IL-15 prodrugs and uses thereof

Abstract

The present application provides IL-15 prodrugs comprising a masking polypeptide (MP) and a cleavable moiety (CM). An isolated nucleic acid molecule encoding the IL-15 prodrug; a carrier comprising the nucleic acid molecule; a host cell comprising the nucleic acid molecule or the vector; and a pharmaceutical composition comprising the IL-15 prodrug, the isolated nucleic acid molecule, the carrier, or the host cell. And methods of making and using the IL-15 prodrugs or pharmaceutical compositions.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Application 63 / 370,605, filed on August 5, 2022; U.S. Provisional Application 63 / 370,606, filed on August 5, 2022; U.S. Provisional Application 63 / 370,607, filed on August 5, 2022; and U.S. Provisional Application 63 / 477,993, filed on December 30, 2022, the content of each of which is incorporated herein by reference in its entirety.

[0003] Submission of sequence listing

[0004] The content of the electronic sequence listing (IL - 15 prodrug and its use SEQ.xml; size: 106KB; and recording date: 2023.07.28) is incorporated herein by reference in its entirety. Technical field

[0005] The present invention relates to IL - 15 prodrugs and methods for their preparation and use. The present invention also relates to the cleavage products of the activatable IL - 15 prodrugs and methods for their use. Background art

[0006] Cytokines are potent immunostimulants and are thus considered very promising anti - tumor therapeutic agents. For example, the anti - tumor activity of interleukin - 15 (IL - 15) is currently being studied and has been used in human therapy. However, cytokines have a narrow therapeutic window and a short serum half - life. Thus, therapeutic administration of cytokines results in undesirable systemic effects and toxicity. The above problems are exacerbated because large amounts of cytokines need to be administered at the intended site of action of the cytokine (e.g., the tumor) to achieve the desired cytokine levels.

[0007] Antibodies are regarded as ideal candidates for the treatment of cancer, autoimmune diseases, and chronic inflammatory diseases, but sometimes antibody therapy is limited by its cross - reactivity with healthy tissues. A variety of methods have been described to improve tumor targeting by engineering antibodies to overcome these "off - target" effects, e.g., by preparing masking antibodies that are selectively activated in the tumor microenvironment (see WO2003 / 068934, WO2004 / 009638, WO2009 / 025846, WO2101 / 081173, and WO2014 / 103973).

[0008] This application relates to the use of conditionally activatable prodrugs (e.g., cytokine prodrugs and antibody prodrugs) having a cleavable moiety linked to a masking polypeptide (MP) for treating cancer or other diseases. The masking polypeptide (MP) can exert a steric hindrance effect on the bioactive moiety. The cleavable moiety can be designed to be cleaved by proteases in a specific tissue or diseased tissue, so that the prodrug can be preferentially activated at the desired site (e.g., tumor) to overcome the dose limitation of cytokines or the "off-target" effect of antibodies.

[0009] Summary of the Application

[0010] This application provides an IL-15 prodrug, wherein the IL-15 prodrug comprises: (i) one or more IL-15 cytokines (I), (ii) one or more cleavable moieties (CM), and (iii) one or more masking polypeptides (MP).

[0011] In some embodiments, the IL-15 prodrug provided herein, wherein the masking polypeptide (MP) attenuates the activity of the IL-15 cytokine (I), and the cleavable moiety (CM) is readily cleaved at or near the tumor or target cell.

[0012] In some embodiments, the IL-15 prodrug provided herein, wherein the IL-15 cytokine (I) and the masking polypeptide (MP) are linked by the cleavable moiety (CM).

[0013] In some embodiments, the IL-15 prodrug provided herein, wherein the IL-15 prodrug further comprises IL-15Rα or a functional fragment (S) thereof, and wherein the IL-15Rα or a functional fragment thereof is selected from the extracellular domain of IL-15Rα or the sushi domain or a functional analogue.

[0014] In some embodiments, the IL-15 prodrug provided herein, wherein the IL-15Rα or a functional fragment (S) thereof and the masking polypeptide (MP) are linked by the cleavable moiety (CM).

[0015] In some embodiments, the IL-15 prodrug provided herein, wherein the IL-15 prodrug further comprises one or more half-life extending moieties (C).

[0016] In some embodiments, the IL-15 prodrug provided herein, wherein the IL-15 cytokine (I) and the half-life extending moiety (C) are linked.

[0017] In some embodiments, the IL-15 prodrug provided herein, wherein the IL-15Rα or a functional fragment (S) thereof and the half-life extending moiety (C) are linked.

[0018] In some embodiments, provided herein is an IL-15 prodrug, wherein the masking polypeptide (MP) and the half-life extending moiety (C) are linked by the cleavable moiety (CM).

[0019] In some embodiments, provided herein is an IL-15 prodrug, wherein 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 the cleavable moiety (CM).

[0020] In some embodiments, provided herein is an IL-15 prodrug, wherein 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 the cleavable moiety (CM).

[0021] In some embodiments, provided herein is an IL-15 prodrug, wherein the IL-15Rα or a functional fragment thereof (S) and the half-life extending moiety (C) are linked, the IL-15 cytokine (I) and the IL-15Rα or a functional fragment thereof (S) are linked, and the masking polypeptide (MP) and the IL-15 cytokine (I) are linked by the cleavable moiety (CM).

[0022] In some embodiments, provided herein is an IL-15 prodrug, wherein 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-15Rα or a functional fragment thereof (S) are linked by the cleavable moiety (CM).

[0023] In some embodiments, provided herein is an IL-15 prodrug, wherein the masking polypeptide (MP) and the half-life extending moiety (C) are linked by the cleavable moiety (CM), the IL-15Rα or a functional fragment thereof (S) and the half-life extending moiety (C) are linked, and the IL-15 cytokine (I) and the IL-15Rα or a functional fragment thereof (S) are linked.

[0024] In some embodiments, the IL-15 prodrugs provided herein, wherein the masking polypeptide (MP) and the half-life extension moiety (C) are linked by the cleavable moiety (CM), the IL-15 cytokine (I) is linked to the half-life extension moiety (C), and the IL-15Rα or its functional fragment (S) is linked to the IL-15 cytokine (I).

[0025] In some embodiments, the IL-15 prodrugs provided herein are monomers or dimers.

[0026] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises a first half-life extension moiety (C), the IL-15Rα or its functional fragment (S), the IL-15 cytokine (I); and the other monomer comprises a second half-life extension moiety (C), the masking polypeptide (MP) and the cleavable moiety (CM), wherein the masking polypeptide (MP) and the second half-life extension moiety (C) are linked by the cleavable moiety (CM)

[0027] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises a first half-life extension moiety (C), the IL-15Rα or its functional fragment (S), the masking polypeptide (MP) and the cleavable moiety (CM); and the other monomer comprises a second half-life extension moiety (C) and the IL-15 cytokine (I).

[0028] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises a first half-life extension moiety (C), the IL-15 cytokine (I), the masking polypeptide (MP) and the cleavable moiety (CM); and the other monomer comprises a second half-life extension moiety (C) and the IL-15Rα or its functional fragment (S).

[0029] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer and the other monomer each comprise the half-life extension moiety (C), the IL-15 cytokine (I), the IL-15Rα or its functional fragment (S), the cleavable moiety (CM) and the masking polypeptide (MP).

[0030] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein in one monomer: both the IL-15 cytokine (I) and the IL-15Rα or its functional fragment (S) are linked to a first half-life extension moiety (C); and in the other monomer: the masking polypeptide (MP) and a second half-life extension moiety (C) are linked by the cleavable moiety (CM).

[0031] In some embodiments, the IL-15 prodrug provided herein comprises two monomers, wherein in one monomer: the IL-15 cytokine (I) is linked to a first half-life extending moiety (C), and the IL-15Rα or a functional fragment thereof (S) is linked to the IL-15 cytokine (I); and in the other monomer: the masking polypeptide (MP) is linked to a second half-life extending moiety (C) via the cleavable moiety (CM).

[0032] In some embodiments, the IL-15 prodrug provided herein comprises two monomers, wherein in one monomer: the IL-15Rα or a functional fragment thereof (S) is linked to a first half-life extending moiety (C), and the IL-15 cytokine (I) is linked to the IL-15Rα or a functional fragment thereof (S); and in the other monomer: the masking polypeptide (MP) is linked to a second half-life extending moiety (C) via the cleavable moiety (CM).

[0033] In some embodiments, the IL-15 prodrug provided herein comprises two monomers, wherein in one monomer: the IL-15Rα or a functional fragment thereof (S) is linked to a first half-life extending moiety (C), and the masking polypeptide (MP) is linked to the first half-life extending moiety (C) via the cleavable moiety (CM); and in the other monomer: the IL-15 cytokine (I) is linked to a second half-life extending moiety (C).

[0034] In some embodiments, the IL-15 prodrug provided herein comprises two monomers, wherein in one monomer: the IL-15Rα or a functional fragment thereof (S) is linked to a first half-life extending moiety (C), and the masking polypeptide (MP) is linked to the IL-15Rα or a functional fragment thereof (S) via the cleavable moiety (CM); and in the other monomer: the IL-15 cytokine (I) is linked to a second half-life extending moiety (C).

[0035] In some embodiments, the IL-15 prodrug provided herein comprises two monomers, wherein in one monomer: the IL-15 cytokine (I) is linked to a first half-life extending moiety (C), and the masking polypeptide (MP) is linked to the first half-life extending moiety (C) via the cleavable moiety (CM); and in the other monomer: the IL-15Rα or a functional fragment thereof (S) is linked to a second half-life extending moiety (C).

[0036] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein in one monomer: the IL-15 cytokine (I) is linked to a first half-life extending moiety (C), and the masking polypeptide (MP) and the IL-15 cytokine (I) are linked through the cleavable moiety (CM); and in the other monomer: the IL-15Rα or its functional fragment (S) is linked to a second half-life extending moiety (C).

[0037] In some embodiments, the IL-15 prodrugs provided herein, wherein the IL-15Rα or its functional fragment (S) is covalently linked to the IL-15 cytokine (I); or the IL-15Rα or its functional fragment (S) is non-covalently linked to the IL-15 cytokine (I) and forms an IL-15 / IL-15Rα complex.

[0038] In some embodiments, the IL-15 prodrugs provided herein, wherein the IL-15 cytokine (I) comprises one or more amino acid mutations selected from the group consisting of L45D, L45E, Q48K, S51D, L52D, E64K, I67D, I67E, I68D, and N72D.

[0039] In some embodiments, the IL-15 prodrugs provided herein, wherein the IL-15 cytokine (I) comprises the amino acid sequence shown in any of SEQ ID NOs: 22-23 and 67-76, or a variant thereof, and the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 22-23 and 67-76.

[0040] In some embodiments, the IL-15 prodrugs provided herein, wherein the IL-15Rα or its functional fragment (S) comprises the amino acid sequence shown in any of SEQ ID NOs: 24-26, or a variant thereof, and the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 24-26.

[0041] In some embodiments, the IL-15 prodrugs provided herein, wherein the half-life extension moiety (C) comprises an Fc domain; preferably, the 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, the Fc domain is a human IgG1 Fc domain.

[0042] In some embodiments, the IL-15 prodrugs provided herein, wherein the Fc domain is a human IgG1 Fc domain having L234A and L235A mutations, and the mutation sites are numbered according to the EU numbering system.

[0043] In some embodiments, the IL-15 prodrugs provided herein, wherein the Fc domain comprises knobs-into-holes mutations (Fc knob and Fc hole).

[0044] In some embodiments, the IL-15 prodrugs provided herein, wherein the Fc knob comprises a T366W mutation in the Fc domain, and the Fc hole comprises T366S, L368A, and Y407V mutations in the Fc domain, and the mutation sites are numbered according to the EU numbering system.

[0045] In some embodiments, the IL-15 prodrugs provided herein, wherein the Fc knob further comprises an S354C mutation, and the Fc hole further comprises a Y349C mutation, and the mutation sites are numbered according to the EU numbering system.

[0046] In some embodiments, the IL-15 prodrugs provided herein, wherein the masking polypeptide (MP) consists 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).

[0047] In some embodiments, the IL-15 prodrugs provided herein, wherein the masking polypeptide (MP) consists of five amino acids G, S, P, E, and A; further, in the masking polypeptide, the percentage of amino acid residue G is about 15%-30%, preferably 20%; in the masking polypeptide, the percentage of amino acid residue S is about 20%-40%, preferably 40%; in the masking polypeptide, the percentage of amino acid residue P is about 15%-40%, preferably 20%; in the masking polypeptide, the percentage of amino acid residue E is about 1%-20%, preferably 10%; in the masking polypeptide, the percentage of amino acid residue A is about 5%-20%, preferably 10%; when the number of amino acids is not an integer, take the integer value.

[0048] In some embodiments, the IL-15 prodrugs provided herein, wherein the masking polypeptide (MP) consists of four amino acids S, P, E, and G; further, in the masking polypeptide, the percentage of amino acid residue S is about 20%-40%, preferably 23%; in the masking polypeptide, the percentage of amino acid residue P is about 15%-40%, preferably 29%; in the masking polypeptide, the percentage of amino acid residue E is about 1%-20%, preferably 18%; in the masking polypeptide, the percentage of amino acid residue G is about 15%-30%, preferably 30%; when the number of amino acids is not an integer, take the integer value.

[0049] In some embodiments, the IL-15 prodrugs provided herein, wherein the masking polypeptide (MP) contains about 40 to 720 amino acid residues; preferably, it contains 80 to 320 amino acid residues; more preferably, it contains 80 to 240 amino acid residues.

[0050] In some embodiments, the IL-15 prodrugs provided herein, wherein the masking polypeptide (MP) contains the amino acid sequence SEQ ID NO:6.

[0051] In some embodiments, the IL-15 prodrugs provided herein, wherein the masking polypeptide (MP) contains the amino acid sequence SEQ ID NO:1.

[0052] In some embodiments, the IL-15 prodrugs provided herein, wherein the masking polypeptide (MP) contains the amino acid sequence shown in any one of SEQ ID NOs: 1-5; or a variant thereof, the variant having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1-5; or a variant thereof, the variant containing one or more amino acid substitutions, additions, and / or deletions.

[0053] In some embodiments, the IL-15 prodrugs provided herein, wherein the cleavable moiety (CM) comprises the amino acid sequence MVX 1 X 2 AX 3 TX 4 SG (SEQ ID NO:49), wherein X 1 is selected from P, L, V or A, X 2 is selected from L or S, X 3 is selected from L, V, P or Y, and X 4 is selected from A or V.

[0054] In some embodiments, the IL-15 prodrugs provided herein, wherein the cleavable moiety (CM) comprises a substrate sequence of urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP) 1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, fibroblast activation protein (FAP), matriptase, cathepsin, caspase, thrombin, metalloprotease, serine protease, cysteine protease, aspartic protease, legumain, kallikrein, cathepsin A, cathepsin B, chymotrypsin, a protease located at the tumor site or its surrounding environment, or any combination thereof.

[0055] In some embodiments, the IL-15 prodrugs provided herein, wherein the cleavable moiety (CM) comprises an amino acid sequence shown in any of SEQ ID NOs: 8-16, or a variant thereof, the variant having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with an amino acid sequence shown in any of SEQ ID NOs: 8-16.

[0056] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein, in one monomer, the IL-15Rα or its functional fragment(s) is linked to the first Fc domain, and, in the other monomer, the IL-15 cytokine (I) is linked to the second Fc domain, and the masking polypeptide (MP) and the IL-15 cytokine (I) are linked by the cleavable moiety (CM).

[0057] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO: 33 or a variant thereof, the variant having 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: 33; and the other monomer comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, the variant having 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: 32.

[0058] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO: 34 or a variant thereof, the variant having 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: 34; and the other monomer comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, the variant having 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: 32.

[0059] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO: 36 or a variant thereof, the variant having 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: 36; and the other monomer comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, the variant having 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: 32.

[0060] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO: 37 or a variant thereof, the variant having 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: 37; and the other monomer comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, the variant having 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: 32.

[0061] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO: 38 or a variant thereof, the variant having 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: 38; and the other monomer comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, the variant having 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: 32.

[0062] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO: 39 or a variant thereof, the variant having 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: 39; and the other monomer comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, the variant having 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: 32.

[0063] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO: 45 or a variant thereof, the variant having 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: 45; and the other monomer comprises the amino acid sequence SEQ ID NO: 46 or a variant thereof, the variant having 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: 46.

[0064] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO: 47 or a variant thereof, the variant having 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: 47; and the other monomer comprises the amino acid sequence SEQ ID NO: 40 or a variant thereof, the variant having 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: 40.

[0065] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO: 33 or a variant thereof, the variant having 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: 33; and the other monomer comprises the amino acid sequence SEQ ID NO: 40 or a variant thereof, the variant having 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: 40.

[0066] In some embodiments, the IL-15 prodrug provided herein comprises two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO:62 or a variant thereof, the variant having 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:62; and the other monomer comprises the amino acid sequence SEQ ID NO:63 or a variant thereof, the variant having 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:63.

[0067] In some embodiments, the IL-15 prodrug provided herein comprises two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO:41 or a variant thereof, the variant having 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:41; and the other monomer comprises the amino acid sequence SEQ ID NO:63 or a variant thereof, the variant having 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:63.

[0068] In some embodiments, the IL-15 prodrug provided herein comprises two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO:64 or a variant thereof, the variant having 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:64; and the other monomer comprises the amino acid sequence SEQ ID NO:65 or a variant thereof, the variant having 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:65.

[0069] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises the amino acid sequence of SEQ ID NO: 62 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 62; and the other monomer comprises the amino acid sequence of SEQ ID NO: 65 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 65.

[0070] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41; and the other monomer comprises the amino acid sequence of SEQ ID NO: 65 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 65.

[0071] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises the amino acid sequence of SEQ ID NO: 66 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 66; and the other monomer comprises the amino acid sequence of SEQ ID NO: 65 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 65.

[0072] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO: 43 or a variant thereof, the variant having 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: 43; and the other monomer comprises the amino acid sequence SEQ ID NO: 46 or a variant thereof, the variant having 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: 46.

[0073] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO: 39 or a variant thereof, the variant having 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: 39; and the other monomer comprises the amino acid sequence SEQ ID NO: 40 or a variant thereof, the variant having 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: 40.

[0074] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof, the variant having 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: 41; and the other monomer comprises the amino acid sequence SEQ ID NO: 87 or a variant thereof, the variant having 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: 87.

[0075] In some embodiments, the IL-15 prodrug provided herein comprises two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof, the variant having 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: 41; and the other monomer comprises the amino acid sequence SEQ ID NO: 91 or a variant thereof, the variant having 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: 91.

[0076] In some embodiments, the IL-15 prodrug provided herein comprises two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof, the variant having 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: 41; and the other monomer comprises the amino acid sequence SEQ ID NO: 93 or a variant thereof, the variant having 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: 93.

[0077] In some embodiments, the IL-15 prodrug provided herein comprises two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof, the variant having 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: 41; and the other monomer comprises the amino acid sequence SEQ ID NO: 94 or a variant thereof, the variant having 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: 94.

[0078] In some embodiments, the IL-15 drug provided herein comprises two monomers, wherein in one monomer, the IL-15Rα or its functional fragment(s) is linked to the first Fc domain, and in the other monomer, the IL-15 cytokine (I) is linked to the second Fc domain.

[0079] In some embodiments, the IL-15 drugs provided herein comprise two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO: 43 or a variant thereof, and 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: 43; and the other monomer comprises the amino acid sequence SEQ ID NO: 83 or a variant thereof, and 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: 83.

[0080] In some embodiments, the IL-15 drugs provided herein comprise two monomers, wherein one monomer comprises the amino acid sequence SEQ ID NO: 43 or a variant thereof, and 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: 43; and the other monomer comprises the amino acid sequence SEQ ID NO: 84 or a variant thereof, and 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: 84.

[0081] Also provided are isolated nucleic acid molecules encoding any of the IL-15 prodrugs or drugs provided herein, vectors comprising the nucleic acid molecules, host cells (e.g., CHO cells, HEK 293 cells, Hela cells or COS cells) comprising the nucleic acid molecules or the vectors, compositions (e.g., pharmaceutical compositions), kits, and articles of manufacture comprising any of the masking polypeptides, cleavable moieties, prodrugs or drugs provided herein. Also provided are methods of using any of the IL-15 prodrugs or drugs or their pharmaceutical compositions provided herein for treating a disease (e.g., a tumor) in an individual (e.g., a human). Brief Description of the Drawings

[0082] Figure 1A Shown is the sequence alignment of the amino acid sequences MP80 and MP100, and the identical amino acid sequences between MP80 and MP100 are marked in the boxes (the amino acid sequence marked in the first box is SEQ ID NO: 6).

[0083] Figure 1B Shown is the amino acid sequence alignment of MP80 and MP163 repeated twice. The amino acid sequence marked in the first box is the sequence of MP80, i.e., MP163 contains the amino acid sequence of MP80.

[0084] Figure 1C Shown is the amino acid sequence alignment of MP80 and MP240 repeated three times. The amino acid sequence marked in the first box is the sequence of MP80, that is, MP240 contains 3 copies of the amino acid sequence of MP80.

[0085] Figure 2A Shown is an exemplary IL-15 prodrug, which has an Fc domain as a half-life extension moiety. Its IL-15Rα_sushi domain is linked to the C-terminus of one Fc domain, optionally via an uncleavable linker. IL-15 is linked to the C-terminus of another Fc domain, optionally via an uncleavable linker. The masking polypeptide (MP) is linked to IL-15 via a cleavable moiety (CM).

[0086] Figure 2B Shown is an exemplary schematic diagram showing the process of activation of the IL-15 prodrug in target tissues (e.g., tumors with high levels of MMPs) by releasing the masking polypeptide (MP).

[0087] Figure 3 Shown are non-reducing and reducing SDS-PAGE gel electrophoresis diagrams for analyzing the purity of exemplary prodrugs SB1902-C2 and SB1902-C7, and the drug SB1902-C1 (without masking polypeptide).

[0088] Figure 4 Shown are SEC-HPLC result diagrams for analyzing the homogeneity of exemplary prodrug SB1902-C2 and drug SB1902-C1 (without masking polypeptide).

[0089] Figure 5A Shown is a polyacrylamide gel diagram for analyzing an exemplary cleavable moiety in the prodrug SB1902-C2 that is sensitive to MMP2 enzyme.

[0090] Figure 5B Shown is a polyacrylamide gel diagram for analyzing an exemplary cleavable moiety in the prodrug SB1902-C2 that is sensitive to MMP9 enzyme.

[0091] Figure 5C Shown is a polyacrylamide gel diagram for analyzing SB1902-C4 with an uncleavable (G4S)2 linker instead of a cleavable linker, and the uncleavable (G4S)2 linker remains intact after digestion with the enzyme MMP2.

[0092] Figure 5D Shown is a polyacrylamide gel diagram for analyzing SB1902-C4 with an uncleavable (G4S)2 linker instead of a cleavable linker, and the uncleavable (G4S)2 linker remains intact after digestion with the enzyme MMP9.

[0093] Figure 5E Shown is a polyacrylamide gel for analyzing exemplary cleavable moieties in the prodrug SB1902-C5 that is sensitive to the MMP9 enzyme.

[0094] Figure 5F Shown is a polyacrylamide gel for analyzing exemplary cleavable moieties in the prodrug SB1902-C5 that is sensitive to the MMP2 enzyme.

[0095] Figure 6A Shown is that the drug SB1902-C1 (without a masked polypeptide and a cleavable moiety), prodrugs SB1902-C2, SB1902-C3, and SB1902-C4 (without a cleavable moiety) do not non-specifically bind to human serum proteins.

[0096] Figure 6B Shown is that the drug SB1902-C1 (without a masked polypeptide), prodrugs SB1902-C2, SB1902-C3, and SB1902-C4 (without a cleavable moiety) do not non-specifically bind to cynomolgus monkey serum proteins.

[0097] Figure 6C Shown is that the drug SB1902-C1 (without a masked polypeptide), prodrugs SB1902-C2, SB1902-C3, and SB1902-C4 (without a cleavable moiety) do not non-specifically bind to rat serum proteins.

[0098] Figure 6D Shown are the bands of prodrugs SB1902-C2, SB1909-C3, and SB1902-C4 (without a cleavable moiety) on a WB membrane before incubation with plasma or PBS buffer.

[0099] Figure 6E Shown are that prodrugs SB1902-C2, SB1909-C3, and SB1902-C4 (without a cleavable moiety) have no detectable degradation bands on a WB membrane after incubation with human plasma (abbreviated as plas in the figure) or PBS buffer.

[0100] Figure 7A Shown is the binding affinity of the exemplary prodrug SB1902-C2 and the exemplary drug SB1902-C1 to the IL-2 / IL-15Rβγ receptor; Figure 7B-7D Shown is the binding affinity of the exemplary IL-15 drug SB1902-C1-variant3 with wild-type IL-15 and IL-15 variants to the IL-2 / IL-15Rβγ receptor; Figure 7E-7FShown is the binding affinity of the exemplary IL-15 prodrug SB1902-C9-variant2 with wild-type IL-15 and IL-15 variant to the IL-2 / IL-15Rβγ receptor.

[0101] Figure 8 Shown are the results of the immunogenicity detection test in Balbc mice, which indicate that the MP80 masking polypeptide is not immunogenic.

[0102] Figure 9 Shown are the results of the Mo7e cell proliferation assay, which indicate that compared with the drug SB1902-C1, the prodrugs SB1902-C2, SB1902-C6, and SB1902-C7 significantly reduce the function of IL-15 in stimulating Mo7e cell proliferation.

[0103] Figure 10A-10Q Shown are the results of the exemplary IL-15 drug and IL-15 prodrug in the CD8+ T cell activation assay.

[0104] Figure 11 Shown are the results of the exemplary prodrug SB1902-C2 and SB1902-C2 digested by MMP (with its masking polypeptide removed) in the CD8+ T cell activation assay.

[0105] Figure 12A Shown are the results of the IFN-γ production assay in PBMCs, Figure 12B Shown are the results of the granzyme B production assay in PBMCs. Figure 12C and Figure 12D Shown are the results of the IL-15 drug and IL-15 prodrug with wild-type IL-15 and IL-15 variant in the IFN-γ production assay. Figure 12E and Figure 12F Shown are the results of the IL-15 drug and IL-15 prodrug with wild-type IL-15 and IL-15 variant in the granzyme B production assay.

[0106] Figure 13 Shown are the results of the IFN-γ production assay in Balb / c mice, which indicate that compared with the drug SB1902-C1, the mice treated with the prodrug SB1902-C2 produce less IFN-γ.

[0107] Figure 14A-14B Shown is the treatment of animals with WEHI-164 tumors at a dose of 3 mg / kg with the IgG1 subtype control antibody MOPC-21 ( Figure 14A ) or the prodrug SB1902-C2 ( Figure 14B ). Compared with the tumor growth of the animals treated with the control antibody, the tumor growth in the animals treated with SB1902-C2 is significantly inhibited.

[0108] Figure 14C-14H Shown are the anti-tumor activities of different molecules in animals bearing WEHI-164 tumors, which were treated with the IgG1 isotype control antibody MOPC-21( Figure 14C ), SB1902-C4 without a cleavable moiety( Figure 14D ), the drug SB1902-C1( Figure 14E ), the prodrug SB1902-C2( Figure 14F ), the prodrug SB1902-C5( Figure 14G ), or the prodrug SB1902-C3( Figure 14H ) on days 0, 4, 7, 10, and 14. All 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.

