WO2022178103A1 - Il-2 receptor beta subunit mutants - Google Patents

Abstract

The present disclosure provides novel interleukin-2 receptor β subunit mutants and IL-2 and IL-5 fusion molecules comprising thereof. Also provided are methods of making and using the fusion molecules in stimulating the immune system, or treating cancer, an autoimmune disease, or an infectious disease.

Description

IL-2 RECEPTOR BETA SUBUNIT MUTANTS

CROSS REFERENCE TO RELATED APPLICATION [0001] The present application claims priority from U.S. Provisional Application No. 63/150,568, filed on February 17, 2021. The contents of the priority application are incorporated herein by reference in their entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 18, 2022, is named 025471_W0009_SL.txt and is 460,154 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Interleukin-2 (IL-2) and Interleukin- 15 (IL-15) plays play important roles in the proliferation, differentiation, and survival of immune cells. IL-2 plays a central role in lymphocyte generation, survival, and homeostasis. It has 133 amino acids and consists of four antiparallel, amphipathic alpha-helices that form a quaternary structure essential for its function (Smith, Science (1988) 240:1169-76; Bazan, Science (1992) 257:410-13). IL-2 exerts its activities by binding to IL-2 receptors (IL-2R), which consist of up to three individual subunits. Association of the a (CD25 or Tac antigen), b (CD122), and γC subunits results in a trimeric, high-affinity receptor for IL-2 (KD ~ 0.01 nM). Dimeric IL-2 receptor consisting of the b and g subunits is termed intermediate-affinity IL-2R (KD ~ 1 nM). The a subunit alone forms the monomeric low affinity IL-2 receptor (KD ~ 10 nM). See, e.g., Kim et al., Cytokine Growth Factor Rev. (2006) 17:349-66). Although the dimeric intermediate- affinity IL-2 receptor binds IL-2 with approximately 100-fold lower affinity than the trimeric high-affinity receptor, both the dimeric and trimeric IL-2 receptors can transmit signal upon IL-2 binding (Minami et al. , Annu Rev Immunol. (1993) 11:245-68).

[0004] IL-15 is a cytokine with structural similarities to IL-2. IL-15 is secreted by mononuclear phagocytes and other immune cells following viral infection. IL-15 induces proliferation of natural killer (NK) and other cells of the immune system and is involved in the killing of virally infected cells and cancer cells. Like IL-2, IL-15 binds to the IL-2 receptor (IL-2R) b/g complex, the intermediate affinity receptor, with a KD of about 1 nM (Giri et al., EMBO J (1994) 13:2822-30). IL-15 binds to IL-15 receptor (IL-15R) a with a much higher affinity (KD ~ 0.05 nM). IL-15Ra can associate with the IL-2Rβ/γ complex to form an IL-15-specific, functional high-affinity (abg) receptor (Minami et al.. Annu. Rev. Immunol. (1993) 11:245-67; Giri et al. , J Leukoc Biol. (1995) 5745:763-6; and Lehours et al., Eur Cytokine Netw. (2000) 11:207-15).

[0005] The above cytokines, their muteins, and fusion proteins have been investigated for their potential as therapeutics, with recombinant IL-2 being the first cytokine approved for cancer therapy. However, these cytokine drugs and drug candidates have significant side effects. In addition, their in vivo half-lives are often short, even when presented as antibody- cytokine fusion molecules, potentially due to the “PK sink” formed by the receptors of the cytokines on the immune cells.

[0006] Thus, there remains a need to develop cytokine-based cancer therapeutics that are more tumor site-selective and have improved PK and efficacy, while causing fewer side effects.

SUMMARY OF THE INVENTION

[0007] The present disclosure provides novel isolated proteins comprising a human IL-2 receptor b subunit (IL-2Rβ ) extracellular domain (ECD), wherein the IL-2Rβ ECD comprises, relative to wildtype IL-2Rβ ECD, D1-5 (deletions of the first five amino acids) and/or one or more mutations at position(s) selected from Fll, V21, L28, W38, L51, P52, V53, 163, P67, 177, V88, W90, V92, M93, 195, M107, 1110, VI 15, R137, H150, W152,

P156, L157, K161, Q162, Q164, W166, C168, P174, L187, F191, W194, P196, W197, P200, and P207 (numbering according to SEQ ID NO:4).

[0008] In some embodiments, the IL-2Rβ ECD comprises one or more mutations at position(s) selected from V92, H150, W152, Q162, Q164, W166, C168, and L187. For example, the IL-2Rβ ECD comprises one or more mutations selected from V92S, H150E, W152S/N, Q162S, Q164E, W166N/S/E, C168T/S, and L187S. In particular embodiments, the IL-2Rβ ECD comprises Q162S; Q164E; C168S; Q162S and Q164E; Q162S and C168S; Q164E and C168S; and Q162S, Q164E, and C168S.

[0009] In some embodiments, the isolated protein is an isolated IL-2 or IL-15 fusion molecule, comprising a carrier moiety, a cytokine moiety, and a masking moiety, wherein the cytokine moiety is fused to the carrier moiety or to a masking moiety, directly or indirectly via a peptide linker, the masking moiety is fused to the carrier moiety or to the cytokine moiety, directly or indirectly via a peptide linker, the cytokine moiety comprises an IL-2 or IL-15 polypeptide, optionally wherein the IL-2 or IL-15 polypeptide is a variant of wildtype IL-2 or IL-15 polypeptide, the masking moiety binds to the cytokine moiety and inhibits binding of the cytokine moiety to IL-2Rβ and/or IL-2Rγ on immune cells and comprises the IL-2Rβ ECD.

[0010] In another aspect, the present disclosure provides a polynucleotide encoding the isolated protein herein, an expression vector comprising the polynucleotide, and a host cell comprising the expression vector. Also included are methods of making the isolated protein by using the host cell.

[0011] In another aspect, the present disclosure provides a pharmaceutical composition comprising the isolated protein herein and a pharmaceutically acceptable excipient; and a method of treating a cancer or an infectious disease, stimulating the immune system, or treating an autoimmune disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition. Also provided are the IL-2 and IL-15 fusion molecules (IL-2 and IL-15 prodrugs) for use in treating a cancer or an infectious disease, stimulating the immune system, or treating an autoimmune disease, and for the manufacture of a medicament for treating a cancer or an infectious disease, stimulating the immune system, or treating an autoimmune disease.

[0012] Other features, objects, and advantages of the invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the invention, is given by way of illustration only, not limitation. Various changes and modification within the scope of the invention will become apparent to those skilled in the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS [0013] FIGs. 1A-1C show the design of the IL-2Rβ muteins to abolish binding to IL-2Rγ. FIG. 1A shows the crystal structure (PDB: 2B5I) of the tetrameric complex of IL-2 with IL-2 receptors a, b and y. The key residues of IL-2Rβ located on the interface to IL-2Rγ were labeled. FIG. 1B shows the inter-species alignment of IL-2Rβ extracellular domain (ECD), which indicates that Q162, Q164 and W166 are highly conserved. FIG. 1B discloses SEQ ID NOs:4 and 113-119, respectively, in order of appearance. FIG. 1C shows a table of mutations of the key interface residues of IL-2Rβ.

[0014] FIGs. 2A-2G show screening of the IL-2Rβ ECD mutations. FIG. 2A is a schematic illustration of a IL-2 prodrug comprising a carrier moiety, a masking moiety, and a cytokine moiety; wherein the carrier moiety comprises an antibody against human PD-1, the masking moiety comprises an IL-2Rβ ECD or a mutant thereof, and the cytokine moiety comprises an IL-2 mutein (IL2v); and wherein the IL-2Rβ ECD is fused to the C-terminus of one of the heavy chains of the PD-1 antibody through a cleavable peptide linker, and the IL2v is fused to the C-terminus of the other heavy chain of the PD-1 antibody directly or indirectly through a non-cleavable peptide linker. There are no knobs-into-holes mutations in the Fc portion of the PD1 antibody. “3xG4S” is disclosed as SEQ ID NO:92. FIG. 2B is a summary of the binding affinity (KD) of the IL-2 prodrugs. FIG. 2C and 2D show a size- exclusion/high performance liquid chromatography (SEC-HPLC) of the IL-2 prodrug samples by Protein A affinity chromatography. FIG. 2E shows binding of the IL-2 prodrugs to IL-2Rγ. FIG. 2F shows an SDS-PAGE gel analysis of the IL-2 prodrugs comprising an IL-2Rβ ECD masking moiety with a C168S mutation or an Q162S/Q164E mutation prior to and after cleavage with protease hMMP2. FIG. 2G shows CTLL2 assays of the IL-2 prodrugs comprising an IL-2Rβ ECD masking moiety with a C168 S mutation or an Q162S/Q164E mutation prior to and after cleavage with protease hMMP2.

[0015] FIGs. 3A-3F show assessment of IL-2 prodrugs comprising a mutant IL-2Rβ ECD masking moiety. FIG. 3A shows a schematic drawing of the IL-2 prodrugs comprising a mutant IL-2Rβ ECD masking moiety. The IL-2 prodrug comprises a carrier moiety, a masking moiety, and a cytokine moiety; wherein the carrier moiety comprises an antibody against human PD-1, the masking moiety comprises an IL-2Rβ ECD with two or more point mutations, and the cytokine moiety comprises an IL-2 mutein (IL2v); and wherein the IL- 2RP ECD masking moiety is fused to the C-terminus of one of the heavy chains of the PD-1 antibody through a cleavable peptide linker, and the IL2v is fused to the C-terminus of the other heavy chain of the PD-1 antibody directly or indirectly via a non-cleavable peptide linker. “3xG4S” is disclosed as SEQ ID NO:92. FIG. 3B shows a summary of the binding affinity (KD) of the IL-2 prodrugs. FIG. 3C shows the results of SEC-HPLC analysis of the IL-2 prodrug samples purified by Protein A affinity chromatography. FIG. 3D shows an SDS-PAGE gel analysis of the IL-2 prodrugs prior to and after cleavage by hMMP2. FIG.

3E shows a CTLL2 assay of the IL-2 prodrugs prior to and after cleavage by hMMP2. FIG. 3F shows a CTLL2 assay of the IL-2 prodrugs prior to and after the activation by hMMP2 digestion. The prodrugs comprise IL-2Rβ ECD with mutations W166N/C168S, W166N/C168T, and Q164E/W166N/C168S. All the IL-2Rβ ECD variants preserved some levels of masking capability, with the mutation W166N/C168S having the lowest masking capability.

[0016] FIGs. 4A-4F show the assessment of the IL-2 prodrugs comprising a mutant IL- 2Rβ ECD that use an Fc domain as the carrier moiety. FIG. 4A shows a schematic drawing of the IL-2 prodrugs. The IL-2 prodrugs comprise a carrier moiety, a masking moiety, and a cytokine moiety; wherein the carrier moiety comprises an Fc domain, the masking moiety comprises an IL-2Rβ ECD with mutations, and the cytokine moiety comprises an IL-2 mutein (IL2v); and wherein the IL-2Rβ ECD is fused to the N-terminus of one of the heavy chain polypeptides of the Fc domain through a non-cleavable peptide linker, and the IL2v is fused to the N-terminus of the other Fc heavy chain polypeptide via a cleavable peptide linker. “3xG4S” is disclosed as SEQ ID NO:92. FIG. 4B shows a summary of the binding (KD) of the IL-2 prodrugs. FIG. 4C shows an SDS-PAGE gel analysis of the IL-2 prodrugs under non-reduced and reduced conditions. FIG. 4D shows binding of the IL-2 prodrugs to IL-2Rγ. FIGs. 4E and 4F show CTLL2 assays of the IL-2 prodrugs. All the b muteins are able to mask the IL-2 activities.

[0017] FIGs. 5A-5G show the assessment of the IL-2 prodrugs comprising a mutant IL- 2Rβ ECD that use a half-antibody as the carrier moiety. FIG. 5A shows a schematic drawing of the half-antibody IL-2 prodrug comprising an Fab domain and an Fc domain, wherein a IL-2 agonist polypeptide is fused to the N-terminus of one of the heavy chains of the Fc domain directly or indirectly, via a non-cleavable peptide, and the mutant IL-2Rβ ECD masking moiety is fused to the N-terminus of the cytokine moiety, optionally via a cleavable or non-cleavable peptide linker. More specifically, the half-antibody comprises three polypeptide chains: a light chain of an antibody against PD-1, a heavy chain of an antibody against PD-1, and an Fc fusion polypeptide chain comprising, from N-terminal to C-terminal, an IL-2Rβ ECD mutant, a cleavable linker, an IL-2 agonist polypeptide, and an Fc polypeptide. FIG. 5B shows a summary of the binding affinity (KD) of the prodrugs with IL-2Rγ. FIG. 5B discloses SEQ ID NOs: 110-112, respectively, in order of appearance.

