands obtained after cleavage of CM1 in the prodrug SB1902-C2 after in vitro digestion with MMP2 and MMP9 according to the standard procedure described above. The mutated substrate CM1 can be cleaved in vitro by the MMP2 and MMP9 enzymes in an enzyme dose-dependent manner.

[0447] Figures 5E-5F show the bands obtained after cleavage of CM4 in the prodrug SB1902-C5 after in vitro digestion with MMP2 and MMP9 according to the standard procedure described above. The mutated substrate CM4 can be cleaved in vitro by MMP2 and MMP9 enzymes in an enzyme dose-dependent manner.

[0448] Other cleavable moieties (e.g., CM2, CM5-CM10) could also be cleaved by MMP2 and MMP9 in vitro (data not shown). These results indicate that the cleavable moieties of the designed prodrugs can be cleaved in target tissues (e.g., tumors) in vivo.

[0449] However, as shown in Figures 5C-5D, the non-cleavable G4S linker of SB1902-C4 is resistant to both MMP2 and MMP9 digestion.

[0450] Example 4: In vitro characterization of prodrugs and drugs 4.1 Nonspecific serum protein binding: Direct ELISA was used to evaluate the binding of exemplary prodrugs SB1902-C2, SB1902-C3, and SB1902-C4 (without a cleavable linker) and the drug SB1902-C1 to human, cynomolgus monkey, or rat serum proteins. Briefly, human, cynomolgus monkey, or rat serum was coated onto a 96-well Maxisorp plate (Corning) at 25 μL per well and left overnight at 4 °C. The plate was then placed in PBS containing 1% BSA and blocked at room temperature for 1 hour. SB1902-C2, SB1902-C3, SB1902-C4, or SB1902-C1 were serially diluted in PBS containing 0.5% BSA (ELISA buffer) and placed on the plate for 1 hour, after which the plate was washed with PBS (wash buffer). Bound SB1902-C2, SB1902-C3, SB1902-C4, and SB1902-C1 were detected using AP-conjugated anti-human IgGFc specific antibody (SouthernBiotech, catalog no. 2014-04) in ELISA buffer. Plates were incubated at room temperature with agitation for 1 hour, washed six times with wash buffer, and developed for 3–10 minutes by adding 50 μL / well of freshly prepared PNPP substrate (Sigma-Aldrich, catalog no. N2770). Enzyme development was stopped with 50 μL / well of TMB stop solution (VWR, catalog no. 95059-200). Analysis was performed at 405 nm using a BioTekGen5 microplate reader (BioTek).

[0451] As shown in Figures 6A to 6C, no nonspecific binding was detected between the prodrugs SB1902-C2, SB1902-C3, and SB1902-C4 and the drug SB1902-C1 and serum proteins from humans, cynomolgus monkeys, and rats.

[0452] 4.2 Plasma stability: The in vitro serum stability of the prodrugs was determined in human, cynomolgus monkey, and rat plasma. Blood samples were collected from humans, cynomolgus monkeys, or rats, and blood cells were separated from the plasma by centrifugation. The upper plasma layer was carefully transferred to a new centrifuge tube and centrifuged at 10,000 rcf for 10 minutes. 500 μL of human plasma, 500 μL of cynomolgus monkey plasma, 500 μL of rat plasma, or 500 μL of PBS was added to each of the prodrugs or drugs. The centrifuge tubes were sealed and incubated at 40°C for 7 days. Five μl of each of the incubated prodrugs and drugs was separated using a 4-20% pre-polyacrylamide gel (ThermoFisher, catalog number XP04205BOX). Proteins separated on the polyacrylamide gel were transferred to a PVDF membrane. After blocking and washing, the blots were incubated with AP-conjugated anti-His6 polyclonal antibody (SouthernBiotech, catalog no. SB194b) for 1 hour and washed. Blot protein bands were developed using a color development reagent (Bio-Rad, catalog no. 1706432) according to the manufacturer's instructions.

[0453] Representative Western blot results are shown in Figures 6D–6E. Figures 6D and 6E show bands on WB membranes before and after incubation of the prodrugs SB1902-C2, SB1902-C3, and SB1902-C4 with plasma and PBS buffer. This indicates that no degradation bands were detected after incubation of the prodrugs SB1902-C2, SB1902-C3, or SB1902-C4 with human plasma or PBS buffer. Furthermore, when the drug SB1902-C1 and the prodrugs SB1902-C2, SB1902-C3, and SB1902-C4 were incubated with cynomolgus monkey or rat plasma, respectively, no detectable degradation bands were found on the WB membrane (data not shown). These results indicate that the prodrugs are stable in human, cynomolgus monkey, and rat plasma.