[0109] Figure 15A Shown is a schematic structural diagram of the anti-TNFR2 antibody. Figure 15B-15D Shown are schematic structural diagrams of the masked antibody prodrugs Pepbody-SB1901-H, Pepbody-SB1901-L, and Pepbody-SB1901-HL, respectively.

[0110] Figure 16A Shown are the antigen-binding results of exemplary anti-TNFR2 antibody prodrugs and anti-TNFR2 antibodies.

[0111] Figure 16B Shown are the results of the exemplary anti-TNFR2 antibody prodrug Pepbody-SB1901-H in a human primary Treg cell proliferation assay compared to the anti-TNFR2 antibody SB1901-72. DETAILED DESCRIPTION OF THE INVENTION

[0113] Disclosed herein are IL-15 prodrugs having a masking polypeptide (MP) and a cleavable moiety (CM). The IL-15 prodrugs overcome the toxicity problems that severely limit the clinical application of IL-15. The activity of IL-15 in the prodrug is attenuated. The cleavable moiety in the prodrug includes a protease cleavage site, and the masking polypeptide in the prodrug is excised by a protease associated with a desired site (such as in a tumor or tumor microenvironment) to restore the activity of IL-15.

[0114] DEFINITIONS

[0115] Unless otherwise defined in the context, the practice of the present invention will employ conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA techniques within the skill of the art. The foregoing methods will be described in detail below for illustrative purposes. Such techniques are well explained in the literature. See, for example, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (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); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. 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.

[0116] As described herein, "treatment" or "treating" is a method of obtaining a beneficial or desired result, including a clinical result. For the purposes of this application, the beneficial or desired result includes a clinical result, but is not limited to one or more of the following: alleviating one or more symptoms caused by a disease, reducing the severity of the disease, stabilizing the disease (e.g., preventing or delaying disease progression), preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, alleviating the disease (partially or completely), reducing the dosage of one or more other drugs required to treat the disease, delaying the progression of the disease, improving or enhancing the quality of life, and / or extending the survival period. At the same time, "treatment" also includes a reduction in the pathological outcome of the disease. The methods of this application contemplate any one or more aspects of these treatments. 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 reducing the symptoms caused by the disease, improving the quality of life of the patient suffering from the disease, reducing the dosage of other drugs required to treat the disease, and / or extending the survival period of an individual.

[0117] The terms "prevent" and similar words such as "prevented", "preventing", etc. denote methods of preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or disorder. It also refers to delaying the occurrence or recurrence of a disease or disorder or delaying the occurrence or recurrence of the symptoms of a disease or disorder. As described herein, "prevent" and similar words also include reducing the intensity, impact, symptoms, and / or burden of a disease or disorder before its recurrence.

[0118] As described herein, "delaying" the development of a disease means postponing, hindering, slowing, retarding, stabilizing, and / or delaying the development of the disease. Depending on the disease history and / or the individual being treated, the time of delay may vary. A method of "delaying" the development of a disease is a method that reduces the probability of disease development and / or reduces the severity of the disease within a given time frame compared to not using the method. This comparison is typically based on clinical studies using a statistically significant number of individuals.

[0119] 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, such as to improve, alleviate, attenuate and / or delay one or more symptoms. In some embodiments, the effective amount is an amount sufficient to delay the progression of a disease. In some embodiments, the effective amount is an amount sufficient to prevent or delay the onset or recurrence of a disease. The effective amount may be administered in one or more administrations. In the case of a disease such as cancer, the effective amount may be an amount sufficient to delay the onset or progression of cancer (e.g., reduce the tumor growth rate and / or delay or prevent tumor angiogenesis, metastasis or infiltration of cancer cells into peripheral organs), reduce the number of epitheloid cells, cause regression of cancer (e.g., shrink or eradicate the tumor), and / or prevent or delay the onset or recurrence of cancer. The effective amount may be administered in one or more administrations.

[0120] As used herein, "individual" or "subject" refers to a mammal, including but not limited to a human, bovine, horse, cat, dog, rodent or primate. In some embodiments, the individual is a human.

[0121] The term "antibody" includes full-length antibodies and antigen-binding fragments thereof. In some embodiments, a full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically include 3 hypervariable loops, referred to as complementarity determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2 and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2 and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein may be defined or identified by the Kabat, Chothia or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The 3 CDR regions of the heavy or light chain are inserted between flanking segments referred to as framework regions (FRs), which are more conserved than the CDR regions and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit a variety of effector functions. Antibodies are classified or typed based on the amino acid sequence of their heavy chain constant regions. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG and IgM, which are characterized by having α, δ, ε, γ and μ heavy chains, respectively. Several of the main antibody classes are 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).

[0122] As used herein, the term "antigen-binding fragment" includes antibody fragments, including, for example, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), VHH, single-chain Fv (scFv), scFv dimers (bivalent diabodies), multispecific antibodies composed of antibody fragments comprising one or more CDRs, single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment capable of binding to an antigen but not containing the complete antibody structure. Antigen-binding fragments also include fusion proteins comprising antibody fragments as described above. An antigen-binding fragment is capable of binding the same antigen as the parent antibody or parent antibody fragment (e.g., parent scFv). In some embodiments, an antigen-binding fragment may comprise one or more CDRs from a particular human antibody that are grafted into framework regions from one or more different human antibodies.

[0123] As used herein, the term "CDR" or "complementary determining region" refers to the non - contiguous antigen - binding sites found within the variable domains of heavy and light chain polypeptides. These particular regions have been described in the literature Kabat et al., J. Biol. Chem. 252:6609 - 6616 (1977); Kabat et al., U.S. Dept. 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); MacCallum et al., 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 Plückthun, J. Mol. Biol., 309:657 - 670 (2001), where these definitions include the overlap or subsets of amino acid residues when compared to each other. However, using any of these definitions to indicate the CDRs of an antibody or a graft - antibody or its variants is included within the scope of the term as defined and used herein. Table A lists the positions of the amino acid residues included in the CDRs defined by each of the above - cited references for comparison. Algorithms for CDR prediction and binding interfaces 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 content of the references cited in this paragraph is incorporated herein by reference in its entirety for use in this application and in one or more claims that may be included herein.

[0124] Table A: CDR Definitions

[0125]

[0126] 1 The amino acid residue numbering refers to the nomenclature method in Kabat et al. mentioned above

[0127] 2 The amino acid residue numbering refers to the nomenclature method in Chothia et al. mentioned above

[0128] 3 The amino acid residue numbering refers to the nomenclature method in MacCallum et al. mentioned above

[0129] 4 The amino acid residue numbering refers to the nomenclature method in Lefranc et al. mentioned above

[0130] 5 The amino acid residue numbering refers to the nomenclature method in Honegger and Plückthun mentioned above

[0131] The term "constant domain" refers to a part of an immunoglobulin molecule that has a more conserved amino acid sequence relative to another part of the immunoglobulin molecule that contains the variable domain of the antigen-binding site. The constant domain contains the C H 1, C H 2, and C H 3 domains (collectively referred to as C H ) and the C L domain of the light chain. Immunoglobulins can be classified into different classes or subtypes according to the amino acid sequence of the constant domain of the immunoglobulin heavy chain (C H ). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains being α, δ, ε, γ, and μ, respectively. γ and α are further divided into subclasses based on relatively minor differences in the CH sequence and function. For example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0132] 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 the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as starting from the amino acid residue at position Cys226 or from Pro230 and extending to its carboxy terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during the production or purification of the protein or by recombinant engineering of the nucleic acid encoding the protein. Suitable native sequence Fc regions for the constructs described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0133] As used herein, the term IgG "subtype" or "subclass" refers to any subclass of immunoglobulin defined by the chemical and antigenic properties of the constant domains. Immunoglobulins are mainly divided into five major classes: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (subtypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different immunoglobulin classes are designated 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 the fourth edition of Cellular and Molecular Immunology by Abbas et al. (W.B. Saunders, Co., 2000).

[0134] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region in a structure containing Fc (e.g., an antibody or a protein containing an Fc domain, hereinafter referred to as an Fc fusion protein). Preferred FcRs are native sequence human FcRs. In addition, preferred FcRs are FcRs that bind IgG antibodies (a γ receptor), including receptor subclasses such as FcγRI, FcγRII, and FcγRIII, as well as allelic variants and alternatively spliced forms of these receptors. The FcγRII receptor includes FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibitory receptor"), which have similar amino acid sequences and differ mainly in their cytoplasmic domains. The 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. 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 Haas et al., J.Lab.Clin.Med. 126:330-41 (1995). As used herein, the term "FcR" encompasses other FcRs, including FcRs yet to be identified.

[0135] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for transporting maternal IgG to the fetus. Guyer et al., J.Immunol. 117:587 (1976); and Kim et al., J.Immunol. 24:249 (1994). Methods for determining 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); Hinton et al., J.Biol.Chem. 279(8):6213-6 (2004); WO 2004 / 92219 (Hinton et al.)). The half-life of binding of human FcRn high affinity binding polypeptides to FcRn in vivo and in serum can be determined, for example, in transgenic mice expressing human FcRn or transfected human cell lines, or in primates administered polypeptides having variant Fc regions. WO2004 / 42072 (Presta) details antibody variants that enhance or reduce binding to FcRs. See Shield et al., J.Biol.Chem. 9(2):6591-6604 (2001).

[0136] "Antibody effector function" refers to the biological activities caused by the Fc region (native sequence Fc region or amino acid sequence variant Fc region) in a structure containing Fc (e.g., an antibody or Fc fusion protein), and varies with 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 receptor) and B cell activation. "Reducing or minimizing" 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 structure containing Fc (e.g., an antibody or Fc fusion protein). Measuring antibody effector function can be readily determined and measured by those of ordinary skill in the art. In a preferred embodiment, the antibody effector functions of complement binding, complement-dependent cytotoxicity and antibody-dependent cytotoxicity are affected. In some embodiments, the effector function is eliminated by eliminating glycosylation through mutations in the constant domain, e.g., "effector function null mutations". In some embodiments, the effector function null mutant is the N297A or DANA mutation (D265A + N297A) in the C H 2 region. Shields et al., J. Biol. Chem. 276(9):6591-6604(2001). Additionally, other mutations that result in reduced or eliminated effector function include: K322A and L234A / L235A (LALA). Additionally, the effector function can be reduced or eliminated by production techniques, such as expressing in a host cell that does not perform glycosylation (e.g., E. coli) or a host cell that results in an altered glycosylation pattern that is ineffective or less effective in promoting effector function (e.g., Shinkaw et al., J. Biol. Chem. 278(5):3466-3473(2003)).

[0137] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells such as natural killer cells (NK), neutrophils, and macrophages, enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells using cytotoxins. The antibody "arms" the cytotoxic cells and is required for this killing. Among the major cell types mediating ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a target molecule, an in vitro ADCC assay can be performed as described in U.S. Patent No. 5,500,362 or 5,821,337. Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK). Optionally, or in addition, the ADCC activity of the target molecule can also be evaluated in vivo, for example, in an animal model as disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

[0138] "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 antibody (subclass with the appropriate structure) that has bound homologous antigen. To assess complement activation, a CDC assay can be performed as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Polypeptide variants with altered amino acid sequences in the Fc region and increased or decreased C1q binding ability are described in U.S. Patent No. 6,194,551 B1 and WO 99 / 51642. The contents of these patent publications are hereby expressly incorporated by reference. See also Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0139] As used herein, the terms "specifically bind", "specifically recognize", or "specifically for" refer to a measurable and reproducible interaction, such as the binding between a ligand and a receptor, and 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 a greater affinity, avidity, is more likely and / or has a longer duration of binding to the target receptor compared to binding to other receptors. In some embodiments, as determined by, for example, radioimmunoassay (RIA), the degree of binding of the ligand to an irrelevant receptor is less than 10% of the degree of binding of the ligand to the target receptor. In some embodiments, the equilibrium dissociation constant (Kd) of a ligand that specifically binds to a target receptor is ≤ 10 -5 M, ≤ 10 -6 M, ≤ 10 -7 M, ≤ 10 -8 M, ≤ 10 -9 M, ≤ 10 -10 M, ≤ 10 -11 M or ≤ 10 -12 M. In some embodiments, the ligand specifically binds to a receptor that is conserved among different species. In some embodiments, specific binding can include, but does not require, exclusive binding. The binding specificity of a ligand can be determined experimentally using methods known in the art. Such as including but not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORETM-tests, and peptide scanning.

[0140] As used herein, when referring to a protease (e.g., a metalloprotease), the term "substrate" refers to any material or substance on which the protease (e.g., a metalloprotease) acts. The material or substance can be, for example, a naturally or non-naturally occurring organic compound or macromolecule, such as a polypeptide or peptidomimetic. In some embodiments, a metalloprotease substrate specifically interacts with one or more metalloproteases and is cleaved by the metalloprotease. The metalloprotease cleaves at least one molecule of the substrate within an experimental time frame under appropriate conditions. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the substrate can be cleaved by the metalloprotease.

[0141] The term "functional analogue" refers to a molecule that has the same biological specificity (e.g., binds to the same ligand) and / or activity (e.g., activates or inhibits a target cell) as a control molecule.

[0142] The term "prodrug" refers to a therapeutic molecule that is inactive until it is activated in vivo.

[0143] The term "modulate" includes "increase", "enhance" or "stimulate" as well as "decrease" or "reduce", typically with a statistically or physiologically significant amount or degree relative to a control group.

[0144] The term "variant" relative to a control polypeptide or polynucleotide includes one or more substitutions, additions, deletions, and / or insertions. As used herein, a variant of a polypeptide or polynucleotide comprises an amino acid or nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, similarity or homology to the control sequence, and substantially retains the activity of the control sequence. Also included are sequences that are compositionally or otherwise different from the control sequence by the addition, deletion, insertion or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more amino acids or nucleotides, and that substantially retain at least one activity of the control sequence. In certain embodiments, the addition or deletion includes C-terminal and / or N-terminal addition and / or deletion.

[0145] The term "wild-type" refers to the gene or gene product (e.g., polypeptide) most frequently observed in a population and is thus designated the "normal" or "wild-type" form of the gene.

[0146] The term "linked" includes covalently linked or non-covalently linked, and refers to a first part, such as a first amino acid sequence or nucleotide sequence, being covalently or non-covalently linked to a second part, such as a second amino acid sequence or nucleic acid sequence, respectively. The first part can be directly linked to the second part or adjacent to each other (hereinafter referred to as direct linkage, for example, in the case of a polypeptide, through a peptide bond), or, optionally, the first part is linked to the second part using an intervening part (for example, a peptide linker) (hereinafter referred to as indirect linkage), and can be expressed as the first part being linked to the second part through the intervening part. In the case of a polypeptide or protein, the term "linked" includes not only the linkage at the C-terminus and / or N-terminus of the first part (or the second part), but also the linkage of the entire first part (or the second part) to any position in the second part (or the first part) (for example, an amino acid residue not located at the terminus). In one aspect, the first part is linked to the second part through a peptide bond or a linker. In some embodiments, the first part can be linked to the second part through a phosphodiester bond or a linker. In some embodiments, the term "linker" refers to a molecule (including but not limited to unmodified or modified nucleic acid or amino acid) or a group of molecules (for example, 2 or more, such as 2, 3, 4, 10, 30, 50, 100 or more) or any chemical group that connects two parts, such as two polypeptides.

[0147] As described herein, a "covalent bond" refers to a stable bond formed between two atoms by sharing one or more electrons. Examples of covalent bonds include but are not limited to peptide bonds and disulfide bonds. As described herein, a "peptide bond" refers to a covalent bond formed between the carboxyl group of an amino acid and the amine group of an adjacent amino acid. As described herein, a "disulfide bond" refers to a covalent bond formed between two sulfur atoms, such as two Fc fragments being bound by one or more disulfide bonds. One or more disulfide bonds between two fragments may be formed by linking the thiol groups in the two fragments. In some embodiments, one or more disulfide bonds may be formed between one or more cysteines of two Fc fragments. Oxidation of two thiol groups forms a disulfide bond. In some embodiments, a covalent linkage is directly formed by a covalent bond. In some embodiments, a covalent linkage is directly formed by a peptide bond or a disulfide bond.

[0148] When referring to two polypeptide sequences, the terms "fused" or "fusion" refer to the joining of two polypeptide fragments by a backbone peptide bond. The two polypeptides can be fused directly or through a peptide linker that contains one or more amino acids. A fusion protein is a polypeptide that contains two or more regions derived from different or heterologous proteins or peptides. Conventional techniques of enzymatic cleavage and ligation of fragments from the desired sequences are used to prepare fusion proteins. PCR techniques with synthetic oligonucleotides can be used to prepare and / or amplify the desired fragments. Overlapping synthetic oligonucleotides representing the desired sequences can also be used to prepare a DNA construct encoding the fusion protein. The fusion protein can contain multiple sequences, including a leader (or signal peptide) sequence, a linker sequence, a leucine zipper sequence or other sequences that form oligomers, and sequences encoding highly antigenic portions that allow for convenient purification or rapid detection of the fusion protein. The fusion protein can be prepared from an encoding sequence by recombinant techniques, the encoding sequence containing the encoding sequences of two components of the fusion protein, with or without a peptide linker therebetween. In some embodiments, the fusion includes chemical conjugation.

[0149] As used 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.

[0150] The half-maximal inhibitory concentration (IC 50 ) is a measure of the effectiveness of a substance (e.g., a ligand) in inhibiting a specific biological or biochemical function. It represents how much of a particular drug or other substance (inhibitor, e.g., ligand) is required to inhibit a given biological process by half. The value is typically expressed as a molar concentration. IC 50 is comparable to the "EC 50 " of an agonist drug or other substance (e.g., ligand). EC 50 also represents the plasma concentration required to obtain 50% of the maximal effect in vivo. As used herein, "IC 50 " is used to denote the effective concentration of a ligand required to neutralize 50% of the receptor biological activity in vitro. IC 50 or EC 50 can be determined by biological assays, such as inhibition of ligand binding by FACS analysis (competitive binding assay), cell-based cytokine release assays, or amplified luminescence homogeneous enzyme-linked immunosorbent assay (AlphaLISA).

[0151] The "percent amino acid sequence identity (%)" and "homology" of a peptide or polypeptide sequence are defined as the percentage of identical amino acid residues in a candidate sequence to those in a specific polypeptide or polypeptide sequence, after alignment of the sequences and introduction of gaps (if necessary) to maximize the percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. To determine the percent amino acid sequence identity, multiple alignment means within the skill in the art can be used, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN TM (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared.

[0152] As used herein, the "C-terminus" of a polypeptide refers to the last amino acid residue of the polypeptide, which residue confers its amino group to form 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, which residue confers its carboxyl group to form a peptide bond with the amino group of an adjacent amino acid residue.

[0153] As used herein, the term "moiety" refers to a part of a molecule that has a different function within the molecule, and that function can be exercised by that part in another molecule. A moiety can be a chemical entity with a specific function, or a part of a biomolecule with a specific function.

[0154] As used herein, the terms "polypeptide", "peptide", and "protein" are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer can be linear or branched, can include modified amino acids, and can be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term "amino acid" refers to natural and / or non-natural 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 single-letter or three-letter abbreviations are used to denote amino acids.

[0155] A "separated" polypeptide refers to a polypeptide that has been identified, separated, and / or recovered from the components of its production environment (e.g., natural or recombinant). Preferably, the separated polypeptide is not associated with all other components in its production environment. Contaminating components in the production environment, such as those produced by recombinant transfected cells, usually interfere with the research, diagnosis, or treatment of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide will be purified to: (1) greater than 95% by weight polypeptide content, as determined by the Lowry method, and in some embodiments, greater than 99% by weight; (2) to an extent sufficient to obtain at least 15 N-terminal residues or internal amino acid sequences by using a spinning cup sequencer; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or preferably silver staining. Separated polypeptides include polypeptides in situ within recombinant cells because at least one element in the polypeptide's natural environment is absent. However, typically, a separated polypeptide will have undergone at least one purification step.

[0156] As used herein, the terms "polynucleotide", "nucleic acid", "nucleotide", and "oligonucleotide" are used interchangeably. They refer to polymeric forms of nucleotides of any length, deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci (locus) 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 can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure can be modified before or after polymer assembly. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by binding to a labeling component.

[0157] An "isolated" nucleic acid molecule encoding a coding construct (such as the masking polypeptide described herein) is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule, which is usually associated with the environment in which it is produced. Preferably, the isolated nucleic acid is unassociated with all components in the environment in which it is produced. The isolated nucleic acid molecule encoding a polypeptide as described herein exists in a form or configuration that is not the same as that in which it is found in nature. Thus, an isolated nucleic acid molecule is different from the nucleic acid encoding the polypeptide described herein that naturally occurs in a cell. An isolated nucleic acid includes the nucleic acid molecule contained in the cell containing the nucleic acid molecule, but the nucleic acid molecule is extrachromosomal or at a chromosomal location different from its natural chromosomal location.

[0158] The term "control sequence" means a DNA sequence that is necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes, for example, include a promoter, optionally, an operator sequence, and a ribosome binding site. It is known that eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.

[0159] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein 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 the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, for a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0160] As used herein, the term "vector" means a nucleic acid molecule capable of amplifying another nucleic acid molecule to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are introduced into the genome of a known host cell. Certain vectors are capable of directing the expression of nucleic acids to which they are linked. Such vectors are referred to herein as "expression vectors".

[0161] As used herein, the term "transfection" or "transformation" or "transduction" means the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. The cells include the primary subject cells and their progeny.

[0162] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", including primary transformed cells and progeny therefrom, regardless of the number of passages. The progeny may not be completely identical to the parental cell in terms of nucleic acid and may contain mutations. This document includes screening or selection of mutant progeny in the original transformed cells that have the same function or biological activity as them.

[0163] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a formulation that is in a form effective for the biological activity of the active ingredient and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such a formulation is sterile. A "sterile" formulation is free of microorganisms and their spores.

[0164] The examples of the present application described herein should be understood to include examples of "consisting of" and / or "consisting essentially of".

[0165] As used herein, the reference to "about" a numerical value or parameter includes (and describes) variations to that value or parameter itself. For example, a description involving "about X" includes a description of "X".

[0166] As used herein, the reference to "not" a numerical value or parameter generally means and describes "other than" a certain numerical value or parameter. For example, the method cannot be used to treat disease type X, which means that the method is generally used to treat other types of diseases other than disease type X.

[0167] As used herein, the term "about X - Y" has the same meaning as "about X to about Y".

[0168] Unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" as used in this document and the claims herein include plural referents.

[0169] Masking polypeptide (MP)

[0170] The masking polypeptide (MP) provided herein refers to a moiety that can block the activity of a bioactive moiety (B). In some embodiments, the masking polypeptide (MP) can inhibit the ability of a cytokine to bind and / or activate its receptor. In some embodiments, the masking polypeptide (MP) can inhibit the ability of an antibody or antigen - binding fragment to bind to its target.

[0171] In some embodiments, the masking polypeptide (MP) has a hydrodynamic radius that is larger than its actual molecular weight. In some embodiments, the masking polypeptide can only form a random coil lacking secondary structure. In some embodiments, the masking polypeptide has a steric masking effect, that is, due to its relative size, it can usually inhibit or block the activity of the bioactive moiety when approaching the bioactive moiety.

[0172] In some embodiments, the masking polypeptide (MP) is composed 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).

[0173] In some embodiments, the masking polypeptide (MP) is composed of four amino acid residues selected from the group consisting of proline (P), alanine (A), serine (S), and glutamic acid (E).

[0174] In some embodiments, the masking polypeptide (MP) is composed of four amino acid residues selected from the group consisting of proline (P), glycine (G), serine (S), and glutamic acid (E).

[0175] In some embodiments, the masking polypeptide (MP) is composed of five amino acid residues selected from the group consisting of proline (P), alanine (A), serine (S), glycine (G), and glutamic acid (E).

[0176] In some embodiments, the net charge in the masking polypeptide (MP) is very important. Generally, a negative charge is more preferred than a positive charge. The net negative charge of the MP can avoid its potential interaction with tissues and cell membranes. If it has a net positive charge, it may "sink" before reaching the destination. However, too much negative charge may increase the possibility of interaction with positively charged proteins. The percentage of the net charge also depends on the object fused with the MP.

[0177] In some embodiments, the masking polypeptide (MP) is composed of five amino acids G, S, P, E, and A; wherein, in the masking polypeptide, the percentage of the amino acid residue G is about 15%-30%; in the masking polypeptide, the percentage of the amino acid residue S is about 20%-40%; in the masking polypeptide, the percentage of the amino acid residue P is about 15%-40%; in the masking polypeptide, the percentage of the amino acid residue E is about 1%-20%; in the masking polypeptide, the percentage of the amino acid residue A is about 5%-20%; when the number of amino acids is not an integer, an integer value is taken.

[0178] In some embodiments, the masking polypeptide (MP) consists of five amino acids G, S, P, E, and A; wherein, in the masking polypeptide, the percentage of amino acid residue G is about 20%; in the masking polypeptide, the percentage of amino acid residue S is about 40%; in the masking polypeptide, the percentage of amino acid residue P is about 20%; in the masking polypeptide, the percentage of amino acid residue E is about 10%; in the masking polypeptide, the percentage of amino acid residue A is about 10%; when the number of amino acids is not an integer, the integer value is taken.

[0179] In some embodiments, the masking polypeptide (MP) consists of four amino acids S, P, E, and G; wherein, in the masking polypeptide, the percentage of amino acid residue S is about 20% - 40%; in the masking polypeptide, the percentage of amino acid residue P is about 15% - 40%; in the masking polypeptide, the percentage of amino acid residue E is about 1% - 20%; in the masking polypeptide, the percentage of amino acid residue G is about 15% - 30%; when the number of amino acids is not an integer, the integer value is taken.

[0180] In some embodiments, the masking polypeptide (MP) consists of four amino acids S, P, E, and G; wherein, in the masking polypeptide, the percentage of amino acid residue S is about 23%; in the masking polypeptide, the percentage of amino acid residue P is about 29%; in the masking polypeptide, the percentage of amino acid residue E is about 18%; in the masking polypeptide, the percentage of amino acid residue G is about 30%; when the number of amino acids is not an integer, the integer value is taken.

[0181] In some embodiments, the masking polypeptide (MP) can be adjusted by changing its amino acid chain length and its total net charge to meet certain requirements for certain objects.

[0182] In some embodiments, the masking polypeptide (MP) contains about 40 to 720 amino acid residues. In some embodiments, the masking polypeptide (MP) contains about 80 to 320 amino acid residues. In some embodiments, the masking polypeptide (MP) contains about 80 to 240 amino acid residues.

[0183] In some embodiments, the masking polypeptide (MP) contains the amino acid sequence SEQ ID NO:6.

[0184] In some embodiments, the masking polypeptide (MP) contains the amino acid sequence SEQ ID NO:1.

[0185] In some embodiments, the masking polypeptide (MP) comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-5; or a variant thereof, the variant having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1-5; or a variant thereof, the variant comprising one or more amino acid substitutions, additions and / or deletions.