FIG. 5C shows a ForteBio assay of the JR8.12.3 (Q164E IL-2Rβ ECD mutant) binding to IL-2Rγ. FIG. 5D shows an SDS-PAGE gel analysis of the IL-2 prodrugs under non-reduced and reduced conditions. FIG. 5E shows an SDS-PAGE gel analysis of the IL-2 prodrug JR8.12.3 prior to and after hMMP2 digestion. FIG. 5F shows CTLL2 assays of the JR8.12.3 IL-2 prodrugs prior to and after hMMP2 cleavage. FIG. 5G shows HEK blue reporter assay of JR8.12.3 prior to and after activation by hMMP2. IL-2Rβ ECD mutant are able to mask the IL-2 activities.

[0018] FIGs. 6A-6F show assessment of purified IL-2 prodrugs comprising a IL-2Rβ ECD fused to the carrier moiety via a non-cleavable peptide linker. FIG. 6A shows a schematic drawing of the two IL-2 prodrugs (PW04-88 and MX06-26) and an antibody-IL2v fusion molecule (LL24-68). The IL-2 prodrugs MX06-26 comprises a carrier moiety, a masking moiety, and a cytokine moiety; wherein the carrier moiety comprises an antibody against human PD-1, the masking moiety comprises a mutant IL-2Rβ ECD, and the cytokine moiety comprises an IL-2 mutein (IL2v); and wherein the IL-2Rβ ECD is fused to the C- terminus of one of the heavy chains of the PD-1 antibody through a non-cleavable peptide linker, and the IL2v is fused to the C-terminus of the other heavy chain of the PD-1 antibody directly or indirectly via a non-cleavable linker. FIG. 6B shows a summary of the molecular information of the IL-2 prodrugs comprising a mutant IL-2Rβ ECD and antibody-IL2v fusion molecule. FIG. 6C shows the HEK Blue-IL2 reporter assay of the IL-2 prodrugs and the antibody-IL2v fusion molecule. The results showed that both prodrug molecules had significantly reduced cell-based activities compared with the fusion molecule. FIG. 6D shows NK92 assay of the IL-2 prodrugs and the fusion molecule. FIG. 6E shows CTLL2 assay of the IL-2 prodrugs and the fusion molecule. FIG. 6F shows ForteBio analysis of the IL-2 prodrugs and the antibody-IL2v fusion molecule binding to IL-2Rγ.

DETAILED DESCRIPTION OF THE INVENTION [0019] As used herein and in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise. Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.” Additionally, use of “about” preceding any series of numbers includes “about” each of the recited numbers in that series. For example, description referring to “about X, Y, or Z” is intended to describe “about X, about Y, or about Z.”

[0020] The term “antigen-binding moiety” refers to a polypeptide or a set of interacting polypeptides that specifically bind to an antigen, and includes, but is not limited to, an antibody (e.g., a monoclonal antibody, polyclonal antibody, a multi-specific antibody, a dual specific or bispecific antibody, an anti-idiotypic antibody, or a bifunctional hybrid antibody) or an antigen-binding fragment thereof (e.g., a Fab, a Fab’, a F(ab’)2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), or a diabody), a single chain antibody, and an Fc-containing polypeptide such as an immunoadhesin. In some embodiments, the antibody may be of any heavy chain isotype (e.g., IgG, IgA, IgM, IgE, or IgD) or subtype (e.g., IgGi, IgG2, IgG3, or IgG4). In some embodiments, the antibody may be of any light chain isotype (e.g., kappa or lambda). The antibody may be human, non-human (e.g., from mouse, rat, rabbit, goat, or another non-human animal), chimeric (e.g., with a non-human variable region and a human constant region), or humanized (e.g., with non-human CDRs and human framework and constant regions). In some embodiments, the antibody is a derivatized antibody.

[0021] As used herein, the phrase “against,” “binding to,” or “specifically binding to” in the context of the interaction between two molecules is intended to mean that the binding has a KD not more than 1000 nM (e.g., not more than 100, 10, or 1 nM; such as less than 1 nM, 1-10 nM, 10-100 nM, or 100-1000 nM) as measured by common methods in the art (e.g., surface plasmon resonance, ELISA, and the like).

[0022] The term “cytokine agonist polypeptide” refers to a wildtype cytokine, or an analog thereof. An analog of a wildtype cytokine has the same biological specificity (e.g., binding to the same receptor(s) and activating the same target cells) as the wildtype cytokine, although the activity level of the analog may be different from that of the wildtype cytokine. The analog may be, for example, a mutein (i.e., mutated polypeptide) of the wildtype cytokine, and may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten mutations relative to the wildtype cytokine.

[0023] The term “cytokine antagonist” or “cytokine mask” refers to a moiety (e.g., a polypeptide) that binds to a cytokine, thereby inhibiting the cytokine from binding to its receptor on the surface of a target cell and/or exerting its biological functions while being bound by the antagonist or mask. Examples of a cytokine antagonist or mask include, without limitations, a polypeptide derived from an extracellular domain of the cytokine’s natural receptor that makes contact with the cytokine.

[0024] The term “effective amount” or “therapeutically effective amount” refers to an amount of a compound or composition sufficient to treat a specified disorder, condition, or disease, such as ameliorate, palliate, lessen, and/or delay one or more of its symptoms.

[0025] The term “functional analog” refers to a molecule that has the same biological specificity (e.g., binding to the same ligand) and/or activity (e.g., activating or inhibiting a target cell) as a reference molecule. [0026] The term “fused” or “fusion” in reference to two polypeptide sequences refers to the joining of the two polypeptide sequences through a backbone peptide bond. Two polypeptides may be fused directly or through a peptide linker that is one or more amino acids long. A fusion polypeptide may be made by recombinant technology from a coding sequence containing the respective coding sequences for the two fusion partners, with or without a coding sequence for a peptide linker in between. In some embodiments, fusion encompasses chemical conjugation.

[0027] The term “pharmaceutically acceptable excipient” when used to refer to an ingredient in a composition means that the excipient is suitable for administration to a treatment subject, including a human subject, without undue deleterious side effects to the subject and without affecting the biological activity of the active pharmaceutical ingredient (API).

[0028] The term “subject” refers to a mammal and includes, but is not limited to, a human, a pet (e.g., a canine or a feline), a farm animal (e.g., cattle or horse), a rodent, or a primate. [0029] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from a disease, diminishing the extent of a disease, ameliorating a disease state, stabilizing a disease (e.g., preventing or delaying the worsening or progression of the disease), preventing or delaying the spread (e.g., metastasis) of a disease, preventing or delaying the recurrence of a disease, providing partial or total remission of a disease, decreasing the dose of one or more other medications required to treat a disease, increasing the patient’s quality of life, and/or prolonging survival. The methods of the present disclosure contemplate any one or more of these aspects of treatment.

[0030] It is to be understood that one, some or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described thereunder.

I. Cytokine Prodrugs

[0031] The present disclosure provides recombinant proteins comprising IL-2Rβ ECD mutants. In some embodiments, these recombinant proteins are cytokine prodrugs or fusion molecules (IL-2 or IL-15 prodrugs or IL-2 or IL-15 fusion molecules) that become more active at the site of a disease. These cytokine prodrugs comprise a mutant IL-2Rβ ECD masking moiety and are useful for the treatment of cancer, infectious diseases, inflammatory diseases, and autoimmune diseases, and for stimulating an immune response. The present inventors found that cytokine prodrugs having a wild type IL-2Rβ ECD as the masking moiety bound to IL-2Rγ. Because IL-2Rγ is shared by six cytokines and may be abundantly expressed in immune organs and other tissues, cytokine prodrugs with wild-type IL-2Rβ ECD as their masking moieties may bind to IL-Ry on those tissues or organs. Thus, the inventors sought to identify IL-2Rβ ECD mutations that would eliminate binding of the cytokine prodrugs to IL-2Rγ, while preserving the masking capacity of IL-2Rβ ECD. The inventors surprisingly discovered that a group of IL-2Rβ ECD mutants not only did not bind to IL-2Rγ but also retained their masking capacity.

[0032] An isolated cytokine prodrug may comprise a wild-type cytokine, a variant thereof, or a mutant thereof. In some embodiments, the cytokine is an IL-2 polypeptide, an IL-2 variant (IL2v), or an IL-2 mutein (collectively referred to as an “IL-2 polypeptide”). In other embodiments, the cytokine is an IL-15 wild-type polypeptide or an IL-15 mutein (collectively referred to as “IL-15 polypeptide”). In some embodiments, the cytokine prodrug comprises a cytokine moiety (IL-2 or IL-15 polypeptide), a carrier (carrier moiety), and a cytokine antagonist (masking moiety), wherein the cytokine moiety is fused to the carrier directly or through a cleavable or non-cleavable peptide linker, and the cytokine antagonist is linked to the cytokine moiety or to the carrier through a non-cleavable or cleavable peptide linker. In some embodiments, the masking moiety may be fused via a cleavable linker to the cytokine moiety, which may be fused to the carrier moiety directly or through a non-cleavable linker. [0033] In some embodiments, the cytokine moiety is fused to the carrier through a non- cleavable peptide linker, and the cytokine antagonist is linked to the carrier through a cleavable or non-cleavable peptide linker. For example, an IL-2 antagonist may be fused to the carrier through the non-cleavable peptide linker of SEQ ID NO:93. In some embodiments, the IL-2 polypeptide is a wildtype IL-2 polypeptide. In other embodiments, the IL-2 polypeptide is an IL-2 variant (IL2v) that has reduced or no binding affinity for CD25.

[0034] The present cytokine prodrugs may comprise a cytokine moiety linked to a carrier moiety and masked (bound) by a cytokine antagonist (masking moiety), where the cytokine antagonist is selected from an extracellular domain (ECD) of IL-2R (CD 122), IL-2Rγ ECD (CD 132), a functional analog of IL-2Rβ ECD, and a combination of a functional analog of IL-2Rβ ECD and IL-2Rγ ECD. In some embodiments, the cytokine antagonist inhibits the binding of the cytokine moiety to IL-2Rγ and/or of IL-2Rβ on T cells in a patient in need thereof. In some embodiments, the carrier moiety is selected from a PEG molecule, an albumin, an albumin fragment, a half-antibody, an antibody Fc domain, an antibody, or an antigen-binding fragment thereof.

A. IL-2 Polypeptide

[0035] In the present IL-2 prodrugs, the IL-2 polypeptide may be a wildtype IL-2 polypeptide such as a wildtype human IL-2 polypeptide (SEQ ID NO: 1), or an IL-2 mutein such as an IL-2 mutein derived from a human IL-2. An IL-2 mutein is an IL-2 derivative that retains at least one or more aspects of the IL-2 biological activities. In some embodiments, IL-2 mutein comprises a sequence of amino acids at least 95% identical to SEQ ID NO:2. In certain embodiments, the IL-2 mutein has the same length as SEQ ID NO:2 but differs from it by no more than 7 (e.g., no more than 6, no more than 5, no more than 4, no more than 3, or no more than 2) amino acid residues. The IL-2 mutein may have reduced affinity for CD 122 and/or CD132, and may comprise one or more mutations selected from L12G, L12K, L12Q, L12S, Q13G, El 5 A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D20F, D20G, D20T, D20W, M23R, R81A, R81G, R81S, R81T, D84A, D84E, D84G, D84I, D84M, D84Q D84R, D84S, D84T, S87R, N88A, N88D, N88E, N88F, N88G, N88M, N88R, N88S, N88V, N88W, N90T, N90S, V91D, V91E, V91G, V91S, I92K, I92R, I92T, I92S, E95G, Q126E, Q126F, Q126G, Q126I, Q126L, Q126M, Q126N, Q126R, Q126V, and Q126Y. Unless otherwise indicated, all residue numbers in IL-2 are in accordance with the numbering of SEQ ID NO: 1. In some embodiments, the IL-2 mutein may have mutations that result in enhanced affinity for CD25. Such mutations may be selected from mutations at positions 69 and 74. In some embodiments, the IL-2 mutein may comprise one or more mutations selected from T3A, C125A, C125S, and C125G.

[0036] In particular embodiments, the IL-2 polypeptide is an IL-2 variant (IL2v) with abolished CD25 binding, such as the IL2v of SEQ ID NO: 3 or a functional analog thereof. In other embodiments, the IL2v comprises a sequence at least 95% identical to SEQ ID NO:3.

B. IL-15 Polypeptide

[0037] In the present IL-15 prodrugs, the IL-15 cytokine moiety may be a wildtype IL-15 polypeptide such as a wildtype human IL-15 polypeptide (SEQ ID NO: 103), or an IL-15 mutein, such as an IL-15 mutein derived from a human wildtype IL-15, with reduced affinity for IL-2Rβ (CD 122) compared to wild type IL-15. The IL-15 mutein may have significantly reduced affinity for CD 122 or the dimeric IL-2R, as compared to the wild type IL-15. [0038] In some embodiments, the IL-15 moiety, when masked, has its biological activity reduced by at least 5 times, at least 10 times, at least 20 times, at least 50 times, or at least 100 times; or has its ECso value increased by at least 5 times, at least 10 times, at least 20 times, at least 50 times or at least 100 times.

[0039] In some embodiments, the IL-15 moiety is an IL-15 mutein comprising at least 1, 2, 3, 4, or 5 mutations at positions selected fromNl, N4, 16, S7, D8, K10, Kll, E46, D61, T62, E64, N65, 168, L69, N72, V63, L66, 167, A70, N71, Q108, N112 of human IL-15.

