Variant il-2 proteins

Abstract

Provided are novel interleukin-2 (IL-2) variant proteins, including variant IL-2 heterodimeric Fc-fusion proteins thereof. Also provided are methods for producing the IL-2 variant proteins, methods of using the IL-2 variant proteins, and pharmaceutical compositions comprising the IL-2 variant proteins.

Description

VARIANT IL-2 PROTEINSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 523,908, filed June 28, 2023; U.S. Provisional Patent Application No. 63 / 511,071, filed June 29, 2023; U.S. Provisional Patent Application No. 63 / 519,421, filed August 14, 2023; the disclosure of each of which is hereby incorporated by reference in its entirety for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 10, 2024, is named DFY122WO_SL.xml and is 43,064 bytes in size.FIELD OF THE DISCLOSURE

[0003] The disclosure relates generally to novel interleukin-2 (IL-2) variant proteins, including variant IL-2 heterodimeric Fc-fused proteins thereof. Methods for producing the IL-2 variant proteins, methods of using the IL-2 variant proteins, and pharmaceutical compositions comprising the IL-2 variant proteins are also disclosed herein.BACKGROUND

[0004] Interleukin-2 (IL-2) is a type I a-helical cytokine that functions as a multi-lineage lymphocyte growth factor. IL-2 stimulates strong anti-tumor responses by promoting expansion and activation of cytotoxic tumor-infiltrating immune cells including CD8+T cells and natural killer (NK) cells. IL-2 is primarily produced by CD4+T cells upon antigen activation and, to a lesser extent, by CD8+T cells, as well as NK cells, natural killer T (NKT) cells, activated dendritic cells (DCs), and mast cells.

[0005] On activated lymphocytes and regulatory T cells (Tregs), IL-2 signals through a high-affinity (10 pM) heterotrimeric receptor complex, consisting of the IL-2Ra (CD25), IL- 2RP (CD122), and yc (CD132) chains. In resting lymphocytes, IL-2 signals via the intermediate-affinity (1 nM) heterodimeric receptor complex, consisting of the IL-2RP and yc chains. The IL2-Ra subunit is constitutively expressed in high levels on Tregs and at lower levels on natural killer (NK) cells and resting effector CD8+T cells, resulting in differentialIL-2 potency between different immune cell compartments. From a clinical perspective, both IL-2 agonism and antagonism is of considerable importance and it has been used for more than three decades towards immunotherapies of malignancies.

[0006] Current strategies for targeting IL-2 signaling include the use of small molecules that bind to IL-2 and block signaling, engineering versions of IL-2 with high affinity for IL- 2RP (“superagonists”), and the use of anti-IL-2 antibodies that direct its actions to different immune cell subsets. For example, monoclonal antibodies against IL-2 can bind to a number of different conformational epitopes, to direct the interactions of IL-2 with any of the IL-2R subunits thereby resulting in the proliferation of either Treg or Teir cells. For example, wildtype mouse IL-2 (mIL-2) can be administered in complex with an anti-mouse IL-2 monoclonal antibody (JES6-1) to induce Treg cell proliferation (e.g., rather than Teir proliferation).

[0007] Although IL-2 is known to effectively expand T cells to enhance adoptive immunotherapy and is approved by the FDA for the treatment of certain cancers, IL-2 can also promote pathologic responses such as cytokine storm. Accordingly, a goal for developing IL-2 therapies is to maintain the therapeutic actions of this cytokine while preventing unwanted deleterious responses.

[0008] To this end, development of human IL-2 signaling variants is of great interest for clinical purposes, including the treatment of cancer and autoimmune diseases. Accordingly, there is a need in the art for improved variant IL-2 proteins that maintain strong anti-tumor activity with minimal toxicity.SUMMARY OF THE INVENTION

[0009] The disclosure is based, in part, on the discovery that it is possible to produce a recombinant variant IL-2 protein that maintains anti-tumor activity but does not stimulate the release of as many cytokines associated with cytotoxicity as wild type IL-2. Accordingly, the disclosure relates, in part, to a heterodimeric Fc-fused IL-2 protein comprising a variant IL-2 protein optionally fused, e.g., via a linker, to an Fc domain, wherein the Fc domain comprises a first and second Fc domain polypeptide. The first and second Fc domain polypeptides can include different heterodimerization mutations.

[0010] The disclosure generally relates to novel IL-2 variant proteins, Fc-fused IL-2 variant proteins, pharmaceutical compositions comprising such proteins, and methods of using same, for example, for treating cancer.

[0011] In one aspect, the present disclosure provides a variant human IL-2 protein comprising a substitution of the threonine at position 3 to an alanine, a substitution of the glycine at position 27 to a methionine, a substitution of the isoleucine at position 28 to a leucine, a substitution of the lysine at position 32 to an aspartic acid, and a substitution of the cysteine at position 125 to a serine; wherein the substitutions are relative to the amino acid sequence of wild-type human IL-2 protein represented by SEQ ID NO: 1.

[0012] In some embodiments, the variant human IL-2 protein further comprises a substitution of the leucine at position 72 to a glutamine and a substitution of the arginine at position 81 to an aspartic acid.

[0013] In some embodiments, the variant human IL-2 protein further comprises a substitution of the methionine at position 39 to a leucine, a substitution of the glutamic acid at position 52 to a serine, a substitution of the leucine at position 72 to an alanine, a substitution of the glutamine at position 74 to a glycine, a substitution of the serine at position 75 to an aspartic acid, a substitution of the lysine at position 76 to an aspartic acid, a substitution of the asparagine at position 77 to a proline, a substitution of the phenylalanine at position 78 to a lysine, a substitution of the histidine at position 79 to a threonine, a substitution of the leucine at position 80 to an isoleucine, a deletion of the arginine at position 81, a deletion of the proline at position 82, a substitution of the leucine at position 85 to a valine, a substitution of the isoleucine at position 86 to a valine, and a substitution of the isoleucine at position 92 to a phenylalanine.

[0014] In some embodiments, the present disclosure provides a variant human IL-2 protein comprises a substitution of the threonine at position 3 to an alanine, a substitution of the glycine at position 27 to a methionine, a substitution of the isoleucine at position 28 to a leucine, a substitution of the lysine at position 32 to an aspartic acid, a substitution of the leucine at position 72 to a glutamine, a substitution of the arginine at position 81 to an aspartic acid, and a substitution of the cysteine at position 125 to a serine; wherein the substitutions are relative to the amino acid sequence of wild-type human IL-2 protein represented by SEQ ID NO: 1.

[0015] In some embodiments, the present disclosure provides a variant human IL-2 protein comprising a substitution of the threonine at position 3 to an alanine, a substitution of the glycine at position 27 to a methionine, a substitution of the isoleucine at position 28 to a leucine, a substitution of the lysine at position 32 to an aspartic acid, a substitution of themethionine at position 39 to a leucine, a substitution of the glutamic acid at position 52 to a serine, a substitution of the leucine at position 72 to an alanine, a substitution of the glutamine at position 74 to a glycine, a substitution of the serine at position 75 to an aspartic acid, a substitution of the lysine at position 76 to an aspartic acid, a substitution of the asparagine at position 77 to a proline, a substitution of the phenylalanine at position 78 to a lysine, a substitution of the histidine at position 79 to a threonine, a substitution of the leucine at position 80 to an isoleucine, a deletion of the arginine at position 81, a deletion of the proline at position 82, a substitution of the leucine at position 85 to a valine, a substitution of the isoleucine at position 86 to a valine, a substitution of the isoleucine at position 92 to a phenylalanine, and a substitution of the cysteine at position 125 to a serine; wherein the substitutions are relative to the amino acid sequence of wild-type human IL-2 protein represented by SEQ ID NO: 1.

[0016] In some embodiments, the variant human IL-2 protein comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2 or SEQ ID NO: 3.

[0017] In some embodiments, the variant human IL-2 protein comprises an amino acid sequence of SEQ ID NO: 2.

[0018] In some embodiments, the variant human IL-2 protein comprises an amino acid sequence of SEQ ID NO: 3.

[0019] In another aspect, the present disclosure provides a heterodimeric Fc-fusion protein comprising a variant human IL-2 protein and an IgG Fc domain.

[0020] In some embodiments, the IgG Fc domain comprises a first Fc domain polypeptide. In some embodiments, the IgG Fc domain comprises a first Fc domain polypeptide and a second Fc domain polypeptide.

[0021] In some embodiments, the IgG Fc domain comprises a first Fc domain polypeptide and a second Fc domain polypeptide, and wherein the first and second Fc domain polypeptides each comprise one or more different mutations promoting heterodimerization, and wherein the Fc domain first polypeptide and the second Fc domain polypeptide are bound to each other.

[0022] Depending upon the circumstances, the IgG Fc domain is derived from an IgGl, IgG2, IgG3, or IgG4 Fc domain. In some embodiments, the IgG Fc domain is derived from a human IgGl Fc domain.

[0023] In some embodiments, the one or more different mutations for promoting heterodimerization are selected from the group consisting of: Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, D356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, F405L, Y407A, Y407I, Y407V, K409F, K409W, K409D, K409R, T41 ID, T41 IE, K439D, and K439E, numbered according to the EU numbering system. In some embodiments, the one or more mutations for promoting heterodimerization comprise K360E and K409W, numbered according to the EU numbering system. In some embodiments, the one or more mutations for promoting heterodimerization comprise Q347R, D399V, and F405T, numbered according to the EU numbering system.

[0024] The first Fc domain polypeptide and the second Fc domain polypeptide can each comprise one or more mutations that promote disulfide bond formation between the first Fc domain polypeptide and the second Fc domain polypeptide. In some embodiments, the first or second Fc domain polypeptide comprises heterodimerization mutations Q347R, D399V, and F405T, and the one or more mutations promoting disulfide bond formation comprise S354C, numbered according to the EU numbering system. In some embodiments, the first or second Fc domain polypeptide comprises heterodimerization mutations K360E and K409W, and the one or more mutations promoting disulfide bond formation comprise Y349C, numbered according to the EU numbering system.

[0025] In some embodiments, the first or second Fc domain polypeptide comprises an amino acid sequence of SEQ ID NO: 4. In some embodiments, the first or second Fc domain polypeptide comprises an amino acid sequence of SEQ ID NO: 5.

[0026] In some embodiments, the first Fc domain polypeptide is fused to the variant human IL-2 protein, and the second Fc domain polypeptide is not fused to the variant human IL-2 protein. In some embodiments, the second Fc domain polypeptide is fused to the variant human IL-2 protein, and the first Fc domain polypeptide is not fused to the variant human IL- 2 protein.

[0027] In some embodiments, the first Fc domain polypeptide and the second Fc domain polypeptide each comprise one or more mutations that reduce effector functions. In some embodiments, the first Fc domain polypeptide and the second Fc domain polypeptide eachcomprise one or more mutations at positions 234, 235, 237, 329, 330, and / or 331, according to the EU numbering system. In some embodiments, the first Fc domain polypeptide and the second Fc domain polypeptide each comprise one or more mutations selected from the group consisting of L234A, L235A, L235E, G237A, P329A, A330S, and P331S. In some embodiments, the first Fc domain polypeptide and the second Fc domain polypeptide each comprise mutations L234A, L235A, and P329A.

[0028] In some embodiments, the variant human IL-2 protein is fused to the IgG Fc domain via a linker polypeptide. In some embodiments, the linker polypeptide comprises the amino acid sequence (GGGGS)s (SEQ ID NO: 6).

[0029] In some embodiments, the human IL-2 protein is fused to the C-terminus of the IgG Fc domain.

[0030] In certain embodiments, the present disclosure is directed to a heterodimeric Fc- fusion protein comprising: a first polypeptide comprising an amino acid sequence of SEQ ID NO: 7; and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 8.

[0031] In certain embodiments, the variant human IL-2 protein has decreased capacity to induce pro-inflammatory cytokine production; as compared to a wild-type human IL-2 protein under comparable assay conditions, wherein the pro-inflammatory cytokine production is assayed using a cytokine release assay.

[0032] In some embodiments, the present disclosure is directed to an isolated nucleic acid encoding a variant human IL-2 protein disclosed herein or the heterodimeric Fc-fusion protein disclosed herein.

[0033] In certain embodiments, the present disclosure is directed to an expression vector comprising a nucleic acid disclosed herein.

[0034] Also disclosed herein is a pharmaceutical composition comprising a variant human IL-2 protein or a heterodimeric Fc-fusion protein, and a pharmaceutically acceptable carrier.

[0035] In certain embodiments, the present disclosure is directed to a recombinant cell comprising one or more nucleic acids or one or more vectors disclosed herein.

[0036] Another aspect of the invention provides a method of expanding T cells, comprising contacting the T cells with an effective amount of a variant human IL-2 protein, a heterodimeric Fc-fusion protein, or a pharmaceutical composition disclosed herein.

[0037] In yet another aspect of the invention, the present disclosure is directed to a method of treating a disease in a subject in need thereof comprising administering to the subject a variant human IL-2 protein, a heterodimeric Fc-fusion protein, or the pharmaceutical composition disclosed herein. In some embodiments, the disease is a hyperproliferative disease. In some embodiments, the disease is a cancer.

[0038] In another aspect, the present disclosure is directed to a purified variant human IL- 2 protein or purified heterodimeric Fc-fusion protein. In some embodiments, the variant human IL-2 protein or heterodimeric Fc-fusion protein is purified using a method selected from the group consisting of: centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification, gel filtration, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixedmode chromatography.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The invention can be more completely understood with reference to the following drawings, in which:

[0040] FIGs. 1A-1D illustrate exemplary heterodimeric Fc-fusion proteins comprising a first polypeptide comprising a first Fc domain polypeptide and a second polypeptide comprising a second, different Fc domain polypeptide, wherein the first Fc domain polypeptide or the second Fc domain polypeptide is fused via a linker to a variant IL-2 protein. FIG. 1A illustrates an exemplary heterodimeric Fc-fusion protein comprising a first Fc domain polypeptide and second, different Fc domain polypeptide. An IL-2 variant protein is fused to the N-terminus of the first Fc domain polypeptide via a linker. FIG. IB illustrates an exemplary IL-2 heterodimeric Fc-fusion protein similar to the protein illustrated in FIG. 1A, but the IL-2 variant protein is fused to the N-terminus of the second Fc domain polypeptide via a linker. FIG. 1C illustrates an exemplary heterodimeric Fc-fusion protein comprising a first Fc domain polypeptide and second, different Fc domain polypeptide, wherein an IL-2 variant protein is fused to the C-terminus of the first Fc domain polypeptide via a linker. FIG. ID illustrates an exemplary IL-2 heterodimeric Fc-fused protein similar to the protein illustrated in FIG. 1C, but the IL-2 variant protein is fused to the C-terminus of the second polypeptide via a linker. The first Fc domain polypeptide and the second Fc domain polypeptide each contains different mutations promoting heterodimerization, and adisulfide bond between the first and second Fc domain polypeptides stabilizes the heterodimer.

[0041] FIGs. 2A-2D are graphs depicting Ki-67 expression in Naive CD4+T cells (FIG. 2A), CD8+T cells (FIG. 2B), Tregs (FIG. 2C), and NK cells (FIG. 2D) after stimulation of naive peripheral blood mononuclear cells (PBMCs) from cynomolgus monkeys with varying concentrations of test articles. Test articles used are: recombinant human IL-2 (rhIL-2); Seql5Rev human IL-2 construct C-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to mouse IgG2a “mu_Seql5rev_3XG4S;” Seql5Rev human IL-2 construct C-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain of human IgGl, “huIgGl - Seql5rev;” SeqlRev human IL-2 construct C-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain of human IgGl, “huIgGl - Seqlrev;” Seql5Rev human IL- 2 construct N-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain of human IgGl, “Seql5rev - huIgGl;” and SeqlRev human IL-2 construct N-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain (CH2-CH3 domains) of human IgGl, “Seqlrev - huIgGl.”

[0042] FIGs. 3A-3D are graphs depicting Ki-67 expression in Naive CD4+T cells (FIG. 3A), CD8+T cells (FIG. 3B), Tregs (FIG. 3C), and NK cells (FIG. 3D) after stimulation of human naive PBMCs with varying concentrations of test articles. Test articles used are: recombinant human IL-2 (rhIL-2); Seql5Rev human IL-2 construct C-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to mouse IgG2a “mu_Seql5rev_3XG4S;” Seql5Rev human IL-2 construct C-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain of human IgGl, “huIgGl - Seql5rev;” SeqlRev human IL-2 construct C-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain of human IgGl, “huIgGl - Seqlrev;” Seql5Rev human IL-2 construct N-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain of human IgGl, “Seql5rev - huIgGl;” and SeqlRev human IL-2 construct N-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain (CH2-CH3 domains) of human IgGl, “Seqlrev - huIgGl .”

[0043] FIGs. 4A-4C are graphs depicting phosphorylated STAT5 (pSTAT5) levels in naive CD8 T cells (FIG. 4A), naive Tregs (FIG. 4B), and activated CD8 T cells (FIG. 4C) after stimulation of naive PBMCs from cynomolgus monkeys with varying concentrations of test articles. Test articles used are: recombinant human IL-2 (rhIL-2); Seql5Rev human IL-2 construct C-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to mouse IgG2a “mu_Seql5rev_3XG4S;” Seql5Rev human IL-2 construct C-terminally fused via a (G4S)3(SEQ ID NO: 6) linker to a truncated heavy chain of human IgGl, “huIgGl - Seql5rev;” SeqlRev human IL-2 construct C-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain of human IgGl, “huIgGl - Seqlrev;” Seql5Rev human IL-2 construct N-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain of human IgGl, “Seql5rev - huIgGl;” and SeqlRev human IL-2 construct N-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain (CH2-CH3 domains) of human IgGl, “Seqlrev - huIgGl.”

[0044] FIGs. 5A-5C are graphs depicting phosphorylated STAT5 (pSTAT5) levels in naive CD8 T cells (FIG. 5A), naive Tregs (FIG. 5B), and activated CD8 T cells (FIG. 5C) after stimulation of naive human PBMCs with varying concentrations of test articles. Test articles used are: recombinant human IL-2 (rhIL-2); Seql5Rev human IL-2 construct C- terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to mouse IgG2a “mu_Seql5rev_3XG4S;” Seql5Rev human IL-2 construct C-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain of human IgGl, “huIgGl - Seql5rev;” SeqlRev human IL-2 construct C-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain of human IgGl, “huIgGl - Seqlrev;” Seql5Rev human IL-2 construct N-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain of human IgGl, “Seql5rev - huIgGl;” and SeqlRev human IL-2 construct N-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain (CH2-CH3 domains) of human IgGl, “Seqlrev - huIgGl.”

[0045] FIG. 6A shows the study design to evaluate anti-tumor activity in the syngeneic mouse CT26-Tyrpl tumor model. FIGs. 6B-6E are graphs showing tumor growth curves of BALB / c mice dosed intraperitoneally (IP) once weekly (QW) for 4 weeks with a dose of 1.3 mg / kg or 0.65 mg / kg of their respective IL-2 variant or a monoclonal antibody isotype control (mIgG2a).

[0046] FIG. 7 is a graph showing dose-responsive binding of a heterodimeric IL-2 Fc- fusion protein described in Example 1, DF IL-2-Fc (circles), hFc(si)-rhIL-2 (black squares), or hFc(si)-rhIL-2-F42A (grey squares) on human IL-2Ra (CD25)-expressing Chinese human ovary (CHO) cells. Each point represents the mean fluorescence intensity (MFI) at varying concentrations of IL-2 variants and the error bars represent standard deviation (SD) from the mean.

[0047] FIGs. 8A-8D are graphs depicting flow cytometric analysis of phosphorylated STAT5 (pSTAT5) signaling in regulatory T cells (Tregs) (FIG. 8A), CD8+T cells (FIG. 8B), CD4+T cells (FIG. 8C), and natural killer (NK) cells (FIG. 8D) after stimulation with DF IL-2-Fc or PROLEUKIN® (aldesleukin).

[0048] FIG. 9 is a graph depicting stimulation of recently activated human CD25- expressing CD8+T cells mediated by DF IL-2-Fc, aldesleukin, or human Fc(si)-rhIL-2-F42A.

[0049] FIGs. 10A-10D are graphs depicting flow cytometric analysis of phosphorylated STAT5 (pSTAT5) signaling in regulatory T cells (Tregs) (FIG. 10A), CD8+T cells (FIG.10B), CD4+T cells (FIG. 10C), and natural killer (NK) cells (FIG. 10D) after stimulation of immune cells from cynomolgus monkey peripheral blood mononuclear cells (PBMCs) with DF IL-2-Fc, PROLEUKIN® (aldesleukin), or isotype control human IgGl with Fc-silent modifications [hFc(si)].