[0186] Cleavable moiety (CM)

[0187] The cleavable moiety (CM) is a cleavage site for an enzyme or protease or a polypeptide containing the above site. In some embodiments, the protease includes but is not limited to urokinase-type 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; 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, 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); metalloproteases (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 such as FVIIa, FIXa, FXa, FXla, FXIIa); elastase; granzyme B; guanidobenzoate hydrolase; HtrA1; human neutrophil elastase; lactoferrin; trypsin; 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);

[0188] In some embodiments, the cleavable moiety (CM) comprises a substrate sequence of 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 substrate sequences of MMP2, MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. In some embodiments, the CM comprises a substrate sequence of MMP2. In some embodiments, the CM comprises a substrate sequence of MMP9. In some embodiments, the CM comprises substrate sequences of two or more MMPs. In some embodiments, the CM comprises substrate sequences of at least MMP2 and MMP9. In some embodiments, the CM comprises substrates of two or more identical MMPs. 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.

[0189] In some embodiments, the cleavable moiety (CM) comprises the amino acid sequence MVX 1 X 2 AX 3 TX 4 SG (SEQ ID NO: 49), wherein X 1 is selected from P, L, V, or A, X 2 is selected from L or S, X 3 is selected from L, V, P, or Y, and X 4 is selected from A or V.

[0190] The terms used to describe protease specificity, i.e., its cleavage of peptide bonds of specific amino acids in the vicinity, are based on the terms originally created by Schechter & Berger (1967, 1968) to describe papain specificity. According to this model, along the N-terminal direction of the cleaved bond, the amino acid residues of the cleaved substrate are designated as P1, P2, P3, P4, etc. Similarly, the residues in the C-terminal direction are designated as P1’, P2’, P3’, P4’, etc.

[0191] In some embodiments, the CM comprises an amino acid sequence shown in any of SEQ ID NOs: 8-16 or a variant thereof, which has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 8-16.

[0192] Bioactive moiety (B)

[0193] In some embodiments, the bioactive moiety (B) can be a chemical entity. In some embodiments, the bioactive moiety (B) can be a therapeutic protein. In some embodiments, the bioactive moiety (B) is a cytokine. In some embodiments, the bioactive moiety (B) is an antibody or antigen-binding fragment that targets a prodrug to a site of action (e.g., an inflammatory site or a tumor site).

[0194] Cytokine: "Cytokine" is a term well known in the art and refers to any one of a class of immunomodulatory proteins (such as interleukins or interferons) that are secreted by cells, especially cells of the immune system, and act as regulators of the immune system. In some embodiments, the cytokine includes functional fragments, mutants or variants of the cytokine. Examples of cytokines can include chemokines, interferons, interleukins, lymphokines and tumor necrosis factors. For cytokine prodrugs, the following takes the IL-15 prodrug as an example. However, prodrugs of other cytokines, especially cytokines that are potent immunomodulators and have strong side effects, are also within the scope of consideration of this application. After proteolytic cleavage at the target of the cleavable moiety, the cytokine becomes an active form, which enables it to bind to its cognate receptor or protein with a stronger affinity. Other cytokine prodrugs can be prepared according to the same principle as the IL-15 prodrug shown below.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] In some embodiments, the cytokine is selected from the group consisting of 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.

[0200] 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.

[0201] 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.

[0202] In some embodiments, 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.

[0203] Antibody: For antibody prodrugs, although the anti-TNFR2 antibody prodrug is exemplified below, prodrugs of other antibodies (especially antibodies with sub-optimal selectivity for the intended target) are also contemplated in the present application. These prodrugs are activated by tumor-associated proteases in the tumor microenvironment, thereby restricting the activity to the tumor microenvironment and minimizing "off-tumor" toxicity. In some embodiments, the masking polypeptide (MP) reduces the ability of the antibody or its antigen-binding fragment to bind to the target, such that the dissociation constant (Kd) of the antibody or antigen-binding fragment conjugated to the masking polypeptide (MP) with the target is at least 10-fold higher, at least 100-fold higher, at least 1000-fold higher, at least 10000-fold higher than the Kd of the antibody or antigen-binding fragment without the conjugated masking polypeptide (MP) with the target. After the cleavable moiety (CM) of the target is proteolytically cleaved, the antibody or antigen-binding fragment is activated. Other antibody prodrugs can be prepared according to the same principle as the anti-TNFR2 antibody prodrug described below.

[0204] In some embodiments, the antibody or antigen-binding fragment is specific for regulatory T cells (Tregs), e.g., targeting the CCR4 or CD39 receptor. In some embodiments, the specific antibody or antigen-binding fragment may bind to antigens on the surface of immune cells, e.g., T cells, NK cells, and macrophages. For example, the specific antibody or antigen-binding fragment may bind to PD-1, LAG-3, TIM-3, TIGIT, CTLA-4, or TNF-α. In some embodiments, the specific antibody or antigen-binding fragment may have the ability to activate immune cells and enhance their anti-cancer activity. In some embodiments, the specific antibody or antigen-binding fragment may bind to antigens on the surface of diseased cells or tissues, e.g., tumor cells, and the tumor antigens are 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.

[0205] In some embodiments, the specific antibody or antigen-binding fragment is specific for an immune checkpoint protein. Exemplary immune checkpoint proteins include, but are 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.

[0206] In some embodiments, the specific antibody or antigen-binding fragment is specific for an immune response modulator. Exemplary immune response modulators include, but are 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.

[0207] In some embodiments, the specific antibody or antigen-binding fragment is specific for a cytokine receptor. Exemplary cytokine receptors include, but are not limited to, type I cytokine receptors, such as GM-CSF receptor, G-CSF receptor, Type IIL receptor, Epo receptor, LIF receptor, CNTF receptor, TPO receptor; type II cytokine receptors, such as IFN-alpha receptor (IFNAR1, IFNAR2), IFB-beta receptor, IFN-gamma receptor (IFNGR1, IFNGR2), Type II IL receptor; chemokine receptors, such as CC chemokine receptor, CXC chemokine receptor, CX3C chemokine receptor, XC chemokine receptor; tumor necrosis factor receptor superfamily receptors, such as TNFRSF5 / CD40, TNFRSF8 / CD30, TNFRSF7 / CD27, TNFRSFlA / TNFRl / CD120a, TNFRSF1B / TNFR2 / CD120b; TGF-beta receptors, such as TGF-beta receptor 1, TGF-beta receptor 2; Ig superfamily receptors, such as IF-1 receptor, CSF-1R, PDGFR (PDGFRA, PDGFRB), or SCFR.

[0208] In some embodiments, the antibody or antigen-binding fragment binds to an antigen selected from 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, alpha v integrin, B7H3, B7H4, Her-3, folate receptor, GD-2, CEACAM5, CEACAM6, c-MET, CD266, MUC1, CD10, MSLN, sialyl Tn, Lewis Y, CD63, CD81, CD98, CD166, tissue factor (CD 142), CD55, CD59, CD46, CD164, TGF beta receptor 1 (TGFpRl), TGFpR2, TGFpR3, FasL, MerTk, Axl, Clecl2A, CD352, FAP, CXCR3 and CD5.

[0209] Non-cleavable linker (L)

[0210] In some embodiments, the linker is a non-cleavable linker. Exemplary non-cleavable linkers are stable under physiological conditions and at the site of disease, such as the tumor site or the site of an inflammatory disease. 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 3 residues. Portions of such linkers may be flexible, hydrophilic, and form little or no secondary structure on their own (linker portions or flexible linker portions). Linkers composed of at least 3 amino acids can be used to connect domains and / or regions that are close to each other after molecular assembly. Longer linkers can also be used. In some embodiments, the linker may 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 the various parts of a molecule, the linkers can be the same or different (e.g., the same or different lengths and / or amino acid sequences).

[0211] 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 composed 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 formula (Gly 4 Ser) n , where 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 (Gly 4 Ser) 1 . In some embodiments, a preferred Gly-Ser linker is (Gly 4 Ser) 2 . In some embodiments, a preferred Gly-Ser linker is (Gly 4 Ser) 3 . In some embodiments, a preferred Gly-Ser linker is (Gly 4 Ser) 4 . In some embodiments, a preferred Gly-Ser linker is (Gly 4 Ser) 5 . In other aspects, two or more Gly-Ser linkers are tandemly arranged in the polypeptide linker.

[0212] In some embodiments, the non-cleavable linker is used in the prodrugs described herein, which comprise an immunoglobulin (Ig) / antibody hinge region. In one embodiment, the hinge region is obtained from an IgGl antibody. In one embodiment, the term Ig "hinge" region refers to a polypeptide comprising an amino acid sequence that shares sequence identity or similarity with a portion of the naturally occurring Ig hinge region sequence, the naturally occurring Ig hinge region sequence including cysteine residues at which disulfide bonds are formed to link the two heavy chains of the immunoglobulin.

[0213] In some embodiments, the non-cleavable linker is used to link any component in the prodrugs provided herein.

[0214] In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in any of SEQ ID NOs: 17-21 or a variant thereof, the variant having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 17-21.

[0215] Half-life extension moiety (C)

[0216] Preferably, the prodrug comprises a half-life extension moiety (C). The term "half-life extension moiety" refers to a moiety that extends the half-life of the target component in serum. A long in vivo half-life is important for therapeutic molecules. For example, the half-life of cytokines administered to a subject is usually short because mechanisms such as normal renal clearance and endocytic degradation rapidly clear them from the subject's body. For therapeutic molecules that are short-lived in nature, increasing their in vivo half-life can result in a more acceptable and manageable dosing regimen without sacrificing efficacy. Thus, in the prodrugs provided herein, to achieve the purpose of extending the in vivo half-life, it is preferred to link the half-life extension moiety to the bioactive moiety.

[0217] As described herein, the "half-life extension moiety" increases the in vivo half-life and improves PK, for example, by altering its size (e.g., above the renal filtration limit), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and elimination. An exemplary way to improve polypeptide PK is by expressing a receptor-binding element in the polypeptide chain, where the receptor can recycle to the plasma membrane without degradation in lysosomes, such as the FcRn receptor and transferrin receptor on endothelial cells. Three types of proteins, such as human IgG, HSA (or fragments), and transferrin, persist in human serum for much longer than predicted by their size, which is related to their ability to bind to receptors that recycle in lysosomes without being degraded. These proteins or their fragments that retain FcRn-binding ability are typically linked to other polypeptides to extend their serum half-life.

[0218] In some embodiments, the half-life extension moiety (C) may also 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 an antibody or its antigen-binding fragment include polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies, such as heavy-chain variable domains (V H )、light-chain variable domains (V L ) and camelid nanobodies (V HH ), dAb, etc.

[0219] In some embodiments, the half-life extension moiety (C) can also function as a linker, optionally as a non-cleavable linker (L).

[0220] In some embodiments, the half-life extension 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 recycling, such as any heavy chain polypeptide or a portion thereof capable of participating in FcRn-mediated recycling. In some embodiments, the Fc domain is monomeric. In some embodiments, the Fc domain is dimeric, comprising a first Fc domain and a second Fc domain.

[0221] Fc domain

[0222] In some embodiments, the Fc domain is from any one of IgA, IgD, IgE, IgG, and IgM and their subclasses. Among all immunoglobulins, IgG has the highest serum content and the longest half-life. Different from other immunoglobulins, IgG can be effectively recycled after binding to Fc receptors (FcRs). In some embodiments, the Fc domain is from IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the Fc domain is from human IgG. In some embodiments, the Fc domain comprises CH2 and CH3 domains. In some embodiments, the Fc domain further comprises all or part of the hinge region. In some embodiments, the Fc domain is from human IgG1 or human IgG4. In some embodiments, the two subunits of the Fc domain are dimerized by 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, such as a truncated Fc domain containing fewer N-terminal cysteines to reduce disulfide bond mismatching during dimerization. In some embodiments, the Fc domain is truncated at the N-terminus, e.g., lacking the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the intact immunoglobulin Fc domain.

[0223] In some embodiments, the Fc domain comprises one or more mutations, such as insertions, deletions, and / or substitutions.

[0224] In some embodiments, the Fc domain comprises one or more amino acid mutations that alter effector functions, and the Fc domain is engineered (e.g., comprising one or more amino acid mutations) to alter its binding to FcRs, particularly to alter its binding to Fcγ receptors (responsible for ADCC) and / or to alter effector functions, such as altering antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). Preferably, such amino acid mutations do not reduce the binding to the FcRn receptor (responsible for half-life).

[0225] The Fc domain (e.g., human IgG1 Fc) is mutated to remove one or more effector functions, such as ADCC, ADCP, or CDC, hereinafter referred to as "effectorless" or "nearly effectorless" Fc domain. For example, in some embodiments, the Fc domain is an effectorless human IgG1 Fc, and the human IgG1 Fc contains 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 contains the L234A and L235A ("LALA") mutations. The combination of K322A, L234A, and L235A in IgG1 Fc is sufficient to completely abolish the binding of FcγR and C1q (Hezareh et al., J Virol 75, 12161–12168, 2001). MedImmune found that a set of three mutations, L234F / L235E / P331S, has very similar effects (Oganesyan et al., Acta Crystallographica 64, 700–704, 2008). In some embodiments, the Fc domain contains glycosylation modifications at N297 of the IgG1 Fc domain, which are known to be required for optimal FcR interaction. The Fc domain modification can be any suitable IgG Fc engineering mentioned by Wang et al. ("IgG Fc engineering to modulate antibody effector functions," Protein Cell. 2018 Jan;9(1):63–73), the content of which is incorporated herein by reference in its entirety.

[0226] Glycosylation variant

[0227] In some embodiments, the degree of glycosylation of the construct is increased or decreased by altering the Fc domain. Glycosylation sites can be added or removed in the Fc domain by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0228] Native Fc-containing proteins produced by mammalian cells typically contain a branched-chain biantennary oligosaccharide that is generally N-linked to Asn297 in the C H 2 domain. See Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide can include various carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc on the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharide in the Fc domain can be modified to produce certain improved properties.

[0229] In some embodiments, the Fc domain as described herein has a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc domain. For example, the fucose content in such an Fc domain or an IL-15 prodrug or an anti-TNFR2 antibody prodrug may be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. As described in WO2008 / 077546, the fucose content is determined by MALDI-TOF mass spectrometry of the average fucose content within the sugar chain attached to Asn297 relative to the sum of all sugar structures attached to Asn297 (e.g., complex, hybrid, and high mannose structures). Asn297 refers to the asparagine residue at position 297 of the Fc domain (Fc region residues are according to the EU numbering system); however, due to minor sequence variations in the Fc region, Asn297 may also 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 US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US 2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 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 lacking protein fucosylation function (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003 / 0157108A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and gene knockout cell lines such as CHO cells with knockout of the α-1,6-fucosyltransferase gene, FUT8 gene (see Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0230] Effector function variant

[0231] In some embodiments, the present application contemplates an Fc domain that has some but not all Fc effector functions, making it an ideal candidate for certain applications in which its half-life in vivo is important, but certain effector functions (such as CDC and ADCC) are non-essential or harmful. Cytotoxicity assays can be performed in vitro or in vivo to determine the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the Fc domain lacks FcγR binding (and thus likely lacks ADCC activity), but retains the ability to bind FcRn. The major cells mediating ADCC, natural killer (NK) cells, only express FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. The 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 evaluating the ADCC activity of a target molecule are detailed in U.S. Patent No. 5,500,362 (see Hellstrom, I. et al., Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive detection methods can be employed (see ACTI TM Non-radioactive toxicity assays (Cell Technology, Inc. Mountain View, CA) and CytoTox Non-radioactive toxicity tests (Promega, Madison, WI)). Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMC) and NK cells. In addition, the ADCC activity of the target molecule can also be evaluated in vivo, for example, in an animal model as disclosed in Clynes et al., Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to determine that the Fc domain does not bind to C1q and thus lacks CDC activity. See the C1q and C3c binding enzyme-linked immunosorbent assays in WO2006 / 029879 and WO 2005 / 100402. A CDC assay can be performed to evaluate complement activity (see Gazzano-Santoro et al., 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)). FcRn binding and in vivo clearance / half-life determinations can be performed using methods known in the art (see Petkova, S.B. et al.., Int’l. Immunol. 18(12):1759-1769 (2006)).

[0232] Fc domains with reduced effector function include those having substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc mutants include substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant that replaces residues 265 and 297 with alanine (U.S. Patent No. 7,332,581). Certain antibody variants that enhance or reduce binding to FcRs are described in detail (see U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001). In some embodiments, the Fc region is engineered to alter (i.e., increase or decrease) C1q binding and / or CDC, as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4 178-4184 (2000).

[0233] In some embodiments, the Fc domain comprises one or more amino acid substitutions that increase the half-life and / or enhance binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-life and enhanced binding to FcRn are responsible for transporting maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), and are described in detail in US2005 / 0014934A1 (Hinton et al.). Antibodies that contain an Fc domain with one or more substitutions thus have increased binding of the Fc region to FcRn. Such Fc variants include those with one or more substitutions of Fc domain residues, e.g., substitution of residue 434 in the Fc domain (US Patent No. 7,371,826).

[0234] 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 for other examples of Fc domain variants.

[0235] Cysteine engineered variant

[0236] In some embodiments, it may be desirable to create a cysteine-engineered Fc domain in which one or more residues of the Fc domain are replaced with cysteine residues. In some embodiments, the substituted residues occur at accessible sites on the Fc domain. By replacing these residues with cysteine, reactive thiol groups are thus positioned at accessible sites on the Fc domain and can be used to conjugate the molecule to other moieties, such as a drug moiety or a linker-drug moiety, to create a long-acting drug or prodrug conjugate. In some embodiments, any one or more of the following residues may be replaced with cysteine: heavy chain A118 (numbered according to the EU numbering system) and heavy chain Fc domain S400 (numbered according to the EU numbering system). Cysteine-engineered molecules can be generated as described in U.S. Patent No. 7,521,541.

[0237] 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 natural variant of IgG1 (e.g., IGHG1*03, which contains the double mutations D239E and L241M relative to IGHG1*05). In some embodiments, the Fc domain does not include the hinge region of IgG1 Fc. In some embodiments, the Fc domain contains up to 5 amino acids truncated from the N-terminus of IgG1 Fc, such as the first, first two, first three, first four, or first five amino acids truncated from the N-terminus of IgG1 Fc. In some embodiments, the Fc domain contains one or more silent mutations and / or deglycosylation mutations.

[0238] In some embodiments, the Fc domain is derived from IgG4 Fc. In some embodiments, the Fc domain is derived from human IgG4 Fc. In some embodiments, the Fc domain is wild-type IgG4 Fc. In some embodiments, in some embodiments, the Fc domain is a natural variant of IgG4. In some embodiments, the Fc domain does not include the hinge region of IgG4. In some embodiments, the Fc portion contains up to 5 amino acids truncated from the N-terminus of IgG4, such as the first, first two, first three, first four, or first five amino acids truncated from the N-terminus of IgG4. In some embodiments, the Fc domain contains one or more silent mutations and / or deglycosylation mutations.

[0239] Strategies for forming Fc fusion protein polypeptides or bispecific antibodies are well known (see Spies et al., Mol Imm. (2015) 67(2)(A):95-106). For example, in some embodiments, the first and / or second polypeptide chains of the Fc domain each contain one or more modifications that promote heterodimerization of the first and second Fc domains. Thus, one or more amino acid modifications can be made to the first Fc domain and one or more amino acid modifications can be made to the second Fc region using any strategy available in the art, including any strategy described in Klein et al. (2012), MAbs, 4(6):653-663. Exemplary strategies and modifications are the "knob into holes" method. In some embodiments, the first Fc domain containing the CH3 domain is a heavy chain polypeptide or a fragment thereof. The CH3 domains of the two Fc domains can be altered by the "knob-into-holes" technique (Fc knob and Fc hole), which is detailed in WO 1996 / 027011; Ridgway, J.B. et al. Protein Eng (1996) 9(7):617-621; Merchant, A.M., et al, Nat. Biotechnol. (1998) 16(7):677-681. Also see Klein et al. (2012), MAbs, 4(6):653-663. Using the knob-into-holes method, the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of the two moieties containing the two altered CH3 structures. This is achieved by introducing a bulky residue into the CH3 domain of one of the Fc domains, which serves as the "knob". Then, in order to accommodate the bulky residue, a "hole" is formed in the other Fc region that can accommodate the knob. One of the altered CH3 domains can be the "knob", while the other can be the "hole". Introduction of a disulfide bond further stabilizes the heterodimer (Merchant, A.M., et al, Nat. Biotechnol (1998) 16(7); Atwell, S., et al, J. Mol, Biol. (1997) 270(1):26-35) and increases yield. It is well known that heterodimer formation can be achieved by introducing T366W and / or S354C mutations in the heavy chain to generate the "knob", and by introducing T366S, L368A, Y407V and / or Y349C mutations in the heavy chain to form the "hole" (residues are numbered according to the Kabat EU numbering system).Carter et al. (2001), J. Immunol. Methods, 248:7-15; Klein et al. (2012), MAbs, 4(6):653-663.

[0240] In some embodiments, the Fc domain or a fragment thereof comprises the T366S, L368A, and Y407V mutations to form a 'hole'. In some embodiments, the Fc domain or a fragment thereof comprises the T366W mutation to form a 'knob'. In some embodiments, the Fc domain or a fragment thereof comprises the Y349C, T366S, L368A, and Y407V mutations to form a 'hole'. In some embodiments, the Fc domain or a fragment thereof comprises the S354C and T366W mutations to form a 'knob'. In some embodiments, the first Fc domain or a fragment thereof comprises the hole mutations, and its second Fc domain or a fragment thereof comprises the knob mutations. In some embodiments, the first Fc domain or a fragment thereof comprises the knob mutations, and its second Fc domain or a fragment thereof comprises the hole mutations, and the residue numbering is according to the EU numbering system.

[0241] In some embodiments, in addition to the LALA (L234A and L235A) mutations, the knob-into-holes mutations are also present in the Fc domain.

[0242] In some embodiments, the first Fc domain comprises the amino acid sequence SEQ ID NO:27 or a variant thereof, the variant having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with 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, the variant having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:28.

[0243] In some embodiments, the first Fc domain comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, the variant having 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, the variant having 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.

[0244] In some embodiments, the first Fc domain comprises the amino acid sequence SEQ ID NO:29 or a variant thereof, the variant having 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, the variant having 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.

[0245] In some embodiments, the first Fc domain comprises the amino acid sequence SEQ ID NO:30 or a variant thereof, the variant having 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, the variant having 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.

[0246] Prodrug

[0247] One aspect of the present application provides activatable prodrugs that can be metabolized in vivo into active therapeutic agents, which have fewer side effects and better targeting specificity. In some embodiments, the prodrug comprises one or more bioactive moieties (B), one or more cleavable moieties (CM), and one or more masking polypeptides (MP). In some embodiments, the masking polypeptide (MP) in the prodrug inhibits the biological function of the bioactive moiety (B). At the target site in a patient (e.g., at the tumor site or the surrounding environment), the cleavable moiety (CM) of the prodrug is cleaved by a protease, releasing the masking polypeptide (MP) from the prodrug, exposing the previously masked bioactive moiety (B), and allowing the bioactive moiety (B) to exert its biological function on the target cells, whereby the prodrug is activated.

[0248] In some embodiments, the prodrug further comprises one or more non-cleavable linkers (L).

[0249] In some embodiments, the prodrugs provided herein, wherein the masking polypeptide (MP) is linked to the bioactive moiety (B) via the cleavable moiety (CM).

[0250] In some embodiments, the prodrugs provided herein, wherein the cleavable moiety (CM) is linked to the masking polypeptide (MP), preferably via a non-cleavable linker (L).

[0251] In some embodiments, the prodrugs provided herein, wherein the cleavable moiety (CM) is linked to the bioactive moiety (B), preferably via a non-cleavable linker (L).

[0252] In some embodiments, the prodrug further comprises one or more half-life extending moieties (C).

[0253] In some embodiments, the prodrugs provided herein, wherein the bioactive moiety (B) is linked to the half-life extending moiety (C).

[0254] In some embodiments, the prodrugs provided herein, wherein the bioactive moiety (B) is linked to the half-life extending moiety (C), preferably via a non-cleavable linker (L).

[0255] In some embodiments, the prodrugs provided herein, wherein the masking polypeptide (MP) is linked to the half-life extending moiety (C) via the cleavable moiety (CM).

[0256] In some embodiments, the prodrugs provided herein, wherein the cleavable moiety (CM) is linked to the half-life extending moiety (C), preferably via a non-cleavable linker (L).

[0257] In some embodiments, the prodrugs provided herein, wherein the cleavable moiety (CM) is linked to the masking polypeptide (MP), preferably, via a non-cleavable linker (L).

[0258] In some embodiments, the prodrugs provided herein, wherein the prodrug comprises a construct of B-C-CM-MP from the N- to the C-terminus or C- to N-terminus, wherein "-" represents a covalent bond with or without a non-cleavable linker (L).

[0259] In some embodiments, the prodrugs provided herein, wherein the prodrug comprises a construct of C-B-CM-MP from the N- to the C-terminus or C- to N-terminus, wherein "-" represents a covalent bond with or without a non-cleavable linker (L).

[0260] In some embodiments, the prodrugs provided herein are monomers. In some embodiments, the prodrugs provided herein are dimers. In some embodiments, the dimer is monovalent. In some embodiments, the dimer is divalent. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer is a heterodimer.

[0261] In some embodiments, the prodrugs provided herein are dimers, wherein one monomer comprises a construct of C-B from the N- to the C-terminus or C- to N-terminus, and the other monomer comprises a construct of C-CM-MP from the N- to the C-terminus or C- to N-terminus, wherein "-" represents a covalent bond with or without a non-cleavable linker (L).

[0262] Exemplary antibody prodrug

[0263] This application provides antibody prodrugs having a masking polypeptide (MP) and a cleavable moiety (CM), which are metabolized in vivo into active antibodies with fewer side effects of the masking antibodies. In some embodiments, the masking polypeptide (MP) is linked to the N-terminus or C-terminus of the V H domain via a cleavable moiety (CM). In some embodiments, the masking polypeptide (MP) is linked to the N-terminus or C-terminus of the V L domain via a cleavable moiety (CM). In some embodiments, the masking polypeptide (MP) is linked to the N-terminus of the V H domain and the N-terminus of the V L domain via a cleavable moiety (CM).

[0264] In some embodiments, the antibody prodrug is a TNFR2 antibody prodrug. The schematic structural diagram of the anti-TNFR2 antibody or antibody prodrug is as Figure 15A-15D shown.

[0265] In some embodiments, the anti-TNFR2 antibody prodrugs provided herein comprise (i) an antibody or an antigen-binding fragment thereof that specifically binds to human TNFR2, (ii) one or more masking polypeptides (MPs) as described herein, and (iii) one or more cleavable moieties (CMs) as described herein.

[0266] In some embodiments, compared to an anti-TNFR2 antibody or antigen-binding fragment that does not have a masking polypeptide (MP), the masking polypeptide (MP) reduces the binding affinity of the anti-TNFR2 antibody or antigen-binding fragment for human TNFR2. In some embodiments, compared to an anti-TNFR2 antibody or its antigen-binding fragment that does not have a masking polypeptide (MP), the binding affinity is reduced by at least about 10-fold. In some embodiments, compared to an anti-TNFR2 antibody or its antigen-binding fragment that does not have a masking polypeptide (MP), the binding affinity is reduced by at least about 100-fold. In some embodiments, compared to an anti-TNFR2 antibody or its antigen-binding fragment that does not have a masking polypeptide (MP), the binding affinity is reduced by at least about 200 to 1500-fold. In some embodiments, the masking polypeptide (MP) comprises the amino acid sequence shown in any of SEQ ID NOs: 1-5 or a variant thereof, the variant 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 shown in any of SEQ ID NOs: 1-5.