Exemplary IL-15 muteins are those with one or more mutations selected fromNlA, N1D, N4A, N4D, I6T, S7A, D8A, DAT, D8E, D8N, K10A, K10D, Kll A, K11D, D61A, D61N, T62L, T62A, E64A, E64L, E64K, E64Q, N65A, N65L, N65D, L66D, L66E, I 67D, I67E, I68S, I68E, L69S, L69E, N72A, N72D, V63E, V63D, L66E, L66D, I67E, I67D, Q108E, and N112A. In some embodiments, the IL-15 moiety comprises a mutation or positions selected from E46, V49, L45, S51, and L52. Unless otherwise indicated, all residue numbers in IL-15 and IL-15 muteins described herein are in accordance with the numbering in SEQ ID NO: 103. In other embodiments, the IL-15 moiety comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:103.

[0040] In particular embodiments, the IL-15 mutein contains mutations selected from N1D/D61N, N1D/E64Q, N4D/D61N, N4D/E64Q, D8N/D61N, D8N/E64Q, D30N/E64Q/N65D, D61N/E64Q, E64Q/Q108E, N1D/N4D/D8N, D61N/E64Q/N65D,

N 1 D/D61N/E64Q, N1D/D61N/E64Q/Q108E, and N4D/D61N/E64Q/Q108E.

C. IL-15 Receptor Alpha Sushi Domain

[0041] In some embodiments, the present IL-15 prodrug comprises an IL-15Ra Sushi domain. The Sushi domain may be fused to the carrier directly or to the IL-15 cytokine moiety, optionally through a linker (e.g., a non-cleavable or cleavable peptide linker). The masking moiety may be fused to the Sushi domain or to the carrier through a cleavable or non-cleavable peptide linker. In a particular embodiment, the Sushi domain is fused to the carrier and the cytokine moiety is fused to the Sushi domain through a peptide linker. In the present IL-15 prodrugs, the Sushi domain may be a wild-type Sushi domain, or a Sushi domain comprising an amino acid sequence of SEQ ID NO: 104 or 106. In other embodiments, the Sushi domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 104 or SEQ ID NO: 106. [0042] In some embodiments, the human IL-15 receptor alpha (IL-15Ra) protein has the amino acid sequence set forth in SEQ ID NO: 105. An exemplary IL-15Ra protein of the prodrug outlined herein can comprise or consist of the Sushi domain of SEQ ID NO: 105 (e.g., amino acids 31-95 or 31- 105 of SEQ ID NO: 105), or in other words, the amino acid sequence of SEQ ID NO: 106 or SEQ ID NO: 104. In some embodiments, the IL-15Ra protein has the amino acid sequence of SEQ ID NO:104and an amino acid insertion selected from the group consisting of D96, P97, A98, D96/P97, D96/C97, D96/P97/A98, D96/P97/C98, and D96/C97/A98, wherein the amino acid position is relative to full-length human IL-15Ra protein or SEQ ID NO: 105. For instance, amino acid(s) such as D, P, A, DP, DC, DP A, DPC, or DCA can be added to the C-terminus of the IL-15Ra protein (e.g., SEQ ID NO: 106). In some embodiments, the IL-15Ra protein has the amino acid sequence of SEQ ID NO: 106and one or more amino acid substitutions selected from the group consisting of K34C, A37C, G38C, S40C, and L42C, wherein the amino acid position is relative to SEQ ID NO: 106. In certain embodiments, the IL-15 analog and the Sushi domain have a set of amino acid substitutions or additions selected from the group consisting of E87C: D96/P97/C98; E87C:D96/C97/A98; V49C: S40C; L52C: S40C; E89C: K34C; Q48C: G38C; E53C: L42C; C42S: A37C; and L45C: A37C, respectively (the mutations in IL-15 are shown before the colon; and the mutations in the Sushi domain are shown after the colon).

D. Masking Moieties of the Cytokine Prodrugs [0043] The cytokine antagonist, i.e., the masking moiety, in the present isolated cytokine prodrugs is an IL-2Rβ mutant such as one derived from human IL-2Rβ (e.g., one of SEQ ID NO:4). In some embodiments, the cytokine prodrug comprises at least one masking moiety. For example, the fusion molecule may comprise both an IL-2Rβ ECD and an IL-2Rγ ECD (or functional analogs thereol) or just one of these ECDs. The ECD may comprise the entirety of the extracellular domain of human IL-2Rβ or IL-2Rγ, or contain only a portion thereof, so long as the portion remains able to bind to the cytokine moiety or otherwise inhibiting the cytokine moiety from binding to IL-2Rβ or IL-2Rγ on T cells.

[0044] In particular embodiments, the masking moiety is a mutant of an IL-2Rβ ECD comprising one or more point mutations at position(s) selected from FI 1, R15, V21, L28, W38, R42, L51, P52, V53, 163, P67, S69, 177, W90, V92, F101, M107, Y134, R137, VI 15, H150, W152, V155, K161, Q162, K163, Q164, Q164, W166, C168, E170, L187, and P200 (numbering according to SEQ ID NO:4). In additional embodiments, the IL-2Rβ ECD mutation is selected from one or more R15Q, R15H, W38N, R42F, L51S, V53S, V53E,

S69R, 177, W90N, V92S, F101D, M107S, Y134R, V115S, H150E, W152S, W152N, V155, K161D, Q162S, K163D, Q164D, Q164E, W166E, W166N, W166S, C168T, C168S, E170S, L187S, and P200S (numbering according to SEQ ID NO:4). In some embodiments, the hydrophobic amino acids of IL-2Rβ ECD are mutated to a hydrophilic amino acid or amino acids selected from S, G, N, T, and Q.

[0045] In some embodiments, the IL-2Rβ ECD comprises one or more mutations selected from V92S, H150E, W152S/N (“/” means “or” when used to denote mutations), Q162S, Q164E, W166N/S, W166E, C168T/S, and L187S.

[0046] In some embodiments, the masking moiety comprising IL-2Rβ ECD abolishes or significantly reduces the binding of the prodrug to IL-2Rγ while at the same time preserving the masking ability of the masking moiety. Prodrugs with such masking moieties may have better PK than those with masks comprising wildtype IL-2Rβ ECD.

[0047] In particular embodiments, the IL-2Rβ ECD mutant comprises SEQ ID NO: 107,

108 or 109, or an amino acid sequence that is at least 90% to SEQ ID NO: 107, 108, or 109, or a functional analog thereof.

[0048] A functional analog of an ECD of an IL-2R subunit (b or g) refers to a polypeptide that has an affinity similar to that of the wildtype ECD for IL-2. For example, the functional analog contains the core IL-2 or IL-15 binding region of the wildtype ECD and may have a sequence that is at least 95% (e.g., at least 96, 97, 98, or 99%) identical to the wildtype ECD (e.g., SEQ ID NO:4, supra) across the entire length of the analog.

[0049] In particular embodiments, a mutant ECD of an IL-2Rβ retains its ability to fully mask the IL-2 or IL-15 polypeptide in comparable fashion to its wild-type counterpart. In other particular embodiments, a mutant ECD of an IL-2Rβ prevents binding of the prodrug to IL-2Rγ on non-target cells or tissues.

E. Carrier Moieties of the Isolated Cytokine Prodrugs [0050] The carrier moieties of the present cytokine prodrugs may be an antigen-binding moiety, or a moiety that is not an antigen-binding moiety. The carrier moiety may improve the PK profiles such as serum half-life of the cytokine agonist polypeptide, and may also target the cytokine agonist polypeptide to a target site in the body, such as a tumor site.

1. Antigen-Binding Carrier Moieties

[0051] The carrier moiety may be an antibody or an antigen-binding fragment thereof, or an immunoadhesin. In some embodiments, the antigen-binding moiety is a full-length antibody with two heavy chains and two light chains, a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a Fv fragment, a disulfide linked Fv fragment, a half-antibody, a single domain antibody, a nanobody, or a single-chain variable fragment (scFv). In some embodiments, the antigen-binding moiety is a bispecific antigen-binding moiety and can bind to two different antigens or two different epitopes on the same antigen. The antigen-binding moiety may provide additional and potentially synergetic therapeutic efficacy to the cytokine agonist polypeptide.

[0052] The cytokine moiety and its mask may be fused to the N-terminus or C-terminus of the light chains and/or heavy chains of the antigen-binding moiety. By way of example, the cytokine moiety and its mask may be fused to the antibody heavy chain or an antigen-binding fragment thereof or to the antibody light chain or an antigen-binding fragment thereof. In some embodiments, the cytokine moiety is fused to the C-terminus of one or both of the heavy chains of an antibody, and the cytokine’s mask is fused to the other terminus of the cytokine moiety through a non-cleavable or cleavable peptide linker. In some embodiments, the cytokine moiety is fused to the C-terminus of one of the heavy chains of an antibody, and the cytokine’s mask is fused to the C-terminus of the other heavy chain of the antibody through a non-cleavable or cleavable peptide linker, wherein the two heavy chains contain mutations that allow the specific pairing of the two different heavy chains.

[0053] Strategies of forming heterodimers are well known (see, e.g., Spies et al., Mol Imm. (2015) 67(2)(A):95-106). For example, the two heavy chain polypeptides in the cytokine prodrug may form stable heterodimers through “knobs-into-holes” mutations. “Knobs-into- holes” mutations are made to promote the formation of the heterodimers of the antibody heavy chains and are commonly used to make bispecific antibodies (see, e.g., U.S. Pat. 8,642,745). For example, the Fc domain of the antibody may comprise a T366W mutation in the CH3 domain of the “knob chain” and T366S, L368A, and/or Y407V mutations in the CH3 domain of the “hole chain.” An additional interchain disulfide bridge between the CH3 domains can also be used, e.g., by introducing a Y349C mutation into the CH3 domain of the “knobs chain” and an E356C or S354C mutation into the CH3 domain of the “hole chain” (see, e.g., Merchant et al., Nature Biotech (1998) 16:677-81). In other embodiments, the antibody moiety may comprise Y349C and/or T366W mutations in one of the two CH3 domains, and E356C, T366S, L368A, and/or Y407V mutations in the other CH3 domain. In certain embodiments, the antibody moiety may comprise Y349C and/or T366W mutations in one of the two CH3 domains, and S354C (or E356C), T366S, L368A, and/or Y407V mutations in the other CH3 domain, with the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain, forming an interchain disulfide bridge (numbering always according to EU index of Kabat; Kabat et al., “Sequences of Proteins of Immunological Interest,” 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Other knobs-into-holes technologies, such as those described in EP1870459A1, can be used alternatively or additionally. Thus, another example of knobs- into-holes mutations for an antibody moiety is having R409D/K370E mutations in the CH3 domain of the “knob chain” and D399K/E357K mutations in the CH3 domain of the “hole chain” (Eu numbering).

[0054] In some embodiments, the antibody moiety in the cytokine prodrug L234A and L235A (“LALA”) mutations in its Fc domain. The LALA mutations eliminate complement binding and fixation as well as Fey dependent ADCC (see, e.g.. Hezareh et al. J. Virol. (2001) 75(24): 12161-8). In further embodiments, the LALA mutations are present in the antibody moiety in addition to the knobs-into-holes mutations.

[0055] In some embodiments, the antibody moiety comprises the M252Y/S254T/T256E (“YTE”) mutations in the Fc domain. The YTE mutations allow the simultaneous modulation of serum half-life, tissue distribution and activity of IgGi (see Dall’Acqua et al., J Biol Chem. (2006) 281(33): 23514-24; and Robbie et al., Antimicrob Agents Chemother. (2013) 57(12):6147-53). In further embodiments, the YTE mutations are present in the antibody moiety in addition to the knobs-into-holes mutations. In particular embodiments, the antibody moiety has YTE, LALA and knobs-into-holes mutations or any combination thereof.

[0056] In particular embodiments, the antigen-binding moiety is an antibody, or antigenbinding fragment thereof, that binds to an antigen on the surface of a target cell, such as an immune cell. Immune cells are well known in the art. Non-limiting examples of immune cells include T cells, NK cells, and macrophages. The antigen-binding moiety may have the ability to activate the immune cell and enhance its anti-cancer activity. The antibody may or may not have ADCC activity. The antigen-binding moiety may also be further conjugated to a cytotoxic drug. In some embodiments, the antigen-binding moiety may bind to PD-1, LAG-3, TIM-3, TIGIT, CTLA-4, or TGF-beta. In other embodiments, the antigen-binding moiety may bind to an antigen on the surface of a target cell, such as a tumor cell. For example, the antigen-binding moiety may bind to FAP alpha, 5T4, Trop-2, PD-L1, HER-2, EGFR, Claudin 18.2, DLL-3, GCP3, or carcinoembryonic antigen (CEA).