[0454] 4.3 Receptor binding affinity: Binding affinities of prodrugs or drugs to the IL-2 / IL15Rβγ-Fc fusion protein were detected using ELISA. ELISA plates were coated with 1 μg / ml recombinant IL15Rβγ-Fc and incubated overnight at 4°C. After blocking and washing with 1% BSA in PBS, serially diluted prodrugs SB1902-C2 or SB1902-C1 were added to the plates and incubated for 2 hours. The human IgG1 subtype antibody MOPC21 (abbreviated as hIgG1 in Figure 7) (see Hamlyn PH, Gait MJ, Milstein C. (1981) Complete sequence of an immunoglobulin mRNA using specific priming and thethedideoxynucleotide method of RNA sequencing. Nucleic Acids Res. 9(18):4485-4494) served as a negative control. After washing the plate, a 1:2000 dilution of anti-human IgG-Fc-AP conjugated antibody was added to each well and incubated for 45 min. After washing the plate, AP substrate pNPP (Thermo, USA) was added, and the optical density was measured at 405 nm using a spectrophotometer (BioTec, USA). Data were analyzed and graphed using GraphPad Prism 8 software.

[0455] As shown in Figure 7, the exemplary prodrug SB1902-C2 significantly reduced the binding affinity to IL15Rβγ compared to the drug SB1902-C1, indicating that the masking polypeptide effectively inhibits the binding function of IL-15.

[0456] Example 5: Immunogenicity studies The immunogenicity of the masking polypeptide (MP) was studied by immunizing Balb / c mice with the prodrug intravenously at a dose of 1 mg / kg / week for six consecutive weeks. Mouse sera were collected at week 6 and analyzed for anti-prodrug antibodies by direct ELISA. Plates were coated with the prodrug SB1902-C2 or MBP-MP80, incubated overnight at 4°C, and then blocked with 1% BSA in PBS. After washing the plates, serum from mice treated with the prodrug SB1902-C2 was collected, serially diluted, and added to wells precoated with the prodrugs SB1902-C2 and MBP-MP80. Serum from mice treated with Boco (bococizumab, Pfizer, positive control) was collected, serially diluted, and added to wells precoated with Boco. After incubation and plate washing, a 1:3000 dilution of goat anti-mouse IgGFc-AP was added to the plate and incubated for 45 minutes. After washing the plate, freshly prepared substrate pNPP (Thermo, USA) was added to each well of the plate, and the optical density of each well was obtained using a spectrophotometer (BioTec, USA) at a wavelength of 405 nm and graphed using GraphPadPrism8 software.

[0457] As shown in Figure 8, after weekly injections of the prodrug for a total of 6 weeks, no anti-drug antibodies (ADA) against the MBP-MP80 fusion protein were detected in the serum of five mice by ELISA. In contrast, the serum of three of the five mice injected weekly with the prodrug SB1902-C2 produced significant ADA against the Fc portion of the prodrug. These results indicate that the masking polypeptide MP80 in the prodrug is not immunogenic.

[0458] (Example 6) In vitro functional test: Mo7e cell proliferation test The prodrugs were measured in an in vitro cell proliferation assay. IL-15 primarily transmits signals through binding to IL-2 / IL-15Rβγ. Mo7e cells express both IL-2 / IL-15Rβ and γc on the cell membrane and are known to require IL-2 or IL-15 for their survival and proliferation. To detect IL-15 cytokine-dependent proliferation of Mo7e cells, the following test was performed. Mo7e cells (DSMZ, Cat. No. ACC104) were maintained in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin and streptomycin, and 10 ng / ml GM-CSF (Peprotech, Cat. No. 300-03) and placed in a 37°C (5% CO2) incubator. Mo7e cells were harvested in the logarithmic growth phase and washed twice with medium without GM-CSF. The cells were plated at 1 x 10 4 The medium was added at 100 μL / well, with a total volume of 45 μL per well (GM-CSF-free). The cells were incubated for 2–4 hours in a 37°C (5% CO2) incubator. The prodrugs SB1902-C2, SB1902-C6, SB1902-C7, and SB1902-C1 were diluted 3-fold with medium and 5 μL was added to each well. Each concentration was repeated three times. Cells containing medium alone served as a blank control. The cells were cultured for 3 days in the incubator. Next, 5 μL of WST-8 (WST-8 Cell Proliferation Assay Kit, Cayman, catalog number 10010199) was added to all wells and incubated for 2–3 hours in the incubator to detect Mo7e cell proliferation. The optical density at 450 nm (OD450) was measured. Data were analyzed and graphed using Graphpad Prism 8 software.

[0459] As shown in Figure 9, all exemplary prodrugs SB1902-C2, SB1902-C6, and SB1902-C7, as well as the drug SB1902-C1, stimulated cell proliferation in a dose-dependent manner. However, the activity of the prodrugs SB1902-C2, SB1902-C6, and SB1902-C7 was significantly reduced compared to the drug SB1902-C1. The results indicated that the masking polypeptides MP80, MP96new, and MP100 blocked the binding of IL-15 prodrugs to the receptor IL-2 / IL-15Rβγ, thereby reducing its signaling and downstream functions.