[0050] FIGs. 11A-11D are graphs depicting flow cytometric analysis of phosphorylated STAT5 (pSTAT5) signaling in regulatory T cells (Tregs) (FIG. 11 A), CD8+T cells (FIG.11B), CD4+T cells (FIG. 11C), and natural killer (NK) cells (FIG. 11D) after stimulation of immune cells from Balb / c mouse splenocytes with DF IL-2-Fc or PROLEUKIN® (aldesleukin).

[0051] FIGs. 12A-12D are graphs depicting efficacy of DF IL-2-Fc administered intraperitoneally in mouse CT26 (colon carcinoma) tumor model. FIGs. 12A-12B are graphs showing tumor growth curves of mice engrafted with CT26 cells and dosed intraperitoneally (IP) with human immunoglobulin (hlgGl) isotype or DF IL-2-Fc at 0.225 mg / kg (FIG. 12A) or 0.675 mg / kg (FIG. 12B) (equimolar to 3 or 1 pg of aldesleukin, respectively) once weekly (QW) for 4 weeks. FIG. 12C is a graph depicting group mean body weight changes as mean ± standard error of the mean (SEM). FIG. 12D is a graph depicting Kaplan-Meier survival curves with median survival indicated in brackets (*** = p < 0.001; log-rank test).

[0052] FIGs. 13A-13U are graphs depicting cytokine and chemokine response in serum after Balb / c mice (n = 10 / group) were engrafted with CT26 (colon carcinoma) cells and treated intraperitoneally (IP) with a single dose of DF IL-2-Fc (0.675 mg / kg or 0.225 mg / kg; equimolar to 3 pg or 1 pg aldesleukin), or human IgGl isotype control. As a positive reference control, serum collected from a mouse treated with anti-CD3 antibody for 4 hours was used. Values that were below the minimum detection limit or above the maximum detection limit (dotted lines) were set to the respective limit.

[0053] FIGs. 14A-14V are graphs depicting cytokine and chemokine response in tumor lysates after Balb / c mice (n = 10 / group) were engrafted with CT26 (colon carcinoma) cells and treated intraperitoneally (IP) with a single dose of DF IL-2-Fc (0.675 mg / kg or 0.225 mg / kg; equimolar to 3 pg or 1 pg aldesleukin), or human IgGl isotype control. As a positive reference control, serum collected from a mouse treated with anti-CD3 antibody for 4 hours was used. Values that were below the minimum detection limit or above the maximum detection limit (dotted lines) were set to the respective limit.

[0054] FIGs. 15A-15Q illustrate lymphocyte cell composition in the tumor microenvironment and periphery in Balb / c mice engrafted with CT26 cells and treated intraperitoneally (IP) with a single dose of DF IL-2-Fc (0.675 mg / kg) or human IgGl isotype control. FIG. 15A show plots showing frequencies (% of CD45) (left panel) and absolute cell count quantification (right panel) of indicated immune cells in the tumor microenvironment (TME) after 96 hours post-treatment. Data are shown as mean ± standard error of the mean (SEM). FIG. 15B show plots showing frequencies (% of CD45) (left panel) and absolute cell count quantification (right panel) of indicated immune cells in the periphery after 96 hours post-treatment. Data are shown as mean ± standard error of the mean (SEM). FIGs. 15C-15H depict immune cell populations in tumor. FIGs. 15I-15N depict immune cell populations in periphery. FIGs. 15O-15P show effector cell ratio (CD8 / Treg ratio in FIG. 150 and NK cells / Treg ratio in FIG. 15P) in TME. FIG. 15Q depict CD25+CD8+T cells at 48 hours post-treatment. Significance for DF IL-2-Fc relative to isotype are noted as * (p< 0.05), ** (p < 0.01), ** (p < 0.001), and **** (p < 0.0001) by unpaired t-test.

[0055] FIGs. 16A-16F depict Ki-67 expression in intratumoral immune cells from Balb / c mice engrafted with CT26 cells and treated intraperitoneally (IP) with a single dose of DF IL- 2-Fc (0.675 mg / kg) or human IgGl isotype control. Graphs show Ki-67 expression after 48 hours post-treatment. Significance for DF IL-2-Fc relative to isotype is noted as * (p < 0.05), *** (p < 0.001), and **** (p < 0.0001) by unpaired t test.

[0056] FIGs. 17A-17E depict Ki-67 expression in peripheral immune cells from Balb / c mice engrafted with CT26 cells and treated intraperitoneally (IP) with a single dose of DF IL- 2-Fc (0.675 mg / kg) or human IgGl isotype control. Graphs show Ki-67 expression after 48 hours post-treatment. Significance for DF IL-2-Fc relative to isotype is noted as * (p < 0.05), *** (p < 0.001), and **** (p < 0.0001) by unpaired t test.

[0057] FIGs. 18A-18B depict characterization of tumor-infiltrating CD8+T cells and NK cells following intraperitoneal (IP) administration of a single dose of DF IL-2-Fc (0.675 mg / kg) or human IgGl isotype control to CT26 engrafted Balb / c mice. The graphs show frequency of granzyme B expressing, tumor infiltrating CD8+ T cells (FIG. 18A) and NK cells (FIG. 18B) after 48 hours post-treatment.

[0058] FIGs. 19A-19B depict characterization of tumor-infiltrating CD8+T cells and NK cells following intraperitoneal (IP) administration of a single dose of DF IL-2-Fc (0.675 mg / kg) or human IgGl isotype control to CT26 engrafted Balb / c mice. The graphs show frequency of granzyme B expressing, tumor infiltrating CD8+ T cells (FIG. 19A) and NK cells (FIG. 19B) after 96 hours post-treatment.

[0059] FIGs. 20A-20E are graphs showing tumor growth curves of C57BL / 6 mice engrafted subcutaneously (SC) with Bl 6F 10 melanoma cells and dosed intraperitoneally (IP) after mean tumor volume reaches 50 mm3with isotype controls (1.35 mg / kg hlgGl once weekly [QW], or 10 mg / kg rat IgG2a biweekly), DF IL-2-Fc (1.35 mg / kg or 0.675 mg / kg QW; equimolar to 6 or 3 pg aldesleukin, respectively), anti-PD-1 monoclonal antibody (mAb) (10 mg / kg biweekly), or a combination of DF IL-2-Fc and anti-PD-1 mAb at the same dose regimen as monotherapy. FIG. 20F is a graph depicting group mean tumor volumes. Significance for tumor volumes with DF IL-2-Fc as monotherapy or in combination with anti-PD-1 mAb relative to isotype control is noted as ** (p < 0.01) and *** (p < 0.001) on Day 19 by 2-way analysis of variance (ANOVA). Treatment groups were also compared for each day within graphed time points: p < 0.01 for DF IL-2-Fc (0.675 mg / kg) vs. isotype on Day 16; p < 0.01 for DF IL-2-Fc (1.35 mg / kg) vs. isotype and Combination (0.675 mg / kg) vs. isotype on Days 13, 16, and 19; p < 0.001 for Combination (1.35 mg. kg) vs. isotype on Days 13, 16, 19; and p < 0.05 on Day 9. FIG. 20G is a graph depicting group mean body weight changes (% from baseline) as mean ± standard error of the mean (SEM). FIG. 20H is a graph depicting Kaplan-Meier curves with median survival indicated in brackets (log-rank test). Significance for survival curves: * p < 0.05 for DF IL-2-Fc (0.675 mg / kg and 1.35 mg / kg) vs. isotype, **** p < 0.0001 for Combination (0.675 mg / kg and 1.35 mg / kg) vs. isotype, and ** p < 0.01 for DF IL-2-Fc (1.35 mg / kg) Combination vs. DF IL-2-Fc (1.35 mg / kg) monotherapy, Combo (1.35 mg / kg) and isotype vs. anti-PD-1 mAb.

[0060] FIGs. 21A-21C are graphs showing tumor growth curves of C57BL / 6 mice engrafted subcutaneously (SC) with Bl 6F 10 melanoma cells and dosed intraperitoneally (IP) after mean tumor volume reaches 100 mm3with isotype controls (1.35 mg / kg hlgGl onceweekly [QW], or 10 mg / kg rat IgG2a biweekly), DF IL-2-Fc (1.35 mg / kg or 0.675 mg / kg QW; equimolar to 6 or 3 pg aldesleukin, respectively), anti-PD-1 monoclonal antibody (mAb) (10 mg / kg biweekly), or a combination of DF IL-2-Fc and anti-PD-1 mAb at the same dose regimen as monotherapy. FIG. 21D is a graph depicting group mean tumor volumes. Significance for tumor volumes with DF IL-2-Fc as monotherapy or in combination with anti-PD-1 mAb relative to isotype control is noted as ** (p < 0.01) and *** (p < 0.001) on Day 19 by 2-way analysis of variance (ANOVA). Treatment groups were also compared for each day within graphed time points: p < 0.01 for DF IL-2-Fc (0.675 mg / kg) vs. isotype on Day 16; p < 0.01 for DF IL-2-Fc (1.35 mg / kg) vs. isotype and Combination (0.675 mg / kg) vs. isotype on Days 13, 16, and 19; p < 0.001 for Combination (1.35 mg. kg) vs. isotype on Days 13, 16, 19; and p < 0.05 on Day 9. FIG. 21E is a graph depicting group mean body weight changes (% from baseline) as mean ± standard error of the mean (SEM). FIG. 21F is a graph depicting Kaplan-Meier curves with median survival indicated in brackets (log-rank test). Significance for survival curves: * p < 0.05 for DF IL-2-Fc (0.675 mg / kg and 1.35 mg / kg) vs. isotype, **** p < 0.0001 for Combination (0.675 mg / kg and 1.35 mg / kg) vs. isotype, and ** p < 0.01 for DF IL-2-Fc (1.35 mg / kg) Combination vs. DF IL-2-Fc (1.35 mg / kg) monotherapy, Combo (1.35 mg / kg) and isotype vs. anti-PD-1 mAb.

[0061] FIGs. 22A-22C are graphs showing tumor growth curves of C57BL / 6 mice engrafted subcutaneously (SC) with Bl 6F 10 melanoma cells and dosed intraperitoneally (IP) after mean tumor volumes reaches 100 mm3with isotype controls (1.35 mg / kg hlgGl QW, or 5 mg / kg rat IgG2a biweekly), DF IL-2-Fc (1.35 mg / kg QW), Tyrpl-TriNKET (5 mg / kg biweekly), or a combination of DF IL-2-Fc and Tyrpl-TriNKET at the same dose regimen as monotherapy. FIG. 22D is a graph depicting group mean tumor volumes. Significance for tumor volumes with DF IL-2-Fc as monotherapy or in combination relative to isotype control is noted as ** (p < 0.01) on Day 16 by 2-way analysis of variance (ANOVA). FIG. 22E is a graph depicting group mean body weight changes (% from baseline) as mean ± standard error of the mean (SEM). FIG. 22F is a graph depicting Kaplan-Meier curves with median survival indicated in brackets (log-rank test). Significance for survival curves: ** p < 0.01 for DF IL-2-Fc vs. isotype and Combo vs. DF IL-2-Fc monotherapy, *** p < 0.001 for Combo vs. isotype.

[0062] FIGs. 23A-23K are graphs depicting release of key cytokines induced by DF IL- 2-Fc in isolated human PBMCs compared to clinical grade monoclonal antibodies and positive controls (anti-CD3 at 10 pg / mL, 10 pg / mL LPS, and 25 pg / mL PHA). Data arepresented as means from each donor, with overall mean values across donors and standard deviation (SD) marked with a dash and error bars, respectively. Significance for cytokine response with 300 nM DF IL-2-Fc to 300 nM aldesleukin, and 300 nM DF IL-2-Fc or 300 nM aldesleukin to isotype control is noted as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by 1-way analysis of variance (ANOVA) multiple comparison (multiple comparisons test). Values that were below the minimum detection limit or above the maximum detection limit (horizontal dotted lines) were set to the respective limit.

[0063] FIGs. 24A-24C are graphs showing concentrations of IL-5, IL-IRA, and MCP-1 in cynomolgus monkeys treated with a single dose of DF IL-2-Fc (100 pg / kg or 300 pg / kg). 1501, 1502, 2501, and 2502 denote individual cynomolgus monkey subjects.

[0064] FIG. 25A is a graph depicting expansion of lymphocytes in cynomolgus monkeys after multiple doses of DF IL-2-Fc. FIG. 25B is a graph depicting expansion of eosinophils in cynomolgus monkeys after multiple doses of DF IL-2-Fc.

[0065] FIGs. 26A-26E are graphs depicting Ki-67 expression in immune cells in cynomolgus monkeys after multiple doses of DF IL-2-Fc.

[0066] FIGs. 27A-27E are graphs depicting expansion of immune cell subsets in cynomolgus monkeys after multiple doses of DF IL-2-Fc.

[0067] FIG. 28 is a schematic diagram of the study protocol described in Example 13DETAILED DESCRIPTION

[0068] The disclosure is based, in part, on the discovery that it is possible to produce a recombinant variant IL-2 protein that maintains anti-tumor activity but does not stimulate the release of as many cytokines associated with cytotoxicity as wild type IL-2. Accordingly, the disclosure relates, in part, to a heterodimeric Fc-fused IL-2 protein comprising a variant IL-2 protein optionally fused, e.g., via a linker, to an Fc domain, wherein the Fc domain comprises a first and second Fc domain polypeptide. The first and second Fc domain polypeptides can include different heterodimerization mutations. The disclosure generally relates to novel IL-2 variant proteins, Fc-fused IL-2 variant proteins, pharmaceutical compositions comprising such proteins, and methods of using same, for example, for treating cancer.I. Definitions

[0069] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[0070] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate.

[0071] As used herein, all the amino acid positions in an Fc domain or hinge region are numbered according to EU numbering.

[0072] As used herein, “IL-2” means wild-type IL-2, whether native or recombinant, from any vertebrate source, including mammals (e.g., human) and rodents (e.g., mice), unless the text infers or indicates otherwise. The term also encompasses naturally occurring variants of IL-2, e.g., splice variants or allelic variants.

[0073] Mature human IL-2 occurs as a 133 amino acid sequence (less the signal peptide, consisting of an additional 20 N-terminal amino acids), as described in Fujita, et al., PNAS USA, 80, 7437-7441 (1983). The amino acid sequence of human IL-2, including signal peptide depicted in underlining, (SEQ ID NO: 11) is found in Genbank under accession locator NP_000577.2.MYRMOLLSCIALSLALVTNSAPTSSSTKKTOLOLEHLLLDLQMILNGINNYKNPKLT RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLE LKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 11)

[0074] The amino acid sequence of mature human IL-2 is depicted in SEQ ID NO: 1, namely,APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQ CLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVE FLNRWITFCQSIISTLT (SEQ ID NO: 1)

[0075] As used herein, “IL-2 variant,” “IL-2 mutein,” “variant IL-2,” “mutein IL-2,” or “mutant IL-2” means any mutant IL-2 protein, including full-length IL-2 and truncated IL-2, that comprise at least one amino acid mutation as compared to the wild-type sequence of SEQ ID NO: 1. The mutation may be a substitution, deletion (such as a truncation), insertion, or modification of the wild-type amino acid residue normally located at that position.Exemplary variants can include substitutions, deletions, insertions, or modifications of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. In certain embodiments, such substitutions, deletions, insertions, or modifications result in an IL-2 variant that retains at least a portion of the biological activity of wild-type IL-2 (e.g., at least 50%, 60%, 70%, 80%, 90%, 95% of the activity (e.g., anti-tumor activity) of wild-type IL-2). In certain embodiments, an IL-2 variant comprises at least one amino acid mutation affecting (e.g., decreasing) the interaction of IL-2 with the a subunit of the IL-2 receptor (CD25). In certain embodiments, an IL-2variant comprises at least one amino acid mutation affecting (e.g., increasing) the interaction of IL-2 with the P and / or y subunit of the IL-2 receptor.

[0076] IL-2 variants can also include conservative substitutions. Such conservative substitutions include those described by Dayhoff in The Atlas of Protein Sequence and Structure 5 (1978), and by Argos in EMBO J., 8:779-785 (1989). For example, amino acids belonging to one of the following groups represent conservative changes: Group I: alanine, proline, glycine, glutamine, asparagine, serine, threonine; Group II: cysteine, serine, tyrosine, threonine; Group III: valine, isoleucine, leucine, methionine, alanine, phenylalanine; Group IV: lysine, arginine, histidine; Group V: phenylalanine, tyrosine, tryptophan, histidine; and Group VI: aspartic acid, glutamic acid. Exemplary IL-2 variants can include conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0077] The term “polypeptide,” “protein” or “peptide” refer to any chain of amino acid residues, regardless of its length or post-translational modification (e.g., glycosylation or phosphorylation).

[0078] As used herein, the phrase “percent identity” and “% identity” refers to the extent to which two sequences e.g., two polypeptides or two nucleic acids have the same respective amino acid or nucleotide at the same positions in an alignment. As used herein, “percent identity” between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Similarly, percent “identity” between a nucleic acid sequence and a reference sequence is defined as the percentage of nucleotides in the nucleic acid sequence that are identical to the nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity (e.g., nucleic acid sequence identity or amino acid sequence identity) can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST (Basic Local Alignment Search Tool), BLAST- 2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. For a discussion of basic issues in searching sequence databases see Altschul et al., (1994) NATURE GENETICS 6: 119-129, which is fully incorporated by reference herein. Those skilled in the art can determine appropriate parameters for aligning sequences, including anyalgorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0079] As used herein, unless otherwise indicated, the term “immunoglobulin Fc domain” or “IgG Fc domain” or “Fc domain” or “Fc” refers to a fragment of an immunoglobulin heavy chain constant region which, either alone or in combination with a second immunoglobulin Fc domain, or unconjugated (or not fused), or conjugated (or fused) to an IL-2, or a functional fragment or variant thereof, is capable of binding to an Fc receptor. An immunoglobulin Fc domain may include, e.g., immunoglobulin CH2 and CH3 domains. An immunoglobulin Fc domain may include, e.g., immunoglobulin CH2 and CH3 domains and an immunoglobulin hinge region. For example, as used herein, an “Fc” or “Fc domain” can refer to a polypeptide comprising a CH2 domain, a CH3 domain, and optionally a hinge or a portion thereof. This polypeptide can bind (e.g., dimerize) to another polypeptide comprising a CH2 domain, a CH3 domain, and optionally a hinge or a portion thereof, wherein the dimer is capable of binding to an Fc receptor. Boundaries between immunoglobulin hinge regions, CH2, and CH3 domains are well known in the art, and can be found, e.g., in the PROSITE database (available on the world wide web at prosite.expasy.org).

[0080] As used herein, the terms “fused” and “linked” are used interchangeably.

[0081] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably include humans.

[0082] As used herein, the term “effective amount” refers to the amount of an active agent (e.g., a variant human IL-2 protein and / or a heterodimeric Fc-fusion protein comprising a variant human IL-2 protein and an IgG Fc domain) sufficient to effect beneficial or desired results (e.g., a desired prophylactic or therapeutic effect). An effective amount can be administered in one or more administration(s), application(s) or dosage(s) and is not intended to be limited to a particular formulation or administration route.

[0083] As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0084] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water,emulsions e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] ,

[0085] As used herein, the term “treating” includes any effect, e.g., lessening, reducing, modulating, ameliorating or eliminating, that results in the improvement of the condition, disease, disorder, and the like, or ameliorating a symptom thereof.

[0086] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or modalities. The term administered “in combination,” as used herein, is understood to mean that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0087] In certain embodiments, a method or composition described herein, is administered in combination with one or more additional therapies, e.g., surgery, radiation therapy, or administration of another therapeutic preparation. In certain embodiments, the additional therapy may include chemotherapy, e.g., a cytotoxic agent. In certain embodiments the additional therapy may include a targeted therapy, e.g., a tyrosine kinase inhibitor, a proteasome inhibitor, or a protease inhibitor. In certain embodiments, the additional therapy may include an anti-inflammatory, anti-angiogenic, anti-fibrotic, or anti-proliferative compound, e.g., a steroid, a biologic immunomodulator, a monoclonal antibody, an antibody fragment, an aptamer, an siRNA, an antisense molecule, a fusion protein, a cytokine, a cytokine receptor, a bronchodilator, a statin, an anti-inflammatory agent e.g., methotrexate), or an NSAID. In certain embodiments, the additional therapy may include a combination of therapeutics of different classes.

[0088] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0089] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0090] Further, it should be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present invention and / or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.

[0091] It should be understood that the expression “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression“and / or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0092] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0093] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.