[0057] In some embodiments, the antigen-binding moiety binds to guanyl cyclase C (GCC), carbohydrate antigen 19-9 (CA19-9), glycoprotein A33 (gpA33), mucin 1 (MUC1), insulin-like growth factor 1 receptor (IGF1-R), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), delta-like protein 3 (DLL3), delta-like protein 4 (DLL4), epidermal growth factor receptor (EGFR), glypican-3 (GPC3), c- MET, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), Nectin-4, Liv-1, glycoprotein NMB (GPNMB), prostates- specific membrane antigen (PSMA), Trop-2, carbonic anhydrase IX (CA9), endothelin B receptor (ETBR), six transmembrane epithelial antigen of the prostate 1 (STEAP1), folate receptor alpha (FR-a), SLIT and NTRK-like protein 6 (SLITRK6), carbonic anhydrase VI (CA6), ectonucleotide pyrophosphatase/phosphodiesterase family member 3 (ENPP3), mesothelin, trophoblast glycoprotein (TPBG), CD19, CD20, CD22, CD33, CD40, CD56, CD66e, CD70, CD74, CD79b, CD98, CD123, CD138, CD352, CD47, signal-regulatory protein alpha (SIRPa), Claudin 18.2, Claudin 6, BCMA, or EPCAM. In some embodiments, the antigen-binding moiety binds to an epidermal growth factor (EGF)-like domain of DLL3. In some embodiments, the antigen-binding moiety binds to a Delta/Serrate/Lag2 (DSL)-like domain of DLL3. In some embodiments, the antigen-binding moiety binds to an epitope located after the 374th amino acid of GPC3. In some embodiments, the antigen-binding moiety binds to a heparin sulfate glycan of GPC3. In some embodiments, the antigenbinding moiety binds to Claudin 18.2 and does not bind to Claudin 18.1. In some embodiments, the antigen-binding moiety binds to Claudin 18.1 with at least 10 times weaker binding affinity than to Claudin 18.2.

[0058] Exemplary antigen-binding moieties include trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereol), anti-dPNAG antibody F598, and antigen-binding fragments thereof. In some embodiments, the antigen-binding moiety has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to trastuzumab, rituximab, brentuximab, cetuximab, or panitumumab, GC33 (or a humanized version thereol), anti- EGFR antibody mAb806 (or a humanized version thereol), anti-dPNAG antibody F598, or a fragment thereof. In some embodiments, the antigen-binding moiety comprises an antibody heavy chain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the antibody heavy chain of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereol), anti-EGFR antibody mAb806 (or a humanized version thereol), anti-dPNAG antibody F598, or a fragment thereof. In some embodiments, the antigen-binding moiety has an antibody light chain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the antibody light chain of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereol), anti-EGFR antibody mAb806 (or a humanized version thereol), anti- dPNAG antibody F598, or a fragment thereof. The antigen-binding moiety is fused to an IL- 2 or IL-15 agonist polypeptide. In some embodiments, the antigen-binding moiety comprises the six complementarity-determining regions (CDRs) of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33, anti-EGFR antibody mAb806, or anti- dPNAG antibody F598.

[0059] A number of CDR delineations are known in the art and are encompassed herein. A person of skill in the art can readily determine a CDR for a given delineation based on the sequence of the heavy or light chain variable region. The “Kabat” CDRs are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). “Chothia” CDRs refer to the location of the structural loops (Chothia & Lesk, J. Mol. Biol. (1987) 196:901-917). The “AbM” CDRs represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software. The “Contact” CDRs are based on an analysis of the available complex crystal structures. The residues from each of these CDRs are noted below in Table 1, in reference to common antibody numbering schemes. Unless otherwise specified herein, amino acid numbers in antibodies refer to the Kabat numbering scheme as described in Kabat et al., supra, including when CDR delineations are made in reference to Kabat, Chothia, AbM, or Contact schemes. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a framework region (FR) or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

Table 1. CDR Delineations According to Various Schemes

[0060] In some embodiments, the CDRs are “extended CDRs,” and encompass a region that begins or terminates according to a different scheme. For example, an extended CDR can be as follows: L24— L36, L26— L34, or L26— L36 (VL-CDR1); L46— L52, L46— L56, or L50 — L55 (VL-CDR2); L91— L97 (VL-CDR3); H47— H55, H47— H65, H50— H55,

H53 — H58, or H53 — H65 (VH-CDR2); and/or H93— HI 02 (VH-CDR3).

[0061] In some embodiments, the antigen-binding moiety binds to PDL1, and comprises a light chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:97, or a fragment thereof, and a heavy chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 98, or a fragment thereof. In some embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO:97, and CDR1, CDR2, and CDR3 from SEQ ID NO: 98.

[0062] In some embodiments, the antigen-binding moiety binds to PD-1, and comprises a light chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:99, or a fragment thereof, and a heavy chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 100, or a fragment thereof. In some embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO:99, and CDR1, CDR2, and CDR3 from SEQ ID NO: 100.

[0063] In some embodiments, the antigen-binding moiety binds to PD-1, and comprises a light chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 101, or a fragment thereof, and a heavy chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 102, or a fragment thereof. In some embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO:99, and CDR1, CDR2, and CDR3 from SEQ ID NO: 100.

[0064] In some embodiments, the antigen-binding moiety comprises one, two, or three antigen-binding domains. For example, the antigen-binding moiety is bispecific and binds to two different antigens selected from the group consisting of CD3, HER2, HER3, EGFR, 5T4, FAP alpha, Trop-2, GPC3, VEGFR2, Claudin 18.2 and PD-L1. In some embodiments, the bispecific antigen-binding moiety binds to two different epitopes of HER2. In other embodiments, the antigen-binding moiety is bispecific and binds to two different antigens selected from PD-1, PD-L1, CTLA-4, CD47, CD3, TIM-3, LAG-3 and TIGIT.

2. Other Carrier Moieties

[0065] Other non-antigen-binding carrier moieties may be used for the present isolated cytokine prodrugs. For example, an antibody Fc domain (e.g., a human IgGi, IgG2, IgG3, or IgG4 Fc), a polymer (e.g., PEG), an albumin (e.g., a human albumin) or a fragment thereof, or a nanoparticle can be used.

[0066] By way of example, the cytokine agonist (IL-2, IL2v, or an IL-2 mutein, or IL-15 or an IL-15 mutein) and its antagonist may be fused to an antibody Fc domain, forming an Fc fusion protein. In some embodiments, the cytokine agonist is fused (directly or through a peptide linker) to the C-terminus or N-terminus of one of the Fc domain polypeptide chains, and the cytokine mask is fused to the C-terminus or N-terminus of the other Fc domain polypeptide chain through a non-cleavable or cleavable peptide linker, wherein the two Fc domain polypeptide chains contain mutations that allow the specific pairing of the two different Fc chains. In some embodiments, the Fc domain comprises the holes-into-holes mutations described above. In further embodiments, the Fc domain may comprise also the YTE and/or LALA mutations described above. In some embodiments, the Fc domain comprises a mutation at N297 (Eu numbering).

[0067] The carrier moiety of the cytokine prodrug may comprise an albumin (e.g., human serum albumin) or a fragment thereof. In some embodiments, the albumin or albumin fragment is about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, or about 99.8% or more identical to human serum albumin or a fragment thereof.

[0068] In some embodiments, the carrier moiety comprises an albumin fragment (e.g., a human serum albumin fragment) that is about 10 or more, 20 or more, 30 or more 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 120 or more, 140 or more, 160 or more, 180 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, or 550 or more amino acids in length. In some embodiments, the albumin fragment is between about 10 amino acids and about 584 amino acids in length (such as between about 10 and about 20, about 20 and about 40, about 40 and about 80, about 80 and about 160, about 160 and about 250, about 250 and about 350, about 350 and about 450, or about 450 and about 550 amino acids in length). In some embodiments, the albumin fragment includes the Sudlow I domain or a fragment thereof, or the Sudlow II domain or the fragment thereof.

F. Linker Components of the Cytokine Prodrugs [0069] The cytokine moiety may be fused to the carrier moiety with or without a peptide linker. The peptide linker may be cleavable or non-cleavable. In some embodiments, the peptide linker is selected from SEQ ID NOs: 89-96.

[0070] The masking moiety may be fused to the cytokine moiety or to the carrier through a non-cleavable or cleavable linker or without a peptide linker. The cleavable linker may contain one or more (e.g., two or three) cleavable moieties (CM). Each CM may be a substrate for an enzyme or protease selected from legumain, plasmin, TMPRSS-3/4, MMP-2, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophil elastase, beta-secretase, uPA, and PSA. In some embodiments, the masking moiety is fused to the carrier through a peptide linker, wherein said peptide linker is selected from SEQ ID NOs: 89-96. In some embodiments, the peptide linker comprises at least 10 amino acids, 12 amino acids, 14 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 25 amino acids, 27 amino acids, or 30 amino acids [0071] Specific, nonlimiting examples of IL-2 polypeptides, cytokine masks, carriers, peptide linkers, and isolated IL-2 prodrugs are shown in the Sequences section below.

Further, the isolated fusion molecules of the present disclosure may be made by well-known recombinant technology. For examples, one more expression vectors comprising the coding sequences for the polypeptide chains of the isolated fusion molecules may be transfected into mammalian host cells (e.g., CHO cells), and cells are cultured under conditions that allow the expression of the coding sequences and the assembly of the expressed polypeptides into the isolated IL-2 fusion molecule complex.

II. Pharmaceutical Compositions

[0072] Pharmaceutical compositions comprising the cytokine prodrugs (i.e., the active pharmaceutical ingredient or API) of the present disclosure may be prepared by mixing the API having the desired degree of purity with one or more optional pharmaceutically acceptable excipients (see, e.g., Remington's Pharmaceutical Sciences , 16th Edition., Osol,

A. Ed. (1980)) in the form of lyophilized formulations or aqueous solutions.

Pharmaceutically acceptable excipients (or carriers) are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers containing, for example, phosphate, citrate, succinate, histidine, acetate, or another inorganic or organic acid or salt thereof; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including sucrose, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; saltforming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).

[0073] Buffers are used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. Buffers are preferably present at concentrations ranging from about 50 mM to about 250 mM. Suitable buffering agents for use with the present invention include both organic and inorganic acids and salts thereof, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, and acetate. Additionally, buffers may comprise histidine and trimethylamine salts such as Tris. [0074] Preservatives are added to retard microbial growth, and are typically present in a range from 0.2% - 1.0% (w/v). Suitable preservatives for use with the present invention include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0075] Tonicity agents, sometimes known as “stabilizers” are present to adjust or maintain the tonicity of liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often termed “stabilizers” because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter- and intra-molecular interactions. Tonicity agents can be present in any amount between 0.1% to 25% by weight, or more preferably between 1% to 5% by weight, taking into account the relative amounts of the other ingredients. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0076] Non-ionic surfactants or detergents (also known as “wetting agents”) are present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Non-ionic surfactants are present in a range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.

[0077] Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC^® polyols, TRITON^®, polyoxyethylene sorbitan monoethers (TWEEN^®-20, TWEEN^®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride. [0078] The choice of pharmaceutical carrier, excipient or diluent may be selected with regard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may additionally comprise any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilizing agent(s).

[0079] There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, pharmaceutical compositions useful in the present invention may be formulated to be administered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestible solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.

[0080] In some embodiments, the pharmaceutical composition of the present disclosure is a lyophilized protein formulation. In other embodiments, the pharmaceutical composition may be an aqueous liquid formulation.

III. Methods of Treatment

[0081] The cytokine prodrug or fusion molecules can be used to treat a disease, depending on the antigen bound by the antigen-binding domain. In some embodiments, the cytokine prodrug or fusion molecule is used to treat cancer. In some embodiments, the cytokine prodrug or fusion molecule is used to treat an infection.

[0082] In some embodiments, a method of treating a disease (such as cancer, a parasitic infection, a viral infection, or a bacterial infection) in a subject comprises administering to the subject an effective amount of a cytokine prodrug or a fusion molecule. [0083] In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a blood cancer or a solid tumor. Exemplary cancers that may be treated include, but are not limited to, leukemia, lymphoma, kidney cancer, bladder cancer, urinary tract cancer, cervical cancer, brain cancer, head and neck cancer, skin cancer, uterine cancer, testicular cancer, esophageal cancer, liver cancer, colorectal cancer, stomach cancer, squamous cell carcinoma, prostate cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer, cholangiocarcinoma, breast cancer, and ovarian cancer.

[0084] In some embodiments, the cytokine prodrug or fusion molecule is used to treat a viral infection. In some embodiments, the virus causing the viral infection is hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), or human papilloma virus (HPV). In some embodiments, the antigen-binding moiety binds to a viral antigen. [0085] In some embodiments, the cytokine prodrug or fusion molecule is used to treat a bacterial infection such as sepsis. In some embodiments, the bacteria causing the bacterial infection is drug-resistant bacteria. In some embodiments, the antigen-binding moiety binds to a bacterial antigen.