[0267] In some embodiments, the anti-TNFR2 antibody prodrugs provided herein, wherein the masking polypeptide (MP) is linked to the N-terminus and / or C-terminus of the V H domain by a cleavable moiety (CM).

[0268] In some embodiments, the anti-TNFR2 antibody prodrugs provided herein, wherein the masking polypeptide (MP) is linked to the N-terminus and / or C-terminus of the V L domain by a cleavable moiety (CM).

[0269] In some embodiments, the anti-TNFR2 antibody prodrugs provided herein, wherein the masking polypeptide (MP) is linked to the N-terminus and / or C-terminus of the V H domain by a cleavable moiety (CM); and the masking polypeptide (MP) is linked to the N-terminus and / or C-terminus of the V L domain by a cleavable moiety (CM).

[0270] In some embodiments, the anti-TNFR2 antibody prodrug, wherein the anti-TNFR2 antibody is a full-length antibody. In some embodiments, the isolated anti-TNFR2 antibody is full-length IgG1, IgG2, IgG3, or IgG4.

[0271] 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 as compared to human wild-type IgG1.

[0272] 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 as compared to human wild-type IgG2.

[0273] In some embodiments, the IgG3 is human IgG3. In some embodiments, the IgG3 is human wild-type IgG3. In some embodiments, the IgG3 comprises one or more mutations as compared to human wild-type IgG3.

[0274] 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 as compared to human wild-type IgG4.

[0275] In some embodiments, the anti-TNFR2 antibody prodrug comprises two heavy chains and two light chains, wherein the masking polypeptide (MP) is linked to the N-terminus of one or two heavy chains through a cleavable moiety (CM).

[0276] In some embodiments, the anti-TNFR2 antibody prodrug comprises two heavy chains and two light chains, wherein the masking polypeptide (MP) is linked to the N-terminus of one or two light chains through a cleavable moiety (CM).

[0277] In some embodiments, the anti-TNFR2 antibody prodrug comprises two heavy chains and two light chains, wherein the masking polypeptide (MP) is linked to the N-terminus of two heavy chains through a cleavable moiety (CM); and the masking polypeptide (MP) is linked to the N-terminus of two light chains through a cleavable moiety (CM).

[0278] In some embodiments, the cleavable moiety (CM) is linked to the masking polypeptide (MP), preferably through a non-cleavable linker (L).

[0279] In some embodiments, the cleavable moiety (CM) is linked to the V H domain and / or the V L domain, preferably through a non-cleavable linker (L).

[0280] In some embodiments, the cleavable moiety (CM) described herein comprises a cleavage site that can be cleaved under specific circumstances, thereby separating its N-terminal fragment from its C-terminal fragment. By masking the cleavage site between the masking polypeptide and the heavy or light chain of the antibody, the masking polypeptide is removed at the cleavage site under specific circumstances, thereby releasing a fully functional anti-TNFR2 antibody.

[0281] In some embodiments, screening for a suitable cleavable moiety (CM) depends on the desired site of action of the anti-TNFR2 antibody. For example, when the tumor site is the desired site of action, a cleavage site of a tumor-specific protease is used to construct a masked anti-TNFR2 antibody designed to act on the tumor site. A tumor-specific protease refers to any protease that has a high level and / or activity in the tumor site relative to normal tissue.

[0282] In some embodiments, the protease cleavage site can be a cleavage site of matrix metalloproteinase (MMP). In some embodiments, the protease cleavage site can be a cleavage site of MMP2. In some embodiments, the protease cleavage site can be a cleavage site of MMP9. In some embodiments, the protease cleavage site can be a cleavage site of both MMP2 and MMP9. Other tumor-specific proteases and corresponding cleavage sites are known in the art, as disclosed in Vasiljeva et al., Scientific Reports, 10:5894, 2020, the relevant disclosure of which is incorporated herein by reference and is consistent with the subject matter and purpose of this article. In some embodiments, the cleavable moiety (CM) comprises an amino acid sequence shown in any of SEQ ID NOs: 8-16 or a variant thereof, the variant 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 shown in any of SEQ ID NOs: 8-16.

[0283] In some embodiments, provided herein is an anti-TNFR2 antibody prodrug, wherein the anti-TNFR2 antibody comprises V H , the V H comprising: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:50, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:51, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO:52, or a variant of the V H having up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V LComprising: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:53, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:54, and LC-CDR3, which comprises the amino acid sequence SEQ ID NO:55, or a variant of said V L having up to about 5 amino acid substitutions in its LC-CDRs.

[0284] In some embodiments, the anti-TNFR2 antibody prodrug provided herein, wherein the anti-TNFR2 antibody comprises V H which comprises the amino acid sequence SEQ ID NO:56 or a variant thereof, said variant having 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; and V L which comprises the amino acid sequence SEQ ID NO:57 or a variant thereof, said variant having 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.

[0285] In some embodiments, the anti-TNFR2 antibody prodrug comprises a heavy chain, the heavy chain comprising the construct: from N- to C-terminus is MP-CM-V H -C H 1-hinge-C H 2-C H 3, wherein "-" represents a covalent bond with or without a non-cleavable linker (L). In some embodiments, the anti-TNFR2 antibody prodrug heavy chain comprises the amino acid sequence SEQ ID NO:58 or a variant thereof, said variant having at least about 80%

[0286] (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0287] In some embodiments, the anti-TNFR2 antibody prodrug comprises a light chain, the light chain comprising the construct: from N- to C-terminus is MP-CM-V L -C L, wherein the "-" represents a covalent bond with or without a non-cleavable linker (L). In some embodiments, the anti-TNFR2 antibody prodrug light chain comprises the amino acid sequence SEQ ID NO:61 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO:61.

[0288] In some embodiments, the anti-TNFR2 antibody prodrug comprises two heavy chains and two light chains, wherein each heavy chain comprises the amino acid sequence SEQ ID NO:58 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO:58; and each light chain comprises the amino acid sequence SEQ ID NO:59 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence

[0289] SEQ ID NO:59.

[0290] In some embodiments, the anti-TNFR2 antibody prodrug comprises two heavy chains and two light chains, wherein each heavy chain comprises the amino acid sequence SEQ ID NO:60 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO:60; and each light chain comprises the amino acid sequence SEQ ID NO:61 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence

[0291] SEQ ID NO:61.

[0292] In some embodiments, the anti-TNFR2 antibody prodrug comprises two heavy chains and two light chains, wherein each heavy chain comprises the amino acid sequence SEQ ID NO:58 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO:58; and each light chain comprises the amino acid sequence SEQ ID NO:61 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence

[0293] SEQ ID NO:61 has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0294] Exemplary cytokine prodrug

[0295] This application provides IL-15 prodrugs as examples, which are metabolized in vivo to the active IL-15 cytokine. The IL-15 prodrugs have fewer side effects. In some embodiments, the IL-15 prodrugs have a better PK profile in vivo (e.g., a longer half-life).

[0296] A schematic structural diagram of an exemplary IL-15 cytokine prodrug is as Figure 2A shown.

[0297] In some embodiments, the IL-15 prodrugs provided herein comprise (i) one or more IL-15 cytokines, (ii) one or more masking polypeptides (MPs) as described herein, and (iii) one or more cleavable moieties (CMs) as described herein.

[0298] In some embodiments, the IL-15 prodrugs provided herein, wherein the IL-15 cytokine (I) is linked to the masking polypeptide (MP) by a cleavable moiety (CM).

[0299] In some embodiments, the IL-15 prodrugs provided herein, wherein the cleavable moiety (CM) is linked to the IL-15 cytokine (I), preferably by a non-cleavable linker (L).

[0300] In some embodiments, the IL-15 prodrugs provided herein, wherein the cleavable moiety (CM) is linked to the masking polypeptide (MP), preferably by a non-cleavable linker (L).

[0301] In some embodiments, the IL-15 prodrugs provided herein comprise a construct of I-CM-MP from the N- to the C-terminus or C- to the N-terminus, where "-" represents a covalent bond with or without a non-cleavable linker (L).

[0302] In some embodiments, the IL-15 prodrugs provided herein further comprise one or more half-life extending moieties (C).

[0303] In some embodiments, the IL-15 prodrugs provided herein, wherein the IL-15 cytokine (I) is linked to the half-life extending moiety (C).

[0304] In some embodiments, the IL-15 prodrugs provided herein, wherein the IL-15 cytokine (I) is linked to the half-life extending moiety (C), preferably via a non-cleavable linker (L).

[0305] In some embodiments, the IL-15 prodrugs provided herein, wherein the masking polypeptide (MP) is linked to the half-life extending moiety (C) via a cleavable moiety (CM).

[0306] In some embodiments, the IL-15 prodrugs provided herein, wherein the cleavable moiety (CM) is linked to the masking polypeptide (MP) via a non-cleavable linker (L).

[0307] In some embodiments, the IL-15 prodrugs provided herein, wherein the cleavable moiety (CM) is linked to the half-life extending moiety (C), preferably via a non-cleavable linker (L).

[0308] In some embodiments, the IL-15 prodrugs provided herein comprise a construct of C-I-CM-MP from the N- to the C-terminus or the C- to the N-terminus, wherein "-" represents a covalent bond with or without a non-cleavable linker (L).

[0309] In some embodiments, the IL-15 prodrugs provided herein comprise a construct of I-C-CM-MP from the N- to the C-terminus or the C- to the N-terminus, wherein "-" represents a covalent bond with or without a non-cleavable linker (L).

[0310] In some embodiments, the IL-15 prodrugs provided herein comprise a construct of C-CM-MP-I from the N- to the C-terminus or the C- to the N-terminus, wherein "-" represents a covalent bond with or without a non-cleavable linker (L).

[0311] In some embodiments, the IL-15 prodrugs provided herein are dimers, wherein one monomer comprises a construct of C-I from the N- to the C-terminus or the C- to the N-terminus; one monomer comprises a construct of C-CM-MP from the N- to the C-terminus or the C- to the N-terminus, and wherein "-" represents a covalent bond with or without a non-cleavable linker (L).

[0312] In some embodiments, the IL-15 prodrugs provided herein comprise (i) one or more IL-15 cytokines (I), (ii) one or more IL-15Rα or functional fragments thereof (S), (iii) one or more masking polypeptides (MP) as described herein, and (iv) one or more cleavable moieties (CM) as described herein.

[0313] In some embodiments, provided herein is an IL-15 prodrug, wherein the IL-15 cytokine (I) is linked to the masking polypeptide (MP) via a cleavable moiety (CM).

[0314] In some embodiments, provided herein is an IL-15 prodrug, wherein the cleavable moiety (CM) is linked to the IL-15 cytokine (I), preferably via a non-cleavable linker (L).

[0315] In some embodiments, provided herein is an IL-15 prodrug, wherein the cleavable moiety (CM) is linked to the masking polypeptide (MP), preferably via a non-cleavable linker (L).

[0316] In some embodiments, provided herein is an IL-15 prodrug, wherein the IL-15Rα or a functional fragment thereof (S) is linked to the IL-15 cytokine (I).

[0317] In some embodiments, provided herein is an IL-15 prodrug, wherein the IL-15Rα or a functional fragment thereof (S) is linked to the masking polypeptide (MP) via a cleavable moiety (CM).

[0318] In some embodiments, provided herein is an IL-15 prodrug, wherein the cleavable moiety (CM) is linked to the IL-15Rα or a functional fragment thereof (S), preferably via a non-cleavable linker (L).

[0319] In some embodiments, provided herein is an IL-15 prodrug, wherein the cleavable moiety (CM) is linked to the masking polypeptide (MP), preferably via a non-cleavable linker (L).

[0320] In some embodiments, provided herein is an IL-15 prodrug, wherein the IL-15Rα or a functional fragment thereof (S) is covalently linked to the IL-15 cytokine (I). In some embodiments, the IL-15Rα or a functional fragment thereof (S) is linked to the IL-15 cytokine (I) via a non-cleavable linker (L).

[0321] In some embodiments, provided herein is an IL-15 prodrug, wherein the IL-15Rα or a functional fragment thereof (S) is non-covalently linked to the IL-15 cytokine (I) and forms an IL-15 / IL-15Rα complex.

[0322] In some embodiments, provided herein is an IL-15 prodrug, wherein the IL-15Rα or a functional fragment thereof and the IL-15 cytokine are transfected separately and form an IL-15 / IL-15Rα complex.

[0323] In some embodiments, the IL-15 prodrugs provided herein further comprise one or more half-life extending moieties (C).

[0324] In some embodiments, the IL-15 prodrugs provided herein, wherein the IL-15 cytokine (I) is linked to the half-life extending moiety (C).

[0325] In some embodiments, the IL-15 prodrugs provided herein, wherein the IL-15 cytokine (I) is linked to the half-life extending moiety (C), preferably, via a non-cleavable linker (L).

[0326] In some embodiments, the IL-15 prodrugs provided herein, wherein the IL-15Rα or its functional fragment (S) is linked to the half-life extending moiety (C).

[0327] In some embodiments, the IL-15 prodrugs provided herein, wherein the IL-15Rα or its functional fragment (S) is linked to the half-life extending moiety (C), preferably, via a non-cleavable linker (L).

[0328] In some embodiments, the IL-15 prodrugs provided herein, wherein the masking polypeptide (MP) is linked to the half-life extending moiety (C) via a cleavable moiety (CM).

[0329] In some embodiments, the IL-15 prodrugs provided herein, wherein the cleavable moiety (CM) is linked to the masking polypeptide (MP), preferably, via a non-cleavable linker (L).

[0330] In some embodiments, the IL-15 prodrugs provided herein, wherein the cleavable moiety (CM) is linked to the half-life extending moiety (C), preferably, via a non-cleavable linker (L).

[0331] In some embodiments, the IL-15 prodrugs provided herein, wherein the IL-15 cytokine (I) is linked to the half-life extending moiety (C), the IL-15Rα or its functional fragment (S) is linked to the IL-15 cytokine (I), and the masking polypeptide (MP) is linked to the IL-15Rα or its functional fragment (S) via a cleavable moiety (CM).

[0332] In some embodiments, the IL-15 prodrugs provided herein, wherein the IL-15 cytokine (I) is linked to the half-life extending moiety (C), the IL-15Rα or its functional fragment (S) is linked to the half-life extending moiety (C), and the masking polypeptide (MP) is linked to the IL-15 cytokine (I) via a cleavable moiety (CM).

[0333] In some embodiments, the IL-15 prodrugs provided herein, wherein the IL-15Rα or its functional fragment(s) is / are linked to the half-life extending moiety (C), the IL-15 cytokine (I) is linked to the IL-15Rα or its functional fragment(s), and the masking polypeptide (MP) is linked to the IL-15 cytokine (I) through a cleavable moiety (CM).

[0334] In some embodiments, the IL-15 prodrugs provided herein, wherein the IL-15 cytokine (I) is linked to the half-life extending moiety (C), the IL-15Rα or its functional fragment(s) is / are linked to the half-life extending moiety (C), and the masking polypeptide (MP) is linked to the IL-15Rα or its functional fragment(s) through a cleavable moiety (CM).

[0335] In some embodiments, the IL-15 prodrugs provided herein, wherein the masking polypeptide (MP) is linked to the half-life extending moiety (C) through a cleavable moiety (CM), the IL-15Rα or its functional fragment(s) is / are linked to the half-life extending moiety (C), and the IL-15 cytokine (I) is linked to the IL-15Rα or its functional fragment(s).

[0336] In some embodiments, the IL-15 prodrugs provided herein, wherein the masking polypeptide (MP) is linked to the half-life extending moiety (C) through a cleavable moiety (CM), the IL-15 cytokine (I) is linked to the half-life extending moiety (C), and the IL-15Rα or its functional fragment(s) is / are linked to the IL-15 cytokine (I).

[0337] In some embodiments, the IL-15 prodrugs provided herein, wherein the IL-15Rα or its functional fragment(s) is / are covalently linked to the IL-15 cytokine (I). In some embodiments, the IL-15Rα or its functional fragment(s) is / are linked to the IL-15 cytokine (I) through a non-cleavable linker (L).

[0338] In some embodiments, the IL-15 prodrugs provided herein, wherein the IL-15Rα or its functional fragment(s) is / are non-covalently linked to the IL-15 cytokine (I) and form an IL-15 / IL-15Rα complex.

[0339] In some embodiments, the IL-15 prodrugs provided herein comprise a construct of C-I-S-CM-MP from the N- to C-terminus or C- to N-terminus, wherein "-" represents a covalent bond with or without a non-cleavable linker (L).

[0340] In some embodiments, the IL-15 prodrugs provided herein comprise a construct of C-S-I-CM-MP from the N- to the C-terminus or C- to N-terminus, wherein the "-" represents a covalent bond with or without a non-cleavable linker (L).

[0341] In some embodiments, the IL-15 prodrugs provided herein comprise a construct of I-C-S-CM-MP from the N- to the C-terminus or C- to N-terminus, wherein the "-" represents a covalent bond with or without a non-cleavable linker (L).

[0342] In some embodiments, the IL-15 prodrugs provided herein comprise a construct of S-C-I-CM-MP from the N- to the C-terminus or C- to N-terminus, wherein the "-" represents a covalent bond with or without a non-cleavable linker (L).

[0343] In some embodiments, the IL-15 prodrugs provided herein comprise a construct of S-I-C-CM-MP from the N- to the C-terminus or C- to N-terminus, wherein the "-" represents a covalent bond with or without a non-cleavable linker (L).

[0344] In some embodiments, the IL-15 prodrugs provided herein comprise a construct of I-S-C-CM-MP from the N- to the C-terminus or C- to N-terminus, wherein the "-" represents a covalent bond with or without a non-cleavable linker (L).

[0345] In some embodiments, the IL-15 prodrugs provided herein are monomers.

[0346] In some embodiments, the IL-15 prodrugs provided herein are dimers. In some embodiments, the dimer is monovalent. In some embodiments, the dimer is divalent. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer is a heterodimer.

[0347] In some embodiments, the IL-15 prodrugs provided herein are dimers, wherein one monomer comprises a first half-life extending moiety (C), the IL-15Rα or a functional fragment thereof (S), the IL-15 cytokine (I); the other monomer comprises a second half-life extending moiety (C), the masking polypeptide (MP) and the cleavable moiety (CM), wherein the masking polypeptide (MP) is linked to the half-life extending moiety (C) through the cleavable moiety (CM).

[0348] In some embodiments, the IL-15 prodrugs provided herein are dimers, wherein one monomer comprises a first half-life extending moiety (C), the IL-15Rα or a functional fragment thereof (S), the masking polypeptide (MP), and the cleavable moiety (CM); and the other monomer comprises a second half-life extending moiety (C) and the IL-15 cytokine (I).

[0349] In some embodiments, the IL-15 prodrugs provided herein are dimers, wherein one monomer comprises a first half-life extending moiety (C), the IL-15 cytokine (I), the masking polypeptide (MP), and the cleavable moiety (CM); and the other monomer comprises a second half-life extending moiety (C) and the IL-15Rα or a functional fragment thereof (S).

[0350] In some embodiments, the IL-15 prodrugs provided herein are dimers, wherein one monomer and the other monomer each comprise a half-life extending moiety (C), the IL-15 cytokine (I), the IL-15Rα or a functional fragment thereof (S), the cleavable moiety (CM), and the masking polypeptide (MP).

[0351] In some embodiments, the IL-15 prodrugs provided herein are dimers, wherein in one monomer: the IL-15 cytokine (I) is linked to a first half-life extending moiety (C), and the IL-15Rα or a functional fragment thereof (S) is linked to the first half-life extending moiety (C); and in the other monomer: the masking polypeptide (MP) is linked to a second half-life extending moiety (C) via the cleavable moiety (CM).

[0352] In some embodiments, the IL-15 prodrugs provided herein are dimers, wherein in one monomer: the IL-15 cytokine (I) is linked to a first half-life extending moiety (C), and the IL-15Rα or a functional fragment thereof (S) is linked to the IL-15 cytokine (I); and in the other monomer: the masking polypeptide (MP) is linked to a second half-life extending moiety (C) via the cleavable moiety (CM).

[0353] In some embodiments, the IL-15 prodrugs provided herein are dimers, wherein in one monomer: the IL-15Rα or a functional fragment thereof (S) is linked to a first half-life extending moiety (C), and the masking polypeptide (MP) is linked to the first half-life extending moiety (C) via the cleavable moiety (CM); and in the other monomer: the IL-15 cytokine (I) is linked to a second half-life extending moiety (C).

[0354] In some embodiments, the IL-15 prodrug provided herein is a dimer, wherein in one monomer: the IL-15Rα or its functional fragment(s) is / are linked to a first half-life extension moiety (C), and the masking polypeptide (MP) is linked to the IL-15Rα or its functional fragment(s) through the cleavable moiety (CM); and in the other monomer: the IL-15 cytokine (I) is linked to a second half-life extension moiety (C).

[0355] In some embodiments, the IL-15 prodrug provided herein is a dimer, wherein in one monomer: the IL-15 cytokine (I) is linked to a first half-life extension moiety (C), and the masking polypeptide (MP) is linked to the first half-life extension moiety (C) through the cleavable moiety (CM); and in the other monomer: the IL-15Rα or its functional fragment(s) is / are linked to a second half-life extension moiety (C).

[0356] In some embodiments, the IL-15 prodrug provided herein is a dimer, wherein in one monomer: the IL-15 cytokine (I) is linked to a first half-life extension moiety (C), and the masking polypeptide (MP) is linked to the IL-15 cytokine (I) through the cleavable moiety (CM); and in the other monomer: the IL-15Rα or its functional fragment(s) is / are linked to a second half-life extension moiety (C).

[0357] In some embodiments, the IL-15 prodrug provided herein, wherein the IL-15Rα or its functional fragment(s) is / are covalently linked to the IL-15 cytokine (I). In some embodiments, the IL-15Rα or its functional fragment(s) is / are linked to the IL-15 cytokine (I) through a non-cleavable linker (L).

[0358] In some embodiments, the IL-15 prodrug provided herein, wherein the IL-15Rα or its functional fragment(s) is / are non-covalently linked to the IL-15 cytokine (I) and form an IL-15 / IL-15Rα complex.

[0359] IL-15:

[0360] Wild-type IL-15 is a member of the four α-helix bundle family, with 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), and is produced by mononuclear phagocytes and other immune system cells. IL-15 is crucial for the development and function of natural killer cells (NK), natural killer T cells (NKT), and memory CD8+ T cells.

[0361] IL-15 is a cytokine similar to IL-2 and was initially called T cell growth factor. These two cytokines exert their cell signaling functions by binding to receptors composed of a trimeric complex, which consists of two shared receptor chains (i.e., common γ chain (γc; CD132) and IL-2 receptor β chain (IL-2Rβ; CD122)) and an α-chain receptor specific to each cytokine (IL-2 receptor α (IL-2Rα; CD25) or IL-15 receptor α (IL-15Rα; CD215)).

[0362] IL-15 shares receptor components with IL-2. The α chain of the IL-2 receptor (IL-2R) is not essential, but the β chain and common γ chain are essential for IL-15-mediated biological activities (Giri JG, et al. IL-15, a novel T cell growth factor that shares activities and receptor components with IL-2. J Leukoc Biol. 1995 May; 57(5):763-6.). The IL-15R consists of three subunits: IL-15Rα, IL-2 / IL-15Rβ, and γ chain. IL-15Rα is essential for high-affinity binding but does not transmit the signal of IL-15. IL-15 mainly functions through trans-presentation (TP), during which APCs expressing IL-15 bind IL-15Rα and present the ligand to the βγ receptor heterodimer on surrounding T / NK cells (Kenesei Volkó J, et al. IL-15 Trans-Presentation Is an Autonomous, Antigen-Independent Process. J Immunol. 2021 Nov 15; 207(10):2489-2500).

[0363] As described herein, the term "IL-15 cytokine" or "IL-15" includes wild-type IL-15 or its variants, and also includes its functional fragments. In some embodiments, the IL-15 or IL-15 cytokine of the present application is wild-type IL-15. In some embodiments, the IL-15 or IL-15 cytokine of the present application is an IL-15 variant. In eukaryotic cells, wild-type IL-15 is first synthesized as a 162-amino acid precursor polypeptide, and then processed into 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 an active mature form.

[0364] In some embodiments, the IL-15 cytokine comprises the amino acid sequence SEQ ID NO:22 or a variant thereof, the variant 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 SEQ ID NO:22.

[0365] In some embodiments, the IL-15 cytokine comprises the amino acid sequence SEQ ID NO:23 or a variant thereof, the variant 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 SEQ ID NO:23.

[0366] In some embodiments, the IL-15 cytokine is also an IL-15 variant or its functional fragment. 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 variant thereof that can bind to the interleukin-15 receptor (IL-15R) or its components (e.g., the IL-15Rα, IL-2 / IL-15Rβ and / or γ chain) or otherwise exhibit enhanced or reduced affinity.

[0367] In some embodiments, the IL-15 cytokine is an IL-15 variant that comprises an amino acid sequence obtained by modifying at least one amino acid of the wild-type IL-15 amino acid sequence (SEQ ID NO: 22). Each of the at least one amino acid modifications can be any amino acid modification, such as a mutation, insertion, or deletion. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct product as long as the final construct product has the desired characteristics, e.g., retains / enhances / reduces ligand-receptor binding, retains / enhances / reduces biological activity, etc. In some embodiments, the IL-15 cytokine comprises an amino acid sequence resulting from at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid substitutions on the amino acid sequence SEQ ID NO: 22.

[0368] In some embodiments, the IL-15 cytokine is an IL-15 variant well-known to those skilled in the art that comprises a sequence obtained by making at least one mutation in human native wild-type IL-15, and the mutations include substitutions, deletions, or additions at residues 45, 48, 51, 52, 64, 67, 68, and / or 72 (see US patent 9,493,533B2, and Han KP, et al., IL-15: IL-15 receptor alpha superagonist complex: high-level co-expression in recombinant mammalian cells, purification and characterization. Cytokine. 2011 Dec;56(3):804-10). In some embodiments, the IL-15 variant comprises one or more amino acid mutations selected from the group consisting of L45D, L45E, Q48K, S51D, L52D, E64K, I67D, I67E, I68D, and N72D, wherein the residue numbering is relative to human native wild-type IL-15 (e.g., SEQ ID NO: 22).

[0369] In some embodiments, the IL-15 cytokine comprises an amino acid sequence shown in any of SEQ ID NOs: 67-76.

[0370] IL-15Rα or a functional fragment thereof:

[0371] The IL-15Rα or a functional fragment thereof according to the present application can be an IL-15Rα or a functional fragment thereof of any species.

[0372] In some embodiments, the IL-15Rα or its functional fragment is selected from the extracellular region of human IL-15Rα or the sushi domain or functional analogs thereof.

[0373] Extracellular region of IL-15Rα:

[0374] The extracellular region of the IL-15Rα is generally defined as the sequence of the IL-15Rα extending from its first N-terminal amino acid to the last amino acid of the tail region (or the region rich in glycosylation sites). The tail region of the IL-15Rα sequence can be determined by those skilled in the art, for example, with the help of software.