[0094] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present invention remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0095] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.

[0096] As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.II. Proteins

[0097] The present disclosure provides variant IL-2 proteins and heterodimeric Fc-fusion proteins comprising a variant IL-2 protein and an IgG Fc domain for therapeutic use.A. Variant IL-2 Protein

[0098] In one aspect, the present disclosure provides one or more variant IL-2 proteins that can exhibit one or more biological activities of wild-type IL-2 and contribute to antitumor response. A variant IL-2 protein can comprise at least one mutation (e.g., a substitution, deletion, insertion, or modification of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) relative to a wild-type IL-2. In certainembodiments, such a mutation results in an IL-2 variant that retains at least a portion of the biological activity of wild-type IL-2. Mutations in a variant IL-2 protein can be at one or more amino acid residues that contact the a, P and / or y subunits of the IL-2 receptor. In certain embodiments, an IL-2 variant comprises at least one amino acid mutation affecting (e.g., decreasing) the interaction of IL-2 with the a subunit of the IL-2 receptor (CD25). In certain embodiments, the IL-2 variant has reduced but not abrogated binding to the a subunit of the IL-2 receptor (CD25). In certain embodiments, an IL-2 variant comprises at least one amino acid mutation affecting (e.g., increasing) the interaction of IL-2 with the P and / or y subunit of the IL-2 receptor.

[0099] Exemplary variant IL-2 proteins of the present disclosure can be at least about 50%, at least about 65%, at least about 70%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identical to wild-type IL-2. The mutation can consist of a change in the number or content of amino acid residues. For example, the variant IL-2 can have a greater or a lesser number of amino acid residues than wild-type IL-2. Alternatively, or in addition, an exemplary variant IL-2 can contain a substitution or deletion of one or more amino acid residues that are present in the wild-type IL-2.

[0100] In some embodiments, the present disclosure provides a variant IL-2 protein comprising one or more substitutions relative to a wild-type IL-2. The substituted amino acid residue(s) can be, but are not necessarily, conservative substitutions, which typically include substitutions within the following groups: Group I: alanine, proline, glycine, glutamine, asparagine, serine, threonine; Group IL cysteine, serine, tyrosine, threonine; Group III: valine, isoleucine, leucine, methionine, alanine, phenylalanine; Group IV: lysine, arginine, histidine; Group V: phenylalanine, tyrosine, tryptophan, histidine; and Group VI: aspartic acid, glutamic acid.

[0101] In certain embodiments, the present disclosure provides a variant IL-2 protein comprising one or more amino acid substitutions and / or deletions in amino acid positions selected from the group consisting of: 3, 27, 28, 32, 39, 52, 72, 74, 75, 76, 77, 78, 79, 80, 81, 82, 85, 86, 92, and 125, relative to the amino acid sequence of wild-type human IL-2 protein represented by SEQ ID NO: 1.

[0102] In certain embodiments, the present disclosure provides a variant IL-2 protein comprising a substitution of the threonine at position 3, a substitution of the cysteine atposition 125, and one or more amino acid substitution(s) or deletion(s) selected from a substitution of the glycine at position 27 to a methionine, a substitution of the isoleucine at position 28, a substitution of the lysine at position 32, a substitution of the methionine at position 39, a substitution of the glutamic acid at position 52, a substitution of the glutamine at position 74, a substitution of the serine at position 75, a substitution of the lysine at position 76, a substitution of the asparagine at position 77, a substitution of the phenylalanine at position 78, a substitution of the histidine at position 79, a substitution of the leucine at position 80, a substitution or deletion of the arginine at position 81, a deletion of the proline at position 82, a substitution of the leucine at position 85, a substitution of the isoleucine at position 86, and a substitution of the isoleucine at position 92, wherein the substitutions are relative to the amino acid sequence of wild-type human IL-2 protein represented by SEQ ID NO: 1.

[0103] In certain embodiments, the present disclosure provides a variant IL-2 protein comprising a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and one or more amino acid substitution(s) selected from a substitution of the glycine at position 27 to a methionine, a substitution of the isoleucine at position 28, a substitution of the lysine at position 32, a substitution of the arginine at position 81, wherein the substitutions are relative to the amino acid sequence of wild-type human IL-2 protein represented by SEQ ID NO: 1.

[0104] In certain embodiments, the present disclosure provides a variant IL-2 protein comprising a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and one or more amino acid substitution(s) or deletion(s) selected from a substitution of the glycine at position 27 to a methionine, a substitution of the isoleucine at position 28, a substitution of the lysine at position 32, a substitution of the methionine at position 39, a substitution of the glutamic acid at position 52, a substitution of the glutamine at position 74, a substitution of the serine at position 75, a substitution of the lysine at position 76, a substitution of the asparagine at position 77, a substitution of the phenylalanine at position 78, a substitution of the histidine at position 79, a substitution of the leucine at position 80, a deletion of the arginine at position 81, a deletion of the proline at position 82, a substitution of the leucine at position 85, a substitution of the isoleucine at position 86, and a substitution of the isoleucine at position 92, wherein the substitutions and deletions are relative to the amino acid sequence of wild-type human IL-2 protein represented by SEQ ID NO: 1.

[0105] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a substitution of the glycine at position 27.

[0106] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, a substitution of the isoleucine at position 28.

[0107] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a substitution of the lysine at position 32.

[0108] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a substitution of the leucine at position 72.

[0109] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a substitution of the arginine at position 81.

[0110] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a substitution of the methionine at position 39.[OHl] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a substitution of the glutamic acid at position 52.

[0112] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a substitution of the glutamic acid at position 74.

[0113] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a substitution of the serine at position 75.

[0114] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a substitution of the lysine at position 76.

[0115] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a substitution of the asparagine at position 77.

[0116] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a substitution of the phenylalanine at position 78.

[0117] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a substitution of the histidine at position 79.

[0118] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a substitution of the leucine at position 80.

[0119] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a deletion of the arginine at position 81.

[0120] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a deletion of the proline at position 82.

[0121] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a substitution of the leucine at position 85.

[0122] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a substitution of the isoleucine at position 86.

[0123] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, and a substitution of the isoleucine at position 92.

[0124] In certain embodiments, the variant IL-2 protein comprises a substitution of the threonine at position 3, a substitution of the cysteine at position 125, a substitution of the glycine at position 27, a substitution of the isoleucine at position 28, a substitution of thelysine at position 32, a substitution of the leucine at position 72, and a substitution of the arginine at position 81.

[0125] In certain embodiments, the substitution of the threonine at position 3 is to an alanine, the substitution of the cysteine at position 125 is to a serine, the substitution of the glycine at position 27 is to a methionine, the substitution of the isoleucine at position 28 is to a leucine, the substitution of the lysine at position 32 is to an aspartic acid, the substitution of the leucine at position 72 is to a glutamine, and the substitution of the arginine at position 81 is to an aspartic acid.

[0126] In certain embodiments, the present disclosure provides a variant IL-2 protein comprising a substitution of the threonine at position 3, a substitution of the cysteine at position 125, a substitution of the glycine at position 27, a substitution of the isoleucine at position 28, a substitution of the methionine at position 39, a substitution of the glutamic acid at position 52, a substitution of the leucine at position 72, a substitution of the glutamine at position 74, a substitution of the serine at position 75, a substitution of the lysine at position 76, a substitution of the asparagine at position 77, a substitution of the phenylalanine at position 78, a substitution of the histidine at position 79, a substitution of the leucine at position 80, a deletion of the arginine at position 81, a deletion of the proline at position 82, a substitution of the leucine at position 85, a substitution of the isoleucine at position 86, and a substitution of the isoleucine at position 92.

[0127] In certain embodiments, the substitution of the threonine at position 3 is to an alanine, the substitution of the cysteine at position 125 is to a serine, the substitution of the glycine at position 27 is to a methionine, the substitution of the isoleucine at position 28 is to a leucine, the substitution of the lysine at position 32 is to an aspartic acid, the substitution of the methionine at position 39 is to a leucine, the substitution of the glutamic acid at position 52 is to a serine, the substitution of the leucine at position 72 is to an alanine, the substitution of the glutamine at position 74 is to a glycine, the substitution of the serine at position 75 is to an aspartic acid, the substitution of the lysine at position 76 is to an aspartic acid, the substitution of the asparagine at position 77 is to a proline, the substitution of the phenylalanine at position 78 is to a lysine, the substitution of the histidine at position 79 is to a threonine, the substitution of the leucine at position 80 is to an isoleucine, the substitution of the leucine at position 85 is to a valine, the substitution of the isoleucine at position 86 is to a valine, and the substitution of the isoleucine at position 92 is to a phenylalanine.

[0128] In certain embodiments, the present disclosure provides a variant IL-2 protein comprising an amino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 2. In certain embodiments, the present disclosure provides a variant IL-2 protein comprising an amino acid sequence of SEQ ID NO: 2.

[0129] In certain embodiments, the present disclosure provides a variant IL-2 protein comprising an amino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 3. In certain embodiments, the present disclosure provides a variant IL-2 protein comprising an amino acid sequence of SEQ ID NO: 3.

[0130] TABLE 1 discloses sequences of exemplary IL-2 proteins.TABLE 1: IL-2 Protein SequencesB. Heterodimeric Fc-fusion proteins

[0131] In another aspect, the present disclosure provides a heterodimeric Fc-fusion protein comprising a variant IL-2 protein and an IgG Fc domain. In certain embodiments, the IgG Fc domain comprises a first Fc domain polypeptide. In certain embodiments, the IgG Fcdomain comprises a first Fc domain polypeptide and a second Fc domain polypeptide. In certain embodiments, the first Fc domain polypeptide and the second Fc domain polypeptide are the same. In certain embodiments, the first Fc domain polypeptide and the second Fc domain polypeptide are different.

[0132] In certain embodiments, the IgG Fc domain is derived from an IgGl, IgG2, IgG3, or IgG4 Fc domain. In certain embodiments, the IgG Fc domain is derived from a human IgGl Fc domain.Fc domain mutations for promoting heterodimerization

[0133] The assembly of proteins of the present disclosure can be accomplished by expressing a first polypeptide comprising a variant IL-2 protein linked to a first Fc domain polypeptide sequence, and a second polypeptide comprising a second, different Fc domain polypeptide sequence in the same cell, which leads to the assembly of a heterodimeric Fc- fusion protein according to the disclosure. The assembled proteins have heterodimeric Fc sequences with the first Fc domain polypeptide sequence and the second, different Fc domain polypeptide sequence bound to each other. Promoting the preferential assembly of heterodimers of the Fc can be accomplished by incorporating different mutations in the CH3 domain of each antibody heavy chain constant region as shown in U.S. 8,679,785, U.S.7,695,936, U.S. 9,493,578, U.S. 9,562,109, U.S. 10,047,167, U.S. 8,592,562, U.S. 9,248,182, U.S. 9,951,145, and U.S. 2016 / 0046727. For example, mutations can be made in the CH3 domain based on human IgGl or IgG4 and incorporating distinct pairs of amino acid substitutions within a first Fc domain polypeptide sequence and a second, different Fc domain polypeptide sequence that allow these two chains to selectively heterodimerize with each other. The positions of amino acid substitutions illustrated below are numbered according to the EU index as in Kabat.

[0134] One or more mutation(s) can be incorporated into the constant region as compared to human IgGl constant region, for example at Q347, Y349, L351, S354, D356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and / or K439. Exemplary substitutions include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, D356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, T394W, D399R, D399K,D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I , Y407V, K409F, K409W, K409D, T41 ID, T41 IE, K439D, and K439E.

[0135] Amino acid substitutions can be selected from the following sets of substitutions shown in TABLE 2. In certain embodiments, the first and second Fc domain polypeptides comprise the mutations of Fc domain Polypeptide A and Fc domain Polypeptide B, respectively, in a line of TABLE 2. In certain embodiments, the first and second polypeptide comprise the mutations of Polypeptide B and Polypeptide A, respectively, in a line of TABLE 2TABLE 2: Heterodimerization Fc mutations

[0136] In some embodiments, the first Fc domain polypeptide sequence comprises one or more mutation(s) selected from K360E and K409W, and the second, different Fc domain polypeptide sequence comprises one or more mutation(s) selected from Q347R, D399V, and F405T. In some embodiments, the first Fc domain polypeptide sequence comprises one or more mutation(s) selected from Q347R, D399V, and F405T, and the second, different Fc domain polypeptide sequence comprises one or more mutation(s) selected from K360E and K409W. In some embodiments, the first Fc domain polypeptide sequence comprises mutations K360E and K409W, and the second, different Fc domain polypeptide sequence comprises mutations Q347R, D399V, and F405T. In some embodiments, the first Fc domain polypeptide sequence comprises mutations Q347R, D399V, and F405T, and the second, different Fc domain polypeptide sequence comprises mutations K360E and K409W.

[0137] In certain embodiments, the Fc domain first polypeptide and the second Fc domain polypeptide are bound to each other.

[0138] In certain embodiments, the first Fc domain polypeptide and the second Fc domain polypeptide each comprise one or more mutations that promote disulfide bond formation between the first Fc domain polypeptide and the second Fc domain polypeptide.

[0139] In certain embodiments, the first and second Fc domain polypeptides are IgGl (e.g., human IgGl) antibody sequences. In certain embodiments, a disulfide bridge is introduced between the Fc monomers to improves the stability of the heterodimer. In an exemplary embodiment, the first Fc domain polypeptide sequence comprises a Y349C substitution in the CH3 domain, and the second Fc domain polypeptide sequence comprises a S354C substitution, wherein the two cysteine residues form a disulfide bond. Alternatively, in certain embodiments, the first Fc domain polypeptide sequence comprises a S354C substitution in the CH3 domain, and the second Fc domain polypeptide sequence comprises a Y349C substitution, wherein the two cysteine residues form a disulfide bond.

[0140] In certain embodiments, the first Fc domain polypeptide and / or second Fc domain polypeptide comprises a C220S substitution.Fc domain mutations for reducing effector functions

[0141] Effector functions of Fc, hinge, and hinge portion regions can be mediated by the interaction of the Fc region with one or more Fc receptor(s) (e.g., FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, and / or FcyRIIIB) and / or one or more complement component(s) (e.g., Clq, Clr, and / or Cis).

[0142] The binding of a hinge-Fc or hinge portion-Fc region to CD16, also called FcyRIIIA, is mediated, at least in part, by the hinge or hinge portion and the CH2 domain. For example, within human IgGl, the interaction with CD 16 involves amino acid residues Asp 265 - Glu 269, Asn 297 - Thr 299, Ala 327 - He 332, Leu 234 - Ser 239, and carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see, Sondermann et al., (2000) NATURE, 406 (6793):267-273). The binding of a hinge-Fc or hinge portion-Fc region to Clq is mediated, at least in part, by the CH2 domain. For example, within human IgGl, the interaction with Clq involves amino acid residues Glu 318, Lys 320, Lys 322, and Pro 331 (see Ward and Ghetie (1995) THERAPEUTIC IMMUNOLOGY 2:77-94). Based on the known domains and sites, mutations can be selected to enhance or reduce the binding affinity to CD 16 and / or Clq, such as by using phage-displayed libraries or yeast surface-displayed cDNA libraries, or can be designed based on the known three-dimensional structure of the interaction. Exemplary Fc mutations are disclosed in PCT Application Publication No.WO 1999 / 051642.

[0143] In some embodiments, the CH2 and hinge or hinge portion regions of an IgGl Fc sequence comprise a mutation to reduce effector functions. For example, in certainembodiments, a Fc-fusion protein of the present disclosure comprises an IgGl hinge-Fc or hinge portion-Fc with LALA (L234A and L235A) mutations, LALAPA (L234A, L235A, and P329A) mutations, LALAPG (L234A, L235A, and P329G) mutations, or LALEGAASPS (L234A, L235E, G237A, A330S, and P331S) mutations. In certain embodiments, the first and second polypeptides comprise the same set of mutations that reduce effector functions.

[0144] Further mutations that can be employed in the present disclosure include mutations that facilitate the formation of an inter-chain disulfide bridge. Such mutations are useful in improving the stability of the heterodimer. In an exemplary embodiment, the first Fc domain polypeptide sequence comprises a Y349C substitution in the CH3 domain, and the second Fc domain polypeptide sequence comprises a S354C substitution, wherein the two cysteine residues form a disulfide bond. Alternatively, in certain embodiments, the first Fc domain polypeptide sequence comprises a S354C substitution in the CH3 domain, and the second antibody Fc sequence comprises a Y349C substitution, wherein the two cysteine residues form a disulfide bond.

[0145] A skilled person in the art would appreciate that during production and / or storage of proteins, N-terminal glutamate (E) or glutamine (Q) can be cyclized to form a lactam (e.g., spontaneously or catalyzed by an enzyme present during production and / or storage). Accordingly, in some embodiments where the N-terminal residue of an amino acid sequence of a polypeptide is E or Q, a corresponding amino acid sequence with the E or Q replaced with pyroglutamate is also contemplated herein.

[0146] A skilled person in the art would also appreciate that during protein production and / or storage, the C-terminal lysine (K) of a protein can be removed (e.g., spontaneously or catalyzed by an enzyme present during production and / or storage). Such removal of K is often observed with proteins that comprise a Fc domain at its C-terminus. Accordingly, in some embodiments where the C-terminal residue of an amino acid sequence of a polypeptide (e.g., a Fc domain sequence) is K, a corresponding amino acid sequence with the K removed is also contemplated herein

[0147] Any of the exemplary IgGl antibody Fc sequences provided in TABLE 3 can be employed in the heterodimeric Fc-fused proteins. In certain embodiments, the first or second Fc domain polypeptide comprises an amino acid sequence of SEQ ID NO: 4. In certain embodiments, the first or second Fc domain polypeptide comprises an amino acid sequence of SEQ ID NO: 5.

[0148] Accordingly, the present disclosure provides heterodimeric Fc-fusion protein constructs that can confer a higher serum half-life compared to a native / natural single subunit protein, improve yield of the proteins during production, enhance stability during storage, and / or improve efficacy when used as a therapeutic. TABLE 3: Exemplary Fc Variants*The amino acid sequences can further comprise a lysine (K) at the C-terminus.Linker

[0149] In certain embodiments, a variant IL-2 protein sequence is fused to the IgG Fc domain via a linker polypeptide. In certain embodiments, the linker polypeptide has an optimized length and / or amino acid composition and / or can be flexible or rigid. In certain embodiments, the linker polypeptide includes 4-50 amino acid residues. In certain embodiments, the linker polypeptide consists of 4-50 amino acid residues. In certain embodiments, the linker polypeptide consists of 4-20 amino acid residues.

[0150] In certain embodiments, the linker polypeptide is absent, z.e., the protein sequence of the variant IL-2 protein is linked to the hinge of the Fc domain or directly to the Fc domain via a peptide bond. In certain embodiments, the linker polypeptide is “short,” e.g., consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues. Thus, in certain instances, the linker polypeptide consists of about 12 or fewer amino acid residues. In certain embodiments, the linker polypeptide is “long,” e.g., consists of 15, 20 or 25 amino acid residues. In some embodiments, the linker polypeptide consists of about 3 to about 15, for example, 8, 9 or 10 contiguous amino acid residues. Regarding the amino acid composition of the linker polypeptide, peptides are selected with properties that confer flexibility to the first polypeptide of the Fc-fusion proteins of the present disclosure, do not interfere with the binding of the first and the second Fc domain polypeptides to each other, and resist cleavage from proteases. For example, glycine and serine residues generally provide protease resistance. The linker polypeptide suitable for linking the variant IL-2 protein to the Fc domain in the first polypeptide may include, as at least a part of a full length linker polypeptide, a (GS)n (SEQ ID NO: 31), (GGS)n (SEQ ID NO: 32), (GGGS)n (SEQ ID NO: 33), (GGSG)n(SEQ ID NO: 34), (GGSGG)n (SEQ ID NO: 35), and (GGGGS)n (SEQ ID NO: 36) sequence, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Exemplary linker polypeptide sequences are provided in TABLE 4.TABLE 4: Linker Polypeptides

[0151] In certain embodiments, a variant IL-2 protein sequence is fused to the C-terminus of any one of the polypeptide sequences listed in TABLE 3, to form a heterodimeric Fc- fusion protein of the present disclosure (see, e.g., FIG. 1C and FIG. ID). In certain embodiments, a variant IL-2 protein sequence is fused to the N-terminus of any one of the polypeptide sequences listed in TABLE 3, to form a heterodimeric Fc-fusion protein of the present disclosure (see, e.g., FIG. 1A and FIG. IB). TABLE 5 lists amino acid sequences of exemplary protein constructs of the present disclosure. The amino acid positions in the IL-2 sequence are numbered according to the mature human IL-2 amino acid sequence provided in SEQ ID NO: 1. TABLE 5: Exemplary Heterodimeric Fc-Fusion Proteins. Linker sequence is in bold font; leader sequence (if present) is underlined; IL-2 mutations are underlined and italicized; and human IgG-Fc amino acid substitutions are bold, italicized, and underlined.*The amino acid sequences can further comprise a lysine (K) at the C-terminus.