[0086] In some embodiments, the cytokine prodrug or fusion molecule is used to treat an inflammatory or autoimmune disease. In some embodiments, a method of treating a disease (such an autoimmune disease) in a subject comprises administering to the subject an effective amount of a cytokine prodrug disclosed herein. In some embodiments, the inflammatory or autoimmune disease is selected from the group consisting of asthma, diabetes (e.g., Type I diabetes or latent autoimmune diabetes), lupus (e.g., systemic lupus erythematosus), arthritis (e.g., rheumatoid arthritis), allergy, organ graft rejection, GVHD, Addison’s disease, ankylosing spondylitis, anti-glomerular basement membrane disease, autoimmune hepatitis, dermatitis, Goodpasture’s syndrome, granulomatosis with polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, hemolytic anemia, Henoch-Schonlein purpura (HSP), juvenile myositis, Kawasaki disease, inflammatory bowel diseases (such as Crohn’s disease and ulcerative colitis), multiple sclerosis, myasthenia gravis, neuromyelitis optica, PANDAS, psoriasis, psoriatic arthritis, Sjogren’s syndrome, systemic scleroderma, systemic sclerosis, thrombocytopenic purpura, uveitis, vasculitis, vitiligo, and Vogt- Koyanagi-Harada Disease.

[0087] Generally, dosages and routes of administration of the present pharmaceutical compositions are determined according to the size and conditions of the subject, according to standard pharmaceutical practice. In some embodiments, the pharmaceutical composition is administered to a subject through any route, including orally, transdermally, by inhalation, intravenously, intra-arterially, intramuscularly, direct application to a wound site, application to a surgical site, intraperitoneally, by suppository, subcutaneously, intradermally, transcutaneously, by nebulization, intrapleurally, intraventricularly, intra-articularly, intraocularly, intracranially, or intraspinally. In some embodiments, the composition is administered to a subject intravenously.

[0088] In some embodiments, the dosage of the pharmaceutical composition is a single dose or a repeated dose. In some embodiments, the doses are given to a subject once per day, twice per day, three times per day, or four or more times per day. In some embodiments, about 1 or more (such as about 2, 3, 4, 5, 6, or 7 or more) doses are given in a week. In some embodiments, the pharmaceutical composition is administered weekly, once every 2 weeks, once every 3 weeks, once every 4 weeks, weekly for two weeks out of 3 weeks, or weekly for 3 weeks out of 4 weeks. In some embodiments, multiple doses are given over the course of days, weeks, months, or years. In some embodiments, a course of treatment is about 1 or more doses (such as about 2, 3, 4, 5, 7, 10, 15, or 20 or more doses).

[0089] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is understood that aspects and variations of the invention described herein include “consisting” and/or “consisting essentially of’ aspects and variations. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

EXAMPLES

[0090] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner. The materials and methods of the experiments described in the examples are as follows.

Materials and Methods

IL-2Rγ binding Affinity

[0091] The 2 mΐ/ml biotinylated IL-2Rγ extracellular domain fusions (Aero Biosystems, ILG-H85E8) were immobilized on the streptavidin (SA) biosensors (ForteBio, 18-5019) in the 1 x ForteBio Kinetic buffer (ForteBio, 18-5028) for 60s. After immobilization, the immobilized biosensor tips were then incubated in the wells containing a 2x fold serial dilution of the soluble protein samples. The top concentration was 300 μg/ml. Association for 300s and dissociation for 1200s were used.

[0092] All ForteBio Octet RED96 raw data was processed using the ForteBio Data Analysis software. First, one tip, which was immobilized IL-2Rgamma but no interaction, was defined as the reference and subtracted from the all the other sample curves in the same column. Second, the association and dissociation curves were isolated and aligned to the Y axis. Thirdly, Savitzky-Golay filtering was applied. Fourthly, the association and dissociation curves for each sample were globally fit with a 1:1 binding model to determine the association rate constant k_a(M^-1sec^-1) and the dissociation rates constants kd (sec^-1); the equilibrium dissociation constant K_D> (M) was calculated by k_d/k_a.

Transient Transfection of ExpiCHO Cells

[0093] Expression plasmids were co-transfected into 6 x 10⁶ cell/ml ExpiCHO™ cells at 1 μg/ml according to the manufactural protocol. For Fc-based IL-2 fusion molecules, the ratios for the Fc-IL-2 mutein fusion polypeptide and the Fc-masking moiety fusion polypeptide were in a 1:2 or 1:3. For antibody-based IL-2 fusion molecules, the ratios for HC-IL2v, HC-IL-2Rbeta mutein fusion polypeptide and the LC were in a 1:3:4. The cell cultures were harvested 7 -8 days after transfection by centrifuging at 9,000rpm for 45 min followed by 0.22 μM filtration. Protein Purification

[0094] The clarified culture medium of each fusion molecule was purified by Protein A affinity with CaptivA® Resin (Repligen, Waltham, MA). In certain cases, further purifications were carried out using a mixed mode chromatography with Capto™ Adhere resin or Capto™ Adhere ImpRes resin, followed by an anion exchange chromatography with Sepharose® Q FF resin or Sepharose® Q HP resin. All the Sepharose® and Capto™ resins were ordered from GE Healthcare Life Sciences (now Cytiva, Marlborough, MA). In certain cases, the samples were formulated in 20 mM Histidine, 7% sucrose, 0.03% polysorbate-20, which were stored at -80°C freezer until use.

SEC-HPLC Analysis

[0095] SEC-HPLC was carried out using an Agilent 1100 Series HPLC system with a TSKgel® G3000SWXL column (7.8 mm IDX 30cm, 5 μm particle size) from Tosoh Bioscience. A sample of up to 100 pi was loaded. The column was run with a buffer containing 200 mM K₃PO₄, 250 mM KC1, pH 6.5. The flow rate was 0.5 ml/min. The column was run at room temperature. The protein elution was monitored both at 220 nm and 280 nm.

SDS-PAGE Analysis

[0096] 10 μl of the culture supernatants or 4 μg of purified protein samples were mixed with Bolt™ LDS Sample Buffer (Novex) with or without reduce reagents. The samples were heated at 70°C for 3 min and then loaded to a NuPAGE™ 4-12% BisTris Gel (Invitrogen). The gel was run in NuPAGE™ MOPS SDS Running buffer (Invitrogen) at 200 Volts for 40 min and then stained with Coomassie blue.

Proteolytic Treatment

[0097] One μg of the protease, human MMP-2 (R&D systems), human MMP-9 (R&D systems), mouse MMP-2 (R&D systems), or mouse MMP-9 (R&D systems) was added to 200 μg of the precursor protein in a buffer (20mM HEPES, 150mM NaCl, 10 mM CaCl₂, 2 pM ZnCl₂, pH7.4), and incubated at 37°C overnight.

CTLL2 Assay

[0098] CTLL2 cells were grown in the RPMI 1640 medium supplemented with L- glutamine, 10% fetal bovine serum, 10% non-essential amino acids, 10% sodium pyruvate, and 55 pM beta-mercaptoethanol. CTLL2 cells were non-adherent and maintained at 5 x 10⁴ - 1 x 10⁶ cells/ml in medium with 100 ng/ml of IL-2. Generally, cells were split twice per week. For bioassays, it was best to use cells no less than 48 hours after passage. [0099] Samples were diluted at 2x concentration in 50 mΐ/well in a 96 well plate. The IL-2 standards were titrated from 20 ng/ml (2x concentration) to 3x serial dilutions for 12 wells. Samples were titer tested as appropriate. CTLL2 cells were washed 5 times to remove IL-2, dispensed 5000 cells/well in 50 mΐ and cultured overnight or for at least 18 hours with the samples. Subsequently, 100 mΐ/well Cell Titer Glo reagents (Promega) were added and luminescence was measured.

NK92 Proliferation Assay

[0100] The NK92 cell line is a factor dependent cell line that requires IL-2 for growth and survival. Prior to assay, the cells are washed to remove IL-2 and cultured overnight without growth factor. Cells are harvested and washed again to remove residual growth factor. Cells (20,000/well) are then added to 96 well plates containing serial dilution of test articles and controls. Plates are incubated overnight, and Cell Titer Glo (Promega) is added and luminescence measured. This provides a measure of ATP levels as an indicator of cell viability.

[0101] The assays were carried out using several IL-2 prodrugs masked with IL-2Rβ extra-cellular domain (ECD) variants.

Example 1: Design and Assessment of IL-2Rβ Mutations

[0102] Several IL-2 prodrugs with IL-2Rβ ECD mutations as listed in Fig. 2B were rationally designed based on the crystal structure of the I L-2/I L-2Rβ/I L-2Ry complex (FIG.

1). The carrier moieties of those fusion molecules comprise an anti-PD-1 antibody, wherein the masking moiety and the cytokine moiety were fused to the C-termini of the heavy chains of the antibody (FIG. 2A). The fusion molecules were transiently expressed and purified through Protein A affinity chromatography. Most constructs expressed well in CHO cells and their Protein A pool samples had reasonable SEC-HPLC purity and correct retention times (FIGs. 2C and 2D). All of the samples (except JR8.6.4 (C168V)) had SEC-HPLC main peak purities over 80%. JR8.6.4 (C168V) had higher levels of aggregation than other molecules and its retention time was also different indicating that the prodrug was not correctly expressed. The IL-2 prodrug with IL-2Rβ ECD having one or more of the following mutations showed very little binding to IL-2Rγ: Q162S, Q164E, Q162S/Q164E, C168S, or Q164E/W166N (FIG. 2E). Those results showed that Q162, Q164 and Cl 68 play important roles in the binding interaction of IL-2/ IL-2Rβ ECD complex with IL-2Rγ. The IL-2 prodrug with IL-2Rβ ECD having one or more of the following mutations were still able to bind to IL-2Rγ W152N, W166N, W166S, C168T, or C168A (FIG. 2E). It was surprising that an IL-2 prodrug having an IL-2Rβ ECD with a C168S mutation abolished the binding with IL-2Rγ. while a C168T mutation or C168A mutation did not. Further, a C 168V mutation did not express well.

[0103] IL-2 prodrugs with an IL-2Rβ ECD C168S mutation or Q162S/Q164E mutations were further purified and tested for their protease cleavage-dependent activation (FIGs. 2F and 2G). Activation (cleavage) of the prodrugs was confirmed using SDS-PAGE analysis (FIG. 2F). The activities of the masked IL-2 prodrugs were found to be significantly lower than the unmasked sample, but increased after the protease activation (FIG. 2G). This showed that the C168S mutation and Q162S/Q164E mutations of the IL-2Rβ ECD masking moieties preserved their ability to mask the biological activity of IL-2.

Example 2: Assessment of IL-2 Prodrugs with IL-2RP ECD Mutations [0104] FIG. 3A illustrates the structure of IL-2 prodrugs wherein the carrier comprises an anti -PD- 1 antibody having YTE mutations in its Fc domain. All IL-2Rβ ECD mutations tested showed abolished binding to IL-2Rγ, except the mutations W166N/C168T, as summarized in FIG. 3B. The molecules were all expressed well, though JR8.6.1 with mutations Q164E/W166N/C168S showed higher level of aggregates compared to the other ones (FIG. 3C). Activation (cleavage) of the IL-2 prodrugs were confirmed using SDS- PAGE analysis (FIG. 3D). All IL-2Rβ ECD mutants preserved some level of masking capability, with the IL-2 C168S/162S IL-2Rβ ECD mutations being the strongest among them, followed by C168S/Q162S/Q164E and C168S/Q164E, while the mutation W166N/C168S had the lowest masking capability (FIG. 3E).

Example 3: Assessment of IL-2 Prodrugs with an Fc Domain as Carrier Moiety [0105] IL-2 fusion prodrugs with an Fc domain as the carrier moiety were also tested. These fusion molecules have a masking moiety and a cytokine moiety fused to the N-termini of the Fc domain. The structure of the IL-2 prodrug is illustrated in FIG. 4 A. Mutations shown in FIG. 4B were tested. Similar to the IL-2 prodrugs tested in the above Examples, the IL-2 prodrugs comprising an IL-2Rβ ECD masking moiety with a Q164E mutation also abolished binding with IL-2Rγ. In addition, the IL-2 prodrugs comprising an IL-2Rβ ECD masking moiety with mutations W152N/W166N, W152S/W166S, W166N/V115S, or W166N/L187S also abolished the binding with IL-2Rγ. However, IL-2 prodrugs comprising an IL-2Rβ ECD masking moiety with the W152N mutation, W166N mutation, or the W166N/V92S IL-2Rβ ECD mutations still retained binding to IL-2Rγ (FIG 4D). The purities of the samples were analyzed by reduced and non-reduced SDS-PAGE (FIG. 4C).

The CTLL2 cell-based activity assay showed that all of the IL-2Rβ ECD mutants retained their abilities to mask IL-2 (FIGs. 4E and 4F).

Example 4: Assessment of IL-2 Prodrugs with Half-Antibody as Carrier [0106] IL-2 prodrugs with the structure as illustrated in FIG. 5A were also tested. Similar as the prodrugs tested above, the prodrug comprising an IL-2Rβ ECD with a Q164E mutation also abolished the binding with IL-2Rγ (FIG. 5C). The other two constructs did not express well (FIGs. 5D and 5E). The CTLL2 cell-based activity assay and the HEK Blue-IL2 Reporter assay showed that all the IL-2Rβ ECD mutant with Q164E retained its ability to mask IL-2 (FIGs. 5F and 5G).