[0460] (Example 7) In vitro functional test: CD8+ T cell activity test According to the literature, IL-15 promotes the proliferation, survival, and homeostasis of CD8+ memory T cells, natural killer (NK) cells, and NKT cells. IL-15 induces T cell activation, which is manifested by upregulated expression of CD69 on the cell surface and the release of cytokines, including IFNγ. CD69 is an early marker of T cells, and the percentage of CD8+ T cell activation is reflected by the percentage of CD69 expressed on the cell surface.

[0461] 7.1 Determining CD8+ T cell-based activity of prodrugs prior to activation To compare the activity of IL-15 prodrugs or drugs in activating CD8+ T cells, the basic procedure for this experiment was as follows: Human PBMCs (StemCell Technologies, Cat. No. 70500) were cultured at 2 × 10 cells per well in a 96-well round-bottom cell culture plate containing RPMI-1640 medium. 5The cells were added at 100 μL / well, and the medium was supplemented with 10% FBS, 1% penicillin, and streptomycin. Exemplary drugs SB1902-C1, SB1902-C1-variant1, SB1902-C1-variant2, and SB1902-C1-variant3 (whose Fc has L234A and L235A mutations, i.e., the LALA mutation) were diluted three-fold with medium along with prodrugs SB1902-C2, SB1902-C6, SB1902-C7, SB1902-C9, SB1902-C9-variant4, and SB1902-C10-variant1. Five microliters of each concentration were added to the wells. Each concentration was repeated three times, and blank wells (medium alone) were used as blank controls. The cell culture dishes were incubated in an incubator for 3 days. The cells were then centrifuged and stained with anti-CD3 Ab (biolegend, catalog no. 317306), anti-CD8 Ab (biolegend, catalog no. 344722), and anti-CD69 Ab (biolegend, catalog no. 310906) in FACS buffer for 30 minutes. After washing twice with FACS buffer, cells were acquired using an Attune (ThermoFisher Scientific) flow cytometer. Data were analyzed using FlowJo software. The percentage of CD69+ cells relative to CD8+ T cells was plotted using Graphpad Prism 8 software.

[0462] As shown in FIG. 10A, compared with the drug SB1902-C1, the prodrug SB1902-C7 with the masking polypeptide MP100 has apparently lower activity in stimulating T cell activation.

[0463] As shown in Figure 10B, compared with the drug SB1902-C1, the prodrug SB1902-C2 with the masking polypeptide MP80 has apparently lower activity in stimulating T cell activation.

[0464] As shown in Figure 10C, both the prodrug SB1902-C2 with the masking polypeptide MP80 and the prodrug SB1902-C6 with the masking polypeptide MP96new had low activity in stimulating T cell activation, whereas SB1902-C2 was more effective than SB1902-C6 in inhibiting CD69 activation in CD8+ T cells.

[0465] As shown in Figure 10D, the prodrug SB1902-C9-variant4, which has the masking polypeptide MP163 and the Fc domain LALA mutations (L234A and L235A), has significantly lower activity in stimulating T cell activation compared to SB1902-C1-variant2 and SB1902-C1-variant3. Furthermore, this result also indicates that the LALA mutation does not affect the activity of the drug or prodrug.

[0466] As shown in FIG. 10E, compared with the drug SB1902-C1-variant1, the prodrug SB1902-C10-variant1 with the masking polypeptide MP240 was obviously less active in stimulating T cell activation.

[0467] The above results show that the T cell activation activity of IL-15 cytokine in different prodrug forms with different masking polypeptides was obviously blocked by all masking polypeptides, indicating that the masking polypeptides of the prodrugs prevented the activation of CD8+ T cells by IL-15.

[0468] As shown in Figure 10F, compared with the drug SB1902-C1, both prodrugs SB1902-C2 and SB1902-C9 had significantly lower activity in stimulating T cell activation. In comparison, SB1902-C9 was more effective than SB1902-C2 in inhibiting CD69 activation in CD8+ T cells, indicating that the masking polypeptide MP163 has a better masking effect than MP80.

[0469] Combining the results of Figures 9 and 10F, we found that masking polypeptides of different lengths all had good masking effects. The masking polypeptide lengths in these experiments ranged from 80 to 100 amino acids, and small changes in MP length did not significantly affect activity. However, within the range of 80 to 163 amino acids, the length of the masking polypeptide did have an effect, and increasing the length improved the masking efficiency to some extent.

[0470] 7.2 After the masking polypeptide is removed by MMP2 treatment, the prodrug activity is activated.