[0375] IL-15Rα_sushi domain:

[0376] The extracellular region of the IL-15Rα contains a domain known as the sushi domain (Wei et al. 2001, J. Immunol. 167:277-282). The IL-15Rα_sushi domain has a β-sheet.

[0377] The IL-15Rα_sushi domain has most of the binding affinity for IL-15 and enhances the binding and biological effects (proliferation and protection against apoptosis) of IL-15 through the IL-15Rβγ heterodimer without affecting the binding and function of IL-15 (Mortier E, et al. J Biol Chem. 2006 Jan 20;281(3):1612-9), and can act as an effective IL-15 antagonist.

[0378] 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 starts from the cysteine residue (C1) encoded by the first exon 2 and ends at the cysteine residue (C4) encoded by the fourth exon 2. Looking at the IL-15Rα protein sequence (in the standard N-terminal to C-terminal direction), the sushi domain of IL-15Rα can be defined as starting from the first cysteine residue (C1) after the signal peptide and ending at 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 suitable analysis software such as: Prosite

[0379] (http: / / us.expasy.org / prosite / ), (http: / / www.ebi.ac.uk / lnterProScan / ), SMART (http: / / elm.eu.org / ).

[0380] In some embodiments, the IL-15Rα_sushi domain comprises the amino acid sequence SEQ ID NO:24 or a variant thereof, the variant having 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.

[0381] In some embodiments, the IL-15Rα_sushi domain comprises the amino acid sequence SEQ ID NO:25 or a variant thereof, the variant having 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.

[0382] In some embodiments, the IL-15Rα_sushi domain comprises the amino acid sequence SEQ ID NO:26 or a variant thereof, the variant having 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.

[0383] In some embodiments, the IL-15 prodrug, wherein the IL-15 cytokine or the IL-15Rα or a functional fragment thereof (e.g., the IL-15Rα_sushi domain) has one or more conservative amino acid substitutions.

[0384] "Conservative substitution" means substitution by another amino acid having the same net charge and substantially the same size and shape as the amino acid being replaced. Amino acids with aliphatic or substituted aliphatic amino acid side chains are of substantially the same size when the total number of carbon and heteroatoms in the side chain differs by no more than four. Amino acids are of substantially the same shape when the number of branches in their side chains differs by no more than one. Amino acids having a phenyl or substituted phenyl group in the side chain can be considered to be of substantially the same size and shape. Unless otherwise stated, conservative substitutions preferably apply to natural amino acids.

[0385] As used herein, the term "amino acid" is used in its broadest definition, i.e., it 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. In accordance with this broad definition, those skilled in the art will find that the amino acids described herein include, for example, the natural L-amino acids that form proteins; D-amino acids; chemically modified amino acids such as amino acid analogs and derivatives; natural amino acids that do not form proteins such as norleucine, β-alanine, ornithine, GABA, etc.; and chemically synthesized compounds known in the art having the characteristics of amino acids. As used herein, the term "protein-forming" refers to amino acids that can be synthesized into peptides, polypeptides or proteins of a cell through metabolic pathways.

[0386] Inserting non-natural amino acids, including synthetic non-natural amino acids, substituted amino acids or one or more D-amino acids, into a polypeptide (e.g., the IL-15 cytokine in the IL-15 prodrug described herein) can have various benefits. Polypeptides containing D-amino acids, etc., exhibit higher stability in vitro and in vivo compared to polypeptides containing L-amino acids. Therefore, when better intracellular stability is required, construction of a polypeptide by adding D-amino acids is particularly useful. In particular, D-peptides and their analogs are able to withstand endogenous peptidase and protease activities, thereby enhancing the bioavailability of the molecule and prolonging its lifespan in vivo when needed. In addition, D-peptides and their analogs cannot be effectively processed due to the limited presentation of class II major histocompatibility complex (MHC) to helper T cells, and thus are not likely to induce a humoral immune response in a subject.

[0387] Table B shows conservative substitutions. More substantial substitutions are provided under the heading "Exemplary Substitutions" in Table B, which are further detailed below in the section on amino acid side chain classes. Amino acids can be classified according to common side chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain direction: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions require replacing a member of one of these classes with a member of another class. Amino acid substitutions can be introduced into the protein construct, and products meeting the required activities described above can be screened.

[0388] Table B. Amino Acid Substitutions

[0389]

[0390]

[0391] In some embodiments, the IL-15 prodrug provided herein, wherein the masking polypeptide (MP) reduces the binding affinity of IL-15 or its functional fragment for IL-2 / IL-15Rβγ as compared to IL-15 or its functional fragment without an MP. In some embodiments, the binding affinity is reduced by at least about 10-fold as compared to IL-15 or its functional fragment without an MP. In some embodiments, the binding affinity is reduced by at least about 100-fold as compared to IL-15 or its functional fragment without an MP. In some embodiments, the binding affinity is reduced by at least about 200 to 1500-fold as compared to IL-15 or its functional fragment without an MP. In some embodiments, the masking polypeptide (MP) comprises an amino acid sequence shown in any of SEQ ID NOs: 1-5 or a variant thereof, the variant 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 shown in any of SEQ ID NOs: 1-5.

[0392] 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 specific conditions, thereby separating its N-terminal fragment from its C-terminal fragment. By introducing the cleavable moiety (CM), the masking polypeptide (MP) is removed at the cleavable site under specific conditions, thereby releasing the fully functional IL-15 cytokine.

[0393] In some embodiments, screening for a suitable cleavable moiety (CM) depends on the desired site of action of the IL-15 cytokine. For example, when the tumor site is the desired site of action, a cleavage site of a tumor-specific protease is used to construct an IL-15 prodrug designed to act on the tumor site. A tumor-specific protease refers to any protease that has a high level and / or activity at the tumor site relative to normal tissue.

[0394] In some embodiments, the protease cleavage site can be a cleavage site of matrix metalloproteinase (MMP). In some embodiments, the protease cleavage site can be a cleavage site of MMP2. In some embodiments, the protease cleavage site can be a cleavage site of MMP9. In some embodiments, the protease cleavage site can be a cleavage site of MMP2 and MMP9. Other tumor-specific proteases and corresponding cleavage sites are known in the art, as disclosed in Vasiljeva et al., Scientific Reports, 10:5894, 2020, the relevant disclosure of which is incorporated herein by reference and is consistent with the subject matter and purpose of this article. In some embodiments, the cleavable moiety (CM) comprises an amino acid sequence shown in any of SEQ ID NOs: 8-16 or a variant thereof, and the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 8-16.

[0395] In some embodiments, the IL-15 prodrug provided herein, wherein the half-life extension moiety (C) comprises an Fc domain; preferably, the 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, the Fc domain is a human IgG1 Fc domain.

[0396] In some embodiments, the IL-15 prodrug provided herein, wherein the half-life extension moiety (C) comprises a human IgG1 Fc domain, which comprises L234A and L235A (LALA) mutations, and the mutation sites are numbered according to the EU numbering system.

[0397] In some embodiments, the IL-15 prodrug provided herein, wherein the half-life extension moiety (C) further comprises knobs-into-holes mutations (Fc knob and Fc hole).

[0398] In some embodiments, the IL-15 prodrugs provided herein, wherein the Fc knob contains a T366W mutation in the Fc domain, and the Fc hole contains T366S, L368A, and Y407V mutations in the Fc domain, and the mutation sites are numbered according to the EU numbering system.

[0399] In some embodiments, the IL-15 prodrugs provided herein, wherein the Fc knob further contains an S354C mutation in the Fc domain, and the Fc hole further contains a Y349C mutation in the Fc domain, and the mutation sites are numbered according to the EU numbering system.

[0400] In some embodiments, the IL-15 prodrugs provided herein, wherein the Fc knob comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, and the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:28.

[0401] In some embodiments, the IL-15 prodrugs provided herein, wherein the Fc hole comprises the amino acid sequence SEQ ID NO:27 or a variant thereof, and the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:27.

[0402] In some embodiments, the IL-15 prodrugs provided herein, wherein the Fc knob and Fc hole further contain LALA mutations.

[0403] In some embodiments, the IL-15 prodrugs provided herein, wherein the Fc knob LALA comprises the amino acid sequence SEQ ID NO:30 or a variant thereof, and the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:30.

[0404] In some embodiments, the IL-15 prodrugs provided herein, wherein the Fc hole LALA comprises the amino acid sequence SEQ ID NO:29 or a variant thereof, and the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:29.

[0405] In some embodiments, the IL-15 prodrug comprises two monomers, wherein in one monomer, the IL-15Rα or its functional fragment is linked to the Fc domain; in the other monomer, the IL-15 cytokine (I) is linked to the Fc domain, and the masking polypeptide (MP) is linked to the IL-15 cytokine (I) through the cleavable moiety (CM).

[0406] In some embodiments, the IL-15 prodrugs provided herein comprise the constructs shown in Table 2.

[0407] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO:31 or a variant thereof, the variant having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO:31, and the second monomer comprises the amino acid sequence SEQ ID NO:32 or a variant thereof, the variant having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO:32.

[0408] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO:33 or a variant thereof, the variant having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO:33, and the second monomer comprises the amino acid sequence SEQ ID NO:32 or a variant thereof, the variant having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO:32.

[0409] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO:34 or a variant thereof, the variant having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO:34, and the second monomer comprises the amino acid sequence SEQ ID NO:32 or a variant thereof, the variant having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO:32.

[0410] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO:36 or a variant thereof that 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 second monomer comprises the amino acid sequence SEQ ID NO:32 or a variant thereof that 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.

[0411] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO:37 or a variant thereof that 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 second monomer comprises the amino acid sequence SEQ ID NO:32 or a variant thereof that 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.

[0412] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO:38 or a variant thereof that 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 second monomer comprises the amino acid sequence SEQ ID NO:32 or a variant thereof that 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.

[0413] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO:39 or a variant thereof, the variant having 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 second monomer comprises the amino acid sequence SEQ ID NO:32 or a variant thereof, the variant having 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.

[0414] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO:45 or a variant thereof, the variant having 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 second monomer comprises the amino acid sequence SEQ ID NO:46 or a variant thereof, the variant having 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.

[0415] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO:47 or a variant thereof, the variant having 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 second monomer comprises the amino acid sequence SEQ ID NO:40 or a variant thereof, the variant having 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.

[0416] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO:33 or a variant thereof that 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 second monomer comprises the amino acid sequence SEQ ID NO:40 or a variant thereof that 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.

[0417] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO:62 or a variant thereof that 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:62, and the second monomer comprises the amino acid sequence SEQ ID NO:63 or a variant thereof that 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:63.

[0418] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO:41 or a variant thereof that 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 second monomer comprises the amino acid sequence SEQ ID NO:63 or a variant thereof that 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:63.

[0419] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO: 64 or a variant thereof, the variant having 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: 64, and the second monomer comprises the amino acid sequence SEQ ID NO: 65 or a variant thereof, the variant having 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: 65.

[0420] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO: 62 or a variant thereof, the variant having 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: 62, and the second monomer comprises the amino acid sequence SEQ ID NO: 65 or a variant thereof, the variant having 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: 65.

[0421] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof, the variant having 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 second monomer comprises the amino acid sequence SEQ ID NO: 65 or a variant thereof, the variant having 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: 65.

[0422] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO:66 or a variant thereof, the variant having 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:66, and the second monomer comprises the amino acid sequence SEQ ID NO:65 or a variant thereof, the variant having 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:65.

[0423] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO:43 or a variant thereof, the variant having 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 second monomer comprises the amino acid sequence SEQ ID NO:46 or a variant thereof, the variant having 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.

[0424] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO:39 or a variant thereof, the variant having 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 second monomer comprises the amino acid sequence SEQ ID NO:40 or a variant thereof, the variant having 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.

[0425] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof that 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 second monomer comprises the amino acid sequence SEQ ID NO: 87 or a variant thereof that 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: 87.

[0426] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof that 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 second monomer comprises the amino acid sequence SEQ ID NO: 88 or a variant thereof that 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: 88.

[0427] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof that 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 second monomer comprises the amino acid sequence SEQ ID NO: 89 or a variant thereof that 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: 89.

[0428] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof that 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 second monomer comprises the amino acid sequence SEQ ID NO: 90 or a variant thereof that 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: 90.

[0429] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof that 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 second monomer comprises the amino acid sequence SEQ ID NO: 91 or a variant thereof that 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: 91.

[0430] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof that 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 second monomer comprises the amino acid sequence SEQ ID NO: 92 or a variant thereof that 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: 92.

[0431] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof, the variant having 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 second monomer comprises the amino acid sequence SEQ ID NO: 93 or a variant thereof, the variant having 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: 93.

[0432] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof, the variant having 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 second monomer comprises the amino acid sequence SEQ ID NO: 94 or a variant thereof, the variant having 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: 94.

[0433] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof, the variant having 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 second monomer comprises the amino acid sequence SEQ ID NO: 95 or a variant thereof, the variant having 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: 95.

[0434] In some embodiments, the IL-15 prodrugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof that 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 second monomer comprises the amino acid sequence SEQ ID NO: 96 or a variant thereof that 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: 96.

[0435] On the other hand, the present application also provides an IL-15 drug without a masking polypeptide (MP) and a cleavable moiety (CM), which comprises two monomers, wherein in one monomer, the IL-15Rα or its functional fragment(s) is linked to a first Fc domain, and in the other monomer, the IL-15 cytokine (I) is linked to a second Fc domain.

[0436] In some embodiments, the IL-15 drugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO: 43 or a variant thereof that 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 second monomer comprises the amino acid sequence SEQ ID NO: 77 or a variant thereof that 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: 77.

[0437] In some embodiments, the IL-15 drugs provided herein comprise two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO: 43 or a variant thereof that 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 second monomer comprises the amino acid sequence SEQ ID NO: 81 or a variant thereof that 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: 81.

[0438] In some embodiments, the IL-15 agent provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO: 43 or a variant thereof that 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 second monomer comprises the amino acid sequence SEQ ID NO: 83 or a variant thereof that 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: 83.

[0439] In some embodiments, the IL-15 agent provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence SEQ ID NO: 43 or a variant thereof that 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 second monomer comprises the amino acid sequence SEQ ID NO: 84 or a variant thereof that 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: 84.

[0440] Binding affinity

[0441] The binding affinity of a molecule (e.g., IL-15 or a functional fragment thereof) for its binding partner (e.g., IL-2 / IL-15Rβγ) can be determined by any suitable ligand binding assay or antibody / antigen binding assay known in the art, such as Western blot, enzyme-linked immunosorbent assay (ELISA), Meso Scale Discovery (MSD) electrochemiluminescence, bead-based multiplex immunoassay (MIA), RIA, surface plasmon resonance (SPR), ECL, IRMA, EIA, Biacore assay, Octet analysis, peptide scanning, etc. For example, a simple assay can be performed by labeling IL-15 or a functional fragment thereof or its receptor (e.g., IL-2 / IL-15Rβγ), or a subunit thereof, with various labeling reagents. Similarly, BiacoreX (Amersham Biosciences), an off-the-shelf measurement kit or a similar kit, can be used and operated according to the user manual and experimental procedures accompanying the kit.

[0442] In some embodiments, protein microarrays are used for large-scale analysis of the interaction, function, and activity of IL-15 or its functional fragments described herein with its receptor. The protein microarray has a support surface that binds to a series of capture proteins (e.g., IL-15 receptor or its subunits). Subsequently, a fluorescently labeled probe molecule (e.g., IL-15 or its functional fragment described herein) is added to the array and interacts with the bound capture protein, releasing a fluorescent signal that is read by a laser scanner.

[0443] Binding affinity can also be measured using SPR (Biacore T-200). For example, an anti-human IgG antibody is coupled to the surface of a CM-5 sensor chip using EDC / NHS chemistry. Then, a human IL-2 / IL-15Rβγ-Fc fusion protein is used as the capture ligand on this surface. Serial dilutions of the IL-15 prodrug or drug described herein are allowed to bind to the capture ligand, and the binding and dissociation of the IL-15 prodrug or drug to IL-2 / IL-15Rβγ can be monitored in real time. The dissociation constant (K d ) and the dissociation rate constant can be determined by kinetic analysis using BIA evaluation software.

[0444] Pharmacokinetics (PK)

[0445] Pharmacokinetics (PK) refers to the absorption, distribution, metabolism, and excretion of a drug (e.g., the IL-15 cytokine or IL-15 prodrug described herein) after administration to a subject. Pharmacokinetic parameters that can be used to determine clinical utility include, but are not limited to, serum / plasma concentration, serum / plasma concentration over time, maximum serum / plasma concentration (C max ), time to reach maximum concentration (T max ), half-life (t 1 / 2 ), area under the concentration-time curve during the dosing interval (AUC τ ), etc.

[0446] Techniques for obtaining a drug PK curve are known in the art, for example, the IL-15 cytokine or IL-15 prodrug described herein. See Heller et al., Annu Rev Anal Chem, 11, 2018; and Ghandforoush Sattari et al., J Amino Acids, Article ID 346237, Volume 2010. In some embodiments, the PK curve of the IL-15 cytokine or IL-15 prodrug as described herein is measured in a blood, plasma, or serum sample of an individual. In some embodiments, mass spectrometry techniques (e.g., LC-MS / MS or ELISA) are used to measure the PK curve of the IL-15 cytokine or IL-15 prodrug as described herein in an individual. PK analysis can be performed on the PK curve 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).

[0447] “C” represents the concentration of a drug or prodrug (e.g., IL-15 cytokine or IL-15 prodrug) in a subject's plasma, serum, or any suitable body 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 “serum concentration” or “plasma concentration”. The serum / plasma concentration at any time after administration (e.g., for the IL-15 cytokine or IL-15 prodrug, e.g., intravenous injection, intraperitoneal injection, or subcutaneous injection) is referred to as C time or C t . The maximum serum / plasma drug concentration during administration is referred to as C max ; C min refers to the minimum serum / plasma drug concentration at the end of the dosing interval; C ave refers to the average concentration during the dosing interval.

[0448] The term “bioavailability” refers to the extent or rate at which a drug or prodrug (e.g., IL-15 cytokine or IL-15 prodrug) passes through the systemic circulation and thus reaches the site of action.

[0449] “AUC” is the area under the serum / plasma concentration-time curve and is considered the most reliable measure of bioavailability, such as the area under the concentration-time curve within the dosing interval (AUCτ ), "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 time t after administration (AUC 0-t ), etc.

[0450] The time to peak serum / plasma concentration (T max ) is the time at which the peak serum / plasma concentration (Cmax) is reached after administration of a drug or prodrug (e.g., an IL-15 cytokine or an IL-15 prodrug).

[0451] The half-life (t 1 / 2 ) is the time required for the concentration of a drug or prodrug (e.g., an IL-15 cytokine or an IL-15 prodrug) measured in plasma or serum (or other biological matrix) to decrease to half of its concentration or amount at a specific time point. For example, after intravenous administration, due to the distribution and elimination of the drug, the drug concentration in plasma or serum decreases. In the curve of plasma or serum drug concentration versus time after intravenous administration, the first phase or rapid decline phase is considered to be mainly due to distribution, while the later decline is usually slower and mainly due to elimination, although both processes occur in both phases. Drug distribution can be completed after sufficient time. Generally speaking, the elimination half-life is determined by the terminal or elimination (main) phase of the plasma / serum concentration-time curve. See Michael Schrag and Kelly Regal, "Chapter 3 - Pharmacokinetics and Toxicokinetics" of "Comprehensive Guide to Preclinical Drug Development Toxicology", 2013.

[0452] Stability

[0453] In some embodiments, 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 embodiments, the IL-15 prodrugs and anti-TNFR2 antibody prodrugs described herein have excellent stability under accelerated stress (e.g., high temperature), such as less or no fragmentation, aggregate formation, and / or aggregate increment.

[0454] The stability of proteins, particularly their sensitivity to aggregation, depends primarily on the conformation and colloidal stability of the protein molecule. It is generally believed that the first step in the aggregation of non-native proteins, which is the most common form of aggregation, is a slight perturbation of the molecular structure, e.g., partial unfolding of the protein, i.e., a conformational change. This is determined by the conformational stability of the protein. In the second step, the partially unfolded molecules approach each other driven by diffusion and random Brownian motion to form aggregates. The second step is mainly determined by the colloidal stability of the molecules (see Chi et 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 bioactive substance, such as a protein, or minimizing its degradation, denaturation, aggregation, or unfolding. As used herein, "improved stability" generally means that, under conditions known to cause degradation, denaturation, aggregation, or unfolding, the target protein (e.g., the IL-15 prodrug described herein) exhibits better stability compared to a control protein (e.g., other IL-15 prodrugs).

[0455] Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) are well-known techniques in the art for predicting the stability of protein formulations. Specifically, these techniques can be used to determine the unfolding temperature (T m ) of a protein in a given formulation. It is standard practice in the art to correlate high T m measurements of a protein in a given formulation with more reliable and stable protein formulations that can be used for long-term, stable storage.

[0456] A "stable" masking polypeptide (MP) or prodrug (or formulation), e.g., an IL-15 prodrug or an anti-TNFR2 antibody prodrug as described herein, substantially maintains its physical stability and / or chemical stability and / or biological activity during manufacture and / or storage. There are various analytical techniques in the art for measuring protein stability and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. (1993) Adv. Drug Delivery Rev. 10:29-90. 10:29-90. For example, in one embodiment, the stability of a protein is determined by the percentage of monomeric protein in solution, where the percentage of degraded (e.g., fragmented) and / or aggregated protein is low. Preferably, the protein (or formulation) is stable at room temperature (about 30 °C) or 40 °C for at least 1 month and / or at about 2-8 °C for at least 6 months, or at least 1 year or at least 2 years. In addition, the protein (or formulation) is preferably stable after freezing (e.g., -70 °C) and thawing, hereinafter referred to as "freeze / thaw cycles".

[0457] A prodrug, e.g., an IL-15 prodrug or an anti-TNFR2 antibody prodrug as described herein, "maintains its physical stability" in a formulation if, upon visual inspection of color and / or transparency or by measurement using ultraviolet light scattering or size exclusion chromatography, there are substantially no signs of instability such as aggregation, precipitation, and / or denaturation. Aggregation is the process by which individual protein molecules or complexes bind covalently or non-covalently to form aggregates. Aggregation can proceed to the point of forming visible precipitates.

[0458] A prodrug, e.g., an IL-15 prodrug or an anti-TNFR2 antibody prodrug as described herein, "maintains its chemical stability" in a formulation if its chemical stability over a given time period is such that the protein still retains its biological activity (e.g., as described in the "Biological Activity" subsection above). Chemical stability can be evaluated by, for example, detecting and quantifying different chemical forms of the protein. Chemical changes may involve size alterations (e.g., shearing), which can be evaluated using size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Other types of chemical changes include charge changes (e.g., changes due to deamidation or oxidation), which can be evaluated, for example, by ion exchange chromatography.

[0459] A prodrug, e.g., an IL-15 prodrug or an anti-TNFR2 antibody prodrug as described herein, a protein in a pharmaceutical formulation "retains its biological activity" if the protein 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 the analytical error) of the biological activity shown at the time of formulation preparation.

[0460] As is known to those skilled in the art, the stability of a prodrug (e.g., an IL-15 prodrug or an anti-TNFR2 antibody prodrug as described herein) depends on other characteristics in addition to the composition of the formulation. For example, stability can be affected by temperature, pressure, humidity, pH, and external radiation. The stability of a protein in a protein formulation (e.g., an IL-15 prodrug or an anti-TNFR2 antibody prodrug as described herein) can be determined by a variety of methods. In some embodiments, 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 in its native three-dimensional conformation as described herein from proteins in various denatured states and / or degraded proteins. In SEC, the stationary phase typically consists of inert particles packed in a dense three-dimensional matrix within a glass or steel column. The mobile phase can be pure water, an aqueous buffer, an organic solvent, a mixture thereof, or other solvents. The stationary phase particles have small pores and / or channels that only allow substances smaller than a certain size to enter. Thus, large particles are excluded from these pores and channels, but smaller particles are transferred from the mobile phase. The time that particles are retained in the fixed pores depends to some extent on the depth to which they can penetrate the pores. Their transfer from the mobile phase liquid flow results in a longer time required for their elution from the chromatographic column, and thus separation of the particles based on their size differences.

[0461] In some embodiments, SEC is combined with identification techniques to identify or characterize a protein (e.g., an IL-15 prodrug or an anti-TNFR2 antibody prodrug as described herein) or a fragment thereof. Protein identification and characterization can be accomplished 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, ultraviolet / visible / infrared spectroscopy, Raman spectroscopy, surface enhanced Raman spectroscopy, mass spectrometry, gas chromatography, static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and / or ANS protein binding.

[0462] In some embodiments, optionally, the sample formulation (e.g., comprising the IL-15 prodrug or anti-TNFR2 antibody prodrug described herein) and the control formulation are assayed prior to the processing stage to determine the content of monomers, aggregates, and / or fragmented proteins (and / or percentage increase in fragments, percentage increase in aggregates, etc.). Subsequently, each protein formulation undergoes a processing stage. For example, each protein formulation can be stored for an extended period (e.g., 3 months, 6 months, 12 months, or longer) at a specific temperature (e.g., 40°C, 25°C, or 5°C). In some embodiments, the protein formulation is subjected to physical stress testing, such as a stirring stress test. In some embodiments, the protein formulation is subjected to an accelerated stability test, such as processing under accelerated stress, including high temperature (e.g., 40°C), high humidity, and / or low pH, etc. In some embodiments, the protein formulation undergoes freeze-thaw cycles. In some embodiments, samples of the same protein formulation are subjected to different treatments, e.g., stored at different temperatures for a period of time. After the processing stage, the protein formulation is assayed to determine the content of protein monomers, aggregates, and / or fragments (and / or percentage increase in fragments, percentage increase in aggregates, etc.).

[0463] "Substantial protein aggregation" means that the level of protein aggregation in the protein formulation is significantly higher than that in the control protein formulation. The control protein formulation can be the same protein formulation before storage or before processing (e.g., before being subjected to destabilizing conditions such as high temperature, humidity, pH, and / or long-term storage).

[0464] "Essentially protein aggregation-free" means the protein (or formulation) of the present invention, the level or percentage of whose protein aggregation is not significantly higher than that of the control formulation. In some embodiments, the stability is measured by SEC. In some embodiments, the stability is measured by CE-SDS.

[0465] In some embodiments, stability refers to reduced fragmentation of the IL-15 prodrug or anti-TNFR2 antibody prodrug described herein. The term "low to undetectable fragmentation level" as used herein refers to a sample containing equal to or greater than 80%, 85%, 90%, 95%, 98%, or 99% of the total protein, e.g., in a single peak if determined by HPSEC, or in multiple peaks (e.g., the same number of peaks as the number of subunits) if determined by reduced capillary gel electrophoresis (rCGE), representing the undegraded protein or its undegraded fragments and containing no other single peak that accounts 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. The term "reduced capillary gel electrophoresis" as used herein refers to capillary gel electrophoresis under reducing conditions sufficient to reduce the disulfide bonds in Fc-containing proteins, such as the IL-15 prodrug or anti-TNFR2 antibody prodrug described herein.