Example 5: Assessment of IL-2 Prodrugs with the Masking Moiety Fused to the Carrier Moiety via a Non-cleavable Peptide Linker

[0107] The carrier of the IL-2 prodrug comprises an antibody against PD-1 (FIG. 6A). The masking moiety, IL-2Rβ ECD with mutations Q162S/Q164E, is fused to the C-terminus of one of the heavy chains of the carrier through a non-cleavable peptide linker (FIG. 6A, MX06-26). Two control molecules, an IL-2 prodrug with the wild type IL-2Rβ ECD (FIG. 6A, PW04-88) and an anti-PD-1 antibody-IL-2 fusion molecule (not masked; LL24-68), were also tested. All of the samples were purified by multiple chromatography steps and formulation of the product were made. An HEK Blue-IL2 reporter assay of the IL-2 prodrugs and the antibody-IL2v fusion molecule was performed and the results showed that both prodrug molecules had significantly reduced cell-based activities compared with the antibody-IL2v fusion molecule (FIG. 6C). NK92 and CTLL2 assays of the prodrug molecules and the fusion molecule showed that both prodrug molecules had significantly reduced cell-based activities compared with the antibody-IL2v fusion molecule (FIGs. 6D and E). Hence, the IL-2 prodrugs comprising IL-2Rβ ECD with mutations Q162S/Q164E had reduced IL-2 activities in all three cell-based activity assays (FIGs. 6C, 6D, and 6E). The IL-2 prodrug comprising an IL-2Rβ ECD with Q162S/Q164E mutations abolished the binding to the IL-2Rγ (FIG. 6F). The unmasked antibody-IL-2 fusion molecule did not bind to IL-2Rγ (FIG. 6F), while the IL-2 prodrug with the wild type IL-2Rβ ECD bound to IL-2Rγ (FIG. 6F), as shown above.

[0108] In summary, the studies described above have identified several IL-2Rβ ECD variants which, when used as a mask for an IL-2 prodrug, abolished the binding of the IL-2 prodrug with IL-2Rγ while retaining its ability to mask biological activity of IL-2 (cytokine moiety). These IL-2Rβ ECD include, but are not limited to, a mutation selected from: Q162S, Q164E, C168S, Q162S/Q164E, W152N/W166N, W152S/W166S, W166N/V115S, and W166N/L187S. In addition, the IL-2Rβ ECD variants worked well in prodrugs with various structures or configurations, as illustrated in FIGs. 2A, 3A, 4A, 5A, and 6A.

SEQUENCES

In the sequences below, boxed residues indicate mutations. Underlines in cleavable linkers indicate protease substrate sequences.

PDl-IL2v/mask

PDl-IL2v* with knobs-in-hole (KIH)

SEQ ID NO : 1 - human IL-2

APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT

SEQ ID NO: 3 - IL2V

APASSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTSML TAKFAMPKKA TELKHLQCLE EALKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFSQSIIS TLT

SEQ ID NO: 4 - IL-2Rbeta wild type

AVNGTSQFTC FYNSRANISC VWSQDGALQD TSCQVHAWPD RRRWNQTCEL LPVSQASWAC NLILGAPDSQ KLTTVDIVTL RVLCREGVRW RVMAIQDFKP FENLRLMAPI SLQWHVETH RCNISWEISQ ASHYFERHLE FEARTLSPGH TWEEAPLLTL KQKQEWICLE TLTPDTQYEF QVRVKPLQGE FTTWSPWSQP LAFRTKPAAL GKDT

SEQ ID NO: 5 - PD1-HC-IL2V KNOB

MGWTLVFLFL LSVTAGVHSQ VQLVESGGGV VQPGRSLRLD CKASGITFSN SGMHWVRQAP GKGLEWVAVI WYDGSKRYYA DSVKGRFTIS RDNSKNTLFL QMNSLRAEDT AVYYCATNDD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT KTYTCNVDHK PSNTKVDKRV

SVFLFPPKPK DTLMISRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPCQEEM

VKGFYPSDIA VEWESNGQPE NNYKTTPPVL RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ

KSLSLSLGKG GGGSGGGGSG GGGSAPASSS TKKTQLQLEH LLLDLQMILN GINNYKNPKL TSMLTAKFAM PKKATELKHL QCLEEALKPL EEVLNLAQSK NFHLRPRDLI SNINVIVLEL KGSETTFMCE YADETATIVE FLNRWITFSQ SIISTLT**

SEQ ID NO: 6 - PD1HC-BETA HOLE

MGWTLVFLFL LSVTAGVHSQ VQLVESGGGV VQPGRSLRLD CKASGITFSN SGMHWVRQAP GKGLEWVAVI WYDGSKRYYA DSVKGRFTIS RDNSKNTLFL

KPREEQFNST YRWSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGAGG GGSGGGGSGG GGSAPASSST KKTQLQLEHL LLDLQMILNG INNYKNPKLT SMLTAKFAMP KKATELKHLQ CLEEALKPLE EVLNLAQSKN FHLRPRDLIS NINVIVLELK GSETTFMCEY ADETATIVEF LNRWITFSQS IISTLT**

SEQ ID NO: 10 - HC-IL-2Rbeta wild type

MGVKVLFALI CIAVAEAQV QLVESGGGW QPGRSLRLDC KASGITFSNS GMHWVRQAPG KGLEWVAVIW YDGSKRYYAD SVKGRFTISR DNSKNTLFLQ MNSLRAEDTA VYYCATNDDY WGQGTLVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTK TYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKD TLMISRTPEV TCVWDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST YRWSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGAGG GGSGGGGSGP LGVRGGGGSG GGGSAVNGTS QFTCFYNSRA NISCVWSQDG ALQDTSCQVH AWPDRRRWNQ TCELLPVSQA SWACNLILGA PDSQKLTTVD IVTLRVLCRE GVRWRVMAIQ DFKPFENLRL MAPISLQWH VETHRCNISW EISQASHYFE RHLEFEARTL SPGHTWEEAP LLTLKQKQEW ICLETLTPDT QYEFQVRVKP LQGEFTTWSP WSQPLAFRTK PAALGKDT**

SEQ ID NO: 11 - PD1-LC CLD signal peptide MDMRVPAQLL GLLLLWLPGA KCEIVLTQSP ATLSLSPGER ATLSCRASQS VSSYLAWYQQ KPGQAPRLLI YDASNRATGI PARFSGSGSG TDFTLTISSL EPEDFAVYYC QQSSNWPRTF GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASWCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC**

PDl-IL2Rbeta muteins no KIH

SEQ ID NO: 12 - HC-IL2Rbetav W152N

MGVKVLFALI CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGAGGG GSGGGGSGPL GVRGGGGSGG GGSAVNGTSQ FTCFYNSRAN ISCVWSQDGA LQDTSCQVHA WPDRRRWNQT CELLPVSQAS WACNLILGAP DSQKLTTVDI VTLRVLCREG VRWRVMAIQD FKPFENLRLM APISLQWHV ETHRCNISWE ISQASHYFER HLEFEARTLS LTLKQKQEWI

CLETLTPDTQ YEFQVRVKPL QGEFTTWSPW SQPLAFRTKP AALGKDT* *

SEQ ID NO: 13 HC-IL2Rbetav Q162S MGVKVL FAL I CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGAGGG GSGGGGSGPL GVRGGGGSGG GGSAVNGTSQ FTCFYNSRAN ISCVWSQDGA LQDTSCQVHA WPDRRRWNQT CELLPVSQAS WACNLILGAP DSQKLTTVDI VTLRVLCREG VRWRVMAIQD FKPFENLRLM APISLQWHV ETHRCNISWE ISQASHYFER HLEFEARTLS PGHTWEEAPL CLETLTPDTQ YEFQVRVKPL QGEFTTWSPW SQPLAFRTKP

SEQ ID NO: 14 - HC-IL2Rbetav Q164E MGVKVL FAL I CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGAGGG GSGGGGSGPL GVRGGGGSGG GGSAVNGTSQ FTCFYNSRAN ISCVWSQDGA LQDTSCQVHA WPDRRRWNQT CELLPVSQAS WACNLILGAP DSQKLTTVDI VTLRVLCREG VRWRVMAIQD FKPFENLRLM APISLQWHV ETHRCNISWE ISQASHYFER HLEFEARTLS PGHTWEEAPL CLETLTPDTQ YEFQVRVKPL QGEFTTWSPW SQPLAFRTKP

SEQ ID NO: 15 - HC-IL2Rbetav W166N MGVKVL FAL I CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGAGGG GSGGGGSGPL GVRGGGGSGG GGSAVNGTSQ FTCFYNSRAN ISCVWSQDGA LQDTSCQVHA WPDRRRWNQT CELLPVSQAS WACNLILGAP DSQKLTTVDI VTLRVLCREG VRWRVMAIQD FKPFENLRLM APISLQWHV ETHRCNISWE ISQASHYFER HLEFEARTLS PGHTWEEAPL CLETLTPDTQ YEFQVRVKPL QGEFTTWSPW SQPLAFRTKP

SEQ ID NO: 16 - HC-IL2Rbetav Q164E/W166N MGVKVL FAL I CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGAGGG GSGGGGSGPL GVRGGGGSGG GGSAVNGTSQ FTCFYNSRAN ISCVWSQDGA LQDTSCQVHA WPDRRRWNQT CELLPVSQAS WACNLILGAP DSQKLTTVDI VTLRVLCREG VRWRVMAIQD FKPFENLRLM APISLQWHV ETHRCNISWE ISQASHYFER HLEFEARTLS PGHTWEEAPL CLETLTPDTQ YEFQVRVKPL QGEFTTWSPW SQPLAFRTKP

SEQ ID NO: 17 - HC-IL2Rbetav Q162S/Q164E MGVKVL FAL I CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGAGGG GSGGGGSGPL GVRGGGGSGG GGSAVNGTSQ FTCFYNSRAN ISCVWSQDGA LQDTSCQVHA WPDRRRWNQT CELLPVSQAS WACNLILGAP DSQKLTTVDI VTLRVLCREG VRWRVMAIQD FKPFENLRLM APISLQWHV ETHRCNISWE ISQASHYFER HLEFEARTLS PGHTWEEAPL CLETLTPDTQ YEFQVRVKPL QGEFTTWSPW SQPLAFRTKP

SEQ ID NO: 18 - HC-IL2Rbetav W166S MGVKVL FAL I CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGAGGG GSGGGGSGPL GVRGGGGSGG GGSAVNGTSQ FTCFYNSRAN ISCVWSQDGA LQDTSCQVHA WPDRRRWNQT CELLPVSQAS WACNLILGAP DSQKLTTVDI VTLRVLCREG VRWRVMAIQD FKPFENLRLM APISLQWHV ETHRCNISWE ISQASHYFER HLEFEARTLS PGHTWEEAPL CLETLTPDTQ YEFQVRVKPL QGEFTTWSPW SQPLAFRTKP

SEQ ID NO: 19 - HC-IL2Rbetav Q164E/W166S MGVKVL FAL I CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK

SEQ ID NO: 22 - HC-IL2Rbetav W166N/C168S

MGVKVLFALI CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS

SEQ ID NO: 25 - HC-IL2Rbetav C168S/Q162S/Q164E MGVKVLFALI CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LYITREPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS

SEQ ID NO: 28 - HC-IL2Rbetav Q164E/W166N/C168S MGVKVLFALI CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LYITREPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGAGGG GSGGGGSGPL GVRGGGGSGG GGSAVNGTSQ FTCFYNSRAN ISCVWSQDGA LQDTSCQVHA WPDRRRWNQT CELLPVSQAS WACNLILGAP

SEQ ID NO: 29 - PDl-IL2v YTE

MGVKVL FAL I CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LYITREPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGAGGG GSGGGGSGGG GSAPASSSTK KTQLQLEHLL LDLQMILNGI NNYKNPKLTS MLTAKFAMPK KATELKHLQC LEEALKPLEE VLNLAQSKNF HLRPRDLISN INVIVLELKG SETTFMCEYA DETATIVEFL NRWITFSQSI ISTLT**

SEQ ID NO: 30 - HC-IL2Rbetav C168S MGVKVL FAL I CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LYITREPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGAGGG GSGGGGSGPL GVRGGGGSGG GGSAVNGTSQ FTCFYNSRAN ISCVWSQDGA LQDTSCQVHA WPDRRRWNQT CELLPVSQAS WACNLILGAP DSQKLTTVDI VTLRVLCREG VRWRVMAIQD FKPFENLRLM APISLQWHV ETHRCNISWE ISQASHYFER HLEFEARTLS PGHTWEEAPL LTLKQKQEWI

SEQ ID NO: 31 - HC-IL2Rbetav Q162S/Q164E MGVKVL FAL I CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LYITREPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGAGGG GSGGGGSGPL GVRGGGGSGG GGSAVNGTSQ FTCFYNSRAN ISCVWSQDGA LQDTSCQVHA WPDRRRWNQT CELLPVSQAS WACNLILGAP DSQKLTTVDI VTLRVLCREG VRWRVMAIQD FKPFENLRLM APISLQWHV ETHRCNISWE ISQASHYFER HLEFEARTLS PGHTWEEAPL CLETLTPDTQ YEFQVRVKPL QGEFTTWSPW SQPLAFRTKP