[0152] The variant IL-2 protein sequence can be positioned anywhere in the first polypeptide. In certain embodiments, the variant IL-2 protein sequence is positioned N- terminal to the first Fc domain polypeptide sequence. In certain embodiments, the variant IL- 2 protein sequence is positioned C-terminal to the first Fc domain polypeptide sequence. Accordingly, in certain embodiments, the first polypeptide comprises from the N-terminus to the C-terminus: an IL-2 protein sequence disclosed herein (e.g., as disclosed in TABLE 1), a linker polypeptide disclosed herein (e.g., as disclosed in TABLE 4), and an Fc domain polypeptide sequence disclosed herein (e.g., as disclosed in Table 3 TABLE 3); and the second polypeptide comprises from the N-terminus to the C-terminus: an Fc domain polypeptide sequence disclosed herein (e.g., as disclosed in TABLE 3). In certain embodiments, the first polypeptide comprises from the N-terminus to the C-terminus: an Fc domain polypeptide sequence disclosed herein (e.g., as disclosed in TABLE 3), a linker polypeptide sequence disclosed herein (e.g., as disclosed in TABLE 4), and a variant IL-2 protein sequence disclosed herein (e.g., as disclosed in TABLE 1); and the second polypeptide comprises from the N-terminus to the C-terminus: an Fc domain sequence disclosed herein (e.g., as disclosed in TABLE 3).Functional Characteristics of Heterodimeric Fc-Fused Proteins

[0153] Aldesleukin, a clinical recombinant human IL-2 (rhIL-2) therapy, was the first approved cancer immunotherapy. At high doses, aldesleukin induced tumor regressions by expanding and activating anti-tumor lymphocyte populations, including cytotoxic CD8+T cells and NK cells, overcoming potential immune-suppression by Tregs. However, the therapeutic use of aldesleukin for cancer treatment has overall been limited by its short halflife (~13 or 85 minutes for distribution or elimination half-lives in patients, respectively) and its toxicity profile. Because of the short half-life of aldesleukin, high and frequent doses are required to maintain beneficial effector cell proliferation, as low-dose aldesleukin reverts the PD effect and preferentially expands immunosuppressive Tregs (Yang et al. (2003) J CLIN ONCOL. 21 ( 16): 3127-32). A course of aldesleukin therapy is administered using 2 intense treatment cycles, separated by a 9-day rest period (PROLEUKIN® (aldesleukin) for injection for intravenous use [package insert], San Diego, CA: Prometheus Laboratories Inc.; 2015). In the first 5-day cycle, aldesleukin up to 600,000 International Units (IU) (0.037 mg / kg) is administered via 15-minute intravenous (IV) bolus infusion every 8 hours, for a maximum of 14 doses. In the second cycle, this same dosing schedule is repeated for another 14 doses. This intensive dosing is associated with severe toxicities; in particular, aldesleukin treatmentis limited by severe capillary leak syndrome (CLS) (also known as vascular leakage syndrome [VLS]), which is characterized by loss of vascular tone and extravasation of plasma proteins into the extravascular space, resulting in hypotension and reduced organ perfusion. These effects can be severe, potentially resulting in death. Additional adverse effects that may be associated with CLS include cardiac arrhythmia (supraventricular and ventricular), angina, myocardial infarction (MI), respiratory insufficiency requiring intubation, gastrointestinal (GI) bleeding and / or infarction, renal insufficiency, edema, and changes in mental status. In addition to CLS and CLS-associated adverse effects, aldesleukin is known to cause a wide range of systemic adverse events in patients, including impaired neutrophil function, increased risk of infection (including sepsis and bacterial endocarditis), and effects related to the blood and lymphatic system, cardiac function, endocrine organs, GI system, hepatobiliary system, immune system, musculoskeletal and connective tissue, nervous system, and psychiatric and vascular effects.

[0154] In one aspect, the present disclosure provides variant IL-2 heterodimeric Fc-fused proteins with improved immunotherapeutic properties as compared to a wild-type IL-2 protein. In certain embodiments, the present disclosure provides heterodimeric Fc-fused proteins comprising a variant IL-2 protein comprising an amino acid substitution or deletion that reduces, but does not abrogate, binding affinity of the variant IL-2 protein to the a subunit of the IL-2 receptor compared to a wild-type IL-2 protein. In certain embodiments, the variant IL-2 protein comprises an amino acid substitution or deletion that enhances binding affinity of the variant IL-2 protein for the P and / or y subunits of the IL-2 receptor compared to a wild-type IL-2 protein. IL-2 variants comprising such mutations are useful for enhancing T cell immunity, particularly the activity of CD8+T cells, without substantially activating regulatory T cells.

[0155] In certain embodiments, the variant IL-2 proteins disclosed herein exhibit one or more biological activities of wild-type IL-2 and contribute to anti-tumor response but with decreased capacity for inducing the production of cytokines in a cytokine release assay.

[0156] In certain embodiments, the variant IL-2 proteins disclosed herein can activate, differentiate, and expand CD4+resting conventional T cells (Tconv) and CD8+T cells, which enhances tumor reactivity and mediates tumor clearance.

[0157] In certain embodiments, the variant IL-2 proteins disclosed herein can promote differentiation of CD8+T cells upon antigen recognition into memory and more terminally differentiated effector cells.

[0158] In certain embodiments, the variant IL-2 proteins disclosed herein can reverse exhaustion of CD8+T cells during a tumor response.

[0159] In certain embodiments, the variant IL-2 proteins disclosed herein can enhance anti -tumor activity in combination with anti-PD-1 immunotherapy, which is dependent on high-affinity IL-2R engagement by IL-2.

[0160] In certain embodiments, the variant IL-2 proteins disclosed herein can potentiate the cytotoxicity of CD8+T cells and NK cells against cancer cells by inducing the expression of granzymes and perforin.

[0161] In certain embodiments, the variant IL-2 proteins disclosed herein can induce the production of interferon gamma (IFN-y) in the tumor microenvironment and activate the expression of major histocompatibility complex molecules in the immediate environment, thereby enhancing antigen presentation and recognition of tumor cells.Serum Half-life

[0162] In certain embodiments, a heterodimeric Fc-fusion protein according to the disclosure has an extended half-life. In certain embodiments, a heterodimeric Fc-fusion protein according to the disclosure has a serum half-life greater than the serum half-life of a recombinant IL-2 protein not fused to an Fc sequence (e.g., aldesleukin).

[0163] In certain embodiments, a heterodimeric Fc-fusion protein comprising a variant IL-2 protein according to the present disclosure has a serum half-life that is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% longer than an IL-2 protein (e.g., wild-type or variant) not fused to an Fc sequence. In certain embodiments, a heterodimeric Fc-fusion protein comprising a variant IL-2 protein sequence according to the present disclosure has a serum half-life that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, or at least 20-fold longer than the an IL-2 protein (e.g., wildtype or variant) not fused to an Fc sequence.

[0164] In certain embodiments, a heterodimeric Fc-fusion protein according to the disclosure has a serum half-life of more than 10 minutes, more than 20 minutes, more than 30 minutes, more than 40 minutes, more than 50 minutes, more than one hour, or more than twohours. In certain embodiments, a heterodimeric Fc-fusion protein according to the disclosure has a serum half-life of between about 1 hour to 5 days. For example, between about 1 hour and about 3 hours, between about 3 hours and about 5 hours, between about 5 hours and about 7 hours, between about 7 hours and about 9 hours, between about 9 hours and about 11 hours, between about 11 hours and about 13 hours, between about 13 hours and about 15 hours, between about 15 hours and about 17 hours, between about 17 hours and about 19 hours, between about 19 hours and about 21 hours, between about 21 hours and about 23 hours, between about 1 day and about 2 days, between about 2 days and about 3 days, between about 3 days and about 4 days, between about 4 days and about 5 days.III. Methods of Preparation

[0165] The proteins of the present disclosure can be made using recombinant DNA technology well known to a skilled person in the art. For example, a first nucleic acid sequence encoding a first polypeptide comprising a variant IL-2 protein linked to a first Fc domain polypeptide sequence can be cloned into a first expression vector; a second nucleic acid sequence encoding a second, different Fc domain polypeptide sequence can be cloned into a second expression vector; and the first and the second expression vectors can be stably transfected together into host cells to produce the heterodimeric Fc-fusion proteins.

[0166] To achieve the highest yield of the protein, different ratios of the first and second expression vectors can be explored to determine the optimal ratio for transfection into the host cells. After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limited dilution, ELISA, FACS, microscopy, or Clonepix.

[0167] Clones can be cultured under conditions suitable for bio-reactor scale-up and maintained expression of the proteins of the present disclosure. The proteins can be isolated and purified using methods known in the art including centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.IV. Pharmaceutical Compositions

[0168] The present disclosure also features pharmaceutical compositions that contain an effective amount of a protein described herein. The composition can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipient(s) or carrier(s) can also be included in the composition for proper formulation. Suitableformulations for use in the present disclosure are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer (Science 249: 1527-1533, 1990).

[0169] A heterodimeric Fc-fusion protein of the present disclosure could exist in a liquid aqueous pharmaceutical formulation including an effective amount of the protein in a buffered solution forming a formulation.

[0170] These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as-is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents. The composition in solid form can also be packaged in a container for a flexible quantity.

[0171] In certain embodiments, the present disclosure provides a formulation with an extended shelf life including a heterodimeric Fc-fusion protein of the present disclosure, in combination with sodium citrate, citric acid, sucrose, polysorbate 80, and water.

[0172] In certain embodiments, the present disclosure provides a formulation comprising a heterodimeric Fc-fusion protein of the present disclosure in 20 mM citrate buffer, pH 6.2, with 6% (w / v) sucrose, 0.01% (w / v) polysorbate 80. In certain embodiments, the heterodimeric Fc-fusion protein is included in an amount of about 0.5 mg / mL to about 5 mg / mL (for example, about 1 mg / mL to about 3 mg / mL, about 2 mg / mL).

[0173] In certain embodiments, an aqueous formulation is prepared including the heterodimeric Fc-fusion protein of the present disclosure in a pH-buffered solution. The buffer of the disclosure may have a pH ranging from about 4 to about 8, e.g., from about 4.5 to about 6.0, or from about 4.8 to about 5.5, may have a pH of about 5.0 to about 5.2, or from about 5.3 to about 6.2. Ranges intermediate to the above recited pH's are also intended to be part of the disclosure. For example, ranges of values using a combination of any of the above recited values as upper and / or lower limits are intended to be included. Examples of buffers that will control the pH within this range include acetate (e.g., sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate and other organic acid buffers.

[0174] In certain embodiments, the formulation includes a buffer system which comprises citrate and phosphate to maintain the pH in a range of about 4 to about 8. In certain embodiments the pH range may be from about 4.5 to about 6.0, or from about pH 4.8 to about 5.5, or in a pH range of about 5.0 to about 5.2, or from about pH 5.2 to about 6.2. In certain embodiments, the buffer system includes sodium citrate, citric acid, disodium phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system includes about 0.4 mg / mL of citric acid (e.g., 0.44 mg / mL) and about 5 mg / mL of sodium citrate (e.g., 5.26 mg / mL).

[0175] A detergent or surfactant may also be added to the formulation. Exemplary detergents include nonionic detergents such as polysorbates (e.g., polysorbates 20, 80, etc.) or pol oxamers (e.g., pol oxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated protein and / or minimizes the formation of particulates in the formulation and / or reduces adsorption. In certain embodiments, the formulation may include a surfactant which is a polysorbate. In certain embodiments, the formulation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitanmonooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th ed., 1996). In certain embodiments, the formulation comprises between about 0.01% (w / v) of polysorbate 80.

[0176] In embodiments, the protein product of the present disclosure is formulated as a liquid formulation. The liquid formulation may be presented at a 10 mg / mL concentration in either a USP / Ph Eur type I 50R vial closed with a rubber stopper and sealed with an aluminum crimp seal closure. The stopper may be made of elastomer complying with USP and Ph Eur. In certain embodiments vials may be filled with 61.2 mL of the protein product solution in order to allow an extractable volume of 60 mL. In certain embodiments, the liquid formulation may be diluted with 0.9% saline solution.

[0177] In certain embodiments, the liquid formulation of the disclosure may be prepared as a 2 mg / mL concentration solution in combination with a sugar at stabilizing levels. In certain embodiments the liquid formulation may be prepared in an aqueous carrier. In certain embodiments, a stabilizer may be added in an amount no greater than that which may result in a viscosity undesirable or unsuitable for intravenous administration. In certain embodiments, the sugar may be disaccharides, e.g., sucrose. In certain embodiments, concentration of sucrose is about 60 mg / mL. In certain embodiments, the liquid formulation may also include one or more of a buffering agent, a surfactant, and a preservative.

[0178] In certain embodiments, the pH of the liquid formulation may be set by addition of a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide.

[0179] The aqueous carrier of interest herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation. Illustrative carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.

[0180] A preservative may be optionally added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation.

[0181] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0182] The specific dose can be a uniform dose for each patient, for example, 50-5000 mg of protein. Alternatively, a patient’s dose can be tailored to the approximate body weight or surface area of the patient. Other factors in determining the appropriate dosage can include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex and medical condition of the patient. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those skilled in the art, especially in light of the dosage information and assays disclosed herein. The dosage can also be determined through the use of known assays for determining dosages used in conjunction with appropriate dose-response data. An individual patient's dosage can be adjusted as the progress of the disease is monitored. Blood levels of the targetable construct or complex in a patient can be measured to see if the dosage needs to be adjusted to reach or maintain an effective concentration. Pharmacogenomics may be used to determine which targetable constructs and / or complexes, and dosages thereof, are most likely to be effective for a given individual (Schmitz et al., (2001) CLINICA CHIMICA ACTA 308: 43-53; Steimer et al., (2001) CLINICA CHIMICA ACTA 308: 33-41).

[0183] In general, dosages based on body weight are from about 0.1 mg / kg to about 100 mg / kg, e.g., 1 mg / kg to 100 mg / kg, 1 mg / kg to 10 mg / kg of body weight.

[0184] A dose may be given once or more times daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can estimate repetition rates for dosing based on measured residence times and concentrations of the targetable construct or complex in bodily fluids or tissues. In certain embodiments, a dose is administered once every week, once every two weeks, once every three weeks, once every four weeks. In certain embodiments, a dose is administered once every two weeks (z.e., once every other week). In certain embodiments, the dose is administered once every two weeks (z.e., once every other week) in a four- week treatment cycle.

[0185] The pharmaceutical composition can be formulated for intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, or intracavitary administration, or by perfusion through a catheter or by direct intralesional injection. The pharmaceutical composition can be formulated for administration once or more times daily, once or more times weekly, once or more times monthly, and once or more times annually.V. Therapeutic Applications

[0186] Certain of the heterodimeric Fc-fusion proteins disclosed herein are useful in increasing or modulating the survival, proliferation, and / or activity of an immune cell. Accordingly, in another aspect, the instant disclosure provides a method of increasing or modulating the survival, proliferation, and / or activity of an immune cell, the method comprising contacting the immune cell with a heterodimeric Fc-fusion protein, pharmaceutical composition, or formulation disclosed herein. In certain embodiments, the immune cell is a T cell (e.g., a CD8+T cell, a CD4+T cell, a regulatory T cell, or an NKT cell) or an NK cell.

[0187] The disclosure relates to methods for activating, differentiating, and expanding CD4+resting conventional T cells (Tconv) and CD8+T cells, the method comprising administering a heterodimeric IL-2 Fc fusion protein to a subject in need thereof, wherein administration of the heterodimeric IL-2 Fc fusion protein enhances tumor reactivity and mediates tumor clearance.

[0188] In certain embodiments, the variant IL-2 proteins disclosed herein can activate, differentiate, and expand CD4+resting conventional T cells (Tconv) and CD8+T cells, which enhances tumor reactivity and mediates tumor clearance.

[0189] In certain embodiments, the variant IL-2 proteins disclosed herein can promote differentiation of CD8+T cells upon antigen recognition into memory and more terminally differentiated effector cells.

[0190] In certain embodiments, the variant IL-2 proteins disclosed herein can reverse exhaustion of CD8+T cells during a tumor response.

[0191] In certain embodiments, the variant IL-2 proteins disclosed herein can enhance anti -tumor activity in combination with anti-PD-1 immunotherapy, which is dependent on high-affinity IL-2R engagement by IL-2.

[0192] In certain embodiments, the variant IL-2 proteins disclosed herein can potentiate the cytotoxicity of CD8+T cells and NK cells against cancer cells by inducing the expression of granzymes and perforin.

[0193] In certain embodiments, the variant IL-2 proteins disclosed herein can induce the production of interferon gamma (IFN-y) in the tumor microenvironment and activate the expression of major histocompatibility complex molecules in the immediate environment, thereby enhancing antigen presentation and recognition of tumor cells.

[0194] In certain embodiments, the immune cell is contacted with the heterodimeric Fc- fusion protein, pharmaceutical composition, or formulation in vivo for preventing or treating a disease or disorder. The diseases and disorders suitable for such treatment include but are not limited to cancer. Accordingly, in certain embodiments, the disclosure provides methods for treating cancer using a heterodimeric Fc-fusion protein described herein and / or a pharmaceutical composition described herein. The methods may be used to treat a variety of cancers by administering to a patient in need thereof an effective amount of a heterodimeric Fc-fusion protein described herein.

[0195] In some embodiments, Fc-fusion proteins or compositions disclosed herein can be administered to a subject to treat a disorder associated with abnormal apoptosis or a differentiative process (e.g., cellular proliferative disorders or cellular differentiative disorders, such as cancer, by, for example, producing an active or passive immunity). In the treatment of such diseases, the disclosed Fc-fusion proteins comprising IL-2 may possess advantageous properties, such as reduced vascular leak syndrome. Examples of cellular proliferative and / or differentiative disorders include cancer (e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias). A metastatictumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver.

[0196] The Fc-fusion proteins comprising IL-2 can be used to treat patients who have, who are suspected of having, or who may be at high risk for developing any type of cancer, including renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues.

[0197] In some embodiments, a heterodimeric Fc-fusion protein of the present disclosure (e.g., a heterodimeric Fc-fusion protein comprising IL-2) is used for treating advanced melanoma, renal cell carcinoma, urothelial bladder cancer, and human papillomavirus (HPV)-positive advanced malignancies.

[0198] IL-2 at low or physiological doses is necessary for the expansion and function of regulatory T cells (Tregs). Treatment with low-dose IL-2 can selectively enhance Treg function while avoiding the activation of effector T cells and ameliorate immune inflammation, because Tregs express abundant amounts of IL-2Ra. Accordingly, in some embodiments, the IL-2 comprised in the Fc-fusion protein useful in selectively enhancing Treg function at low doses is wild-type IL-2 or a variant thereof that binds IL-2Ra at an equal or higher affinity.

[0199] In some embodiments, a heterodimeric Fc-fusion protein of the present disclosure (e.g., a heterodimeric Fc-fusion protein comprising IL-2) is used for treating an advanced malignancy as a monotherapy.