[0466] carrier

[0467] 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 prodrug or IL-15 prodrug) described herein, and carriers comprising the nucleic acids encoding the nucleic acids described herein. It also relates to isolated host cells (e.g., CHO cells, HEK 293 cells, Hela cells, or COS cells) comprising the nucleic acids or carriers encoding the nucleic acids described herein. Suitable nucleic acid constructs include, but are not limited to, constructs that can be expressed in eukaryotic or prokaryotic cells. The choice of expression construct is typically made to be compatible with the host cell used. In some embodiments, the carrier encodes a masking polypeptide (e.g., MP80, MP96new, MP100, MP163, or MP240). In some embodiments, the carrier encodes a cleavable moiety (e.g., CM1, CM2, or CM4). In some embodiments, the carrier encodes a non-cleavable linker (e.g., lk, lk1, lk2, lk3, or lk5). In some embodiments, the carrier encodes a protein or a prodrug (e.g., a masking polypeptide or an IL-15 prodrug).

[0468] In some embodiments, the carrier comprising the nucleic acid encoding the prodrug or any prodrug component described herein is suitable for replication and integration in eukaryotic cells, such as mammalian cells (e.g., CHO cells, HEK 293 cells, Hela cells, COS cells). In some embodiments, the carrier is a viral carrier. In some embodiments, the carrier is a non-viral carrier, such as pTT5.

[0469] Numerous 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 derivatives thereof. 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 in other virology and molecular biology manuals. Retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into the vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to engineered mammalian cells in vitro or ex vivo. 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, self-inactivating lentiviral vectors carrying a protein-coding sequence of a construct can be packaged using experimental methods known in the art. The resulting lentiviral vectors can be used to transduce mammalian cells using methods known in the art. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transduction because they allow for long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.

[0470] In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is the 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 the nucleic acid encoding the IL-15 prodrug or the anti-TNFR2 antibody prodrug into a host cell. See Yin H. et al., Nature Rev. Genet. (2014) 15:521-555; Aronovich E. L. et al., “The Sleeping Beauty transposon system: a non-viral vector for gene therapy.” Hum. Mol. Genet. (2011) R1:R14-20 and Zhao S. et al., “PiggyBac transposon vectors: the tools of the human gene editing.” Transl. Lung Cancer Res. (2016) 5(1):120-125, which are incorporated herein by reference. In some embodiments, any one or more nucleic acids or vectors encoding the prodrugs described herein are introduced into a host cell (e.g., CHO, HEK 293, Hela, or COS) by physical methods, including but not limited to electroporation, sonoporation, photoporation, magnetofection, and hydrodynamic transfection.

[0471] In some embodiments, the vector comprises a selectable marker gene or a reporter gene for selecting cells that express the prodrugs described herein from a population of host cells transfected with the vector (e.g., a lentiviral vector, the pTT5 vector). The selectable marker and the reporter gene may both be surrounded by appropriate regulatory sequences to enable their expression in the host cell. For example, the vector may comprise transcription and translation terminators, initiation sequences, and promoters for regulating the expression of the nucleic acid sequence.

[0472] Any molecular cloning method known in the art can be used, including, for example, cloning nucleic acids into a vector using restriction endonuclease sites and one or more selectable markers. In some embodiments, the nucleic acid is operably linked to a promoter. A variety of promoters for gene expression in prokaryotic or eukaryotic cells (e.g., mammalian cells) have been explored, and any promoter known in the art can be used in the present invention. Promoters can be generally classified into constitutive promoters or regulatable promoters, such as inducible promoters.

[0473] 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 heterologous genes (also referred to as transgenes) in host cells. 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 coupled with the CMV early enhancer (CAGG), Rous sarcoma virus (RSV) promoter, polyomavirus enhancer / herpes simplex thymidine kinase (MC1) promoter, β-actin (β-ACT) promoter, "myeloproliferative sarcoma virus enhancer, negative control region deleted, d1587rev primer binding site replaced (MND)" promoter. The efficiencies of these constitutive promoters in driving transgene expression have been widely compared in a large number of studies. In some embodiments, the nucleic acid encoding the prodrug described herein is operably linked to the CMV promoter.

[0474] In some embodiments, the nucleic acid encoding the prodrug described herein is operably linked to an inducible promoter. Inducible promoters fall within the category of regulatable promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of the host cell or the physiological state of the host cell, inducers (i.e., inducing agents) or their compositions. In some embodiments, the inducing conditions do not induce the expression of endogenous genes in the host cell. In some embodiments, the inducing conditions are selected from: inducers, radiation (such as ionizing radiation, light), temperature (such as heat), redox state, and the activation state of the host cell. In some embodiments, the inducible promoter can be the NFAT promoter, promoter or the NFκB promoter.

[0475] Preparation method

[0476] Also provided are methods of preparing any of the masked polypeptides (MPs), any cleavable moieties (CMs), any non-cleavable linkers (Ls), or any prodrugs described herein. Thus, in some embodiments, provided is a method of preparing the masked polypeptide (MP), the cleavable moiety (CM), the non-cleavable linker (L), or the prodrug, comprising: (a) culturing a host cell (e.g., a CHO cell, a HEK 293 cell, a Hela cell, or a COS cell) comprising a nucleic acid or vector encoding the masked 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 masked polypeptide (MP), the cleavable moiety (CM), the non-cleavable linker (L), or the prodrug from the host cell. In some embodiments, the method of step (a) further comprises preparing a host cell comprising a nucleic acid or vector encoding the masked polypeptide (MP), the cleavable moiety (CM), the non-cleavable linker (L), or the prodrug described herein. The masked polypeptides (MPs), the cleavable moieties (CMs), the non-cleavable linkers (Ls), or the prodrugs described herein can be prepared using any method known in the art or described herein.

[0477] In some embodiments, the masked polypeptides (MPs), the cleavable moieties (CMs), the non-cleavable linkers (Ls), or the prodrugs described herein are expressed in eukaryotic cells, such as mammalian cells. In some embodiments, the masked polypeptides (MPs), the cleavable moieties (CMs), the non-cleavable linkers (Ls), or the prodrugs described herein are expressed in prokaryotic cells.

[0478] 1. Recombinant products of prokaryotic cells

[0479] a) Vector construction

[0480] Polynucleotide sequences encoding the protein constructs described in this application can be obtained using standard recombinant techniques. Polynucleotides can be synthesized using a nucleotide synthesizer or PCR technology. Once the sequence encoding the polypeptide 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 for the present invention. The choice of a suitable vector depends mainly on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed by the vector. Each vector contains various components, depending on the function of the vector (amplification or expression of heterologous polynucleotides, or both) and the compatibility between the vector and the particular host cell in which it is to be located. Vector components generally include, but are not limited to: an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence.

[0481] Generally, plasmid vectors contain replicons and control sequences derived from species compatible with the host cell, which are used together with these host cells. The vector usually carries an origin of replication, as well as a marker sequence that can provide phenotypic selection in the transformed cells. For example, Escherichia coli is commonly transformed using pBR322, a plasmid derived from E. coli. pBR322 contains genes encoding resistance to ampicillin (Amp) and tetracycline (Tet), and thus provides a simple method for identifying transformed cells. pBR322, its derivatives, or other bacterial plasmids or bacteriophages may also contain or be modified to contain promoters that can be used by microorganisms to express endogenous proteins. Carter et al., U.S. Pat. No. 5,648,237 details examples of pBR322 derivatives for expressing specific antibodies.

[0482] In addition, phage vectors containing replicons and control sequences compatible with the host microorganism can be used as transformation vectors together with these host cells. For example, phages such as GEM TM -11 can be used to prepare recombinant vectors, which can be used to transform susceptible host cells, such as Escherichia coli LE392.

[0483] A promoter is a non-translated regulatory sequence located upstream (5′) of a cistron that can regulate downstream gene expression. Prokaryotic promoters are generally divided into two categories, inducible and constitutive. An inducible promoter is a promoter that can respond to changes in culture conditions (e.g., the presence or absence of nutrients or changes in temperature), thereby initiating and increasing the transcriptional level of the cistron.

[0484] Many promoters that can be recognized by potential host cells are well known. By removing the promoter from the source DNA using restriction endonucleases and inserting the isolated promoter sequence into the vector of the present application, the selected promoter can be operably linked to the cistron DNA encoding the polypeptide. Both natural promoter sequences and many heterologous promoters can be used to direct the amplification and / or expression of the target gene. In some embodiments, heterologous promoters are utilized because, compared to the natural target polypeptide promoter, heterologous promoters generally allow greater transcription and higher yields of expressing the target gene.

[0485] Promoters suitable for prokaryotic hosts include the PhoA promoter, the β-galactosidase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoter. However, other promoters that function in bacteria (such as other known bacterial or phage promoters) are also applicable. Their nucleic acid sequences have been published, enabling those skilled in the art to use linkers or adaptors to provide any desired restriction sites to ligate them to the cistrons encoding the target light and heavy chains (Siebenlist et al., (1980) Cell 20:269).

[0486] In some embodiments, each cistron within the recombinant vector contains a secretion signal sequence component that directly directs transmembrane transfer of the expressed polypeptide. Generally, the signal sequence can be part of the vector or part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the present invention should be a sequence that can be recognized and processed by the host cell (i.e., cleaved by signal peptidase). For prokaryotic host cells that cannot recognize and process the native signal sequence of a heterologous polypeptide, the signal sequence is replaced by a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, Ipp or the leader of heat-stable enterotoxin II (STII), LamP, PhoE, PelB, OmpA, and MBP.

[0487] In some embodiments, production of the protein constructs of the present application can occur in the cytoplasm of the host cell, and thus there is no need for a secretion signal sequence to be present within each cistron. In some embodiments, the polypeptide components are expressed, folded, and assembled to form the protein constructs within the cytoplasm. Certain host strains (e.g., the E. coli trxB- strain) provide cytoplasmic conditions that favor disulfide bond formation, thereby allowing proper folding and assembly of the expressed protein subunits. See Proba and Pullthun, Gene, 159:203 (1995).

[0488] b) Prokaryotic host cell

[0489] Prokaryotic host cells suitable for expressing the proteins of the present application include archaebacteria and eubacteria, such as Gram-negative or Gram-positive bacteria. Examples of available bacteria include Escherichia coli (e.g., E. coli), Bacillus (e.g., Bacillus subtilis), Enterobacter, 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 are used as the host of the present invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) and its derivatives, including strain 33D3 with the genotype W3110 ΔfhuA (ΔtonA) ptr3 lacIq lacL8 ΔompT Δ(nmpc fepE) degP41 kanR (U.S. Pat. No. 5,639,635). Other strains and their derivatives, such as E. coli 294 (ATCC 31446), E. coli B, E. coli 1776 (ATCC 31537) and E. coli RV308 (ATCC 31608) are also equally applicable. These examples are illustrative rather than restrictive. Methods for constructing bacterial derivatives of any of the above-mentioned known genotypes are known in the art and are detailed, for example, in Bass et al., Proteins, 8: 309-314 (1990). Considering the replicability of the replicon in bacterial cells, it is usually necessary to select a suitable bacterium. For example, when using well-known plasmids such as pBR322, pBR325, pACYC177 or pKN410 to provide the replicon, Escherichia coli, Serratia or Salmonella is suitable for use as the host.

[0490] Generally, the host cells should secrete the least amount of proteolytic enzymes, and it is necessary to appropriately add additional protease inhibitors to the cell culture.

[0491] c) Protein production

[0492] The host cell is transformed with the above expression vector and cultured in a conventionally modified nutrient medium to induce the promoter, select transformants, or amplify the gene encoding the desired sequence. Transformation refers to the introduction of DNA into a prokaryotic host such that the DNA can replicate as an extrachromosomal element or by chromosomal integration. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Calcium treatment with calcium chloride is commonly used for bacterial cells with a substantial cell wall barrier. Another transformation method employs polyethylene glycol / dimethyl sulfoxide. Another technique is electroporation.

[0493] Prokaryotic cells for producing the protein constructs of the present application are known in the art and are suitable for growth in a medium for culturing the selected host cells. Suitable media include luria broth (LB) and necessary nutrient supplements. In some embodiments, the medium further contains a selection agent selected based on the structure of the expression vector to selectively permit the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium for the growth of cells expressing the ampicillin resistance gene.

[0494] Any necessary supplements can also be added, either alone or as a mixture with other supplements or media (such as a complex nitrogen source), at appropriate concentrations in addition to a carbon source, a nitrogen source, and an inorganic phosphate source. Optionally, the medium may contain one or more reducing agents selected from glutathione, cysteine, cysteamine, thiodiglycolic acid, dithioerythritol, and dithiothreitol. The prokaryotic host cells are cultured at a suitable temperature. For example, for the growth of Escherichia 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 can be any pH value between 5 and 9, mainly depending on the host organism. For Escherichia coli, the pH value is preferably 6.8 to 7.4, and more preferably 7.0.

[0495] If 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 aspect of the present application, the PhoA promoter is used to control the transcription of the polypeptide. Therefore, the transformed host cells are cultured in a phosphate-limited medium for induction. Preferably, the phosphate-limited medium is the C.R.A.P medium (see Simmons et al., J. Immunol. Methods (2002), 263: 133-147). Depending on the vector construct employed, a variety of other inducers known in the art can be used, which are known in the art.

[0496] The protein construct expressed in this application is secreted into the periplasm of the host cell and recovered therefrom. Protein recovery typically involves disrupting the microorganism, usually by means of 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 therein. Cells can be removed from the culture medium, and the culture supernatant can be filtered and concentrated to further purify the resulting protein. The expressed polypeptide can be further separated and identified by common methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0497] Alternatively, the protein is produced on a large scale by a fermentation process. Various large-scale fed-batch fermentation procedures can be used to produce recombinant proteins. The capacity of large-scale fermentation is at least 1000 liters, preferably 1000 to 100,000 liters. These fermenters use agitator impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon / energy source). Small-scale fermentation generally refers to fermentation in a fermenter with a volume capacity not exceeding 100 liters, ranging from 1 liter to 100 liters.

[0498] During the fermentation process, usually after the cells have grown to the desired density under suitable conditions, protein expression is induced. For example, when the OD 550 is approximately 180 - 220, at which time the cells are in the early stationary phase. Depending on the vector construct employed, various inducers known in the art and described above can be used. The cells can be grown for a shorter period of time before induction. The cells are usually induced for about 12 - 50 hours, although induction times that may be longer or shorter can be used.

[0499] To improve the yield and quality of the protein constructs of the present application, various fermentation conditions can be modified. For example, to enhance the correct assembly and folding of secreted polypeptides, an additional vector overexpressing a chaperone protein can be used to co-transform a host prokaryotic cell, such as a Dsb protein (DsbA, DsbB, DsbC, DsbD, or DsbG) or FkpA (peptidyl-prolyl cis-trans isomerase with chaperone activity). Chaperone proteins have been shown to assist in promoting the correct folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al., (1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No. 6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275:17106-17113; Arie et al., (2001) Mol. Microbiol. 39:199-210.

[0500] To minimize the hydrolysis of the expressed heterologous proteins, especially proteolysis-sensitive proteins, certain host strains lacking proteolytic enzymes can be used in the present invention. For example, the host cell strain can be modified such that the genes encoding known bacterial proteases are mutated, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and their combinations. Some Escherichia coli protease-deficient strains can be used, which are described in detail in Joly et al., (1998), supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0501] An Escherichia coli strain lacking proteolytic enzymes and transformed with a plasmid overexpressing one or more chaperone proteins can be used as a host cell in an expression system encoding the protein constructs described in the present application.

[0502] d) Protein purification

[0503] The protein constructs produced herein are further purified to obtain a substantially homogeneous preparation for further analysis and use. Standard protein purification methods known in the art can be employed. The following procedures are examples of applicable purification procedures: fractionation on an immunoaffinity column or an ion-exchange column, ethanol precipitation, reverse-phase liquid chromatography HPLC, silica or cation-exchange resin (such as DEAE) chromatography, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration, for example, Sephadex G-75.

[0504] In some embodiments, Protein A immobilized on a solid phase is used for immunoaffinity purification of the protein construct that comprises the Fc region described herein. Protein A is a 42 kDa surface protein from Staphylococcus aureus that has a high binding affinity for Fc-containing constructs, such as, for example, the IL-15 prodrug or the anti-TNFR2 antibody prodrug described herein. Lindmark et al., (1983) J. Immunol. Meth. 62:1-1. The solid phase to which Protein A is immobilized preferably comprises a column with a glass or silica surface, more preferably a controlled pore glass column or a silica column. In certain applications, the chromatography column is coated with a reagent, such as glycerol, to prevent non-specific adhesion of contaminants. The solid phase is then washed to remove contaminants that non-specifically bind to the solid phase. Finally, the target protein construct is recovered from the solid phase by elution.

[0505] 2. Recombinant products of eukaryotic cells

[0506] For eukaryotic expression, vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence.

[0507] a) Signal sequence element

[0508] Vectors for eukaryotic hosts can also be an insert that encodes a signal sequence or other polypeptide that has a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected is preferably a sequence that is recognized and processed by the host cell (i.e., cleaved by signal peptidase). In mammalian cell expression, mammalian signal sequences as well as viral secretion leaders can be obtained, for example, the herpes simplex gD signal, which are useful. The DNA of this precursor region is ligated in-frame with the DNA encoding the protein construct of the present application.

[0509] b) Replication origin

[0510] Generally, mammalian expression vectors do not require an origin of replication element (the SV40 origin is usually only used because it contains an early promoter).

[0511] c) Selection gene element

[0512] Expression and cloning vectors may contain selectable genes, also called selectable markers. Typical selectable genes encode the following proteins: (a) proteins that confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate or tetracycline, (b) complement nutritional deficiency proteins or (c) proteins that provide key nutrients not available in complex media, such as the gene encoding Bacillus D-alanine racemase.

[0513] One example of a selection scheme is to use a drug to prevent the growth of host cells. Cells in which the heterologous gene has been successfully transformed produce a protein that confers resistance and thus survive the selection scheme. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

[0514] Another example of a selectable marker suitable for mammalian cells is a selectable marker that can identify cells capable of carrying the nucleic acid encoding the protein construct described in this application, such as DHFR, thymidine kinase, metallothionein-I and -II, preferably the primate metallothionein gene, adenosine deaminase, ornithine decarboxylase, etc.

[0515] For example, cells transformed with the DHFR selectable gene are first cultured in a medium containing methotrexate (Mtx) for identification, where methotrexate is a competitive antagonist of DHFR. When using wild-type DHFR, a suitable host cell is a Chinese hamster ovary (CHO) cell line lacking DHFR activity (e.g., ATCC CRL-9096).

[0516] Alternatively, host cells (especially wild-type hosts containing endogenous DHFR) transformed or co-transformed with a DNA sequence encoding a polypeptide, wild-type DHFR protein and another selectable marker, such as aminoglycoside 3'-phosphotransferase (APH), can be selected by cell growth in a medium containing a selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin or G418. See U.S.Pat.No.4,965,199.

[0517] d) Promoter element

[0518] Expression and cloning vectors generally contain a promoter that is recognized by the host and operably linked to a nucleic acid encoding the desired polypeptide sequence. Almost all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site. Another sequence found 70 to 80 bases upstream of the transcription start site in many genes is the CNCAAT region, where N can be any nucleotide. There is an AATAAA sequence at the 3′ end of most eukaryotes, 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 section "Vectors" above.

[0519] Transcription of polypeptides in mammalian host cell vectors is controlled by promoters, for example, promoters obtained from viral genomes such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably simian virus 40 (SV40), promoters from heterologous mammals, for example, the actin promoter or immunoglobulin promoter from a heat shock promoter, provided that such promoters are compatible with the host cell system.

[0520] The early and late promoters of the SV40 virus are readily available as SV40 restriction fragments that also contain the SV40 virus origin of replication. The immediate early promoter of the human cytomegalovirus is readily available as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papillomavirus as a vector is disclosed in U.S. Pat. No. 4,419,446. Improvements to this system are detailed in U.S. Pat. No. 4,601,978. See Reyes et al., Nature 297:598-601 (1982), regarding the expression of human interferon cDNA in mouse cells under the control of the herpes simplex virus thymidine kinase promoter. Alternatively, the long terminal repeat sequence of Rous sarcoma virus can be used as a promoter.

[0521] e) Enhancer element

[0522] Transcription of DNA encoding the protein constructs of the present application in higher eukaryotes is usually increased by inserting enhancer sequences into the vector. Many enhancer sequences have been found in mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, enhancers from eukaryotic cell viruses are commonly used. Examples include the SV40 enhancer at the end of the origin of replication (100 - 270 bp), the cytomegalovirus early promoter enhancer, the polyomavirus enhancer at the end of the origin of replication, and the adenovirus enhancer. See Yaniv, Nature 297:17 - 18 (1982) regarding enhancer elements that activate eukaryotic promoters. Enhancers can be spliced into the vector at the 5′ or 3′ of the polypeptide coding sequence, but are preferably located at the 5′ of the promoter.

[0523] f) Transcription termination element

[0524] Expression vectors used in eukaryotic host cells (nucleated cells of yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) also contain sequences necessary for transcription termination and mRNA stabilization. These sequences are usually obtained from the 5′ untranslated regions of eukaryotic or viral DNA or cDNA, and occasionally from the 3′ end. These regions contain nucleotide segments that are transcribed as polyadenylation segments in the untranslated portion of the mRNA encoding the polypeptide. A suitable transcription termination element is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vectors disclosed therein.

[0525] g) Selection and transformation of host cells

[0526] Suitable host cells for cloning or expressing the DNA in the vectors described herein include the higher eukaryotic cells described herein, including vertebrate host cells. The culture and propagation of vertebrate cells (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); the COS fibroblast-like cell line derived from monkey kidney tissue; the human embryonic kidney line (293 or 293 cell subclones for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); the baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC-ccl2); dog kidney cells (MDCK, ATCC-ccl34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells and the human hepatoma cell line (Hep G2).

[0527] Transform host cells with the above-described expression or cloning vectors to produce protein constructs and culture them in a suitably modified conventional nutrient medium to induce the promoter, select transformants or amplify the gene encoding the desired sequence.

[0528] h) Culturing host cells

[0529] The host cells for producing the protein constructs of the present application can be cultured in a variety of media. Commercial 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 host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655 or 5,122,469, WO 90 / 03430, WO 87 / 00195 or U.S. Pat. Re. 30,985 can be used as the medium for host cells. Any of these media can be supplemented 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 gentamicin TM drug), trace elements (defined as inorganic compounds that are usually present at a final concentration in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements can also be added at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc. are those previously used for host cell expression and are obvious to those of ordinary skill in the art.

[0530] i) Protein purification

[0531] When recombinant techniques are used, the protein constructs of the present invention can be produced intracellularly, in the periplasm, 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 lysed fragments) by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) details the procedure for isolating antibodies secreted into the periplasm of Escherichia coli. Briefly, cell bodies are lysed for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. When the protein construct is secreted into the culture medium, the supernatant of such an expression system is usually first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration device. Protease inhibitors such as PMSF can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0532] Protein compositions prepared from cells can be purified using, for example, 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-containing protein construct. Protein A can be used to purify Fc-containing proteins based on human immunoglobulins with 1, 2, or 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse subtypes and human type 3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which the affinity ligand is attached is usually agarose, but there are other matrices. Compared to agarose, mechanically stable matrices such as controlled pore glass or poly(styrene-divinyl)benzene can achieve faster flow rates and shorter processing times. Bakerbond ABXTM resin can be used to purify protein constructs containing the C H 3 domain (J.T. Baker, Phillipsburg, N.J.). Other protein purification techniques are also applicable, such as ion exchange column fractionation, ethanol precipitation, reverse phase liquid chromatography HPLC, silica gel chromatography, heparin SEPHAROSE TM chromatography, anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, depending on the protein construct to be recovered.

[0533] After any initial purification step, low pH hydrophobic interaction chromatography can be performed on a mixture containing the target protein construct and contaminants, with an elution buffer pH of about 2.5 - 4.5, preferably at a low salt concentration (e.g., from 0 - 0.25 M salt).

[0534] Pharmaceutical Compositions

[0535] Further provided are pharmaceutical compositions comprising a prodrug described herein (e.g., an IL-15 prodrug or an anti-TNFR2 antibody prodrug), and optionally a pharmaceutically acceptable carrier. The pharmaceutical compositions can be prepared by mixing a prodrug of the required purity described herein with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of a lyophilized formulation or an aqueous solution.

[0536] A recombinant formulation can be prepared by dissolving the lyophilized prodrug in a diluent to uniformly disperse the protein. Examples of pharmaceutically acceptable (safe and non-toxic for administration to humans) diluents suitable for use in this application include, but are not limited to, sterile water, bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution or glucose solution, or an aqueous solution of salts and / or buffers.

[0537] In some embodiments, the pharmaceutical composition comprises a homogeneous population of the prodrug described herein (e.g., an IL-15 prodrug). A homogeneous population means that the prodrugs are identical to each other, 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 any), and the same Fc domain. In some embodiments, at least 70% (such as any one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of the IL-15 prodrugs in the pharmaceutical composition are homogeneous.

[0538] The pharmaceutical composition is preferably stable, and the protein contained therein substantially maintains its physical and chemical stability and integrity during storage. There are various analytical techniques in the art for measuring protein stability, which are reviewed in Peptide and Protein Drug Delivery, 247 - 301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (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 accelerated screening, the formulation can be stored at 40 °C for 2 weeks to 1 month, during which stability is measured. For example, the degree of aggregation during storage can be used as an indicator of protein stability.

[0539] In some embodiments, the pharmaceutical composition has a shelf life of at least 15 days, such as at least 20 days, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years or longer, for example, at 2-25 °C (such as 2-8 °C). As used herein, "shelf life" refers to the storage period during which, when a pharmaceutical formulation is stored under specific storage conditions, such as 2-8 °C, the active ingredient in the pharmaceutical formulation, such as a therapeutic protein (e.g., the IL-15 prodrug or anti-TNFR2 antibody prodrug described herein), undergoes minimal degradation (e.g., no more than 5% degradation, such as no more than 4%, 3%, or 2% degradation). Exemplary techniques for evaluating protein or formulation stability include size exclusion chromatography (SEC)-HPLC to detect, for example, aggregation, reverse phase liquid chromatography (RP)-HPLC to detect, for example, protein fragments, ion exchange HPLC to detect, for example, protein charge changes, mass spectrometry, fluorescence spectroscopy, circular dichroism (CD) spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and Raman spectroscopy to detect protein conformational changes. All of these techniques can be used alone or in combination to evaluate the degradation of the protein in the pharmaceutical formulation and determine the shelf life of the formulation.

[0540] Acceptable carriers, excipients or stabilizers are non-toxic to the subject at the dosages and concentrations used and include buffers, antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives, isotonic agents (such as sodium chloride), stabilizers, metal complexes (e.g., zinc-protein complexes); chelating agents, such as EDTA and / or non-ionic surfactants.

[0541] Examples of physiologically acceptable carriers include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; o-catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or non-ionic surfactants such as Tween TM , polyethylene glycol (PEG) and PLURONICS TM .

[0542] Buffers are used to control the pH value within a range that optimizes the therapeutic effect, especially in cases where stability depends on the pH value. Buffers suitable for this application include organic acids and inorganic acids and their salts. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. In addition, the buffer may include histidine and trimethylamine salts such as Tris.

[0543] Preservatives are added to prevent microbial growth. For example, the addition of preservatives can facilitate the production of multi-use (multi-dose) formulations. Preservatives suitable for this application include octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butanol or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid, such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol, 3-pentanol and m-cresol.