SEQ ID NO: 32 - HC-IL2Rbetav C168S YTE noncleavable 5 ' BamHI , 3' Swal

MGVKVLFALI CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LYITREPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGAGGG GSGGGGSAAG GGGSGGGGSA VNGTSQFTCF YNSRANISCV WSQDGALQDT SCQVHAWPDR RRWNQTCELL PVSQASWACN LILGAPDSQK LTTVDIVTLR VLCREGVRWR VMAIQDFKPF ENLRLMAPIS LQWHVETHR CNISWEISQA SHYFERHLEF EARTLSPGHT WEEAPLLTLK LTPDTQYEFQ VRVKPLQGEF TTWSPWSQPL AFRTKPAALG

SEQ ID NO: 33 - HC-IL2Rbetav Q162S/Q164E YTE noncleavable 5' BamHI , 3 ' Swal

MGVKVLFALI CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LYITREPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGAGGG GSGGGGSAAG GGGSGGGGSA VNGTSQFTCF YNSRANISCV WSQDGALQDT SCQVHAWPDR RRWNQTCELL PVSQASWACN LILGAPDSQK LTTVDIVTLR VLCREGVRWR VMAIQDFKPF ENLRLMAPIS LQWHVETHR CNISWEISQA SHYFERHLEF EARTLSPGHT WEEAPLLTLK LTPDTQYEFQ VRVKPLQGEF TTWSPWSQPL AFRTKPAALG

SEQ ID NO: 34 - HC-IL2Rbetav Q162S/Q164E YTE noncleavable MGVKVLFALI CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LYITREPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGAGGG GSGGGGSAVN GTSQFTCFYN SRANISCVWS QDGALQDTSC QVHAWPDRRR WNQTCELLPV SQASWACNLI LGAPDSQKLT TVDIVTLRVL CREGVRWRVM AIQDFKPFEN LRLMAPISLQ WHVETHRCN ISWEISQASH YFERHLEFEA RTLSPGHTWE VKPLQGEFTT WSPWSQPLAF

SEQ ID NO: 35 - N-terminal IL2Rbeta muteins Fc

MGVKVL FAL I CIAVAEAAVN GTSQFTCFYN SRANISCVWS QDGALQDTSC QVHAWPDRRR WNQTCELLPV SQASWACNLI LGAPDSQKLT TVDIVTLRVL CREGVRWRVM AIQDFKPFEN LRLMAPISLQ WHVETHRCN ISWEISQASH YFERHLEFEA RTLSPGHTWE

QEWICLETLT PDTQYEFQVR VKPLQGEFTT WSPWSQPLAF RTKPAALGKD TGGGGSGGGG SAAGGGGSGG GGSAESKYGP PCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVW DVSQEDPEVQ FNWYVDGVEV HNAKTKPREE QFNSTYRWS VLTVLHQDWL NGKEYKCKVS NKGLPSSIEK TISKAKGQPR EPQVCTLPPS QEEMTKNQVS LSCAVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLVS RLTVDK SRWQEGNVFS CSVMHEALHN HYTQKSLSLS LGK**

SEQ ID NO: 52 - IL2R K163D _IgG4 Fc hole MGVKVL FAL I CIAVAEAAVN GTSQFTCFYN SRANISCVWS QDGALQDTSC QVHAWPDRRR WNQTCELLPV SQASWACNLI LGAPDSQKLT TVDIVTLRVL CREGVRWRVM AIQDFKPFEN LRLMAPISLQ WHVETHRCN ISWEISQASH YFERHLEFEA RTLSPGHTWE

PDTQYEFQVR VKPLQGEFTT WSPWSQPLAF RTKPAALGKD TGGGGSGGGG SAAGGGGSGG GGSAESKYGP PCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVW DVSQEDPEVQ FNWYVDGVEV HNAKTKPREE QFNSTYRWS VLTVLHQDWL NGKEYKCKVS NKGLPSSIEK TISKAKGQPR EPQVCTLPPS QEEMTKNQVS LSCAVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLVS RLTVDK SRWQEGNVFS CSVMHEALHN HYTQKSLSLS LGK**

SEQ ID NO: 53 - IL2R R42F_IgG4 Fc hole MGVKVL FAL I CIAVAEAAVN GTSQFTCFYN SRANISCVWS QDGALQDTSC WNQTCELLPV SQASWACNLI LGAPDSQKLT TVDIVTLRVL

CREGVRWRVM AIQDFKPFEN LRLMAPISLQ WHVETHRCN ISWEISQASH YFERHLEFEA RTLSPGHTWE EAPLLTLKQK QEWICLETLT PDTQYEFQVR VKPLQGEFTT WSPWSQPLAF RTKPAALGKD TGGGGSGGGG SAAGGGGSGG GGSAESKYGP PCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVW DVSQEDPEVQ FNWYVDGVEV HNAKTKPREE QFNSTYRWS VLTVLHQDWL NGKEYKCKVS NKGLPSSIEK TISKAKGQPR EPQVCTLPPS QEEMTKNQVS LSCAVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLVS RLTVDK SRWQEGNVFS CSVMHEALHN HYTQKSLSLS LGK**

SEQ ID NO: 54 - IL2R S69R _IgG4 Fc hole MGVKVL FAL I CIAVAEAAVN GTSQFTCFYN SRANISCVWS QDGALQDTSC QVHAWPDRRR WNQTCELLPV SQASWACNLI

TVDIVTLRVL CREGVRWRVM AIQDFKPFEN LRLMAPISLQ WHVETHRCN ISWEISQASH YFERHLEFEA RTLSPGHTWE EAPLLTLKQK QEWICLETLT PDTQYEFQVR VKPLQGEFTT WSPWSQPLAF RTKPAALGKD TGGGGSGGGG SAAGGGGSGG GGSAESKYGP PCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVW DVSQEDPEVQ FNWYVDGVEV HNAKTKPREE QFNSTYRWS VLTVLHQDWL NGKEYKCKVS NKGLPSSIEK TISKAKGQPR EPQVCTLPPS QEEMTKNQVS LSCAVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLVS RLTVDK SRWQEGNVFS CSVMHEALHN HYTQKSLSLS LGK**

SEQ ID NO: 55 - IL2R W166S _IgG4 Fc hole MGVKVL FALI CIAVAEAMGV KVLFALICIA VAEAAVNGTS QFTCFYNSRA NISCVWSQDG ALQDTSCQVH AWPDRRRWNQ TCELLPVSQA SWACNLILGA PDSQKLTTVD IVTLRVLCRE GVRWRVMAIQ DFKPFENLRL MAPISLQWH VETHRCNISW EISQASHYFE RHLEFEARTL SPGHTWEEAP

ICLETLTPDT QYEFQVRVKP LQGEFTTWSP WSQPLAFRTK PAALGKDTGG GGSGGGGSAA GGGGSGGGGS AESKYGPPCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRWSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VCTLPPSQEE MTKNQVSLSC AVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLV SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGK**

SEQ ID NO: 56 - IL2R R15Q _IgG4 Fc hole MGVKVLFALI CIAVAEAMGV KVLFALICIA VAEAAVNGTS QFTCFYNS(Q|A NISCVWSQDG ALQDTSCQVH AWPDRRRWNQ TCELLPVSQA SWACNLILGA PDSQKLTTVD IVTLRVLCRE GVRWRVMAIQ DFKPFENLRL MAPISLQVVH VETHRCNISW EISQASHYFE RHLEFEARTL SPGHTWEEAP LLTLKQKQEW ICLETLTPDT QYEFQVRVKP LQGEFTTWSP WSQPLAFRTK PAALGKDTGG GGSGGGGSAA GGGGSGGGGS AESKYGPPCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRWSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VCTLPPSQEE MTKNQVSLSC AVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLV SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGK**

SEQ ID NO: 57 - IL2R F101D _IgG4 Fc hole MGVKVLFALI CIAVAEAAVN GTSQFTCFYN SRANISCVWS QDGALQDTSC QVHAWPDRRR WNQTCELLPV SQASWACNLI LGAPDSQKLT TVDIVTLRVL CREGVRWRVM

LRLMAPISLQ WHVETHRCN ISWEISQASH YFERHLEFEA RTLSPGHTWE EAPLLTLKQK QEWICLETLT PDTQYEFQVR VKPLQGEFTT WSPWSQPLAF RTKPAALGKD TGGGGSGGGG SAAGGGGSGG GGSAESKYGP PCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVW DVSQEDPEVQ FNWYVDGVEV HNAKTKPREE QFNSTYRWS VLTVLHQDWL NGKEYKCKVS NKGLPSSIEK TISKAKGQPR EPQVCTLPPS QEEMTKNQVS LSCAVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLVS RLTVDK SRWQEGNVFS CSVMHEALHN HYTQKSLSLS LGK**

SEQ ID NO: 58 - IL2R Y134R _IgG4 Fc hole MGVKVLFALI CIAVAEAAVN GTSQFTCFYN SRANISCVWS QDGALQDTSC QVHAWPDRRR WNQTCELLPV SQASWACNLI LGAPDSQKLT TVDIVTLRVL CREGVRWRVM AIQDFKPFEN LRLMAPISLQ WHVETHRCN ISWEISQASH

RTLSPGHTWE EAPLLTLKQK QEWICLETLT PDTQYEFQVR VKPLQGEFTT WSPWSQPLAF RTKPAALGKD TGGGGSGGGG SAAGGGGSGG GGSAESKYGP PCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVW DVSQEDPEVQ FNWYVDGVEV HNAKTKPREE QFNSTYRWS VLTVLHQDWL NGKEYKCKVS NKGLPSSIEK TISKAKGQPR EPQVCTLPPS QEEMTKNQVS LSCAVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLVS RLTVDK SRWQEGNVFS CSVMHEALHN HYTQKSLSLS LGK**

SEQ ID NO: 59 - IL2R Q164D _IgG4 Fc hole MGVKVLFALI CIAVAEAAVN GTSQFTCFYN SRANISCVWS QDGALQDTSC QVHAWPDRRR WNQTCELLPV SQASWACNLI LGAPDSQKLT TVDIVTLRVL CREGVRWRVM AIQDFKPFEN LRLMAPISLQ WHVETHRCN ISWEISQASH YFERHLEFEA RTLSPGHTWE EAPLLTLKQK

PDTQYEFQVR VKPLQGEFTT WSPWSQPLAF RTKPAALGKD TGGGGSGGGG SAAGGGGSGG



LSCAVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLVS RLTVDK SRWQEGNVFS CSVMHEALHN HYTQKSLSLS LGK

SEQ ID NO: 68 - IL2R M107S _IgG4 Fc hole

MGVKVLFALI CIAVAEAAVN GTSQFTCFYN SRANISCVWS QDGALQDTSC QVHAWPDRRR WNQTCELLPV SQASWACNLI LGAPDSQKLT TVDIVTLRVL CREGVRWRVM AIQDFKPFEN

WHVETHRCN ISWEISQASH YFERHLEFEA RTLSPGHTWE EAPLLTLKQK QEWICLETLT PDTQYEFQVR VKPLQGEFTT WSPWSQPLAF RTKPAALGKD TGGGGSGGGG SAAGGGGSGG GGSAESKYGP PCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVW DVSQEDPEVQ FNWYVDGVEV HNAKTKPREE QFNSTYRWS VLTVLHQDWL NGKEYKCKVS NKGLPSSIEK TISKAKGQPR EPQVCTLPPS QEEMTKNQVS LSCAVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLVS RLTVDK SRWQEGNVFS CSVMHEALHN HYTQKSLSLS LGK

SEQ ID NO: 69 - IgG4-YTE-knob-IL2C125A (5' NheI , 3 ' PmeI ) MGVKVLFALI CIAVAEAAES KYGPPCPPCP APEAAGGPSV FLFPPKPKDT

CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPCQEEMTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGAGGG GSGGGGSGGG GSAPTSSSTK KTQLQLEHLL LDLQMILNGI NNYKNPKLTR MLTFKFYMPK KATELKHLQC LEEELKPLEE VLNLAQSKNF HLRPRDLISN INVIVLELKG SETTFMCEYA DETATIVEFL N

ISTLT**

SEQ ID NO: 70 - IgG4-YTE-knob-IL-2 C125A/V69A/Q74 P 5' NheI ,

3 ' Pme I )

MGVKVLFALI CIAVAEAAES KYGPPCPPCP APEAAGGPSV FLFPPKPKDT LYITREPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPCQEEMTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGAGGG GSGGGGSGGG GSAPTSSSTK KTQLQLEHLL LDLQMILNGI NNYKNPKLTR MLTFKFYMPK KATELKHLQC LEEELKPLEE ALNLAPSKNF HLRPRDLISN INVIVLELKG SETTFMCEYA DETATIVEFL NRWITFAQSI ISTLT**

SEQ ID NO: 71 - IgG4-YT E-hole IL-2Rbeta C168S (5' BamHI , 3' SwaI ) MGVKVLFALI CIAVAEAAES KYGPPCPPCP APEAAGGPSV FLFPPKPKDT

CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVCT LPPSQEEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGAGGG GSGGGGSAAG GGGSGGGGSA VNGTSQFTCF YNSRANISCV WSQDGALQDT SCQVHAWPDR RRWNQTCELL PVSQASWACN LILGAPDSQK LTTVDIVTLR VLCREGVRWR VMAIQDFKPF ENLRLMAPIS LQWHVETHR CNISWEISQA SHYFERHLEF EARTLSPGHT WEEAPLLTLK