[0471] To render the prodrug into a fully functional drug, the prodrug's masking polypeptide (MP) must be removed. To this end, recombinant human MMP-2 (rhMMP2) (R&D Systems, catalog number 902-MP-010) was activated according to the manufacturer's instructions. Next, 1 μg of activated MMP2 was placed in an Eppendorf tube containing 250 μg of purified prodrug SB1902-C2, and enzyme digestion buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w / v) Brij 35, pH 7.5) was added to bring the enzyme digestion volume to 0.5 mL. The test tube containing the enzyme / prodrug mixture was incubated at 37 °C for 5 h. After enzymatic hydrolysis, samples were taken and the efficiency of enzymatic hydrolysis was evaluated using SDS-PAGE. The results showed that approximately 90% of the prodrug SB1902-C2 was digested by the enzyme. The digested SB1902-C2 was purified with Protein A beads to remove the enzyme, followed by buffer exchange and concentration. The final concentrate was used in a human PBMC activation assay and compared with undigested prodrug SB1902-C2 and drug SB1902-C1.

[0472] As shown in FIG. 11, compared to the drug SB1902-C1, removal of the masking polypeptide from the exemplary prodrug SB1902-C2, followed by MMP digestion, restored the activity of SB1902-C2 in stimulating CD8+ T cell activation.

[0473] (Example 8) In vivo IFN-γ production test T cell-derived IFN-γ is a key cytokine stimulating innate immune responses, and IL-15 plays a key role as an activator of T cell function. Studies have shown that IL-15 can induce IFN-γ expression (Strengell M, et al. IL-21 synergy with IL-15 or IL-18 enhances IFN-γ production in human NK and T cells. J Immunol. 2003 Jun 1;170(11):5464-9). In vivo, overproduction of the pro-inflammatory cytokine IFN-γ can cause unwanted side effects / toxicity. To determine whether the masking polypeptide in the prodrug affects IL-15 cytokine-dependent IFN-γ production, the following study was performed.

[0474] For example, the drug SB1902-C1 or the prodrug SB-1902-C2 was intravenously injected into Balb / c mice at the same molar concentration. Plasma was collected 6 and 24 hours after injection. IFN-γ was detected using an ELISA kit (ThermoFisherScientific, catalog number KMC4021C) according to the manufacturer's instructions.

[0475] As shown in Figure 12, mice treated with the exemplary prodrug SB1902-C2 produced less IFNγ compared to the drug SB1902-C1, which does not contain a masking polypeptide. The results indicate that the reduced IFNγ production by IL-15 is due to inhibition of IL-15 function by the masking polypeptide in the prodrug. This may mean that the use of SB1902-C2 in vivo reduces systemic immune responses.

[0476] Example 9: In vivo tumor model for evaluating prodrug and drug activity The ability of IL-15 prodrugs and drugs without the masking polypeptide to promote tumor elimination and inhibit metastasis was evaluated in vivo using a murine WEHI-164 tumor model. The human IgG1 subtype antibody MOPC21 (see Hamlyn PH, Gait MJ, Milstein C. (1981) Complete sequence of an immunoglobulin mRNA using specific priming and the thedideoxynucleotide method of RNA sequencing. Nucleic Acids Res. 9(18):4485-4494) was used as a control in this experiment.

[0477] A. In vivo activity of prodrugs in the WEHI-164 subcutaneous tumor model Animals and care This study used female mice aged 7–9 weeks. They were fed irradiated Harlan 2918.15 rat chow and had free access to water. For easy identification, the animals' ears were marked, and the left dorsal area was shaved in preparation for cell transplantation. Animals were housed in Innovive disposable ventilated cages with corncob bedding and 60 complete air changes per hour. The environment was controlled at 70°±2°F and humidity between 30–70%. All procedures in this experiment were performed by technicians, and the operation complied with all laws, regulations, and guidelines of the National Institutes of Health (NIH) and was approved by Biomere's Animal Care and Use Committee (Richmond, CA).

[0478] Cell production and transplantationWEHI-164 cells were obtained from ATCC (CAT#: CRL-1751™). WEHI-164 cells were cultured and expanded in Dulbecco's modified Eagle's medium (DMEM) containing 2 mM L-glutamine, 10% fetal bovine serum (FBS), and 1% 100x penicillin / streptomycin (PS). The incubator was maintained at 37°C with 5% CO2. Upon completion of expansion, cells were trypsinized using 0.25% trypsin-EDTA solution, washed, and counted. Cell viability was >95% before transplantation. Cells were resuspended in Dulbecco's phosphate-buffered saline (DPBS). The transplantation site of the experimental animals was disinfected with alcohol swabs, and 0.1 mL of the medium was subcutaneously implanted on day 0 using a 25-gauge needle and a 1 mL syringe.

[0479] Measurement and Processing : Tumor 70~150mm 3 After the mice reached a tumor mass of 100 mg / kg, they were randomly assigned to groups. The mice were assigned to groups to ensure that the average weight of mice in all groups was within 10% of the overall average tumor burden of the study population. The mice were intravenously injected with human IgG1 subtype antibody or the prodrug SB1902-C2 at a dose of 3 mg / kg twice a week for two weeks, and tumor volume was monitored.