[0544] Tonicity agents, sometimes called "stabilizers", are used to regulate or maintain the tonicity of the liquid in the composition. When used with large charged biomolecules such as proteins, they are usually called "stabilizers" because they can interact with the charged groups of amino acid side chains, thereby reducing the likelihood of intermolecular and intramolecular interactions. Considering the relative content of other components, the tonicity agent can be present in any amount between 0.1% and 25% (by weight), preferably 1% to 5%. Preferred tonicity agents include polyhydric alcohols, preferably trihydric or higher polyhydric alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0545] Other excipients include one or more of the following formulations: (1) fillers, (2) solubility enhancers, (3) stabilizers, and (4) formulations that prevent denaturation or adhesion to the container wall. Such excipients include: polyhydric alcohols (as described above); amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, inositol, inositol, galactose, galactitol, glycerol, cyclohexanol (such as inositol), polyethylene glycol; sulfur-containing reducing agents, such as urea, glutathione, lipoic 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.

[0546] The presence of a nonionic surfactant or detergent (also referred to as a “wetting agent”) aids in dissolving the protein and protects the protein from agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active protein.

[0547] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), polyols, polyoxyethylene sorbitan monoethers ( etc.), laureth 400, polyoxyethylene 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic detergents that can be used include sodium dodecyl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0548] For a pharmaceutical composition to be useful for in vivo administration, it must be sterile. The pharmaceutical composition can be made sterile by filtration through a sterile filter membrane. The pharmaceutical composition is typically placed in a container having a sterile access port, e.g., an intravenous solution bag or vial having a stopper that can be pierced by a hypodermic needle.

[0549] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing an antagonist, the matrix being in the form of a shaped article, e.g., a film or a microcapsule. 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), copolymers of L-glutamic acid and L-glutamic acid ethyl ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT TM (injectable microspheres composed of a lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid.

[0550] Depending on the needs of the particular indication being treated, the pharmaceutical compositions described herein may also contain more than one active compound, preferably compounds having complementary activities that do not adversely affect one another. Such molecules are combined in an effective amount to achieve the desired purpose.

[0551] The active ingredient can also be encapsulated in, for example, microcapsules prepared by gel technology or interfacial polymerization, such as in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in hydroxy methyl cellulose microcapsules, gelatin microcapsules, and polymethyl methacrylate microcapsules in a coarse emulsion. These techniques are disclosed in Remington's Pharmaceutical Sciences.

[0552] In some embodiments, the pharmaceutical composition is contained in a single-use vial, such as a disposable sealed vial. In some embodiments, the pharmaceutical composition is contained in a reusable vial. In some embodiments, the pharmaceutical composition is bulked in a container. In some embodiments, the pharmaceutical composition is cryopreserved.

[0553] Methods of treating diseases

[0554] There is further provided a method of treating a subject having or at risk of having a disease, such as a proliferative disease, a neoplastic disease, an inflammatory disease, an immune disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, or graft-versus-host disease. The methods disclosed herein administer an effective amount of an activatable prodrug to a subject in need thereof, typically as a pharmaceutical composition, wherein the prodrug is activated upon enzymatic cleavage. In some embodiments, the method further comprises selecting a subject having or at risk of having such a disease or disorder. In some embodiments, the prodrug is activated in the tumor microenvironment. The prodrug has therapeutic activity after cleavage from the masking polypeptide. Thus, in some embodiments, the active agent is the cleavage product. In some embodiments, the prodrug can rely on the binding of an antigen to an antigen-binding domain to treat the disease.

[0555] In some embodiments, there is provided a method of treating a disease (e.g., a tumor, a viral infection, or a bacterial infection) in an individual (e.g., a human), comprising administering to the individual an effective dose of any of the prodrugs described herein (e.g., an IL-15 prodrug or an anti-TNFR2 antibody prodrug) or a pharmaceutical composition thereof. In some embodiments, the prodrug (or a pharmaceutical composition thereof) is administered by intravenous injection, intramuscular injection, or subcutaneous injection. In some embodiments, the method of treatment further comprises administering an additional therapeutic agent in combination with the prodrug (before, after, or simultaneously). The additional agent may be an antibody or an antigen-binding fragment thereof, a small molecule drug, or other types of therapeutic agents.

[0556] In some embodiments, the IL-15 prodrug or anti-TNFR2 antibody prodrug is used to treat cancer or tumors in a subject, including administering to the subject a therapeutically effective amount of the IL-15 prodrug or anti-TNFR2 antibody prodrug. As described herein, in some embodiments, the term "tumor or cancer" refers to all types of cancers, tumors, or malignancies in mammals, including leukemia, lymphoma, melanoma, neuroendocrine tumors, carcinomas, and sarcomas. Exemplary cancers treatable 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, ductal carcinoma, lobular carcinoma, primary, metastatic), ovarian cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer, squamous cell lung cancer, adenocarcinoma, large cell lung cancer, small cell lung cancer, carcinoid, 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 (e.g., head and neck or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma. Other examples include thyroid cancer, endocrine system 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 tumor, cancer, malignant insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, urogenital cancer, malignant hypercalcemia, endometrial cancer, adrenocortical cancer, endocrine or exocrine pancreatic tumors, medullary thyroid cancer, medullary thyroid cancer, melanoma, colorectal cancer, papillary thyroid carcinoma, 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.

[0557] In some embodiments, the IL-15 prodrug is used for treating bacterial infections, such as sepsis. In some embodiments, the bacterium causing the bacterial infection is a drug-resistant bacterium. In some embodiments, the antigen-binding portion binds to a bacterial antigen.

[0558] In some embodiments, the IL-15 prodrug is used for treating viral infections. 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 portion binds to a viral antigen.

[0559] The prodrug or its pharmaceutical composition described herein can be administered in any convenient manner, including by injection or infusion. The administration route follows known and recognized methods, such as by single or multiple boluses or by prolonged infusion in a suitable manner. The prodrug or its pharmaceutical composition can be administered orally, subcutaneously, intravenously, intracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, intraocularly, topically, transarterially, intradermally, intraarticularly, intracavitarily, or intramedullarily, intrathecally, intraventricularly, intracerebrally, intraspinally, intrathecally, intralesionally, or intraocularly. In some embodiments, the prodrug or its pharmaceutical composition is administered systemically. In some embodiments, the prodrug or its pharmaceutical composition is administered to an individual by infusion (such as intravenous infusion). The 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 its pharmaceutical composition 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 its pharmaceutical composition. However, there are also other devices for administering the prodrug or its pharmaceutical composition, such as injection devices; injection pens; autoinjector devices, needle-free devices; and transdermal patch delivery systems. In some embodiments, the prodrug or its pharmaceutical composition is administered by intravenous injection. In some embodiments, the prodrug or its pharmaceutical composition is directly injected into the brain or spinal column. In some embodiments, the prodrug or its pharmaceutical composition is administered by sustained release or extended release techniques.

[0560] The dosage and required drug concentration of the pharmaceutical composition of the present invention may vary according to the specific intended use. Determining the appropriate dosage or route of administration is entirely within the technical scope of those skilled in the art. Animal experiments provide reliable guidance for determining the effective dose for human treatment. Interspecies extrapolation of the effective dose can be carried out according to the principles in Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp. 42 - 46.

[0561] When the prodrug or its pharmaceutical composition is administered in vivo, the dosage administered varies depending on the route of administration and the type of mammal. Within the scope of the present application, different formulations will be effective for different treatments and different diseases, and the mode of administration intended to treat a particular organ or tissue may be different from that for another organ or tissue. In addition, the dosage can be administered by a single or multiple separate administrations or by continuous infusion. For repeated administration over several days or longer, depending on the condition, the treatment continues until the disease symptoms reach the desired degree of suppression. However, other dosage regimens may be useful. The progress of such treatment can be easily monitored by conventional techniques and analysis.

[0562] In some embodiments, the prodrug or its pharmaceutical composition is administered once (e.g., by bolus injection). In some embodiments, the prodrug or its pharmaceutical composition is administered multiple times (such as 2, 3, 4, 5, 6 or more times). If administered multiple times, it can be by the same or different routes and can be at the same site or other sites. The prodrug or its pharmaceutical composition can be administered once daily to once a year. The interval between two administrations can be from 24 hours to any time during the year. The interval may also be irregular (e.g., as the tumor progresses). In some embodiments, there is no interruption in the dosing schedule. The optimal dose and treatment regimen for a particular patient can be determined by those skilled in the medical field by monitoring the patient's disease signs and adjusting accordingly.

[0563] In some embodiments, the prodrug or its pharmaceutical composition is administered in divided doses, such as 2, 3, 4, 5 or more doses. In some embodiments, the divided doses are administered over a period of more than 1 week, 1 month, 2 months, 3 months or longer. In some embodiments, the doses are equal. In some embodiments, the divided doses are 20%, 30% and 50% of the total dose. In some embodiments, the interval between successive divided dose administrations is 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months or longer. For repeated administrations over several days or longer, depending on the condition, treatment continues until an expected degree of suppression of the disease symptoms occurs. However, other dosing regimens may be useful. The progress of such treatment can be easily monitored by conventional techniques and analysis.

[0564] Articles and kits

[0565] There are further provided kits, unit doses and articles comprising any of the prodrugs described herein. In some embodiments, there are provided kits comprising any of the prodrug pharmaceutical compositions described herein, and preferably instructions for use thereof, such as for treating the diseases described herein (e.g., tumors).

[0566] The kits of the present invention include one or more containers comprising the prodrug described herein, e.g., for treating a disease. For example, it includes instructions describing the administration of the prodrug for treating a disease (such as a tumor). The kit may further include a description for selecting a suitable individual (e.g., a human) for treatment based on identifying whether the individual has the disease and the disease stage. The instructions related to the use of the prodrug generally include information about the dose, dosing schedule and route of administration of the intended treatment. The container can be a unit dose, bulk packaging (e.g., multi-dose packaging) or sub-unit dose. The instructions provided in the kits of the present invention are generally written instructions on a label or package insert (e.g., a sheet included in the kit), but machine-readable instructions (e.g., instructions stored on a disk or optical disc) are also acceptable. The kits of the present application are packaged appropriately. Appropriate packaging includes but is not limited to vials, bottles, jars, flexible packages (e.g., sealed mylar or plastic bags), etc. Packaging in combination with a specific device is also contemplated, such as an infusion device like a micropump. The kit may have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a subcutaneous injection needle). At least one active agent in the composition is a prodrug as described herein. The container may further contain a second pharmaceutical active agent. The kit may optionally provide additional components, such as buffers and interpretive information. Generally, the kit comprises a container and a label or package insert on or associated with the container.

[0567] Accordingly, the present application also provides articles, including vials (such as sealed vials), bottles, cans, flexible packages, etc. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be made of a variety of materials, such as glass or plastic. Generally, the composition contained in the container can effectively treat the diseases or disorders described herein (such as tumors), and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The label or package insert indicates that the composition is for treating a specific disorder of an individual. The label or package insert further contains instructions for administering the composition to the individual. The label may note the reconstitution and / or usage instructions. The container containing the pharmaceutical composition can be a multi-use vial, allowing repeated administration of the reconstituted formulation (for example, 2 - 6 administrations). A package insert refers to the instructions usually included in the commercial packaging of a therapeutic product, which contains information on indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic product. Additionally, the article may further comprise 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 required materials may further be included, including other buffers, diluents, filters, needles, and syringes.

[0568] The kit or article includes multiple unit doses of a pharmaceutical composition and instructions for use, in a packaging quantity sufficient for storage and use in a pharmacy, such as a hospital pharmacy and a compounding pharmacy. Examples

[0569] The following examples are merely intended to be illustrative of the present invention and should not be construed as limiting the invention in any way. The following examples and detailed description are provided in an illustrative, rather than a limiting, sense.

[0570] Example 1: Preparation of Masking Polypeptide (MP)

[0571] Composition of the masking polypeptide:

[0572] Amino acids are selected for a chemically stable and predominantly unstructured masking polypeptide that comprises at least 40 contiguous amino acids and is substantially incapable of non-specific binding 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 peptide conforms to a certain ratio: the percentage of amino acid residue A in the masking polypeptide is about 5%-20%, the percentage of amino acid residue E in the masking polypeptide is about 1%-20%, the percentage of amino acid residue G in the masking polypeptide is about 15%-30%, the percentage of amino acid residue P in the masking polypeptide is about 15%-40%, and the percentage of amino acid residue S in the masking polypeptide is about 20%-40%. The masking polypeptide comprises at least 40 amino acids, and the length of the sequence can be extended with non-repetitive, unstructured polypeptides. The exemplary masking polypeptide MP80 was first designed as the sequence SEQ ID NO:1, and the masking polypeptides MP163 and MP240 comprise the amino acid sequence of MP80. According to the above rules, masking polypeptides can be designed to, but are not limited to, the masking polypeptides shown in Table 1, and the masking activity is tested. Figure 1A-1C Shown are the sequence alignments of MP100 with MP80, MP163 with MP80 repeated twice, and MP240 with MP80 repeated three times. MP80 and MP100 comprise the amino acid sequence SEQ ID NO:6, and both MP163 and MP240 comprise the amino acid sequence of MP80.

[0573] All sequences were analyzed using silicon T cell epitope prediction tests, B cell immunogenicity tests, and secondary structure predictions. No detectable T- and B-cell epitopes and no detectable protease cleavage sites were found in the masking polypeptides.

[0574] Table 1

[0575]

[0576]

[0577] Example 2: Expression and Characterization of IL-15 Prodrug

[0578] To reduce the toxicity of IL-15-related therapeutic agents, an IL-15 prodrug with a masking polypeptide was constructed and recombinantly expressed in HEK293 cells. The constructs of the IL-15 prodrug or drug are shown in Table 2. Figure 2A Schematic diagram of an exemplary structure of an IL-15 prodrug comprising a masking polypeptide. Figure 2BShown is an exemplary schematic diagram that illustrates the process by which a masking polypeptide (MP) is released from an IL-15 prodrug by a target tissue (e.g., a tumor with a high level of MMPs), thereby activating the prodrug. Before the masking polypeptide is cleaved by the protease of the target tissue, the activity of the prodrug is very low. The main difference between the IL-15 drug and the prodrug is that the drug does not have a masking polypeptide and a cleavable moiety (e.g., SB1902-C1, SB1902-C1-variant1, SB1902-C1-variant2, and SB1902-C1-variant3). In some embodiments, IL-15_L45D, IL-15_L45E, IL-15_Q48K, IL-15_S51D, IL-15_L52D, IL-15_E64K, IL-15_I67D, IL-15_I67E, IL-15_I68D, or IL-15_N72D refers to an IL-15 mutant that contains the L45D, L45E, Q48K, S51D, L52D, E64K, I67D, I67E, I68D, or N72D mutation, respectively, relative to human wild-type IL-15. Thus, taking SB1902-C1-variant3_L45D as an example, it refers to an IL-15 drug in SB1902-C1-variant3 that contains the IL-15 L45D mutant rather than human native wild-type IL-15. The drug SB1902-C1 without a masking polypeptide and the non-activatable IL-15 cytokine construct without a cleavable moiety were used as controls in the following experiments, such as SB1902-C4.

[0579] Table 2

[0580]

[0581]

[0582]

[0583] The prodrug SB1902-C2 (hIgG Fc(hole)-lk1-IL15-lk2-CM1-lk2-MP80 / hIgG Fc(knob)-lk5-IL15Rαsushi) was used as an example for plasmid construction. Similar methods were also used for other prodrugs and drugs. The prodrug expression vectors were cloned using standard molecular techniques. Gene fragments of the masking polypeptide (e.g., MP80), human IL-15, and human IL-15Rα_sushi were commercially synthesized (Genscripts USA) and digested with the corresponding restriction enzymes accordingly. Human IgG1 Fc(hole) or Fc(knob) was amplified by PCR and digested with restriction enzymes. The cleavable part CM1 was synthesized by annealing the sense and antisense nucleotide single strands with the corresponding restriction enzyme sites at both the 5′ and 3′ ends at 50 °C. 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 cultured overnight at 37 °C in LB medium containing carbenicillin. The recombinant plasmids were extracted using a commercial kit (Qiagen, Cat No. 27104) and sequenced using T7 forward and BGH reverse primers. The entire coding sequence was verified by DNA sequencing. Exemplary sequences of the cleavable parts (CM1-CM10) are shown in Table 3, non-cleavable linkers (lk, lk1, lk2, lk3, and lk5) are shown in Table 4, exemplary sequences of human wild-type IL-15 (mature form or precursor form), IL-15 variants, and IL-15Rα_sushi (long form or short form) are shown in Table 5, sequences of human IgG1 Fc(hole), Fc(knob), Fc(knob-LALA), and Fc(hole-LALA) are shown in Table 6, and exemplary sequences of the IL-15 prodrug constructs are shown in Table 7. In Table 7, the human IL-15 or IL-15Rα_sushi domains are italicized, the non-cleavable linkers are bolded, the cleavable parts are Single underline labeled, the masking polypeptide is Double underline labeled, and the introduced restriction enzyme recognition sites are indicated by dashed lines.

[0584] For characterization and evaluation, expression constructs of the prodrugs and drugs with an N-terminal 8×His tag were cloned and verified accordingly.

[0585] Exemplary cleavable parts (e.g., CM1, CM2, or CM4) in the activatable cytokines used in this application can be cleaved by matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9).

[0586] Table 3

[0587]

[0588]

[0589] Table 4

[0590] Non-cleavable linker SEQ ID NO. Sequence lk 17 GSG lk1 18 (G4S)1 lk2 19 (G4S)2 lk3 20 (G4S)3 lk5 21 (G4S)5

[0591] Table 5

[0592]

[0593]

[0594] Table 6

[0595]

[0596]

[0597] Table 7

[0598]

[0599]

[0600]

[0601]

[0602]

[0603]

[0604]

[0605]

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615]

[0616]

[0617]

[0618]

[0619]

[0620]

[0621] Preparation: Transiently transfect a vector encoding the IL-15 prodrug, IL-15 drug, or non-activatable IL-15 cytokine shown in Table 2 (i.e., SB1902-C4, which does not have a cleavable moiety), and express the protein in Expi293 cells (Thermo Fisher Scientific, Waltham, MA, USA) according to the method recommended by the manufacturer. Clarify the cultured supernatant medium by centrifugation and 0.2 μm membrane filtration. Purify the exemplary IL-15 prodrugs, exemplary IL-15 drugs, and non-activatable IL-15 cytokine SB1902-C4 shown in Table 2 by a two-step purification process according to the method recommended by the manufacturer, including using a pre-packed MabSelect SuRe pcc column (Cytiva lifescience, Cat No. 17549112) and size exclusion chromatography (Superdex200, Cytiva, USA). MBP-MP80 is a fusion protein of maltose binding protein and the masking polypeptide MP80 (SEQ ID NO:1), and is purified using a pre-packed amylose resin column (NEB, Cat No. E8021L, USA) according to the manufacturer's standard operation.

[0622] SDS-PAGE analysis: Purity analysis of the exemplary purified prodrugs SB1902-C2, SB1902-C7, and SB1902-C1 was performed using 4-20% polyacrylamide SDS-PAGE under reducing or non-reducing conditions, and the gel was stained with SimplyBlue SafeStain ( Figure 3 )

[0623] SEC-HPLC analysis: The prodrug SB1902-C2 and the drug SB1902-C1 were analyzed by analytical SEC-HPLC for homogeneity evaluation ( Figure 4)。HPLC analysis was performed using a TSKgel G3000SWxl column according to the method recommended by the manufacturer. All purified drugs and prodrugs were analyzed by SDS-PAGE and SEC-HPLC in the same manner.

[0624] The results of SDS-PAGE and HPLC are shown in Figure 3 and Figure 4 which demonstrate that the constructs described herein were successfully prepared, and the purified prodrugs SB1902-C2 and SB1902-C7, as well as the drug SB1902-C1, have good purity and homogeneity.

[0625] Example 3: Proteolytic cleavage of prodrugs by MMP2 and MMP9

[0626] This experiment was related to the discovery of polypeptide substrates sensitive to MMP2 and MMP9, and the substrate cleavage efficiency could be measured by different methods.

[0627] 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 US Patent Pub. NO.: US 2009 / 0253896A1) as a template. After mutagenesis and screening, a series of MMP2 and MMP9 substrate sequences (cleavable parts) with appropriate sensitivity were obtained, named CM1-CM10, and the sequences and results of in vitro cleavage efficiency are shown in Table 8.

[0628] To test the in vitro cleavage efficiency, an in vitro enzyme assay was performed. Briefly, recombinant human MMP2 (rhMMP2) (R&D Systems, Cat. No. 902-MP-010) and recombinant human MMP9 (rhMMP9) (R&D Systems, Cat. No. 911-MP-010) were diluted to 100 μg / mL with assay buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w / v) Brij 35, pH 7.5). 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 taken into an Eppendorf tube, and 4-aminophenylmercuric acetate (APMA) (Sigma, Cat. No. a-9563) was added to a final concentration of 1 mM. For rhMMP2, it was incubated at 37 °C for 2 hours, and for rhMMP9 it was incubated overnight to activate rhMMP2 and rhMMP9. The activated rhMMP-2 or rhMMP9 was diluted to 1 ng / μL in assay buffer. 16 μL of the activated rhMMP2 and rhMMP9 were respectively loaded into the first tube, and 4 μL of assay buffer was added. Serial two-fold dilutions were made 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 pre-loaded with activated rhMM2 or rhMM9 at different enzyme concentrations, and 1.5 μg of SB1902-C2, SB1902-C5 or SB1902-C4 was added thereto for protease digestion, and digestion was carried out at 37 °C for 5 hours. After incubation for 5 hours, the prodrug or drug before and after digestion was analyzed using 4-20% polyacrylamide gel.

[0629] Table 8

[0630]

[0631] Figure 5A-5B Describes the bands obtained after cleavage of CM1 in the prodrug SB1902-C2 by MMP2 and MMP9 in vitro according to the above standard operation. The mutant substrate CM1 can be cleaved by MMP2 and MMP9 enzymes in vitro and shows enzyme-dose dependence.

[0632] Figure 5E-5F Describes the bands obtained after cleavage of CM4 in the prodrug SB1902-C5 by MMP2 and MMP9 in vitro according to the above standard operation. The mutant substrate CM4 can be cleaved by MMP2 and MMP9 enzymes in vitro and shows enzyme-dose dependence.

[0633] Other cleavable moieties (such as CM2, CM5 - CM10) can also be cleaved by MMP2 and MMP9 in vitro (data not shown). These results indicate that the cleavable moieties in the designed prodrugs can be cleaved at the target tissues (e.g., tumors) in vivo.

[0634] However, as Figure 5C-5D shown, the non - cleavable G4S linker in SB1902 - C4 is resistant to both MMP2 and MMP9 cleavage.

[0635] Example 4: In vitro characterization of prodrugs and drugs

[0636] 4.1 Non-specific serum protein binding:

[0637] The binding of exemplary prodrugs SB1902 - C2 and SB1902 - C3, and SB1902 - C4 (without a cleavable linker) and the drug SB1902 - C1 to human, cynomolgus monkey, or rat serum proteins was evaluated using a direct ELISA assay. Briefly, human, cynomolgus monkey, or rat serum was separately coated onto 96 - well Maxisorp plates (Corning) at 25 μL per well and incubated overnight at 4 °C. The plates were then placed in PBS containing 1% BSA and blocked for 1 hour at room temperature. SB1902 - C2, SB1902 - C3, SB1902 - C4, or SB1902 - C1 was serially diluted in PBS (ELISA buffer) containing 0.5% BSA for 1 hour and washed with PBS (washing buffer). Bound SB1902 - C2, SB1902 - C3, SB1902 - C4, and SB1902 - C1 were detected using an AP - conjugated anti - human IgG Fc - specific antibody (Southern Biotech, Cat. No. 2014 - 04) in ELISA buffer. The plates were incubated with stirring at room temperature for 1 hour, washed 6 times with the washing buffer, and developed with 50 μL / well of freshly prepared PNPP substrate (Sigma Aldrich, No. bern2770) for 3 - 10 minutes. Enzymatic color development was blocked with 50 μL / well of TMB stop solution (VWR, Cat. No. 95059 - 200). Analysis was performed at 405 nm using a BioTek Gen5 microplate reader (BioTek).

[0638] As Figure 6A-6C shown, no non - specific binding of the prodrugs SB1902 - C2, SB1902 - C3, SB1902 - C4, and the drug SB1902 - C1 to human, cynomolgus monkey, and rat serum proteins was detected.

[0639] 4.2 Plasma stability:

[0640] The in vitro serum stability of the prodrug was detected in human plasma, cynomolgus monkey plasma, and rat plasma, respectively. Blood was collected from humans, cynomolgus monkeys, or rats, and the blood cells in the plasma were separated by centrifugation. The upper-layer plasma was carefully transferred to a new centrifuge tube and centrifuged continuously 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 taken, and 5 micrograms of the prodrug or the drug was added to each respectively. The centrifuge tube was sealed and incubated at 40 °C for 7 days. 5 μL of the incubated prodrug and the drug were taken and separated by 4-20% precast polyacrylamide gel (Thermo Fisher, Cat No. XP04205BOX). The separated proteins on the polyacrylamide gel were transferred to a PVDF membrane. After blocking and washing, it was incubated with an AP-conjugated anti-His6 polyclonal antibody (SouthernBiotech, Cat No. SB194b) for 1 hour and then washed. According to the manufacturer's instructions, the blotted protein bands were developed with a chromogenic reagent (Bio-Rad, Cat No. 1706432).

[0641] As Figure 6D-6E shown are the representative western blot results. The bands of the prodrugs SB1902-C2, SB1902-C3, and SB1902-C4 on the WB membrane before and after incubation with plasma and PBS buffer are as Figure 6D and Figure 6E shown, indicating that no degradation bands were detected after the prodrugs SB1902-C2, SB1902-C3, and SB1902-C4 were incubated with human plasma or PBS buffer. Moreover, no detectable degradation bands were found on the WB membrane after the drug SB1902-C1 and the prodrugs SB1902-C2, SB1902-C3, and SB1902-C4 were incubated with cynomolgus monkey or rat plasma, respectively (data not shown). These results indicate that the prodrugs are stable in human, cynomolgus monkey, and rat plasma.

[0642] 4.3 Receptor binding affinity:

[0643] The binding affinity of the prodrug or drug to the IL-2 / IL15Rβγ-Fc fusion protein was detected by ELISA. The ELISA plate was coated with 1 μg / ml recombinant IL15Rβγ-Fc and incubated overnight at 4°C. After blocking and washing the plate with PBS containing 1% BSA, serial dilutions of the exemplary prodrug or drug were added to the plate 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 the dideoxynucleotide method of RNA sequencing. Nucleic Acids Res. 9(18):4485-4494), was used as a negative control. After washing the plate, a 1:2000 dilution of anti-human IgG-Fc-AP conjugate antibody was added to each well of the plate and incubated for 45 min. After washing the plate, the AP substrate pNPP (Thermo, USA) was added, and the optical density was obtained at 405 nm using a spectrophotometer (BioTec, USA). The data were analyzed and plotted using GraphPad Prism 8 software.