[0200] In certain embodiments, the heterodimeric Fc-fusion protein comprising IL-2 is administered at a dose of about 0.1 mg / kg to about 100 mg / kg, e.g., 1 mg / kg to 100 mg / kg, or 1 mg / kg to 10 mg / kg of body weight. In certain embodiments, the heterodimeric Fc-fusion protein comprising IL-2 is administered at a dose of about 10 pg / kg to about 1,000 pg / kg. In certain embodiments, the heterodimeric Fc-fusion protein comprising IL-2 is administered at a dose of about 10 pg / kg to about 30 pg / kg, about 10 pg / kg to about 60 pg / kg, about 10 pg / kg to about 90 pg / kg, about 10 pg / kg to about 150 pg / kg, about 10 pg / kg to about 250 pg / kg, about 10 pg / kg to about 400 pg / kg, about 10 pg / kg to about 500 pg / kg, about 10 pg / kg to about 600 pg / kg, from about 10 pg / kg to about 700 pg / kg, from about 10 pg / kg to 800 pg / kg, about 30 pg / kg to about 60 pg / kg, about 30 pg / kg to about 90 pg / kg, about 30 pg / kg to about150 ig / kg, about 30 pg / kg to about 250 pg / kg, about 30 pg / kg to about 400 pg / kg, about 30 pg / kg to about 500 pg / kg, about 30 pg / kg to about 600 pg / kg, from about 30 pg / kg to about 700 pg / kg, from about 30 pg / kg to 800 pg / kg, about 60 pg / kg to about 90 pg / kg, about 60 pg / kg to about 150 pg / kg, about 60 pg / kg to about 250 pg / kg, about 60 pg / kg to about 400 pg / kg, about 60 pg / kg to about 500 pg / kg, about 60 pg / kg to about 600 pg / kg, from about 60 pg / kg to about 700 pg / kg, from about 60 pg / kg to 800 pg / kg, about 90 pg / kg to about 150 pg / kg, about 90 pg / kg to about 250 pg / kg, about 90 pg / kg to about 400 pg / kg, about 90 pg / kg to about 500 pg / kg, about 90 pg / kg to about 600 pg / kg, from about 90 pg / kg to about 700 pg / kg, from about 90 pg / kg to 800 pg / kg, about 150 pg / kg to about 250 pg / kg, about 150 pg / kg to about 400 pg / kg, about 150 pg / kg to about 500 pg / kg, about 150 pg / kg to about 600 pg / kg, from about 150 pg / kg to about 700 pg / kg, from about 150 pg / kg to 800 pg / kg, about 250 pg / kg to about 400 pg / kg, about 250 pg / kg to about 500 pg / kg, about 250 pg / kg to about 600 pg / kg, from about 250 pg / kg to about 700 pg / kg, from about 250 pg / kg to 800 pg / kg, about 400 pg / kg to about 500 pg / kg, about 400 pg / kg to about 600 pg / kg, from about 400 pg / kg to about 700 pg / kg, from about 400 pg / kg to 800 pg / kg, about 500 pg / kg to about 600 pg / kg, from about 500 pg / kg to about 700 pg / kg, from about 500 pg / kg to 800 pg / kg, about 600 pg / kg to about 700 pg / kg, from about 600 pg / kg to 800 pg / kg, or about 700 pg / kg to 800 Pig / kg-

[0201] A dose may be given once or more times daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can estimate repetition rates for dosing based on measured residence times and concentrations of the targetable construct or complex in bodily fluids or tissues. In certain embodiments, a dose is administered once every week, once every two weeks, once every three weeks, once every four weeks. In certain embodiments, a dose is administered once every two weeks (z.e., once every other week). In certain embodiments, the dose is administered once every two weeks (z.e., once every other week) in a four- week treatment cycle.

[0202] Administration of the heterodimeric Fc-fusion protein of the present disclosure could be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by perfusion through a catheter or by direct intralesional injection. This may be administered once or more times daily, once or more times weekly, once or more times monthly, and once or more times annually.VI. Combination Therapies

[0203] Another aspect of the disclosure provides for combination therapy. A heterodimeric Fc-fusion protein described herein can be used in combination with additional therapeutic agents to treat the cancer.

[0204] In some embodiments, a heterodimeric Fc-fusion protein of the present disclosure (e.g., a heterodimeric Fc-fusion protein comprising IL-2) is used in treating an advanced malignancy in combination with another therapeutic agent selected from: cytotoxic chemotherapy; radiotherapy; an antibody that targets a molecule involved in an anti-tumor immune response, such as CTLA-4, PD-1, PD-L1, or TGF-P; an antibody that acts by ADCC on a tumor-associated antigen; a multispecific antibody binding NKG2D, CD 16, and a tumor- associated antigen, optionally administered in combination with an antibody that targets PD-1 or PD-L1; a personalized cancer vaccine; an oncolytic cancer vaccine; and a personalized vaccine administered in combination with an antibody that targets PD-1 or PD-L1.

[0205] In some embodiments, a heterodimeric Fc-fusion protein of the present disclosure (e.g., a heterodimeric Fc-fusion protein comprising IL-2) is used in treating locally advanced malignancy that can be fully resected, in combination with an antibody that targets PD-1 or PD-L1.Checkpoint Inhibitor Therapy

[0206] In certain embodiments, the heterodimeric Fc-fused protein therapy can be combined with a checkpoint inhibitor therapy. Illustrative immune checkpoint molecules that can be targeted for blocking or inhibition include, but are not limited to, CTLA-4, 4- IBB (CD137), 4-1BBL (CD137L), PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, y5, and memory CD8+ (a|3) T cells), CD 160 (also referred to as BY55), and CGEN-15049. Immune checkpoint inhibitors include antibodies, or antigen binding fragments thereof, or other binding proteins, that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4, CD160, and CGEN- 15049. Illustrative immune checkpoint inhibitors include Illustrative immune checkpoint inhibitors include nivolumamb (anti-PD-1; OPDIVO® - BMS), AMP224 (anti-PD-1; NCI), pembrolizumab (anti-PD-1; MK-3475 / KEYTRUDA® - Merck), pidilizumab (anti-PD-1 antibody; CT-011 - Teva / CureTech), atezolizumab (anti-PD-Ll; TECENTRIQ® - Roche / Genentech), durvalumab (anti-PD-Ll; MEDI4736 / IMFINZI® -Medimmune / AstraZeneca), avelumab (anti-PD-Ll; BAVENCIO® - Pfizer), BMS-936559 (anti-PD-Ll - BMS), ipilimumab (anti-CTLA-4; YERVOY® - BMS), tremelimumab (anti- CTLA-4; Medimmune / AstraZeneca), lirilumab (anti-KIR; BMS), monalizumab (anti- NKG2A; Innate Pharma / AstraZeneca), BY55 (anti-CD160), anti-OX40, anti-TIM3, and anti- LAG3.

[0207] In an exemplary embodiment, the heterodimeric Fc-fused protein therapy can be combined with nivolumab. In an exemplary embodiment, the heterodimeric Fc-fused protein therapy can be combined with pembrolizumab.

[0208] In certain embodiments, the checkpoint inhibitor therapy is used in combination with the heterodimeric Fc-fused protein therapy to treat subjects known or suspected of having cancer.

[0209] In some embodiments, a heterodimeric Fc-fusion protein of the present disclosure (e.g., a heterodimeric Fc-fusion protein comprising IL-2) is administered to a patient (e.g., a cancer patient) who has progressed after treatment with a checkpoint inhibitor. In some embodiments, a heterodimeric Fc-fusion protein of the present disclosure (e.g., a heterodimeric Fc-fusion protein comprising IL-2) is administered to a patient (e.g., a cancer patient) who has received a treatment regimen that includes an anti-PD-1 inhibitor. In some embodiments, the patient has progressed after receiving a treatment regimen that includes an anti-PD-1 inhibitor. In certain embodiments, the PD-1 inhibitor is an anti-PD-1 antibody.Multi-specific Bindins Protein (“TriNKET”) Therapy

[0210] In certain embodiments, the heterodimeric Fc-fused protein therapy is combined with a therapy comprising a multi-specific binding protein, which comprises: (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds a tumor- associated antigen; and (c) an antibody Fc domain or a portion thereof sufficient to bind CD 16, or a third antigen-binding site that binds CD 16 (“TriNKET”) (for example, multispecific binding proteins comprising various NKG2D-binders and tumor-associated antigenbinding sites described in International Publication No. WO 2019 / 157332, the entire contents of which are incorporated by reference herein), to treat subjects known or suspected of having cancer. In some embodiments, the tumor-associated antigen comprises Tyrpl.

[0211] The amount of heterodimeric Fc-fusion protein and additional therapeutic agent and the relative timing of administration may be selected in order to achieve a desired combined therapeutic effect. For example, when administering a combination therapy to apatient in need of such administration, the therapeutic agents in the combination, or a pharmaceutical composition or compositions comprising the therapeutic agents, may be administered in any order such as, for example, sequentially, concurrently, together, simultaneously and the like. Further, for example, a heterodimeric Fc-fusion protein may be administered during a time when the additional therapeutic agent(s) exerts its prophylactic or therapeutic effect, or vice versa.

[0212] The description above describes multiple aspects and embodiments of the disclosure. The patent application specifically contemplates all combinations and permutations of the aspects and embodiments.EXAMPLES

[0213] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.Example 1 - Exemplary Variant IL-2 Heterodimeric Fc-Fusion ProteinsMethod of Preparation

[0214] The amino acid sequence of variant IL-2 constructs, SeqlRev (SEQ ID NO: 3) and Seql5Rev (SEQ ID NO: 2), together with the linker polypeptide sequence, G4S3 (SEQ ID NO: 6), were back translated into nucleotide sequences with Integrated DNA Technologies (IDT) codon optimization tool. The DNA fragments for human Fc fusion domain were codon optimized based on codon usage of Cricetulus Greseus for optimal expression in Chinese Hamster Ovary (CHO) cell expression system. Sequence coding murine IgK signal peptide was added to N-terminal of expression cassette after ATG start codon for secretion expression of fusion protein. In addition, 25-30 base pairs of overhang homology sequences were added to 5’-end and 3’- end of each fragment to facilitate cloning with Gibson assembling method. The DNA fragments coding the human IgGl Fc variants of SEQ ID NOs: 4 and 5 were also codon optimized with IDT codon optimization tool. All double stranded DNA fragments were synthesized by IDT.

[0215] Protein expression constructs were generated in a pcDNA3.4 vector backbone with Gibson assembling methods. A DNA fragment comprising Seql5Rev was assembled with the human IgGl Fc variant of SEQ ID NO: 4 and cloned into pCDNA3.4 under control of CMV promoter following HiFi DNA assembly protocol (New England Biolabs Cat# E2621) (“RVT-Fc-IL-2”). The cloning mixture was transformed to E.coli DH5a through head shock. The E.coli colony comprising the expression constructs was identified bysequencing and propagated for large scale DNA preparation. The human IgGl Fc variant comprising SEQ ID NO: 5 was also generated with pCDNA3.4 vector backbone with same process (“mono-EW-Fc”).

[0216] The monovalent IL-2 human Fc fusion protein heterodimer was generated by coexpression of RVT-Fc-IL-2 with mono-EW-Fc using ExpiCHO™ transient expression system (Life Technologies). The plasmids encoding RVT-Fc-IL-2 and mono-EW-Fc were transiently cotransfected in pair at 1 : 1 ratio in shaker flask following standard protocol provided from kit (Life Technologies Cat# A29130). Expression supernatant was harvested 7 days post transfection through centrifugation and filtration (“Harvest Supernatant”). The Harvest Supernatant was used for purification the same day or stored at 4 °C for next day purification.Method of Purification

[0217] The human Fc-Seql5RevIL-2 (“DF IL-2-Fc”) was purified by one affinity step with protein A (MabSelect Prisma, Cytiva) followed by size exclusion chromatography (HiLoad 16 / 60 Superdex 200, Cytiva). For affinity chromatography, 0.2 mL of MabSelect Prisma resin per 100 mL of Harvest Supernatant was added to the Harvest Supernatant and incubated at 4 °C for 90 minutes on stir plate with stir bar. After incubation, the sample was added to a filtration unit (Rapid-Flow Sterile Disposable Filter Units, Nalgene) and the resin was washed with -100 mL Phosphate Buffered Saline (PBS). The resin was recovered and added to a disposable column (10 mL centrifuge column, Pierce). Target protein was eluted in 5 column volumes of 200 mM sodium chloride, 100 mM glycine, pH 3.0. Protein solution was neutralized by adding 1 / 6 of 1 M Tris-HCl, pH 8.3. Target protein was concentrated and filtered prior to loading on a HiLoad Superdex 200 column (Cytiva) equilibrated with PBS.

[0218] The final preparation of DF IL-2-Fc comprised 100% monomer (as determined on a Superdex 200 Increase 5 / 150 G (Cytiva) in PBS at 25 °C) and had 0.1 endotoxin unit (EU) / mg endotoxin (as determined by Lumulus Amebocyte Lysate (LAL) assay on a Endosafe nexgen-MCS (Charles River) used according to the manufacturer's instruction). The production yield was 50 mg / L. Mass spectrometry analysis revealed the assembled molecule with traces (<5%) of other molecules.Exemplary IL-2 Variant Heterodimeric Fc-Fused Protein, DF IL-2-Fc

[0219] DF IL-2-Fc is a modified monovalent human interleukin 2 (IL-2)-Fc (fragment crystallizable region) C-terminus fusion protein that binds to the IL-2 receptor (IL-2R). DFIL-2-Fc is a heterodimer consisting of 2 polypeptide chains: (1) Chain A is the monovalent novel IL-2 unit C-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain (CH2-CH3 domains); (2) Chain B is a truncated heavy chain (CH2-CH3 domains).

[0220] The amino acid sequences for each chain are shown below, with amino acids of the putative leader sequences (signal sequence of hlgK) underlined. The corresponding mutein amino acid residues in the Fc region are in bold and underlined italics.

[0221] Chain A: engineered human IL-2-Fc, with (G4S)3 (SEQ ID NO: 6) linker in bold font, leader sequence underlined, IL-2 in italics, IL-2 substitutions in underlined italics highlighted in grey, and human IgG-Fc amino acid substitutions are bold, italicized, and underlined.MDMRVPAOLLGLLLLWLPGARCDI<THTCPPCPAPEAAGGPSVFLFPPI<PI<DTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPEVYTLPPCRDELTKNQVSLTC LVKGF YPSDIAVEWESNGQPENNYKTTPPVL ESDGSF 7L YSKLTVDKSRWQQGNVF S CSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSd / SNS7 7G / .A7 / / . / . / . DLQMILNMLNNY NPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVL^AQSKNF HL PRDLISNINl ’lVLELKGSETTFMCEYADETATIVEFLNRWITF^JSIISTLT (SEQ ID NO: 9)

[0222] Chain B: Engineered human IgGl, with leader sequence underlined and humanIgG-Fc amino acid substitutions are bold, italicized, and underlined.MDMRVPAOLLGLLLLWLPGARCDI<THTCPPCPAPEAAGGPSVFLFPPI<PI<DTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGI<EYI<CT<VSNI<ALA APIEI<TISI<AI<GQPREPQVCTLPPSRDELTENQVSLTC LVI<GFYPSDIAVEWESNGQPENNYI<TTPPVLDSDGSFFLYSIFLTVDI<SRWQQGNVF SCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 10)Example 2 - Characterization of C-terminal IL-2 Constructs and N-terminal IL-2 ConstructsProliferation of Immune Cell Subsets

[0223] The objective of this study was to evaluate the ability of the IL-2 variants to proliferate immune cell subsets from isolated human or cynomolgus monkey PBMCs.Briefly, isolated naive peripheral blood mononuclear cells (PBMCs) from human or cynomolgus monkeys were counted, plated, and stimulated with different concentrations of recombinant human IL-2 (rhIL-2); Seql5Rev human IL-2 construct C-terminally fused via a(G4S)3 (SEQ ID NO: 6) linker to mouse IgG2a “mu_Seql5rev_3XG4S”; and different IL-2 variants (listed below) and incubated for 4 days at 37 °C / 5% CO2.Tested IL-2 Variants:

[0224] (a) C-terminal IL-2 variants: (i) Seql5Rev human IL-2 construct C-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain of human IgGl, “huIgGl - Seql5rev;”

[0225] (ii) SeqlRev human IL-2 construct C-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain of human IgGl, “huIgGl - Seqlrev;”

[0226] (b) N-terminal IL-2 variants: (i) Seql5Rev human IL-2 construct N-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain of human IgGl, “Seql5rev - huIgGl;”

[0227] (ii) SeqlRev human IL-2 construct N-terminally fused via a (G4S)3 (SEQ ID NO: 6) linker to a truncated heavy chain (CH2-CH3 domains) of human IgGl, “Seqlrev - huIgGl ”

[0228] After 4 days, cells were stained with various surface and intracellular markers and analyzed via flow cytometry. Within each cell subset analyzed (Naive CD8+T cells, CD4+T cells, Tregs, and NK cells), the % positive Ki-67 population for each concentration was plotted against the logarithm of the test article concentration and the ECso of proliferation was calculated based on a 4-parameter nonlinear regression analysis. As shown in FIGs. 2A-2D and 3A-3D, both C-terminal and N-terminal IL-2 variants showed comparable PBMC proliferation in vitro.In Vitro Potency of IL-2 Variants

[0229] The objective of this study was to evaluate the potency of different IL-2 variants in stimulating different interleukin-2 receptor (IL-2R)-expressing immune cells (naive CD8 T cells, naive Tregs, and activated CD8 T cells) compared to that of recombinant human IL-2 (rhIL-2). In particular, the potency of C-terminal IL-2 variants were compared with N- terminal IL-2 variants (as described above).

[0230] Immune cell activation induced by the IL-2 variants after a 20-minute stimulation was assessed using intracellular detection of phosphorylated STAT5 (pSTAT5) levels via flow cytometry in naive human peripheral blood mononuclear cells (PBMCs) and cynomolgus monkey PBMCs.

[0231] Additionally, in vitro potency was assessed in anti-CD3 / anti-CD28-coated bead- stimulated CD8+T cells from both human and cynomolgus monkeys. Within each cell subsetanalyzed (Naive CD8+T cells, Tregs, and activated CD8+T cells), the pSTAT5 mean fluorescence intensity (MFI) for each concentration was plotted against the logarithm of the test article concentration and the ECso of signaling was calculated based on a 4-parameter nonlinear regression analysis. As shown in FIGs. 4A-4C and 5A-5C, both C-terminal and N-terminal IL-2 variants showed equivalent potencies on naive CD8 T cells, Tregs, and activated CD8 T cells.Anti-Tumor Activity of IL-2 Variants

[0232] This study evaluated the ability of the N- and C-terminal IL-2 variants (listed above) to elicit anti -turn or activity in the syngeneic mouse CT26-Tyrpl tumor model. BALB / c mice (n = 10 / group) were dosed intraperitoneally (IP) once weekly (QW) for 4 weeks with a dose of 1.3 mg / kg or 0.65 mg / kg of their respective IL-2 variant or a monoclonal antibody isotype control (mIgG2a). Treatment was initiated when tumor bearing mice within the study had an average tumor volume of ~50 mm3 (Day 5). Tumor volumes were assessed 3 times per week using digital calipers. Tumor volumes were calculated as length(mm) x width(mm) x height(mm).

[0233] As shown in FIGs. 6B and 6D, C-terminal IL-2 variants appear to induce stronger anti-tumor responses at lower concentrations than their respective N-terminal IL-2 variants.Example 3 - Biological Characterization of DF IL-2-Fc Interactions with IL-2R Subunits and IL-2RBYC Complex

[0234] This example describes the in vitro binding characterization of DF IL-2-Fc. The exemplary DF IL-2-Fc has been designed to stimulate effector cells expressing the high- affinity IL-2 receptor with reduced Treg activation compared to that seen with aldesleukin.

[0235] The binding kinetics and affinity of DF IL-2-Fc for purified recombinant IL-2R a and P subunits and IL-2R complexes from humans, cynomolgus monkeys, and mice were obtained using surface plasmon resonance (SPR). Measurements were performed using a Biacore 8K SPR instrument. As shown in TABLE 6, the biochemical interaction of DF IL- 2-Fc with the low-affinity human IL 2Ra subunit demonstrated reduced binding (KD = 1.1 ± 0.0 pM [mean ± standard deviation (SD)]) by 50-fold compared to that of aldesleukin (KD = 20.5 ± 0.7 nM), which is in line with reported binding affinities of aldesleukin to IL 2Ra in the literature (KD = 10-14 nM) (see Wang et al. (1987) “The interleukin 2 receptor.Functional consequences of its biomolecular structure.” J EXP MED. 166(4): 1055-69, Minami et al. (1993) “The IL-2 receptor complex: its structure, function, and target genes”, ANNUREV IMMUNOL. 11 :245-68). In contrast, the binding affinity of DF IL-2-Fc to the IL 2RP human subunit was 2.7-fold increased (KD = 186.2 ± 2.0) when compared to aldesleukin (KD = 513.2 ± 19.0). DF IL-2-Fc binds with an affinity of 16.9 ± 2.1 pM to the IL-2RPyc complex, which is in a similar range as the binding affinity of aldesleukin (KD = 19.0 ± 2.9 pM). A similar pattern was observed with the nonclinical species, with reduced binding between DF IL-2-Fc and the respective monkey and mouse IL 2Ra subunits. Binding to monkey IL-2RP and the IL-2RPy complex was in a similar range between DF IL-2-Fc and aldesleukin.TABLE 6: Kinetics and Affinity of DE IL-2-Fc Binding to IL-2 Receptor Subunits and Complexes Evaluated by SPRka. association rate constant; kd'. dissociation rate constant; KD: equilibrium dissociation equilibrium constant; NA: not available; SPR: surface plasmon resonance.Note: Values are presented as mean ± standard deviation (SD) of n > 3. Mouse data for IL-2RP and IL-2RPy are not presented as mouse IL-2RP was not functional (i.e., positive control for mouse IL-2 did not show binding to mouse IL- 2Rp protein in SPR).Example 4 - Dose-Responsive Binding of DF IL-2-Fc to human IL-2Rot

[0236] This example describes reduced cell binding of DF IL-2-Fc to human IL-2Ra- expressing CHO cells as compared to an Fc(si)-rhIL-2 tool molecule that abrogates IL-2Ra binding (“non-alpha tool molecule”).