[0480] Evaluation of side effects All animals were observed at least daily for clinical signs or toxicity. Animals were weighed weekly. Individual animals were weighed daily if they showed obvious signs of distress or had lost 15% of their body weight. Animals were euthanized if they lost more than 20% of their body weight or showed other clinical signs requiring euthanasia. If an individual animal's tumor volume reached 2000 mm 3 If the serotonin level reached or exceeded the threshold, the animals were euthanized.

[0481] resultAs shown in Figures 13A-13B, WEHI-164 tumor animals were treated with a human IgG1 subtype control antibody or the exemplary prodrug SB1902-C2. Throughout the treatment process, none of the animals in the study showed any obvious signs of systemic toxicity. Tumor growth was significantly inhibited in animals treated with SB1902-C2 (Figure 13B) compared to animals treated with the subtype control antibody (Figure 13A). These results indicate that the prodrug can play a role in tumor-inducing mice, with the masking polypeptide being cleaved in situ and IL-15 being released in the tumor microenvironment.

[0482] B. In vivo activity of prodrugs with various cleavable fragments (CM) in the WEHI-164 subcutaneous tumor model The procedure for detecting the activity of different prodrug constructs with different cleavable moieties in the WEHI-164 tumor mouse model was the same as described above. In this experiment, exemplary prodrugs SB1902-C2, SB1902-C3, and SB1902-C5, drugs SB1902-C1, and SB1902-C4 without a cleavable moiety (CM) were tested. In this experiment, the human IgG1 subtype antibody MOPC21 (see Hamlyn PH, Gait MJ, Milstein C. (1981) Complete sequence of an immunoglobulin mRNA using specific priming and thedideoxynucleotide method of RNA sequencing. Nucleic Acids Res. 9(18):4485-4494) was used as a control. Except for the drug SB1902-C1 group, which was administered at a dose of 0.3 mg / kg (according to previous administration studies, this dose was the MTD), the prodrugs in the other groups were administered intravenously at a dose of 1 mg / kg. The administration times were days 0, 4, 7, 10, and 14. Tumor volume was monitored.

[0483] resultAs shown in Figures 13C-13H, there was no significant difference in tumor size between the subtype control antibody group (Figure 13C) and the SB1902-C4 group (Figure 13D), indicating that the IL-15 in SB1902-C4 plays no role because the non-cleavable linker in SB1902-C4 is not cleaved by enzymes in the tumor, and therefore the masking peptide is not removed from the conjugate. However, tumor growth was clearly reduced in the SB1902-C2 group (Figure 13F), the SB1902-C3 group (Figure 13H), and the SB1902-C5 group (Figure 13G), with no significant difference between the groups (except for one outlier in the SB1902-C5 group). These results strongly suggest that in these experimental groups, the cleavable moiety is cleaved in the tumor microenvironment, activating the IL-15 prodrug and inhibiting tumor cells by the activated IL-15 prodrug. Furthermore, tumor growth in the SB1902-C1 group (Figure ​(Figure13E)​)​ was only partially inhibited, indicating that the maximum tolerated IL-15 dose (MTD) was not sufficient to achieve optimal antitumor efficacy. Therefore, these results demonstrate that the masking polypeptide not only reduces the toxicity of IL-15, but also that the CM in the prodrug is cleavable at the expected location in the tumor microenvironment.

[0484] Example 10: Construction and Expression of Masked Anti-TNFR2 Antibody Prodrugs As previously described, the role of masking polypeptides in cytokine prodrugs has been demonstrated. In the following experiments, the role of masking polypeptides in antibody prodrugs was determined, for example, to test whether masking polypeptides can block antibody binding to antigen.

[0485] The anti-TNFR2 antibody SB1901-72 provided herein is described in US Patent Application No. 63 / 219,796, the entire contents of which are incorporated herein by reference.

[0486] In the case of the masked antibody construct, the masking polypeptide is linked to the N-terminus of the SB1901-72 heavy chain via a cleavable moiety (CM) and is designated as Pepbody-SB1901-H. The masking polypeptide is linked to the N-terminus of the SB1901-72 light chain via a cleavable moiety (CM) and is designated as Pepbody-SB1901-L. The masking polypeptide is simultaneously linked to the N-terminus of the SB1901-72 heavy chain and the SB1901-72 light chain via a cleavable moiety (CM) and is designated as SB1901-HL. The antibody prodrug construct was recombinantly expressed in HEK293 cells and purified as described in Example 2. The CDR sequences of the SB1901-72 antibody are shown in Table 9. CDR numbering is based on the EU numbering system of Kabat. The V of the SB1901-72 antibody H Arrays and V L The sequences are shown in Table 10. The masked anti-TNFR2 antibody prodrug sequences are shown in Table 12. The structural schematic of the anti-TNFR2 antibody is shown in Figure 14A, and the structural schematic of the masked antibody prodrugs: Pepbody-SB1901-H, Pepbody-SB1901-L, and Pepbody-SB1901-HL are shown in Figures 14B-14D.