[0644] 4.3.1 Effect of the masking polypeptide in the IL-15 prodrug on IL-15 receptor binding affinity:

[0645] This experiment detected the exemplary prodrug SB1902-C2 and the drug SB1902-C1. As Figure 7A shown, compared with the drug SB1902-C1, the binding affinity of the exemplary prodrug SB1902-C2 to IL-2 / IL-15Rβγ was significantly reduced. This result indicates that the masking polypeptide effectively inhibits the receptor-binding function of IL-15.

[0646] 4.3.2 Binding affinity of the IL-15 drug or prodrug:

[0647] This experiment detected the exemplary drug SB1902-C1-variant3 with wild-type IL-15 and the exemplary drug with an IL-15 variant. As Figure 7B-7D shown, compared with the drug SB1902-C1-variant3, the exemplary drugs with an IL-15 variant: SB1902-C1-variant3_L45D, SB1902-C1-variant3_L52D, SB1902-C1-variant3_I67D, and SB1902-C1-variant3_I67E all showed higher affinity for IL-2 / IL-15Rβ.

[0648] This experiment also detected an exemplary prodrug (SB1902-C9-variant2) with wild-type IL-15 and an exemplary prodrug with an IL-15 variant. However, as shown in Figure 7E-7F Figure Figure 7E-7F , compared with the prodrug SB1902-C9-variant2, the exemplary IL-15 prodrugs SB1902-C9-variant2_L45D, SB1902-C9-variant2_L52D, SB1902-C9-variant2_I67D, and SB1902-C9-variant2_I67E showed reduced binding affinity to IL-2 / IL-15Rβ.

[0649] The above results indicate that in the prodrugs with IL-15 variants, the masking polypeptide can also effectively block the binding ability of the IL-15 variant.

[0650] Example 5: Immunogenicity study

[0651] Balb / c mice were immunized by continuous intravenous injection of the prodrug at a dose of 1 mg / kg / week for 6 weeks to study the immunogenicity of the masking peptide (MP). Serum samples from all mice 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 overnight at 4 °C and then blocked with PBS containing 1% BSA. After washing the plates, sera from mice treated with the prodrug SB1902-C2 were serially diluted and added to wells pre-coated with the prodrug SB1902-C2 and MBP-MP80. Sera from mice treated with Boco (Bococizumab, Pfizer, as a positive control) were also serially diluted and added to wells pre-coated with Boco. After incubation and washing, 1:3000 diluted goat anti-mouse IgG Fc-AP was added to the plates and incubated for 45 minutes. After washing the plates, 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. GraphPad Prism 8 software was used for plotting.

[0652] As shown in Figure 8 Figure Figure 8 , after weekly injection of the prodrug for a total of 6 weeks, no anti-drug antibodies (ADA) against the MBP-MP80 fusion protein were detected in the sera of 5 mice by ELISA. In contrast, among 5 mice injected weekly with the prodrug SB1902-C2, sera from 3 mice produced significant ADA against the Fc portion of the prodrug. The results indicate that the masking polypeptide MP80 in the prodrug is not immunogenic.

[0653] Example 6: In vitro functional assay: Mo7e cell proliferation assay

[0654] The prodrugs were assayed in an in vitro cell proliferation assay. It is known that IL-15 mainly transmits signals through its binding to IL-2 / IL-15Rβγ. Mo7e cells express both IL-2 / IL15Rβ and γc on the cell membrane, and their survival and proliferation require IL-2 or IL-15.

[0655] To detect the proliferation of IL-15 cytokine-dependent Mo7e cells, the following experiment was conducted: Mo7e cells (DSMZ, Cat No. ACC 104) were stored 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 an incubator at 37 °C (5% CO 2 ) The Mo7e cells were harvested during the logarithmic growth phase and washed twice with medium without GM-CSF.

[0656] The cells were added to a 384-well plate at 1×10 4 / well, and the medium volume was 45 μL / well (without GM-CSF). The cells were incubated in an incubator at 37 °C (5% CO 2 ) for 2 - 4 hours. 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 with only medium were used as blank controls. The cells were cultured in the incubator for 3 days. Then, 5 μL of WST-8 (WST-8 Cell Proliferation Assay Kit, Cayman, Cat No. 10010199) was added to all wells and incubated in the incubator for 2 - 3 hours to detect the proliferation of Mo7e cells. The absorbance at 450 nm (OD450) was measured. The data were analyzed and plotted using Graphpad Prism 8 software.

[0657] As Figure 9 shown, all exemplary prodrugs SB1902-C2, SB1902-C6, and SB1902-C7, as well as the drug SB1902-C1, had a dose-dependent stimulatory effect on cell proliferation. However, compared with the drug SB1902-C1, the activities of the prodrugs SB1902-C2, SB1902-C6, and SB1902-C7 were significantly reduced. The results indicate that the masking polypeptides MP80, MP96new, and MP100 blocked the binding of the IL-15 prodrug to its receptor IL-2 / IL-15Rβγ, reducing its signal transduction and downstream functions.

[0658] Example 7: In vitro functional assay: CD8+ T cell activity assay

[0659] It is documented in the literature that IL-15 promotes the proliferation, survival, and homeostasis of CD8+ memory T cells, natural killer cells (NK), and NKT cells. IL-15 induces T cell activation, which can be indicated by the upregulated expression of CD69 on the cell membrane surface and the release of cytokines including IFNγ. CD69 is an early marker of T cells. The percentage of CD8+ T cell activation is reflected by the percentage of CD69 expressed on the cell surface.

[0660] 7.1 Activity of IL-15 drug or prodrug related to CD8+ T cells

[0661] To compare the activities of IL-15 prodrug and IL-15 drug in activating CD8+ T cells, the basic operations of this assay are as follows: Human PBMCs (Stem Cell Technologies, Cat No. 70500) were added to a 96-well round-bottom cell culture plate containing RPMI-1640 medium at a cell density of 2×10 5 / well. 10% FBS, 1% penicillin, and streptomycin were added to the medium. Exemplary IL-15 drug and prodrug were serially diluted 3-fold with the medium, and 5 μL was taken and added to the wells. Each concentration was repeated three times, and blank wells (containing only the medium) served as blank controls. The cell culture dishes were incubated in an incubator for 3 days. Then the cells were centrifuged and stained with anti-CD3 Ab (biolgend, cat#317306), anti-CD8 Ab (biolgend, cat#344722), and anti-CD69 Ab (biolgend, cat#310906) in FACS buffer for 30 minutes. After washing twice with FACS buffer, the cells were obtained using an Attune (ThermoFisher Scientific) flow cytometer. The data was analyzed using FlowJo software. Graphpad Prism 8 software was used to plot the percentage of CD69+ cells in CD8+ T cells.

[0662] 7.1.1 Function of the masking polypeptide in the IL-15 prodrug:

[0663] This experiment detected the exemplary prodrugs SB1902-C2, SB1902-C6, SB1902-C7, SB1902-C2-variant1, SB1902-C9-variant0, SB1902-C9-variant1, SB1902-C9-variant2, SB1902-C9-variant3, SB1902-C9-variant4, and SB1902-C10-variant1, as well as the exemplary drugs SB1902-C1, SB1902-C1-variant1, SB1902-C1-variant2, and SB1902-C1-variant3.

[0664] As Figure 10A shown, compared with the drug SB1902-C1, the prodrug SB1902-C7 with the masking polypeptide MP100 has significantly lower activity in stimulating T cell activation.

[0665] As Figure 10B shown, compared with the drug SB1902-C1, the prodrug SB1902-C2 with the masking polypeptide MP80 has significantly lower activity in stimulating T cell activation.

[0666] As Figure 10C shown, both the prodrug SB1902-C2 with the masking polypeptide MP80 and the prodrug SB1902-C6 with the masking polypeptide MP96new have lower activities in stimulating T cell activation. In contrast, SB1902-C2 is superior to SB1902-C6 in inhibiting CD69 activation in CD8+ T cells.

[0667] As Figure 10D shown, compared with the drugs SB1902-C1-variant 2 and SB1902-C1-variant 3, the prodrug SB1902-C9-variant4 with the masking polypeptide MP163 and the Fc domain LALA mutations (L234A and L235A) has significantly lower activity in stimulating T cell activation.

[0668] As Figure 10E shown, compared with the drug SB1902-C1-variant1, the prodrug SB1902-C10-variant1 with the masking polypeptide MP240 has significantly lower activity in stimulating T cell activation.

[0669] As Figure 10F shown, compared with the drug SB1902-C1, the prodrug SB1902-C2-variant1 with the masking polypeptide MP80 has significantly lower activity in stimulating T cell activation.

[0670] AsFigure 10G As shown, compared with the drug SB1902-C1-variant1, the prodrug SB1902-C9-variant0 with the masking polypeptide MP163 has significantly lower activity in stimulating T cell activation.

[0671] As Figure 10H shown, compared with the drug SB1902-C1, the prodrug SB1902-C9-variant2 with the masking polypeptide MP163 has significantly lower activity in stimulating T cell activation.

[0672] As Figure 10I shown, the prodrugs SB1902-C9-variant1, SB1902-C9-variant2, and SB1902-C9-variant3 with the masking polypeptide MP163 have significantly lower activity in stimulating T cell activation.

[0673] The above results indicate that among different prodrug forms with different masking polypeptides of the IL-15 cytokine, all the masking polypeptides significantly block the activity of activating T cells, indicating that the masking polypeptides of the prodrugs can prevent IL-15 from activating CD8+ T cells.

[0674] 7.1.2 Masking polypeptides (MPs) of different lengths in the IL-15 prodrug:

[0675] This experiment was conducted as described above, and the results of the prodrugs SB1902-C9 and SB1902-C2 are as Figure 10J shown. The results of the prodrugs SB1902-C9-variant2 and SB1902-C2-variant2 ...

Claims

1. An IL-15 prodrug, comprising: (i) one or more IL-15 cytokines (I), (ii) one or more cleavable moieties (CM), and (iii) one or more masking polypeptides (MP).

2. The IL-15 prodrug according to claim 1, wherein the masking polypeptide (MP) attenuates the activity of the IL-15 cytokine (I), and the cleavable moiety (CM) is readily cleaved at or near the tumor or target cell.

3. The IL-15 prodrug according to claim 1 or 2, wherein the IL-15 cytokine (I) and the masking polypeptide (MP) are linked by the cleavable moiety (CM).

4. The IL-15 prodrug according to any one of claims 1-3, wherein the IL-15 prodrug further comprises IL-15Rα or a functional fragment thereof (S), and wherein the IL-15Rα or a functional fragment thereof is selected from the extracellular domain of IL-15Rα or the sushi domain or a functional analogue.

5. The IL-15 prodrug according to any one of claims 1-4, wherein the IL-15Rα or a functional fragment thereof (S) and the masking polypeptide (MP) are linked by the cleavable moiety (CM).

6. The IL-15 prodrug according to any one of claims 1-5, wherein the IL-15 prodrug further comprises one or more half-life extending moieties (C).

7. The IL-15 prodrug according to claim 6, wherein: (i) the IL-15 cytokine (I) and the half-life extending moiety (C) are linked; and / or (ii) the IL-15Rα or a functional fragment thereof (S) and the half-life extending moiety (C) are linked; and / or (iii) the masking polypeptide (MP) and the half-life extending moiety (C) are linked by the cleavable moiety (CM).

8. The IL-15 prodrug according to claim 7, wherein: (i) 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 the cleavable moiety (CM); or (ii) 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 the cleavable moiety (CM); or (iii) the IL-15Rα or a functional fragment thereof (S) and the half-life extending moiety (C) are linked, the IL-15 cytokine (I) and the IL-15Rα or a functional fragment thereof (S) are linked, and the masking polypeptide (MP) and the IL-15 cytokine (I) are linked by the cleavable moiety (CM); or (iv) The IL-15 cytokine (I) is linked to the half-life extending moiety (C), the IL-15Rα or its functional fragment (S) is linked to the half-life extending moiety (C), and the masking polypeptide (MP) and the IL-15Rα or its functional fragment (S) are linked through the cleavable moiety (CM); or (v) The masking polypeptide (MP) and the half-life extending moiety (C) are linked through the cleavable moiety (CM), the IL-15Rα or its functional fragment (S) is linked to the half-life extending moiety (C), and the IL-15 cytokine (I) and the IL-15Rα or its functional fragment (S) are linked; or (vi) The masking polypeptide (MP) and the half-life extending moiety (C) are linked through the cleavable moiety (CM), the IL-15 cytokine (I) is linked to the half-life extending moiety (C), and the IL-15Rα or its functional fragment (S) and the IL-15 cytokine (I) are linked.

9. The IL-15 prodrug according to any one of claims 1-8, wherein the prodrug is a monomer or a dimer.

10. The IL-15 prodrug according to claim 9, wherein the prodrug is a dimer and comprises two monomers, wherein: (i) One monomer comprises a first half-life extending moiety (C), the IL-15Rα or its functional fragment (S), the IL-15 cytokine (I); and the other monomer comprises a second half-life extending moiety (C), the masking polypeptide (MP) and the cleavable moiety (CM), wherein the masking polypeptide (MP) and the second half-life extending moiety (C) are linked through the cleavable moiety (CM); or (ii) One monomer comprises a first half-life extending moiety (C), the IL-15Rα or its functional fragment (S), the masking polypeptide (MP) and the cleavable moiety (CM); and the other monomer comprises a second half-life extending moiety (C) and the IL-15 cytokine (I); or (iii) One monomer comprises a first half-life extending moiety (C), the IL-15 cytokine (I), the masking polypeptide (MP) and the cleavable moiety (CM); and the other monomer comprises a second half-life extending moiety (C) and the IL-15Rα or its functional fragment (S); or (iv) One monomer and the other monomer respectively comprise the half-life extending moiety (C), the IL-15 cytokine (I), the IL-15Rα or its functional fragment (S), the cleavable moiety (CM) and the masking polypeptide (MP).

11. The IL-15 prodrug according to claim 10, wherein: (i) In one monomer: both the IL-15 cytokine (I) and the IL-15Rα or its functional fragment (S) are linked to the first half-life extending moiety (C); and in the other monomer: The masking polypeptide (MP) and the second half-life extension moiety (C) are linked by the cleavable moiety (CM); or (ii) In one monomer: the IL-15 cytokine (I) is linked to the first half-life extension moiety (C), and the IL-15Rα or its functional fragment (S) is linked to the IL-15 cytokine (I); and, in another monomer: the masking polypeptide (MP) and the second half-life extension moiety (C) are linked by the cleavable moiety (CM); or (iii) In one monomer: the IL-15Rα or its functional fragment (S) is linked to the first half-life extension moiety (C), and the IL-15 cytokine (I) is linked to the IL-15Rα or its functional fragment (S); and, in another monomer: the masking polypeptide (MP) and the second half-life extension moiety (C) are linked by the cleavable moiety (CM); or (iv) In one monomer: the IL-15Rα or its functional fragment (S) is linked to the first half-life extension moiety (C), and the masking polypeptide (MP) and the first half-life extension moiety (C) are linked by the cleavable moiety (CM); and, in another monomer: the IL-15 cytokine (I) is linked to the second half-life extension moiety (C); or (v) In one monomer: the IL-15Rα or its functional fragment (S) is linked to the first half-life extension moiety (C), and the masking polypeptide (MP) and the IL-15Rα or its functional fragment (S) are linked by the cleavable moiety (CM); and, in another monomer: the IL-15 cytokine (I) is linked to the second half-life extension moiety (C); or (vi) In one monomer: the IL-15 cytokine (I) is linked to the first half-life extension moiety (C), and the masking polypeptide (MP) and the first half-life extension moiety (C) are linked by the cleavable moiety (CM); and, in another monomer: the IL-15Rα or its functional fragment (S) is linked to the second half-life extension moiety (C); or (vii) In one monomer: the IL-15 cytokine (I) is linked to the first half-life extension moiety (C), and the masking polypeptide (MP) and the IL-15 cytokine (I) are linked by the cleavable moiety (CM); and, in another monomer: the IL-15Rα or its functional fragment (S) is linked to the second half-life extension moiety (C); or (viii) One monomer and another monomer each comprise any one of the constructs described in claim 8.

12. The IL-15 prodrug according to any one of claims 5-11, wherein the IL-15Rα or its functional fragment (S) and the IL-15 cytokine (I) are covalently linked; or the IL-15Rα or its functional fragment (S) and the IL-15 cytokine (I) are non-covalently linked and form an IL-15 / IL-15Rα complex.

13. The IL-15 prodrug according to any one of claims 1-12, wherein the IL-15 cytokine (I) comprises one or more amino acid mutations selected from the group consisting of L45D, L45E, Q48K, S51D, L52D, E64K, I67D, I67E, I68D, and N72D.

14. The IL-15 prodrug according to any one of claims 1-13, wherein the IL-15 cytokine (I) comprises the amino acid sequence shown in any of SEQ ID NOs: 22-23 and 67-76, or a variant thereof, and the variant has at least 90% sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 22-23 and 67-76.

15. The IL-15 prodrug according to any one of claims 1-12, wherein the IL-15Rα or its functional fragment (S) comprises the amino acid sequence shown in any of SEQ ID NOs: 24-26, or a variant thereof, and the variant has at least 90% sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 24-26.

16. The IL-15 prodrug according to any one of claims 6-15, wherein the half-life extension moiety (C) comprises an Fc domain; preferably, the Fc domain is selected from the group consisting of human IgG1 Fc domain, human IgG2 Fc domain, human IgG3 Fc domain, human IgG4 Fc domain, IgA Fc domain, IgD Fc domain, IgE Fc domain, and IgM Fc domain; more preferably, the Fc domain is the human IgG1 Fc domain.

17. The IL-15 prodrug according to claim 16, wherein the Fc domain is a human IgG1 Fc domain having L234A and L235A mutations, and the mutation sites are numbered according to the EU numbering system.

18. The IL-15 prodrug according to claim 16 or 17, wherein the Fc domain comprises a knobs-into-holes mutation (Fc knob and Fc hole).

19. The IL-15 prodrug according to claim 18, wherein the Fc knob comprises a T366W mutation in the Fc domain, and the Fc hole comprises T366S, L368A, and Y407V mutations in the Fc domain, and the mutation sites are numbered according to the EU numbering system.

20. The IL-15 prodrug according to claim 19, wherein the Fc knob further comprises an S354C mutation, and the Fc hole further comprises a Y349C mutation, and the mutation sites are numbered according to the EU numbering system.

21. The IL-15 prodrug according to any one of claims 1-12, wherein the masking polypeptide (MP) consists 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).

22. The IL-15 prodrug according to claim 21, wherein the masking polypeptide (MP) consists of five amino acids G, S, P, E, and A; further, in the masking polypeptide, the percentage of amino acid residue G is about 15%-30%, preferably 20%; in the masking polypeptide, the percentage of amino acid residue S is about 20%-40%, preferably 40%; in the masking polypeptide, the percentage of amino acid residue P is about 15%-40%, preferably 20%; in the masking polypeptide, the percentage of amino acid residue E is about 1%-20%, preferably 10%; in the masking polypeptide, the percentage of amino acid residue A is about 5%-20%, preferably 10%; when the number of amino acids is not an integer, take the integer value.

23. The IL-15 prodrug according to claim 21, wherein the masking polypeptide (MP) consists of four amino acids S, P, E, and G; further, in the masking polypeptide, the percentage of amino acid residue S is about 20%-40%, preferably 23%; in the masking polypeptide, the percentage of amino acid residue P is about 15%-40%, preferably 29%; in the masking polypeptide, the percentage of amino acid residue E is about 1%-20%, preferably 18%; in the masking polypeptide, the percentage of amino acid residue G is about 15%-30%, preferably 30%; when the number of amino acids is not an integer, take the integer value.

24. The IL-15 prodrug according to any one of claims 1-23, wherein the masking polypeptide (MP) contains about 40 to 720 amino acid residues; preferably, it contains 80 to 320 amino acid residues; more preferably, it contains 80 to 240 amino acid residues.

25. The IL-15 prodrug according to any one of claims 1-24, wherein the masking polypeptide (MP) contains the amino acid sequence SEQ ID NO:

6.

26. The IL-15 prodrug according to any one of claims 1-25, wherein the masking polypeptide (MP) contains the amino acid sequence SEQ ID NO:

1.

27. The IL-15 prodrug according to any one of claims 1-26, wherein the masking polypeptide (MP) contains the amino acid sequence shown in any one of SEQ ID NOs: 1-5; or a variant thereof, the variant having at least 90% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1-5; or a variant thereof, the variant containing one or more amino acid substitutions, additions, and / or deletions.

28. The IL-15 prodrug according to any one of claims 1-20, wherein the cleavable moiety (CM) comprises the amino acid sequence MVX 1 X 2 AX 3 TX 4 SG (SEQ ID NO:49), wherein X 1 is selected from P, L, V or A, X 2 is selected from L or S, X 3 is selected from L, V, P or Y, and X 4 is selected from A or V.

29. The IL-15 prodrug according to claim 28, wherein the cleavable moiety (CM) comprises a substrate sequence of urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP) 1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, fibroblast activation protein (FAP), stromelysin, cathepsin, caspase, thrombin, metalloprotease, serine protease, cysteine protease, aspartic protease, legumain, kallikrein, cathepsin A, cathepsin B, chymotrypsin, a protease located at the tumor site or its surrounding environment, or any combination thereof.

30. The IL-15 prodrug according to claim 28 or 29, wherein the cleavable moiety (CM) comprises an amino acid sequence shown in any of SEQ ID NOs: 8-16, or a variant thereof, the variant having at least 90% sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 8-16.

31. The IL-15 prodrug according to any one of claims 1-30, wherein the prodrug comprises two monomers, wherein, in one monomer, the IL-15Rα or its functional fragment(s) is linked to the first Fc domain, and in the other monomer, the IL-15 cytokine (I) is linked to the second Fc domain, and the masking polypeptide (MP) and the IL-15 cytokine (I) are linked through the cleavable moiety (CM).

32. The IL-15 prodrug according to claim 31, wherein: (1) one monomer comprises the amino acid sequence SEQ ID NO: 33 or a variant thereof, the variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 33; and the other monomer comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, the variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 32; or (2) one monomer comprises the amino acid sequence SEQ ID NO: 34 or a variant thereof, the variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 34; and the other monomer comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, the variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 32; or (3) One monomer comprises the amino acid sequence SEQ ID NO: 36 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 36; and another monomer comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 32; or (4) One monomer comprises the amino acid sequence SEQ ID NO: 37 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 37; and another monomer comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 32; or (5) One monomer comprises the amino acid sequence SEQ ID NO: 38 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 38; and another monomer comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 32; or (6) One monomer comprises the amino acid sequence SEQ ID NO: 39 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 39; and another monomer comprises the amino acid sequence SEQ ID NO: 32 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 32; or (7) One monomer comprises the amino acid sequence SEQ ID NO: 45 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 45; and another monomer comprises the amino acid sequence SEQ ID NO: 46 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 46; or (8) One monomer comprises the amino acid sequence SEQ ID NO: 47 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 47; and another monomer comprises the amino acid sequence SEQ ID NO: 40 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 40; or (9) One monomer comprises the amino acid sequence SEQ ID NO: 33 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 33; and another monomer comprises the amino acid sequence SEQ ID NO: 40 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 40; or (10) One monomer comprises the amino acid sequence SEQ ID NO: 62 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 62; and another monomer comprises the amino acid sequence SEQ ID NO: 63 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 63; or (11) One monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 41; and another monomer comprises the amino acid sequence SEQ ID NO: 63 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 63; or (12) One monomer comprises the amino acid sequence SEQ ID NO: 64 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 64; and another monomer comprises the amino acid sequence SEQ ID NO: 65 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 65; or (13) One monomer comprises the amino acid sequence SEQ ID NO: 62 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 62; and another monomer comprises the amino acid sequence SEQ ID NO: 65 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 65; or (14) One monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 41; and another monomer comprises the amino acid sequence SEQ ID NO: 65 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 65; or (15) One monomer comprises the amino acid sequence SEQ ID NO: 66 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 66; and another monomer comprises the amino acid sequence SEQ ID NO: 65 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 65; or (16) One monomer comprises the amino acid sequence SEQ ID NO: 43 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 43; and another monomer comprises the amino acid sequence SEQ ID NO: 46 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 46; or (17)One monomer comprises the amino acid sequence SEQ ID NO: 39 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 39; and another monomer comprises the amino acid sequence SEQ ID NO: 40 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 40; or (18)One monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 41; and another monomer comprises the amino acid sequence SEQ ID NO: 87 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 87; or (19)One monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 41; and another monomer comprises the amino acid sequence SEQ ID NO: 91 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 91; or (20)One monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 41; and another monomer comprises the amino acid sequence SEQ ID NO: 93 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 93; or (21)One monomer comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 41; and another monomer comprises the amino acid sequence SEQ ID NO: 94 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:

94.

33. An IL-15 drug, wherein the drug comprises two monomers, wherein, in one monomer, the IL-15Rα or its functional fragment(s) is linked to the first Fc domain, and in another monomer, the IL-15 cytokine (I) is linked to the second Fc domain.

34. The IL-15 drug according to claim 33, wherein: (1)One monomer comprises the amino acid sequence SEQ ID NO: 43 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 43; and another monomer comprises the amino acid sequence SEQ ID NO: 83 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 83; or (2)One monomer comprises the amino acid sequence SEQ ID NO: 43 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 43; and another monomer comprises the amino acid sequence SEQ ID NO: 84 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 84; or (3)One monomer comprises the amino acid sequence SEQ ID NO: 43 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 43; and another monomer comprises the amino acid sequence SEQ ID NO: 44 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:

44.

35. An isolated nucleic acid molecule encoding the IL-15 prodrug according to any one of claims 1-32, or the IL-15 drug according to claim 33 or 34.

36. A vector comprising the isolated nucleic acid molecule according to claim 35.

37. An isolated host cell comprising the IL-15 prodrug according to any one of claims 1-32, the IL-15 drug according to claim 33 or 34, the nucleic acid molecule according to claim 35, or the vector according to claim 36.

38. A method for preparing the IL-15 prodrug or drug, comprising: a) culturing the host cell according to claim 37 under conditions effective for expressing the IL-15 prodrug or drug; and b) obtaining the expressed IL-15 prodrug or drug from the host cell.

39. A pharmaceutical composition comprising the IL-15 prodrug according to any one of claims 1-32, the IL-15 drug according to claim 33 or 34, the nucleic acid according to claim 35, the vector according to claim 36, the isolated host cell according to claim 37, or the IL-15 prodrug prepared by the method according to claim 38, and a pharmaceutically acceptable carrier or excipient.

40. A method for treating a disease or disorder in an individual in need thereof, comprising administering to the individual an effective amount of the IL-15 prodrug according to any one of claims 1-32, the IL-15 drug according to claim 33 or 34, the nucleic acid according to claim 35, the vector according to claim 36, the isolated host cell according to claim 37, the prodrug or drug prepared by the method according to claim 38, or the pharmaceutical composition according to claim 39.

41. The method according to claim 40, wherein the disease or disorder is cancer or an infectious disease or stimulates the immune system in a patient in need thereof.

42. The method according to claim 41, wherein the cancer is selected from the group consisting of prostate cancer, colon cancer, kidney cancer, melanoma, lung cancer, breast cancer, thyroid cancer, bladder cancer, gastric and esophageal cancer, pancreatic cancer, liver cancer, brain cancer, head and neck cancer, neuroblastoma, soft tissue cancer, lymphoma, leukemia, multiple myeloma, or any metastasis thereof.

Images