[0237] To verify the reduced affinity of DF IL-2-Fc to the IL-2Ra subunit (CD25), binding of DF IL-2-Fc to human CD25-expressing CHO cells was assessed by flow cytometry in comparison to aldesleukin and a non-alpha rhIL-2 variant that contains an F42A modification that abrogates IL-2Ra binding (referred to as rhIL-2-F42A). For the detection of binding with a phycoerythrin (PE), conjugated anti-human Fc antibody, aldesleukin and rhIL-2-F42A were C-terminally fused to an Fc having the same human Fey silencing modifications. Aldesleukin and rhIL-2-F42A C-terminally fused to the Fc having the human Fey silencing modifications were referred to as hFc(si)-rhIL-2 and hFc(si)-rhIL-2-F42A (non- alpha tool molecule), respectively.

[0238] Chinese human ovary (CHO) cells were incubated with a dose titration of test articles (DF IL-2-Fc, hFc(si)-rhIL-2, and hFc(si)-rhIL-2-F42A) from 500 nM in 1 :3 dilutions for 1 hour on ice. Cell binding was detected via flow cytometry using an anti-human Fc secondary antibody conjugated with the fluorophore phycoerythrin (PE). Each data point was run in duplicate. Each point and error bars represent mean ± standard deviation (SD).

[0239] As shown in FIG. 7, DF IL-2-Fc showed reduced cell binding to human IL-2Ra- expressing CHO cells compared to the Fc(si)-rhIL-2 tool molecule. However, binding of DF IL-2-Fc to IL-2Ra was greater than that of the non-alpha tool molecule hFc(si)-rhIL-2-F42A.Example 5 - Fc Receptor and FcRn Binding Characteristics of DF IL-2-Fc

[0240] This example describes the binding profiles of DF IL-2-Fc to recombinant FcyRs and FcRn were obtained using SPR (Biacore 8K, Cytiva).

[0241] The experimental IgGl isotype control trastuzumab demonstrated expected dosedependent binding at the concentrations tested, while DF IL-2-Fc did not demonstrate any quantifiable binding to the FcyRs tested: recombinant human CD64 (FcyRI), CD32a (FcyRIIa) H131 and R131 alleles, CD16a (FcyRIIIa) V158 and F158 alleles, CD32b (FcyRIIb), and CD16b (FcyRIIIb); and recombinant cynomolgus monkey CD64 (FcyRI) and CD 16 (FcyRIII) (data not shown).

[0242] As shown in TABLE 7, both human and cynomolgus monkey FcRn binding were not affected by FcyR-silencing modifications. DF IL-2-Fc and IgGl isotype control trastuzumab were comparable in their binding affinity values for human and cynomolgus FcRn at pH 6.0 and similar in their lack of quantifiable binding at the concentrations tested at pH 7.4. Each molecule had nearly identical affinity for FcRn across humans and cynomolgus monkeys at pH 6.0.TABLE 7: DF IL-2-Fc Binding to FcRn Evaluated by SPRFcRn: neonatal Fc receptor; KD: equilibrium dissociation equilibrium constant.Note: Values are presented as mean ± standard deviation (SD) of n = 2 (pH 7.4) or 4 (pH 6.0).Example 6 - In Vitro Cellular Activity of DF IL-2-FcDF IL-2 -Fc Demonstrates Increased Potency on CD8+T Cells and NK Cells With Reduced Potency on Tregs in Human PBMCs

[0243] The amino acid substitutions in the variant IL-2 moiety of DF IL-2-Fc result in different binding affinities to IL-2Rs compared to those seen with aldesleukin. To evaluate if these alternate binding affinities influence the ability of DF IL-2-Fc to stimulate different IL- 2R-expressing immune cells, immune activation induced by DF IL-2-Fc and aldesleukin was assessed by intracellular detection of phosphorylated STAT5 (pSTAT5) levels using flow cytometry in human peripheral blood mononuclear cells (PBMCs).

[0244] Briefly, human PBMCs were stimulated for 20 minutes with DF IL-2-Fc or aldesleukin. Test articles were dose titrated from 400 nM to 6.1 pM in 1 :4 dilutions and from 6.1 pM to 6.1 fM in 1 : 10 dilutions. After stimulation, cells were fixed, permeabilized, and stained with immune cell-specific antibodies. Percentage of pSTAT5-positive populations was analyzed using Flow Jo™. Dose-response curves were fit with a nonlinear 4-parameterregression model in GraphPad Prism. FIGs. 8A-8D are graphs depicting flow cytometric analysis of phosphorylated STAT5 (pSTAT5) signaling in regulatory T cells (Tregs) (FIG. 8A), CD8+T cells (FIG. 8B), CD4+T cells (FIG. 8C), and natural killer (NK) cells (FIG. 8D) after stimulation with DF IL-2-Fc or PROLEUKIN® (aldesleukin). The plots depicted are data from a single donor (assay was run as single points), representative of data from 9 healthy human PBMC donors.

[0245] DF IL-2-Fc and aldesleukin dose-dependently activated human immune cells. DF IL-2-Fc demonstrated a ~10-fold reduction in activation of Tregs, which express high levels of IL-2Ra, with a concentration resulting in half-maximal response (ECso) of 9.5 ± 17 pM compared to that of aldesleukin (ECso = 1.1 ± 1.3 pM). When compared to aldesleukin, DF IL-2-Fc showed increased activation of IL-2Rp / y naive CD8+T cells and NK cells. DF IL-2- Fc demonstrated a 4-fold increase in potency on naive CD8+T cells (ECso = 0.26 ± 0.19 nM) and a 6-fold increase on naive NK cells (ECso = 0.054 ± 0.054 nM) compared to aldesleukin (ECso = 0.94 ± 0.70 nM for naive CD8+T cells and EC50 = 0.29 ± 0.28 nM for naive NK cells). In contrast, DF IL-2-Fc showed a 3-fold reduction of potency on naive conventional CD4+T cells (CD4+Tconv cells), with an EC50 of 0.15 ± 0.057 nM compared to aldesleukin (ECso = 0.053 ± 0.036 nM).

[0246] Overall, DF IL-2-Fc differentiates in stimulation of naive human PBMCs from aldesleukin by shifting the potency towards cytotoxic effector cells while reducing expansion of immunosuppressive Tregs.DF IL-2 -Fc Stimulates Activated Human CD8+T Cells Similar to Aldesleukin

[0247] The amino acid substitutions in the IL-2 moiety of DF IL-2-Fc resulted in reduced binding affinity to the IL-2Ra subunit and increased binding affinity to the ZL-2RPy complex compared to that seen with aldesleukin. To evaluate if these alternate binding affinities influence the ability of DF IL-2-Fc to stimulate recently T cell receptor (TCR)-activated CD8+T cells expressing high levels of the high-affinity IL-2R (consisting of the a, P, and yc chains), STAT5 activation by DF IL-2-Fc was compared to that of aldesleukin and a nonalpha tool molecule, referred to as hFc(si)-rhIL-2-F42A, using flow cytometry.

[0248] Briefly, human peripheral blood mononuclear cells (PBMCs) were stimulated with anti-CD3 / anti-CD28 beads for 3 days. After removing activation beads, cells were rested overnight for 16 hours prior to IL-2 treatment. Activated immune cells were stimulated for 20 minutes with DF IL-2-Fc, aldesleukin, or human Fc(si)-rhIL-2-F42A. Testarticles were dose-titrated from 400 nM to 6.1 pM in 1 :4 dilutions and from 6.1 pM to 0.61 fM in 1 : 10 dilutions. After stimulation, cells were fixed, permeabilized, and stained with immune cell-specific antibodies. Percentage of pSTAT5-positive, CD25-expressing CD8+T cells was analyzed using FlowJo™ Dose-response curves were fit with a nonlinear 4- parameter regression model in GraphPad Prism. The plots depicted are data from a single donor (assay was run as single points), representative of data from 6 human PBMC donors.

[0249] As shown in FIG. 9, DF IL-2-Fc stimulated recently activated CD8+T cells expressing the high-affinity IL-2R in a similar manner to aldesleukin (ECso = 2.4 ± 1.4 pM and 2.6 ± 1.9 pM, respectively). In contrast, the non-alpha tool molecule hFc(si)-rhIL-2- F42A showed an ~8-fold reduced potency on activated CD8+T cells(ECso = 25.50 ± 21.66 pM) when compared to aldesleukin (TABLE 8). The EC50 of DF IL- 2-Fc (ECso = 2.4 ± 1.4 pM) on activated CD8+T cells was lower than that on naive Tregs (ECso = 9.5 ± 17 pM). Thus, DF IL-2-Fc more potently stimulates activated CD8+T cells than it does Tregs, while aldesleukin stimulates activated CD8+T cells and Tregs similarly.

[0250] TABLE 8: Summary of DF IL-2-Fc-Mediated STAT5 Activation in TCR-Activated Human CD8+T CellsEC50: concentration resulting in half-maximal response; PBMC: peripheral blood mononuclear cell; rhIL-2: recombinant human interleukin-2; SD: standard deviation; TCR: T cell receptor.DF IL-2 -Fc Demonstrates Enhanced Potency on Effector Cells and Reduced Potency on Tregs in Cynomolgus Monkey PBMCs

[0251] To evaluate cross-reactivity of DF IL-2-Fc in cynomolgus monkeys, STAT5 signaling in cynomolgus monkey PBMCs was assessed using flow cytometry.

[0252] Briefly, cynomolgus monkey PBMCs were stimulated for 20 minutes with DF IL- 2-Fc, PROLEUKIN® (aldesleukin), or isotype control human IgGl with Fc-silent modifications [hFc(si)]. Test articles were dose-titrated from 200 to 0.195 nM in 1 :4 dilutions and from 0.195 nM to 1.95 fM in 1 : 10 dilutions. After stimulation, cells were fixed, permeabilized, and stained with immune cell-specific antibodies. Percentage of pSTAT5-positive populations was analyzed using Flow Jo™ Dose-response curves were fit with a nonlinear 4-parameter regression model in GraphPad Prism. The plots (FIGs. 10A- 10D) depicted are data from a single donor (assay was run as single points), representative of data from 4 cynomolgus monkey PBMC donors.

[0253] As shown in FIGs. 10A-10D, DF IL-2-Fc showed dose-dependent activation of cynomolgus immune cells from PBMCs. When compared to aldesleukin, DF IL-2-Fc demonstrated similar activation of effector cells, including CD8+T cells and NK cells, with reduced activation of Tregs (~50-fold reduced potency) and CD4+T cells (~2-fold reduced potency) in cynomolgus monkey PBMCs.DF IL-2-Fc Demonstrates Enhanced Activation of Effector Cells and Reduced Activation of Tregs in Mouse Splenocytes

[0254] To evaluate cross-reactivity of DF IL-2-Fc in mice, STAT5 signaling in mouse splenocytes was assessed using flow cytometry.

[0255] Briefly, mouse splenocytes isolated from a single Balb / c mouse were stimulated for 20 minutes with DF IL-2-Fc or PROLEUKIN® (aldesleukin). Test articles were dosetitrated from 200 nM to 12.2 pM in 1 :4 dilutions and from 12.2 pM to 1.22 fM in 1 : 10 dilutions. After stimulation, cells were fixed, permeabilized, and stained with immune cellspecific antibodies. Percentage of pSTAT5-positive populations was analyzed using FlowJo™. Dose-response curves were fit with a non-linear 4-parameter regression model in GraphPad Prism. The plots (FIGs. 11A-11D) depicted are data from splenocytes isolated from a single Balb / c mouse (assay was run as single points), representative of data from 3 mice.

[0256] As shown in FIGs. 11A-11D, DF IL-2-Fc showed dose-dependent activation of mouse immune cells from Balb / c splenocytes. When compared to aldesleukin, DF IL-2-Fc demonstrated increased (~2-fold) activation of effector cells including CD8+T cells and NK cells, with reduced activation of Tregs in mouse splenocytes. Similar stimulation of CD4+T cells in mouse splenocytes was observed with DF IL-2-Fc compared to aldesleukin.However, dose-response curves for mouse CD4+T cell stimulation were not saturated at the highest test article concentration.Example 8 -Anti-Tumor Activity of DF IL-2-Fc Monotherapy in the CT26 Syngeneic Mouse Model

[0257] To evaluate the ability of DF IL-2-Fc to elicit anti-tumor activity in vivo, an efficacy study in the syngeneic mouse CT26 tumor model was performed.

[0258] Briefly, Balb / c mice (n = 10 / group) were engrafted with 5 * 105CT26 (colon carcinoma) cells and dosed intraperitoneally (IP) with human immunoglobulin (hlgGl) isotype or DF IL-2-Fc at 0.675 mg / kg or 0.225 mg / kg (equimolar to 3 or 1 pg of aldesleukin, respectively) once weekly (QW) for 4 weeks. Individual tumor volumes are shown in FIGs. 12A-B, with treatment days and frequencies as indicated (dotted line). Group mean body weight changes are depicted in FIG. 12C as mean ± standard error of the mean (SEM). Kaplan-Meier survival curves are shown in FIG. 12E, with median survival indicated in brackets (*** = p < 0.001; log-rank test.

[0259] As shown in FIGs. 12A-12B, DF IL-2-Fc treatment resulted in a strong anti -turn or response in the CT26 model, with 2 out of 10 and 5 out of 10 complete responders (CRs) at 0.225 mg / kg and 0.675 mg / kg, respectively. DF IL-2-Fc was well tolerated at both dose levels tested, with no body weight loss at the 0.225 mg / kg dose level and only 8% body weight loss observed after initial dosing at the 0.675 mg / kg level. Additional body weight loss was not observed after repeated dosing, nor were there clinical observations.

[0260] To understand the mode of action of DF IL-2-Fc, CT26 tumor-bearing mice were treated IP once with either isotype hlgGl or DF IL-2-Fc at 0.675 mg / kg or 0.225 mg / kg (equimolar to 3 or 1 pg aldesleukin), and samples were analyzed for soluble and cellular changes.Cytokine and Chemokine Response by DF IL-2 -Fc in Serum and Tumor Lysates after 24 Hours

[0261] Chemokines and cytokines were measured in serum and tumor lysates 24 hours after treatment with isotype or DF IL-2-Fc at 0.675 mg / kg and 0.225 mg / kg, using the multiplex bead-based Luminex technology. As a positive reference control, serum collected from a mouse treated with anti-CD3 antibody for 4 hours was used. As shown in FIGs. 13A- 13U and 14A-14V, a single dose of DF IL-2-Fc increased peripheral and intratumoral levels of several cytokines and chemokines 24 hours post-treatment: e.g., a broad cytokine responsein the periphery including INF-y, TNF-a, IL-5, and others, and relatively small changes in just a handful of cytokines (e.g., IFN-y, GM-CSF, IL-5, and MIP-la [CCL3]) in the tumor microenvironment (TME).Lymphocyte Cell Composition in the Tumor Microenvironment and Periphery Following DF IL-2-Fc Treatment

[0262] Immune cell populations were analyzed by flow cytometry in the TME and periphery at 2 and 4 days after treatment. Overall, increased lymphocytes were shown in both the tumor (FIG. 15A) and periphery (FIG. 15B). DF IL-2-Fc treatment significantly increased tumor-infiltrating CD8+T cell and NK cells relative to isotype control (FIGs. 15C- 15H) without increasing the Treg population in the TME when compared to isotype control, resulting in increased ratios of CD8+T cells:Tregs and NK cells:Tregs (FIGs. 15O-15P). In contrast, DF IL-2-Fc increased peripheral Tregs, CD8+T cells, NK cells, and NKT cells 96 hours after treatment compared to isotype-treated mice (FIGs. 15I-15N). In addition, DF IL- 2-Fc induced expansion of recently antigen-activated CD25-expressing CD8+T cells in the TME when compared to isotype control (FIG. 15Q).Ki-67 Expression in Immune Cells in the TME and Periphery Following DF IL-2 -Fc Treatment

[0263] Furthermore, Ki-67 expression after DF IL-2-Fc treatment was significantly increased in both intratumoral and peripheral immune cells, including CD8+T cells, CD4+Tconv cells, Tregs, NK cells, and NKT cells, relative to that seen after isotype control treatment 2 days after treatment (FIGs. 16A-16F and 17A-17E). In addition, DF IL-2-Fc significantly increased Ki-67 expression in antigen-activated CD25 -expressing CD8+T cells in the TME when compared to isotype control.Characterization of Tumor -Infiltrating CD8+T Cells and NK Cells Following DF IL-2 -Fc Treatment

[0264] DF IL-2-Fc increased the cytolytic activity of tumor-infiltrating NK cells and CD8+T cells, as measured by increased granzyme B expression, compared to that seen in isotype control -treated mice (FIGs. 18A-18B and FIGs. 19A-19B).Example 9 - Combination of DF IL-2-Fc and PD-1 blockade in B16F10 mouse model

[0265] Combination therapy of DF IL-2-Fc and PD-1 blockade was performed to analyze whether anti -tumor immune response can be amplified in established B16F10 tumors.

[0266] Briefly, C57BL / 6 mice (n = 10 per group) were engrafted subcutaneously (SC) with 2 x 105B16F10 melanoma cells. Once mean tumor volumes reached 50 mm3, mice were dosed intraperitoneally (IP) with isotype controls (1.35 mg / kg hlgGl once weekly [QW], or 10 mg / kg rat IgG2a biweekly), DF IL-2-Fc (1.35 mg / kg or 0.675 mg / kg QW, equimolar to 6 or 3 pg aldesleukin, respectively), anti-PD-1 monoclonal antibody (mAb) (10 mg / kg biweekly), or a combination of DF IL-2-Fc and anti-PD-1 mAb at the same dose regimen as monotherapy. Individual tumor volumes are shown in FIGs. 20A-20E, with treatment days and frequencies as indicated (dotted line [DF IL-2-Fc] or arrow [anti-PD-1]).

[0267] Both monotherapies resulted in delayed tumor progression, with 1 of 10 CRs at 1.35 mg / kg DF IL-2-Fc. Both monotherapies significantly increased median survival compared to isotype control (p < 0.05). Combination of DF IL-2-Fc and PD-1 blockade delayed tumor progression further compared to both monotherapies, resulting in 60% survival rate for 1.35 mg / kg DF IL-2-Fc combination treatment and 20% for 0.675 mg / kg DF IL-2-Fc combination treatment at Day 60 post-tumor inoculation. Combination treatment with DF IL-2-Fc at 1.35 mg / kg significantly increased median survival compared to DF IL-2-Fc monotherapy at the same dose level (p < 0.01). There were no synergistic or additive adverse effects on tolerability, body weights, or clinical observations with the combination regimen of DF IL-2-Fc and anti-PD-1 mAb. This study therefore provides evidence that DF IL-2-Fc therapy can further enhance anti-tumor efficacy when administered in combination with PD-1 blockade.

[0268] A subsequent study was conducted in the Bl 6F 10 melanoma model using more advanced established tumors, which are known to be largely resistant to PD-1 blockade (Mosely et al. (2017) “Rational selection of syngeneic preclinical tumor models for immunotherapeutic drug discovery,” CANCER IMMUNOL RES. 5(1):29— 41), in order to analyze whether the anti-tumor immune response could be amplified. When tumor volume averaged -100 mm3, B16F10 tumor-bearing mice were dosed IP with 1.35 mg / kg DF IL-2-Fc (equimolar to 6 pg aldesleukin) alone, 10 mg / kg anti-PD-1 biweekly alone, or the combination. DF IL-2-Fc exhibited potent synergy with PD-1 blockade, enhancing antitumor responses over monotherapies, with 4 of 10 CRs with DF IL-2-Fc combined with anti- PD-1 (FIGs. 21A-21F). Both the monotherapies and combination therapies were well tolerated, with no additional body weight loss observed over DF IL-2-Fc monotherapy when combined with anti-PD-1.Example 10 - Combination of DF IL-2-Fc with Tyrpl-TriNKET in B16F10 mouse model

[0269] Combination therapy of DF IL-2-Fc and Tyrpl-Tri-specific NK cell EngagerTherapy (TriNKET™) (“Tyrpl-TriNKET”) was performed to analyze whether an anti -turn or response can be amplified in established B16F10 tumors. Tyrpl-TriNKET is an asymmetric antibody on mouse IgG2a backbone with one antibody binding domain specific to mouse Tyrpl tumor associated antigen and the other to murine NKG2D, generated by control Fabarm exchange reaction.