[0487] [Table 11]

[0488] [Table 12]

[0489] [Table 13]

[0490] [Table 14] TIFF2025527215000023.tif230166

[0491] Example 11: Masked antibody prodrugs reduce antibody affinity and function A. Effect of masking peptide on antibody binding: To evaluate the ability of the masking polypeptide within the antibody prodrug to block or inhibit anti-TNFR2 antibody binding, a binding ELISA assay was performed. Plates were coated with 50 μL / well of 1 μg / mL human TNFR2 and incubated overnight at 4°C. After incubation, 200 μL of blocking buffer (PBS containing 1% BSA) was added and incubated at room temperature for 60 min. After washing the plate, 50 μL of serially diluted Pepbody-SB1901-H, Pepbody-SB1901-L, Pepbody-SB1901-HL, or SB1901-72 (prepared anti-TNFR2 antibodies) was added to each well and incubated for 120 min at 37°C. After washing the plate, anti-musFC-AP conjugated antibodies were diluted 1:2000 in PBS containing 1% BSA, added to the wells, and incubated for 90 min at 37°C. After washing the plate, 50 μL of freshly prepared substrate (pNPP in Tris buffer) was added to each well. The absorbance at 405 nm was obtained with a microplate reader (BioTec, USA).

[0492] As shown in Figure 15A, compared to the SB1901-72 antibody, the prodrugs Pepbody-SB1901-H, Pepbody-SB1901-L, and Pepbody-SB1901-2-HL all significantly reduced binding to TNFR2, indicating that the masking polypeptide can also block the binding of the antibody SB1901-72 to its target TNFR2, demonstrating that the masking polypeptide can be used in the form of an antibody to block or reduce the binding of the antibody to its target.

[0493] B. Effect of masking polypeptide on TNFR2 antibody (SB1901-72) in human primary Treg cell proliferation assay: TNFR2 plays an important role in the proliferation of human primary Treg cells. Blocking the TNFR2 signaling pathway with anti-TNFR2 antibodies can inhibit TNFα-induced Treg cell proliferation. This study was used to test whether a masking peptide could block the effect of the anti-TNFR2 antibody SB1901-72.

[0494] Human PBMCs were incubated with 200 U / mL IL-2 (Peprotech, Catalog No. 200-02) and 20 ng / mL TNFα (Peprotech, Catalog No. 300-01A) in the presence or absence of SB1901-72 or Pepbody-SB190-H. The incubation was performed in complete RPMI medium in 96-well plates at 37°C for 72 hours. Cells were stained with fluorescently conjugated anti-CD3 antibody (BD Biosciences, Catalog No. 557705) and anti-CD4 antibody (Biolegend, Catalog No. 317424) in FACS buffer, followed by fixation / permeabilization and intracellular staining with fluorescently conjugated anti-Foxp3 antibody (FisherScientific, Catalog No. 50-151-75). Cells were fixed with 2% PFA and analyzed by flow cytometry. The percentage of Foxp3+ cells among CD4+ cells was analyzed using FlowJo software.

[0495] As shown in Figure 15B, Pepbody-SB1901-H reduced the inhibitory effect of anti-TNFR2 antibody SB1901-72 on Treg cell proliferation to some extent. This data further demonstrates that masking polypeptides can be used in antibody formats to block or reduce antibody function.

Claims

1. A synthetic masking polypeptide (MP) consisting of four or five types of amino acid residues, said amino acid residues being selected from the group consisting of proline (P), alanine (A), serine (S), glycine (G), and glutamic acid (E).

2. The masking polypeptide (MP) of claim 1, wherein the masking polypeptide consists of five types of amino acids: G, S, P, E, and A; further wherein the percentage of G amino acid residues in the masking polypeptide is about 15% to 30%, preferably 20%; the percentage of S amino acid residues in the masking polypeptide is about 20% to 40%, preferably 40%; the percentage of P amino acid residues in the masking polypeptide is about 15% to 40%, preferably 20%; the percentage of E amino acid residues in the masking polypeptide is about 1% to 20%, preferably 10%; and the percentage of A amino acid residues in the masking polypeptide is about 5% to 20%, preferably 10%; and the number of amino acids, if not an integer, takes an integer value.