LTPDTQYEFQ VRVKPLQGEF TTWSPWSQPL AFRTKPAALG KDT**

SEQ ID NO: 72 - IgG4-YT E-hole IL-2Rbeta Q162S/Q164E ( 5' BamHI ,

3' Swal )

KVSNKGLPSS IEKTISKAKG QPREPQVYTL PPCQEEMTKN QVSLWCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK* *

SEQ ID NO: 88 - PD1 HC HOLE YTE

MGVKVLFALI CIAVAEAQVQ LVESGGGWQ PGRSLRLDCK ASGITFSNSG MHWVRQAPGK GLEWVAVIWY DGSKRYYADS VKGRFTISRD NSKNTLFLQM NSLRAEDTAV YYCATNDDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LYITREPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVCT LPPSQEEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK**

SEQ ID NO: 89-96 Peptide Linker

(GGGGS ) _n (SEQ ID NO: 89 ) , wherein n =1 , 2 , 3 , and 4 GGGGS (SEQ ID NO: 90 )

GGGGSGGGGS (SEQ ID NO: 91 )

GGGGS GGGGS GGGGS (SEQ ID NO: 92 )

GGGGS GGGGSXXGGGGS GGGGS ( SEQ ID NO : 93 ) , X = A or N GGGGS GGGGS GGGGSXXGGGGS GGGGS ( SEQ ID NO : 94 ) , X = A or N GGGGS GGGGS GGGGSXXGGGGS GGGGS GGGGS (SEQ ID NO: 95 ) , X = A or N GGGGS GGGGXGGGGYGGGGS (SEQ ID NO: 96) , X = S , A or N, and Y = A or N

SEQ ID NO: 97 - Anti-PDLl atezolizumab LC

DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLI YS

AS FLYSGVPS

RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YLYHPATFGQ GTKVEIKRTV AAPSVFI FPP

SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

SEQ ID NO: 98 - Anti-PDLl atezolizumab HC EVQLVESGGG LVQPGGSLRL SCAASGFTFS DSWIHWVRQA PGKGLEWVAW ISPYGGSTYY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSWTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVWDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYAST YRWSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK

SEQ ID NO: 99 - PD1-LC

EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLI YD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKRTV AAPSVFI FPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

SEQ ID NO: 100 - Anti-PD-1 nivolumab HC

QVQLVESGGG WQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYY ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPS VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVS WNS GALT SGVHTFPAVL QSSGLYSLSS WTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP PCPAPEFLGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRWSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGK

SEQ ID NO: 101 - Anti-PD-1 pembrolizumab LC EIVLTQSPAT LSLSPGERAT LSCRASKGVS TSGYS YLHWY QQKPGQAPRL LIYLASYLES GVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRDLPL TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

SEQ ID NO: 102 - Anti-PD-1 pembrolizumab HC QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMYWVRQA PGQGLEWMGG INPSNGGTNF NEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRD YRFDMGFDYW GQGTTVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNS GALT SGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CWVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK

SEQ ID NO: 103 - Human IL-15

NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIH DTVENLIILA NNSLSSNGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INT

SEQ ID NO: 104 - IL-15 receptor alpha subunit Sushi domain ITCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPA

SEQ ID NO: 105 - Amino acid sequence of IL-15 receptor alpha MAPRRARGCR TLGLPALLLL LLLRPPATRG ITCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTEISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTVAIST STVLLCGLSA VSLLACYLKS RQTPPLASVE MEAMEALPVT WGTSSRDEDL ENCSHHL

SEQ ID NO: 106 - Amino acid sequence of IL-15 receptor alpha Sushi domain ITCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIR

SEQ ID NO: 107 - Human IL-2 Receptor Beta Subunit Extracellular Domain Δ1-5 (https : //www . uniprot . org/uniprot/P14784 )

SQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAP DSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEIS

QASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEF TTWSPWSQPLAFRTKPAALGKDT

SEQ ID NO: 108 - Human IL-2 Receptor Beta Subunit Extracellular Domain Δ1-5, N45Q (https : //www. uniprot . org/uniprot/P14784 )

SQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWQQTCELLPVSQASWACNLILGAP DSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEIS

QASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEF TTWSPWSQPLAFRTKPAALGKDT

SEQ ID NO: 109 - Human IL-2 Receptor Beta Subunit Extracellular Domain mutant

X_aa2 is amino acid V or deleted; X_aa3 is amino acid N or deleted; X_aa4 is amino acid G or deleted; X_aa5 is amino acid T or deleted; X_aa38 is amino acid W, S, N or G; X_aa45 is amino acid N or Q; X_aa90 is amino acid W, S, N or G; X_aa150 is amino acid H or K; X_aa152 is amino acid W, S, N or G; X_aa166 is amino acid W, S, N or G; X_aa194 is amino acid W, S, N or G; and X_aa197 is amino acid W, S, N or G.

Claims

1. An isolated protein comprising a human IL-2 receptor b subunit ( IL-2Rβ ) extracellular domain (ECD), wherein the IL-2Rβ ECD comprises, relative to wildtype IL-2Rβ ECD, A 1-5 (deletions of the first five amino acids) and/or one or more mutations at position(s) selected from FI 1, V21, L28, W38, L51, P52, V53, 163, P67, 177, V88, W90,

V92, M93, 195, M107, 1110, VI 15, H150, R137, W152, P156, L157, K161, Q162, Q164, W166, C168, P174, L187, F191, W194, P196, W197, P200, and P207 (numbering according to SEQ ID NO:4).

2. The isolated protein of claim 1, wherein the IL-2Rβ ECD comprises one or more mutations at position(s) selected from V92, H150, W152, Q162, Q164, W166, C168, and L187.

3. The isolated protein of claim 2, wherein the IL-2Rβ ECD comprises one or more mutations selected from V92S, H150E, W152S/N, Q162S, Q164E, W166N/S/E, C168T/S, and L187S.

4. The isolated protein of claim 3 wherein the IL-2Rβ ECD comprises one, two, or three mutations selected from Q162S, Q164E, and C168S.

5. The isolated protein of claim 1, wherein the IL-2Rβ ECD comprises mutations at the following positions: a. Fll and F191; b. L51, P52, and V53; c. V92, M93, and 195; d. M107, P196, and 1110; or e. P156 and L157.

6. The isolated protein of claim 1, wherein the IL-2Rβ ECD comprises the following mutations: a. F11S and F191G; b. L51S, P52G, and V53S; c. V92S, M93G, and I95G; d. M107G, P196S, and I110G; e. P156S and L157G f. W166N g. Q162S; h. Q164E; i. V115S and W166N; j. W152N; k. W152N and W166N l. V92S; m. V92S and W166N; n. L157S; o. W157S and W165N; p. W152S and W166S; q. W166N and L187S; r. Q162S and C168S; s. Q164E and C168S; t. Q162S and Q164E; u. Q162S, Q164E, and C168S; v. C168S; or w. Q164E and W166N.

7. The isolated protein of any one of the preceding claims, wherein the isolated protein is an isolated IL-2 or IL-15 fusion molecule, comprising a carrier moiety, a cytokine moiety, and a masking moiety, wherein the cytokine moiety is fused to the carrier moiety or to a masking moiety, directly or indirectly via a peptide linker, the masking moiety is fused to the carrier moiety or to the cytokine moiety, directly or indirectly via a peptide linker, the cytokine moiety comprises an IL-2 or IL-15 polypeptide, optionally wherein the IL-2 or IL-15 polypeptide is a variant of wildtype IL-2 or IL-15 polypeptide, and the masking moiety binds to the cytokine moiety and inhibits binding of the cytokine moiety to IL-2Rβ and/or IL-2Rγ on immune cells and comprises the IL-2Rβ ECD.

8. The isolated protein of claim 7, wherein the cytokine moiety comprises an IL-2 variant that does not bind CD25 (IL-2Rα).

9. The isolated protein of claim 7 or 8, wherein the cytokine moiety comprises an IL-2 polypeptide comprising (i) a Cl 25 A or Cl 25 S substitution, or (ii) an IL-2 amino acid sequence comprising one or more substitutions selected from T3A, C125S, V69A, and Q74P (numbering according to SEQ ID NO: 1).

10. The isolated protein of any one of claims 7-9, wherein the isolated protein is an isolated IL-2 fusion molecule and the IL-2 polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, optionally wherein the amino acid sequence is SEQ ID NO: 1, 2, or 3.

11. The isolated protein of claim 7, wherein the isolated protein is an isolated IL-15 fusion molecule and the IL-15 polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 103, optionally wherein the amino acid sequence is SEQ ID NO:103.

12. The isolated protein of claim 7 or 11, comprising an IL-15 cytokine moiety and a Sushi domain, wherein the masking moiety is fused to the carrier moiety, the Sushi domain is fused to the carrier moiety, and the IL-15 cytokine moiety is fused to the Sushi domain.

13. The isolated protein of claim 7 or 11, comprising an IL-15 cytokine moiety and a Sushi domain, wherein the IL-15 cytokine moiety is fused to the carrier moiety, the Sushi domain is fused to the carrier moiety, and the masking moiety is fused to the Sushi domain.

14. The isolated protein of any one of claims 7-13, wherein the cytokine moiety is fused to the carrier moiety or the masking moiety through a non-cleavable or cleavable peptide linker, and the masking moiety is fused to the carrier moiety or the cytokine moiety through a non-cleavable or cleavable peptide linker.

15. The isolated protein of any one of claims 7-15, wherein the carrier moiety is selected from a PEG molecule, an albumin, an albumin fragment, an antibody Fc domain, a halfantibody, an antibody, or an antigen-binding fragment thereof.

16. The isolated protein of claim 15, wherein the carrier moiety comprises an antibody Fc domain with a mutation at N297 and/or mutations L234A and L235A (“LALA”) (Eu numbering).

17. The isolated protein of claim 15 or 16, wherein the carrier moiety comprises an antibody Fc domain, and wherein the cytokine moiety and the masking moiety are fused to different polypeptide chains of the antibody Fc domain.

18. The isolated protein of any one of claims 15-17, wherein the cytokine moiety and the masking moiety are fused to the C-termini of the two different polypeptide chains of the Fc domain or to the C-termini of the two different heavy chains of the antibody, optionally via a cleavable or non-cleavable peptide linker.

19. The isolated protein of any one of claims 15-17, wherein the cytokine moiety and the masking moiety are fused to the N-termini of the two different polypeptide chains of the Fc domain, optionally via a cleavable or non-cleavable peptide linker.

20. The isolated protein of claim 19, wherein the carrier is a half-antibody comprising one Fab domain and an Fc domain, and wherein the cytokine moiety is fused to the N-terminus of one of the heavy chains of the Fc domain directly or indirectly, via a non-cleavable peptide, and the masking moiety is fused to the N-terminus of the cytokine moiety, optionally via a cleavable or non-cleavable peptide linker.

21. The isolated protein of any one of claims 14-20, wherein the cleavable peptide linker comprises a substrate sequence of urokinase-type plasminogen activator (uPA), matrix metallopeptidase (MMP) 2, or MMP9, optionally wherein the cleavable peptide linker comprises substrate sequences of (i) both uPA and MMP2, (ii) both uPA and MMP9, or (iii) uPA, MMP2 and MMP9.

22. The isolated protein of any one of claims 14-20, wherein the noncleavable peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 89-96.

23. The isolated protein of any one of claims 15-22, wherein the carrier moiety comprises an antibody Fc domain comprising knobs-into-holes mutations.

24. The isolated protein of claim 23, wherein the knobs-into-holes mutations comprise a T366Y “knob” mutation on a polypeptide chain of the Fc domain, and a Y407T “hole” mutation in the other polypeptide of the Fc domain (Eu numbering).

25. A polynucleotide encoding the isolated protein of any one of claims 1-24.

26. An expression vector comprising the polynucleotide of claim 25.

27. A host cell comprising the expression vector of claim 26, optionally wherein the gene(s) encoding uPA, MMP-2, and/or MMP-9 are knocked out in the host cell.

28. A method of making the isolated protein of any one of claims 1-24, comprising culturing the host cell of claim 27 under conditions that allow expression of the isolated protein, and isolating the isolated protein.

29. A pharmaceutical composition comprising the isolated protein of any one of claims 1- 24 and a pharmaceutically acceptable excipient.

30. A method of treating a cancer or an infectious disease, stimulating the immune system, or treating an autoimmune disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of claim 29.

31. A cytokine IL-2 or IL-15 fusion molecule of any one of claims 7-24 for use in treating a cancer or an infectious disease, stimulating the immune system, or treating an autoimmune disease in the method of claim 30.

32. Use of an IL-2 or IL-15 fusion molecule of any one of claims 7-24 for the manufacture of a medicament for treating a cancer or an infectious disease, stimulating the immune system, or treating an autoimmune disease in the method of claim 30.

33. The method of claim 30, the IL-2 or IL-15 fusion molecule for use of claim 31, or the use of claim 32, wherein the patient has HIV infection, or a cancer selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer, and stomach cancer.