[0270] Briefly, C57BL / 6 mice (n = 10 / group) were engrafted subcutaneously (SC) with 2 x 105B16F10 melanoma cells. Once mean tumor volumes reached 100 mm3, mice were dosed intraperitoneally (IP) with isotype controls (1.35 mg / kg hlgGl QW, or 5 mg / kg rat IgG2a biweekly), DF IL-2-Fc (1.35 mg / kg QW), Tyrpl-TriNKET (5 mg / kg biweekly), or a combination of DF IL-2-Fc and Tyrpl-TriNKET at the same dose regimen as monotherapy. Individual tumor volumes are shown in FIGs. 22A-22C, with treatment days and frequencies as indicated (dotted line [DF IL-2-Fc] or arrow [Tyrpl-TriNKET]).

[0271] As shown in FIGs. 22A-22F, DF IL-2-Fc exhibited potent synergy with Tyrpl-TriNKET against Bl 6F 10 tumors, resulting in durable responses compared to either monotherapy. Combination therapy significantly increased median survival to 60 days with 20% of CRs compared to median survival of 19 days with isotype control or Tyrpl-TriNKET treatment and 26 days with DF IL-2-Fc monotherapy. There were no synergistic or additive adverse effects on tolerability, body weights, or clinical observations observed with the combination treatment of DF IL-2-Fc and Tyrpl-TriNKET. This study therefore provided evidence that DF IL-2-Fc therapy can also enhance anti-tumor efficacy when administered in combination with a TriNKET.Example 11 - Cytokine Release by DF IL-2-Fc Compared to Aldesleukin

[0272] One of the most dangerous side effects caused by the administration of immune- modulating therapies is the potential for cytokine release syndrome (CRS), which is a large, rapid release of cytokines into the blood from immune cells after treatment. The ability of DF IL-2-Fc to induce cytokine release in vitro was assessed with human PBMCs in a non- GLP study using both soluble and immobilized (wet-coated) DF IL-2-Fc.In Vitro Cytokine Release With DF IL-2 -Fc in a Soluble Format

[0273] In a preliminary assay, isolated PBMCs from 5 healthy human volunteers were stimulated in vitro for 48 hours with DF IL-2-FC up to 300 nM at 37 °C. An hlgGl-Fc(si) control and HERCEPTIN® (trastuzumab) were used as negative reference controls. In addition, aldesleukin, anti-CD3 mAh, PHA, and lipopolysaccharide (LPS) were used as positive reference controls. A Luminex platform-based 12-plex assay was used for the detection of 12 cytokine levels in PBMC supernatant samples: G-CSF, IFN-y, IL-IRA, IL- 1P, IL-2, IL-5, IL-6, IL-10, IL-12p70, MCP-1 (CCL2), MIP-ip (CCL4), and TNF-a. Positive and negative controls performed as expected, except for IL-12p70. Neither of the positive controls for IL-12p70 worked, so the analysis of the IL-12p70 response was excluded.

[0274] In general, aldesleukin demonstrated pan cytokine increases starting at the lowest tested concentration of 0.3 nM. No statistical differences between DF IL-2-FC and isotype control were observed due to high donor variability; however, there was a trend with DF IL- 2-FC for cytokine increase. Starting at 3 nM, cytokines were elevated by soluble DF IL-2-FC in a dose-dependent manner (FIGs. 23A-23K), except for G-CSF, IL-6, and IL-ip. DF IL-2- FC also induced cytokine secretion in the wet-coated assay format (data not shown), with changes similar to those observed in the soluble format.

[0275] Based on the data, 3 nM was defined as the Minimal Biologically Active Concentration for the cytokine release assay. The cytokines most related to IL-2-driven toxicity (IL-5, IL-6, MCP-1, TNF-a, and IL-ip) were generally detected at lower levels in the presence of DF IL-2-Fc compared to reference controls, with statistically significant differences between DF IL-2-Fc and aldesleukin seen in the release of IL-6, MCP-1, TNF-a, and IL-ip in the soluble assay format. Therefore, preliminary human in vitro cytokine release assays with DF IL-2-Fc up to 300 nM demonstrated a reduced potential for cytokine release compared to a molar equivalent of aldesleukin. The results of the CRA (soluble format) are summarized in TABLE 9.

[0276] TABLE 9: In Vitro Cytokine Release With DE IL-2-Fc in a Soluble FormatCone.: concentration; NA; not applicable; SD: standard deviation.Example 12 - In Vivo Monkey Safety Pharmacology and Pharmacodynamics

[0277] Briefly, DF IL-2-Fc was administered by IV bolus from 30 jj.g / kg up to 900 pg / kg, with the maximum tolerated dose (MTD) being 300 pg / kg. Pharmacodynamic (PD) endpoints included assessment of lymphocyte expansion by hematology and immunophenotyping, as well as measurement of cytokine induction. Safety pharmacology endpoints included assessment of neurological function, body temperature assessment, blood pressure, and cardiac function.Cytokine release in cynomolgus monkeys treated with a single dose of DF IL-2 -Fc

[0278] After a single dose in cynomolgus monkeys up to 300 pg / kg, DF IL-2-Fc caused an increase in IL-IRA, IL-5, and MCP-1 from the 24- to 96 / 120-hour time point, which resolved (FIGs. 24A-24C). There was a potential increase in IL- 10 at 48 hours postdose that was of uncertain test article relationship. There was no increase in GM-CSF, IFN-y, IL-12 / 23 (p40), IL-13, IL- 17 A, IL-ip, MIP-ip, IL-6, IL-8, or TNF-a. There was a transient increase in IL-2 thought to be due to DF IL-2-Fc cross-reactivity to the IL-2 enzyme-linked immunosorbent assay (ELISA). In a repeat-dose setting, DF IL-2-Fc demonstrated similar cytokine kinetics as those observed in the single-dose monkey study, although an increase in MIP-ip was also observed. DF IL-2-Fc also caused an increase in peripheral cytokines (MCP-1, IL-5, IL- IRA, and MIP-ip) in monkeys that was associated with lymphocyte expansion and moderate IL-2Ra pharmacology but did not increase most cytokines tested orcytokines associated with CRS (e.g., TNF-a or IL-6). The cytokine results were generally similar up to 900 pg / kg, although a smaller cytokine panel was used for analysis.Expansion of Lymphocytes and Eosinophils in Cynomolgus Monkeys And Ki-67 Expression in Immune Cells in Cynomolgus Monkeys After Multiple Doses ofDFIL-2-Fc

[0279] After a single dose of DF IL-2-Fc in cynomolgus monkeys, a decrease in lymphocytes (nadir) was observed 24 hours after dosing that subsequently recovered and then expanded, with peak lymphocytes at Day 8 that generally returned to baseline by 14 days postdose (FIG. 25A). Immunophenotyping demonstrated a dose-dependent increase in Ki-67 expression (FIGs. 26A-26E) and expansion of lymphocytes, including Tregs, NK cells, and CD4+and CD8+T cells, with peak increase on Day 8 (FIGs. 27A-27E). The lymphocyte expansion generally resolved by 14 days postdose. In a repeat-dose setting, there was profound lymphocyte expansion with earlier peak expansion on Days 5-6 after repeat dosing, including Tregs, NK cells, and CD4+and CD8+T cells, with no evidence of attenuation or impact of anti-drug antibodies (ADA).

[0280] Eosinophils also demonstrated nadir at 24 hours after initial dose with a later peak expansion on -Day 14 postdose (FIG. 25B). In a repeat-dose setting, eosinophils remained elevated in some monkeys and at some dose levels. The increase in eosinophils correlated to increased IL-5 and known IL-2 alpha pharmacology (Van Gool et al. (2014) “Interleukin-5- producing group 2 innate lymphoid cells control eosinophilia induced by interleukin-2 therapy.” BLOOD 124(24):3572-6). NO negative outcomes were observed in monkeys that were attributed to the increase in eosinophils, as there was no evidence of capillary leakage syndrome (CLS), hypersensitivity, or infiltration of eosinophils into tissues as assessed by microscopy.Example 13 - Treatment of Advanced Solid Tumors Using DF IL-2-Fc

[0281] This example describes a phase 1 / lb clinical study to assess the safety, tolerability, pharmacokinetics, biological and clinical activity of DF IL-2-Fc in patients with advanced solid tumors. Patients who have advanced solid tumors have a poor prognosis and no approved or established treatment options. Given the significant unmet medical need, the risk-benefit relationship has been carefully considered in the design of this first-in-human study of DF IL-2-Fc, as well as in the selection of the population. Design of this clinical trial is illustrated in FIG. 26.Objectives

[0282] The primary objectives of the study are to determine the maximum tolerated dose (MTD) and clinical activity of DF IL-2-Fc. The secondary objectives of the study are to assess the safety and tolerability, the pharmacokinetics (PK), and the immunogenicity of DF IL-2-Fc. Exploratory objectives of the study are to characterize the pharmacodynamics (PD) of DF IL-2-Fc, to assess the relationship between DF IL-2-Fc exposure and clinical activity and safety, and to assess overall survival (OS).Study Design

[0283] This study is a Phase 1 / lb, first-in-human, multi-part, open-label, dose escalation, and dose expansion study, to investigate the safety, tolerability, PK, PD, biological and clinical activity of DF IL-2-Fc in patients with advanced (unresectable, recurrent, or metastatic) solid tumors. The study schema is presented in FIG. 28. This study consists of 2 parts:1. Part 1 Dose Escalation is composed of 2 subparts: a. 3+3 Dose Escalation b. Safety / PK / PD2. Part 2 Efficacy Expansion

[0284] Patients will receive DF IL-2-Fc as an intravenous (IV) infusion administered over 30 minutes in a volume of 50-250 mL. DF IL-2-Fc will be administered once every other week (Q2W) in 4-week treatment cycles. In the 3+3 Dose Escalation subpart, the MTD is defined as the highest dose level (DL) at which < 33% of evaluable patients experience a dose-limiting toxicity (DLT) event.Inclusion Criteria

[0285] Patients in all cohorts have the following criteria:1. Patients have signed written informed consent.2. Patients are male or female aged > 18 years3. Patients have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 at study entry and an estimated life expectancy of at least 3 months.4. Patients have adequate hematological function defined by white blood cell (WBC) count > 3 x 109 / L, with absolute neutrophil count (ANC) > 1.5 x 109 / L, lymphocyte count > 0.5 x 109 / L, platelet count > 75 x 109 / L, and hemoglobin > 9 g / dL (may have been transfused).have adequate cardiac function defined by the absence of the following: a) A clinically relevant abnormality on the electrocardiogram (ECG). b) Clinically relevant coronary artery disease or uncontrolled congestive heart failure. c) Medically uncontrolled angina pectoris. d) An implantable pacemaker or automatic implantable cardioverter-defibrillator. e) A history of risk factors for ventricular tachycardia, torsades de pointes, fainting, unexplained loss of consciousness, or convulsions. f) A history of heart failure, congestive heart failure, myocardial infarction, cardiomyopathy, uncontrolled hypokalemia, hypomagnesemia, or hypoglycemia; any evidence of conduction abnormality (e.g., increased QRS complex). g) Congenital long QT syndrome or a prolonged QTc interval mean on screening ECG. h) QTc more than 470 msec. i) History of myocardial infarction within 6 months before the first dose of study drug. j) Received sotalol within 10 days of the first dose of treatment, or received a medication known to prolong the ECG QT interval within 14 days of the first dose of treatment. k) A heart rate of <50 or >100 bpm at rest on screening ECG. l) Patients aged more than 50 years should have a cardiac stress test. m) Note: Patients with history of coronary artery disease should have a thallium stress test and be cleared to participate in the study. have normal pulmonary function, established as follows: a) Patients aged less than 40 years without medical history of pulmonary disease: no testing. b) Patients aged more than 40 years, history of smoking, or pulmonary disease: determination of FEV (minimal FEV required: 75%). have adequate hepatic function defined by: a) A total bilirubin level < 1.5x ULN, AST level < 2.5* ULN, and ALT level < 2.5x ULN. b) Patients with documented metastatic disease to the liver: AST and ALT levels < 5x ULN. c) Patients with known Gilbert Disease who have serum bilirubin level < 3x ULN.8. Patients have adequate renal function, defined by a creatinine clearance > 60 mL / min according to the Cockcroft-Gault formula, or another suitable formula such as CKD-EPI, cystatin C equation or measurement method (Europe 51Cr-EDTA is available, not in USA).9. Patients have effective contraception: a) For women of child-bearing potential (WOCBP) patients as defined by World Health Organization (WHO) guidelines for 1 “highly effective” method or 2 “effective” methods. b) Effective contraception for men: Patients must not donate sperm during the study and for 3 months after the last treatment administration; patients who have partners of childbearing potential must use adequate methods of birth control with barrier protection, deemed acceptable by the Investigator during the study and for 3 months after last study drug administration.

[0286] Patients in the 3+3 Dose Escalation Cohorts have the following criteria:10. Histologically or cytologically proven locally advanced or metastatic solid tumor, for which no standard therapy exists, or standard therapy has failed.11. Evidence of objective disease (but participation does not require a measurable lesion).12. Archived tumor biopsy. If archival tissue is unavailable, a fresh tumor biopsy is required, obtained within the screening window.

[0287] Patients in the Safety / PK / PD Cohorts have the following criteria:13. Histologically or cytologically proven locally advanced or metastatic solid tumor from the following list, where standard therapy does not exist or has failed: a) Melanoma b) HPV-positive advanced malignancies c) Ovarian cancer d) Head and neck cancer e) Lung cancer (non-small-cell lung cancer [NSCLC]) f) Renal cell carcinoma (RCC) g) Other tumor types may be eligible after discussion with the Sponsor medical monitor14. Disease must be measurable with at least 1 unidimensional measurable lesion by Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1).15. A fresh tumor biopsy must be obtained during the screening window and on-treatment:a) Acceptable samples include core needle biopsies for deep tumor tissue (minimum 3 cores) or excisional, incisional, punch, or forceps biopsies for cutaneous, subcutaneous, and / or mucosal lesions. b) Fine-needle aspiration, brushing, cell pellet from pleural effusion, bone metastases, and lavage samples are not acceptable. c) Tumor tissue from bone metastases is not evaluable for PD-L1 expression, therefore not acceptable. d) Patients must be able and willing t o have both a screening and on-treatment biopsy.

[0288] Inclusion Criteria for Efficacy Expansion:16. Disease must be measurable with at least 1 uni dimensional measurable lesion by RECIST 1.1.17. Patients must have received an anti-PD-1 monoclonal antibody (mAb) and a B-Raf inhibitor if the tumor carries a BRAF-activating mutation and have progressed after the last line of treatment. Patients may have received an anti-LAG3 in combination with nivolumab or an anti-CTLA-4. Pre-treatment requisites and disease progression demonstration must meet the following criteria: a) Patients must have documented evidence of progressive disease by RECIST1.1, on or within 12 weeks of receiving the last dose of prior approved anti-PD-1 therapy. b) Patients must have received immune checkpoint inhibitor therapy. c) Patients whose disease is BRAFV600 mutation positive must have objective evidence of disease progression within 24 weeks of the last dose of BRAF and / or MEK inhibitor therapy for unresectable or metastatic melanoma.18. A fresh tumor biopsy must be obtained during the screening window and on-treatment: a) Acceptable samples include core needle biopsies for deep tumor tissue (minimum 3 cores) or excisional, incisional, punch, or forceps biopsies for cutaneous, subcutaneous, and / or mucosal lesions. b) Fine-needle aspiration, brushing, cell pellet from pleural effusion, bone metastases, and lavage samples are not acceptable. c) Tumor tissue from bone metastases is not evaluable for PD-L1 expression, therefore not acceptable. d) Patients must be able and willing to have both a screening and on-treatment biopsy.Exclusion Criteria

[0289] Patients meeting any of the following criteria are not eligible for study participation:

[0290] For All Cohorts:1. Patients receiving chemotherapy, radiotherapy (other than palliative bone-directed radiotherapy), major surgery, or receiving another therapeutic agent within 28 days before the start of study drug or within 5 half-lives of the previous therapeutic agent (if known), whichever is shorter.2. Concurrent anticancer treatment (e.g., cytoreductive therapy, radiotherapy [except for palliative bone-directed radiotherapy], immune therapy, or cytokine therapy [except for erythropoietin]), major surgery (excluding prior diagnostic biopsy), concurrent systemic therapy with steroids or other immunosuppressive agents, or use of any investigational drug within 28 days before the start of treatment or within 5 half-lives of the previous therapeutic agent (if known), whichever is shorter. Short-term administration of systemic steroids (e.g., for allergic reactions or the management of immune-related adverse events [irAEs]) is allowed, a) Note: Patients receiving bisphosphonate or denosumab are eligible, provided treatment was initiated at least 14 days before the first dose of DF IL-2-Fc.3. Previous malignant disease (other than the target malignancy to be investigated in this study) within the last 3 years, with the exception of basal or squamous cell carcinoma of the skin, low-grade prostate cancer (Gleason score < 6 and must be Stage I or II), or cervical carcinoma in situ.4. Life expectancy of less than 3 months.5. Patients with brain metastases are excluded, unless all of the following criteria are met: a) Central nervous system (CNS) lesions are asymptomatic and previously treated. b) Patient does not require ongoing daily steroid treatment for replacement for adrenal insufficiency (except < 10 mg prednisone [or equivalent]). c) Imaging demonstrates stable disease 28 days after last treatment.6. Receipt of any organ transplant, including autologous or allogeneic stem-cell transplantation.7. Patients must not have received aldesleukin or any other experimental IL-2 based drug.8. Significant acute or chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection during the screening window, as well as historic positive for human immunodeficiency virus (HIV).9. Patients with known HIV infection: a) Must have CD4+ T-cell (CD4+) counts > 350 cells / pL. b) Must have no history of AIDS-related opportunistic infections. c) Should be on an established antiretroviral therapy for at least four weeks and have an HIV viral load < 400 copies / mL prior to enrollment.10. Preexisting autoimmune disease (except for patients with vitiligo) needing treatment with systemic immunosuppressive agents for more than 28 days within the last 3 years, or clinically relevant immunodeficiencies (e.g., dysgammaglobulinemia or congenital immunodeficiencies). Patients with a history of immune-related endocrinopathies (e.g., hypothyroidism, hyperthyroidism, type 1 diabetes mellitus, and adrenal insufficiency) that are stable on hormone replacement therapy are eligible for this study.11. Patients with a known medical history of keratitis, ulcerative keratitis, or corneal perforation.12. Known severe hypersensitivity reactions to mAbs (> Grade 3 as defined by NCI-CTCAE v5.0), any history of anaphylaxis, or uncontrolled asthma (z.e., 3 or more features of partly controlled asthma).13. Persisting toxicity related to a prior therapeutic agent > Grade 1 as defined by NCI- CTCAE v5.0 (however, < Grade 2 alopecia, < Grade 2 endocrinopathies, and < Grade 2 sensory neuropathy are acceptable).14. Patients with prior anti-PD-1 or anti-PD-Ll treatment are eligible for the study, unless they have experienced any of the following: a) Grade 3 or 4 treatment-related toxicity during an anti-PD-1 or anti-PD-Ll treatment (excluding Grade 3 or 4 immune-related endocrinopathies adequately controlled with hormone replacement therapy). b) Grade 2 treatment-related toxicity that impacted either the lungs or the nervous system during an anti-PD-1 or anti-PD-Ll treatment (patients with a history of Grade 2 peripheral neuropathy, or paresthesia, not related to anti-PD-1 or anti-PD-Ll treatments are eligible).15. Pregnancy or lactation during the study.16. Known alcohol or drug abuse.17. Severe dyspnea at rest due to complications of advanced malignancy.18. Requiring supplementary oxygen therapy.19. All other significant diseases (e.g., inflammatory bowel disease [IBD]) which, in the opinion of the Investigator, might impair the patient’s ability to participate.20. Any psychiatric condition that would prohibit the understanding of informed consent.21. Legal incapacity or limited legal capacity.22. Incapable of giving signed informed consent, which includes compliance with the requirements and restrictions listed in the ICF and in the protocol.Toxicity AssessmentDosage and AdministrationDF IL-2-FcRational

[0291] The starting dose of 10 pg / kg was selected as it was determined to have a minimal pharmacological effect and safe to use. The patient population planned to be enrolled in the study have advanced cancers, therefore it is also important to minimize the number of patients treated at non-therapeutic doses.