3. The masking polypeptide (MP) of claim 1, wherein the masking polypeptide consists of four types of amino acids: S, P, E, and G; further wherein the percentage of amino acid residues S in the masking polypeptide is about 20% to 40%, preferably 23%, the percentage of amino acid residues P in the masking polypeptide is about 15% to 40%, preferably 29%, the percentage of amino acid residues E in the masking polypeptide is about 1% to 20%, preferably 18%, and the percentage of amino acid residues G in the masking polypeptide is about 15% to 30%, preferably 30%, and the number of amino acids, if not an integer, takes an integer value.

4. A masking polypeptide (MP) according to any one of claims 1 to 3, wherein said masking polypeptide comprises approximately 40 to 720 amino acid residues, preferably 80 to 320 amino acid residues, most preferably 80 to 240 amino acid residues.

5. The masking polypeptide is (i) forming a random coil lacking secondary structure; (ii) appears to be a molecule larger than expected molecular size as detected by size exclusion chromatography; (iii) does not induce immunogenicity in the host or bind nonspecifically to serum proteins; and / or (iv) A masking polypeptide according to any one of claims 1 to 4, which is stable in a buffer or in plasma.

6. A masking polypeptide according to any one of claims 1 to 5, comprising the amino acid sequence SEQ ID NO:

6.

7. A masking polypeptide according to any one of claims 1 to 6, comprising the amino acid sequence SEQ ID NO:

1.

8. 8. The masking polypeptide according to any one of claims 1 to 7, comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 5, or a variant thereof having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 5, or a variant thereof comprising one or more amino acid substitutions, additions and / or deletions.

9. Amino acid sequence MVX １ X ２ AX ３ TX ４ SG (SEQ ID NO: 49), with the proviso that X １ is selected from P, L, V or A; X ２ is selected from L or S, and X ３ is selected from L, V, P or Y, and X ４ is a cleavable moiety (CM) selected from A or V.

10. 10. The cleavable moiety (CM) of claim 9, comprising a substrate sequence for urokinase plasminogen activator (uPA), matrix metalloproteinase (MMP) 1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, fibroblast activation protein (FAP), matrix proteases, cathepsins, caspases, thrombin, metalloproteinases, serine proteases, cysteine proteases, aspartic acid proteases, legumain, kallikrein, cathepsin A, cathepsin B, chymase, proteases located at the tumor site or its surrounding environment, or any combination thereof.

11. 11. The cleavable moiety (CM) of claim 9 or 10, comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 8 to 16, or a variant thereof having at least 90% sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 8 to 16.

12. A prodrug comprising: (i) one or more biologically active moieties (B); (ii) one or more cleavable moieties (CM); and (iii) one or more masking polypeptides (MP).

13. The method of claim 12, wherein the masking polypeptide (MP) is selected from the polypeptides of any one of claims 1 to 8.

14. The prodrug of claim 12, wherein the cleavable moiety (CM) is selected from the polypeptides of any one of claims 9 to 11.

15. 15. The prodrug of any one of claims 12 to 14, wherein the masking polypeptide (MP) reduces the activity of the biologically active moiety (B) and the cleavable moiety (CM) is susceptible to cleavage in or near a tumor, target cell.

16. The prodrug of any one of claims 12 to 15, wherein the masking polypeptide (MP) and the biologically active moiety (B) are linked by the cleavable moiety (CM).

17. 17. The prodrug of any one of claims 12 to 16, wherein the prodrug further comprises one or more non-cleavable linkers (L), and optionally, the non-cleavable linker (L) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 17 to 21.

18. 18. The prodrug of any one of claims 12 to 17, wherein the biologically active moiety (B) is selected from a cytokine, an antigen-binding fragment or an antibody, or a small molecule drug having cytotoxic or cytostatic activity against tumor cells.

19. 19. The prodrug of claim 18, wherein the cytokine is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IFNα, IFNβ, IFNγ, TNFα, TNFβ1, TNFβ2, TNFβ3, lymphotoxin, GM-CSF, CXCL10, CCL19, CCL20, CCL21, or mutants of the above cytokines.

20. 19. The prodrug of claim 18, wherein the antigen-binding fragment or antibody specifically binds a tumor-associated antigen, optionally wherein the antigen-binding fragment or antibody specifically binds one or more antigens, wherein the antigens are selected from the group consisting of TNFR2, CTLA4, PD1, PDL1, LAG3, TIM3, BCMA, HER2, CEA, EGFR, VEGFR1, and VEGFR2.

21. The prodrug of any one of claims 12 to 20, further comprising one or more half-life extending moieties (C).

22. The half-life extending moiety (C) is (i) a serum protein or a molecule that binds to a serum protein, optionally wherein the serum protein is selected from the group consisting of fibronectin, transferrin, and human serum albumin (HSA); or (ii) a biocompatible polymer, optionally selected from PEG or hydroxyethyl starch; or (iii) The prodrug of claim 21, comprising an Fc domain, or an antibody containing an Fc domain, or a fragment involved in FcRn-mediated recycling.

23. (i) said biologically active moiety (B) is linked to said half-life extending moiety (C), or (ii) The prodrug of claim 21 or 22, wherein said masking polypeptide (MP) and said half-life extending moiety (C) are linked by said cleavable moiety (CM).

24. An antibody prodrug comprising: (i) one or more antibodies or antigen-binding fragments; (ii) one or more cleavable moieties (CM); and (iii) one or more masking polypeptides (MP).

25. The antibody prodrug of claim 24, wherein the masking polypeptide (MP) is selected from the polypeptides of any one of claims 1 to 8.

26. 25. The antibody prodrug of claim 24, wherein the cleavable moiety (CM) is selected from the polypeptides of any one of claims 9 to 11.

27. 27. The antibody prodrug of claim 26, wherein the antibody or antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)'2, Fab'-SH, single-chain Fv (scFv), Fv fragment, dAb, Fd, VHH, or diabody.

28. The masking polypeptide (MP) and V Ｈ Domains and / or V Ｌ The antibody prodrug of any one of claims 24 to 27, wherein the N-terminus and / or C-terminus of the domain are linked by the cleavable moiety (CM).

29. 25. The antibody prodrug of claim 24, wherein the antibody or antigen-binding fragment specifically binds TNFR2.

30. The V Ｈ a HC-CDR1 comprising the amino acid sequence SEQ ID NO:50, a HC-CDR2 comprising the amino acid sequence SEQ ID NO:51, and a HC-CDR3 comprising the amino acid sequence SEQ ID NO:52, or any of the above Vs comprising at most about five amino acid substitutions in the HC-CDRs. Ｈ and variants of said V Ｌ a LC-CDR1 comprising the amino acid sequence SEQ ID NO:53, a LC-CDR2 comprising the amino acid sequence SEQ ID NO:54, and a LC-CDR3 comprising the amino acid sequence SEQ ID NO:55, or any of the above Vs comprising at most about five amino acid substitutions in the LC-CDRs. Ｌ 30. The antibody prodrug of any one of claims 24 to 29, comprising a variant of:

31. The V Ｈ comprises the amino acid sequence SEQ ID NO:56 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence SEQ ID NO:56, and Ｌ The antibody prodrug of any one of claims 24 to 30, wherein the antibody prodrug comprises the amino acid sequence SEQ ID NO: 57 or a variant thereof, wherein the variant has at least about 90% sequence identity to the amino acid sequence SEQ ID NO:

57.

32. the antibody prodrug comprises two heavy chains and two light chains, the heavy chains comprising the amino acid sequence SEQ ID NO:58 or SEQ ID NO:60, or a variant thereof, the variant having at least 80% sequence identity to the amino acid sequence SEQ ID NO:58 or SEQ ID NO:60; and 32. The antibody prodrug of any one of claims 24 to 31, wherein the light chain comprises the amino acid sequence SEQ ID NO:59 or SEQ ID NO:61, or a variant thereof, wherein the variant has at least 80% sequence identity to the amino acid sequence SEQ ID NO:59 or SEQ ID NO:

61.

33. An isolated nucleic acid molecule encoding a masking polypeptide (MP) according to any one of claims 1 to 8, or a cleavable moiety (CM) according to any one of claims 9 to 11, or a prodrug according to any one of claims 12 to 32.

34. 34. A vector comprising the isolated nucleic acid molecule of claim 33.

35. 35. An isolated host cell comprising a masking polypeptide (MP) according to any one of claims 1 to 8, a cleavable moiety (CM) according to any one of claims 9 to 11, a prodrug according to any one of claims 12 to 32, a nucleic acid molecule according to claim 33, or a vector according to claim 34.

36. a) culturing the host cell of claim 35 under conditions that allow effective expression of the masking polypeptide (MP), cleavable moiety (CM) or prodrug; b) obtaining the expressed masking polypeptide (MP), cleavable moiety (CM) or prodrug from the host cell.

37. 37. A pharmaceutical composition comprising a masking polypeptide (MP) according to any one of claims 1 to 8, a cleavable moiety (CM) according to any one of claims 9 to 11, a prodrug according to any one of claims 12 to 32, a nucleic acid according to claim 33, or a vector according to claim 34, an isolated host cell according to claim 35, or a masking polypeptide (MP), cleavable moiety (CM) or prodrug produced by the method of claim 36, and a pharmaceutically acceptable carrier.

38. 1. A method of treating a disease or condition in an individual in need thereof, comprising:

38. A method comprising administering to an individual an effective amount of the prodrug of any one of claims 12 to 32, the nucleic acid of claim 33, the vector of claim 34, the isolated host cell of claim 35, the prodrug produced by the method of claim 36, or the pharmaceutical composition of claim 37.

39. 39. The method of claim 38, wherein the disease or condition is cancer or an infectious disease, and optionally, the disease or condition is associated with TNFR2 signaling or aberrant TNFR2 expression.

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