[0292] The starting dose of 10 pg / kg / 2 weeks for DF IL-2-Fc has been calculated by considering all available data.

[0293] DF IL-2-Fc is a recombinant product that is an immune agonist, designed to be used as a treatment for patients with advanced malignancies.

[0294] The DF IL-2-Fc moiety and aldesleukin sequences are very similar, with only 7 amino acid substitutions, and target the same receptors (IL-2Ra [CD25], IL-2RP [CD122], and IL-2YC [CD 132] chains), but with different affinities, as a consequence of the sequence modifications. Specifically, DF IL-2-Fc shows increased stimulation, as measured by STAT5 phosphorylation, of naive CD8+ T cells and NK cells, with reduced potency on Tregs compared to that of rhIL-2. In addition to being able to more potently activate naive T cells, DF IL-2-Fc maintains the critical function of expanding activated CD8+ T cells that express the high affinity IL-2R, with the same potency as rhIL-2, and therefore maintains this critical aspect of anti-tumor responses, despite its increased safety profile.

[0295] Cynomolgus monkeys experience the same toxicity as humans when exposed to aldesleukin. In particular, monkeys recapitulated the CLS phenomenon, which was one of the key drivers of the toxicity of aldesleukin in humans. There was no overt sign of CLS with DF IL-2-Fc at doses up to 900 pg / kg.

[0296] DF IL-2-Fc has a lesser affinity for CD25 than that seen with aldesleukin. This difference is important because a major toxicity of aldesleukin is hypothesized to be caused by the binding of the molecule to the CD25 expressed by endothelial cells.Drug Product and Dosing

[0297] DF IL-2-Fc has a has a theoretical polypeptide mass of 66.9 kDa. Due to N- linked glycosylation in both chains (2 conserved sites), the observed mass of DF IL-2-Fc is approximately 69.8 kDa. The DF IL-2-Fc drug product is a clear, colorless solution free of visible particulates. The drug product consists of 2 mg / mL of DF IL-2-Fc in a 20 mM citrate buffer, pH 6.2, with 6% (w / v) sucrose, 0.01% (w / v) polysorbate 80. DF IL-2-Fc is supplied in a nominal volume of 5 mL. The drug product contains no preservative and is for single use only.

[0298] DF IL-2-Fc drug product is administered as an IV infusion over 30 minutes, in a volume of 50-250 mL. DF IL-2-Fc drug product is administered once every other week (Q2W) in 4-week treatment cycles.

[0299] DF IL-2-Fc drug product is diluted prior to use. Refer to the DF IL-2-Fc Pharmacy Manual for preparation, storage, and administration instructions.

[0300] A starting dose of DF IL-2-Fc of 10 pg / kg 2-weekly has been defined using the in vivo and in vitro data generated with DF IL-2-Fc, including the non-GLP 4-week toxicology study. A dose of 10 pg / kg DF IL-2-Fc would be the molar equivalent of ~2 pg / kg aldesleukin, which is well tolerated (Yang 2003).

[0301] The dose of DF IL-2-Fc is calculated based on the actual body weight of the patient at baseline (C1D1). The patient’s calculated dose needs only be recalculated if the patient’s actual body weight changes by >10% since the time of their last dose calculation.

[0302] Treatment with DF IL-2-Fc will continue until confirmed progression, unacceptable toxicity, withdrawal of consent, or any criterion for withdrawal from the study or treatment occurs for a maximum treatment duration of 2 years.CohortsDose Escalation Cohorts

[0303] Part 1 of this study (Phase 1) is a 3+3 Dose Escalation subpart, followed by a Safety / PK / PD subpart. The target population for each subpart is patients with advanced (unre sectable, recurrent, or metastatic) solid tumors who have progressed on standard-of-care therapy or are intolerant of standard therapy(ies). To be eligible for the 3+3 Dose Escalation subpart of the study, patients must present with a histologically or cytologically provenlocally advanced or metastatic solid tumor, for which no standard therapy exists, or standard therapy has failed.

[0304] Each patient stays on the dose level (DL) assigned at study entry (only dose adjustments for >10% changes in actual body weight are permitted).

[0305] The starting dose of 10 pg / kg, administered as an infusion over 30 minutes, was established based on all available data, primarily:• Results from the non-Good Laboratory Practice (GLP) 4-week repeat-dose DF IL-2-Fc toxicology studies: The duration of the 4-week GLP study was sufficient for hazard identification of DF IL-2-Fc, manifestation of IL-2-related toxi cities, and support of the clinical development plan. The DF IL-2-Fc toxicology profile is consistent with expected IL-2 pharmacology, as seen with aldesleukin and other IL-2 muteins, and is expected to be manageable, monitorable, and reversible. Routine monitoring of clinical pathology and cardiac function is recommended. Overall, the nonclinical studies support the first-in-human investigation of DF IL-2-Fc for the treatment of patients with various solid tumor types and patients with advanced (unresectable, recurrent, or metastatic) melanoma that have progressed after at least 1 line of anti-PD-1 treatment.• Results of in vitro cytokine release assays with DF IL-2-Fc.• Determination of a minimum anticipated biological effect level (MABEL) for DF IL-2- Fc.• Existing clinical data regarding the safety of the administration of PROLEUKIN® (aldesleukin) and other formats of interleukin-2 (IL-2).

[0306] Dose escalations are no greater than a 3-fold increase following a standard 3+3 design. For each 3+3 Dose Escalation cohort, 28-day dose-limiting toxicity (DLT) criteria, safety, and tolerability will be reviewed, and dose escalations will be determined by an SMC composed of Investigators and the Sponsor. If 0 / 3 patients treated at a single DL experience DLTs within the 28-day Cycle 1, the safety monitoring committee (SMC) may recommend dose escalation to the next DL. If 1 / 3 patients experience a DLT during the 28-day Cycle 1, the cohort will be expanded to a total of 6 patients. If 2 or more DLTs are observed in up to 6 patients in any DL, that DL has exceeded the MTD and there will be no further dose escalation. Interim DLs may be evaluated using the same criteria. The highest DL tested that has fewer than 2 DLTs in up to 6 patients is the MTD.

[0307] The 3+3 Dose Escalation design is planned at the following DLs (TABLE 10): TABLE 10aAny DL in dose escalation may be expanded to an additional 3 patients (maximum of 6; the SMC may recommend enrolling more than 6 patients at each DL, based on emerging data) based on emerging data, including in the absence of a safety signal. De-escalation to a lower or intermediate dose may also be considered by the Sponsor in consultation with the SMC. For this purpose, the following dose levels are defined a priori:• DL3 : 60 pg / kg• DL4: 150 pg / kg• DL5: 400 pg / kg• DL6: 600 pg / kg• DL7: 800 pg / kg

[0308] A schematic of the protocol is provided in FIG. 28 with 3+3 Dose Escalation levels DL1, DLla, DL2, DL3, DL4, DL5, and DL6.

[0309] Patients enrolled in the same cohort and at the same DL should receive C1D1 at least 3 days (72 hours) apart.

[0310] The Sponsor and SMC may recommend that any DL at or below the MTD be further expanded to an additional 3 patients during the dose escalation procedure on the basis of emerging data, including in the absence of a safety signal. De-escalation to a lower or intermediate.

[0311] Initially, up to 3 patients are to be enrolled in each cohort. After the first patient is enrolled, the second will be enrolled no sooner than 3 days after the first infusion of DF IL-2- Fc to the first patient. After the second patient is enrolled, the third will be enrolled no sooner than 3 days after the first infusion of DF IL-2-Fc to the second patient.

[0312] There is no predefined interval between treatment start in the event there is a need to enroll more than 3 patients in a particular dose cohort (e.g., in the event of a DLT observed in the first 3 patients), unless a > Grade 2 infusion reaction or CRS, or any other treatment- related toxicity > Grade 3, has been observed during the 2 days following the treatment of any of the first 3 patients. In such an instance, the same predefined intervals for the first 3 patients would be repeated with the second set of 3 patients.

[0313] After 3 patients receive two DF IL-2-Fc doses and have safety evaluations performed through C1D28, if:• None of these 3 patients experience a DLT, then enrollment of the next cohort may commence with approval from the SMC.• 1 of 3 patients within a cohort experiences a DLT, then 3 additional patients are to be enrolled sequentially at that dose level. If none of the additional 3 patients has a DLT (z.e., 1 of 6 patients has a DLT), then enrollment at the next scheduled dose may commence with approval from the SMC.• If greater than 2 patients within a cohort experience a DLT, then the DLT dose level will have been reached and the previous lower dose level will be considered the MTD.Dose-limiting Toxicity (DLT) Definitions

[0314] Toxicities are graded by the Investigator using the National Cancer Institute (NCI) CTCAE v5.0 criteria.

[0315] The DLT observation period is defined as the first 28 days of treatment (Cycle 1), which includes 2 IV administrations of DF IL-2-Fc on Days 1 and 15.

[0316] A DLT during the observation period is defined as a treatment-related adverse event (AE) meeting any of the following criteria:• Any non-hematologic AE > Grade 3, deemed related or possibly related to treatment, occurring within the DLT window that does not resolve to Grade 1 or baseline within 7 days.• Any Grade 4 treatment-related hematologic AE.• Recurrent Grade 3 infusion-related reaction.• Grade 4 infusion-related reaction.• Confirmed QTc prolongation > Grade 3 occurring within the DLT window that does not resolve to Grade 1 or baseline within 7 days.• Any Grade 4 nausea or vomiting.• Any myocarditis or uveitis > Grade 2.• Dose delay of > 14 days for the study drug due to an AE.• Any death not clearly due to the underlying disease or extraneous cause.• Neutropenic fever.• Grade 3 thrombocytopenia with bleeding.• Any pneumonitis Grade 3.• Any neurotoxicity > Grade 2.• Any treatment-related hypotension > Grade 3 lasting > 48 hours following study drug administration.• Any capillary leak syndrome (CLS) > Grade 3.• Pulmonary edemas > Grade 3.• Any aspartate aminotransferase (AST) or alanine aminotransferase (ALT) elevation > 8 times the upper limit of normal (ULN), regardless of duration, and AST or ALT increase between 5 and 8 times the ULN that persists for greater than 2 weeks, all regardless of the presence or absence of liver metastasis.• Drug-induced liver injury, as defined by Hy's Law: o AST or ALT > 3 times the ULN and o Total bilirubin > 2 times the ULN and o Alkaline phosphatases < 2 times the ULN; and o No other reason for liver injury.

[0317] The following Grade 3 or 4 AEs are not be considered a DLT:• Grade 3 endocrinopathy controlled with hormone supplementation and does not result in hospitalization, dose modification, or withholding the study drug.• Grade 3 asymptomatic amylase or lipase elevations.• Grade 3 or 4 lymphopenia < 14 days in duration or not associated with clinical manifestations.• Grade 3 electrolyte imbalances / abnormalities that are not associated with clinical sequelae and are corrected with supplementation / appropriate management within 72 hours of onset.• Any Grade 3 AE of tumor flare (defined as pain at sites of known or suspected tumor) that resolves to < Grade 1 within 72 hours.• Grade 3 nausea or vomiting that can be medically managed to < Grade 2 within 72 hours Grade 3 fatigue that improves to < Grade 2 within 7 days.Safety / PK / PD Cohorts

[0318] Safety / PK / PD cohorts are opened after a DL in the 3+3 Dose Escalation subpart has been cleared. Up to 40 patients may be enrolled across all Safety / PK / PD cohorts.

[0319] The populations eligible for the Safety / PK / PD cohorts are defined as patients with a primary tumor that should be melanoma, HPV-positive advanced malignancies, ovarian cancer, head and neck cancer, lung cancer (non-small-cell lung cancer [NSCLC]), or renalcell carcinoma (RCC). Other tumor types may be eligible after discussion with the Sponsor medical monitor.

[0320] Accrual of up to 40 patients will proceed without a pre-defined DLT observation window between patients or dose levels.

[0321] The safety information generated during the treatment of these patients will be communicated to the SMC as it emerges. An alternative dosing schedule may be considered based on emerging data.

[0322] For patients enrolled in the Safety / PK / PD cohorts, mandatory tumor biopsies are performed at Screening (within 30 days before the first study drug dose) and within 3 days prior to the C1D22 (cycle 1, day 22) visit. The dose escalation procedure is summarized in TABLE 11TABLE 11. Summary of Dose Escalation Procedure

[0323] Decisions regarding dose escalation will be made based on review of data from the DLT evaluation period for patients at the currently enrolling dose level. However, safety data will also be collected from all patients throughout the study, and this will be reviewed periodically by the SMC. Any detected cumulative or late toxicity may require later dose reductions or other action as appropriate, including further refinement of the dose selected for Phase lb.Efficacy Expansion Cohort

[0324] Part two of this study is Efficacy Expansion. Once Phase 1 (3+3 Dose Escalation and Safety / PK / PD cohorts) is completed, 20 patients with advanced melanoma who have progressed after an anti-PD-1 containing regimen are planned to be enrolled into a Phase lb Efficacy Expansion cohort. The development of DF IL-2-Fc in melanoma is justified by existing data that have established that IL2 (and by extension, improved IL2 format) have clinical activity in advanced melanoma.

[0325] For patients enrolled in the Phase lb Efficacy Expansion cohort, mandatory tumor biopsies will be performed at Screening (within 30 days before the first study drug dose) and within 3 days prior to the C1D22 (cycle 1, day 22) visit.Planned Number of PatientsPart 1 Dose Escalation:

[0326] The sample size for the Dose Escalation part is typical of Phase 1 oncology studies in patients with no approved or established treatment options and is designed to minimize exposure to patients at subtherapeutic dosing levels. Therefore, the 3+3 design initiates near the expected therapeutic range.

[0327] Safety / PK / PD cohorts may be opened after a DL in the 3+3 Dose Escalation subpart has been cleared. Up to 40 patients may be enrolled across all Safety / PK / PD cohorts. During the Dose Escalation part, the following numbers of patients may be enrolled:• Up to 42 patients in the 3+3 Dose Escalation subpart• Up to 40 patients in the Safety / PK / PD subpartPart 2 Efficacy Expansion:

[0328] Once Phase 1 (3+3 Dose Escalation and Safety / PK / PD cohorts) is completed, 20 patients with advanced melanoma who have progressed after an anti-PD-1 containing regimen are planned to be enrolled into a Phase lb Efficacy Expansion cohort.

[0329] This sample size will be sufficient to establish an initial efficacy signal within advanced melanoma patients. Among a sample size of 20 patients, there is 80% power with a one-sided type I error rate of 0.025 to exclude a null hypothesis of objective response rate (ORR) of 15% if the true ORR among treated patients is 41.8%. The null hypothesis can be rejected if at least 7 / 20 responses are observed.Treatment Duration

[0330] The planned treatment duration for each patient experiencing stable disease or partial response (PR) is until confirmed disease progression, unacceptable toxicity, or any criterion for withdrawal from the study or treatment occurs or a maximum of 2 years.

[0331] Treatment beyond progression can be considered provided they are clinically stable and do not require urgent evidence based medical intervention.

[0332] Any treatment-related AEs (TRAEs) should be followed until they resolve, return to baseline, or are irreversible.Dose Modification

[0333] The dose of DF IL-2-Fc will be adjusted by the Investigator in the event of a treatment-related adverse event (TRAE).• Grade 1 TRAE: DF IL-2-Fc will be kept at the dose initially assigned to the patient.• Grade 2 TRAE: The subsequent dose of DF IL-2-Fc will be withheld if the Grade 2 TRAE has not resolved or until its grading returns to Grade 1, unless in the event of a myocarditis or uveitis. For these two TRAEs, treatment with DF IL-2-Fc will be discontinued. When possible, treatment with DF IL-2-Fc will be resumed.• Grade 3 TRAE: Grade 3 events listed as possible DLT if they occur during the DLT observation period will trigger the discontinuation of treatment with DF IL-2-Fc. Patients on DL3 and above who have reversible Grade 3 toxicities within 4 weeks may resume one DL lower. If they have further Grade 3 toxicity, they will discontinue all DF IL-2-Fc dosing. The subsequent dose of DF IL-2-Fc will be withheld if the Grade 3 TRAE has not resolved or until its grading returns to Grade 2 or lower.

[0334] The following TRAE will not necessitate a dose reduction:• Grade 3 or 4 endocrinopathy controlled with hormone supplementation and does not result in hospitalization, dose modification, or withholding the study drug.• Grade 3 asymptomatic amylase or lipase elevations.• Grade 3 or 4 lymphopenia < 14 days in duration or not associated with clinical manifestations.• Grade 3 electrolyte imbalances / abnormalities that are not associated with clinical sequelae and are corrected with supplementation / appropriate management within 72 hours of onset.• Any Grade 3 AE of tumor flare (defined as pain at sites of known or suspected tumor) that resolves to Grade < 1 within 72 hours.• Grade 3 nausea or vomiting that can be medically managed to < Grade 2 within 72 hours• Grade 3 fatigue that improves to < Grade 2 within 7 days.

[0335] In the event of Grade 4 TRAE: DF IL-2-Fc should be discontinued in the event of any Grade 4 treatment-related AE or clinically significant laboratory abnormality, with the exception of the following events, which do not require discontinuation:• Grade 4 neutropenia < 7 days.• Grade 4 lymphopenia or leukopenia < 14 days in duration.• Isolated Grade 4 amylase or lipase abnormalities that are not associated with symptoms or clinical manifestations of pancreatitis and decrease to Grade < 4 or return to baseline within 7 days.• Grade 4 endocrinopathy (e.g., diabetic ketoacidosis [DKA] and hypophysitis): o Per the most recent guidance (Schneider 2021) on irAE management, an exception may be made for Grade 4 endocrinopathy e.g., DKA and hypophysitis) that has been treated and patient becomes stable on hormone replacement, with Sponsor approval.

[0336] Retreatment with DF IL-2-Fc is contraindicated in patients who have experienced the following treatment-related toxi cities while receiving an earlier course of therapy:• Sustained ventricular tachycardia (> 5 beats).• Cardiac arrhythmias not controlled or unresponsive to management.• Chest pain with ECG changes, consistent with angina or myocardial infarction• Cardiac tamponade.• Any Grade > 2 cardiac, pulmonary or neurologic TRAE, with the exception of peripheral neuropathy or paresthesia.• Any Grade 3 TRAEs that require interruption of treatment for 42 days or longer.• Patients with progressive disease.Analysis Cut-Off Dates

[0337] The final data cut-off will be 1 year after the last dose of DF IL-2-Fc has been administered or all patients have stopped treatment plus 28 days.Definition of End of Study

[0338] The end of the study will be defined as 1 year after the last patient completes his / her EOT visit.If the study is not terminated for a reason given above, the end of the study is defined as 1 year after the last patient has received the last dose of DF IL-2-Fc.Study EndpointsPrimary Endpoints

[0339] Dose Escalation (Part 1)• Occurrence of DLTs during the first 4 weeks of treatment with DF IL-2-Fc.

[0340] Efficacy Expansion (Part 2)• Objective response rate (ORR), duration of response (DOR), and progression free survival (PFS) as determined per Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria.Secondary Endpoints

[0341] Dose Escalation (Part 1)• Number, severity, and duration of treatment-emergent adverse events (TEAEs), treatment-related adverse events (TRAEs), and serious adverse events (SAEs) per Common Terminology Criteria for Adverse Events (CTCAE) v5.0 (cytokine release syndromes will be reported using American Society for Transplantation and Cellular Therapy [ASTCT] criteria)• Adverse events of special interest (AESI) and adverse events (AEs) leading to study and treatment discontinuation.

[0342] Dose Escalation (Part 1) and Efficacy Expansion (Part 2)• PK parameters (e.g., AUCo-t, Cmax, Cmin, tmax, t' / z).• Immunogenicity determined by serum titers of anti-drug antibodies (AD As) of DF IL- 2-Fc.Exploratory Endpoints

[0343] Dose Escalation (Part 1) and Efficacy Expansion (Part 2)• To evaluate the change from baseline in tumor and peripheral blood pharmacodynamic biomarkers and the relationship to DF IL-2-Fc PK.• To evaluate the association between baseline and on-treatment biomarkers in tumor and peripheral blood, and best objective response ([BOR]; complete response [CR], partial response [PR], stable disease [SD], or progressive disease).• Assess exposure-safety and exposure-efficacy relationships.• Assess potential correlation between PD / safety and PD / efficacy.• OS from initial treatment to death from any cause.

[0344] Additional safety endpoints include:• Clinical laboratory tests• ECGs• Vital signs• ECOG performance statusPharmacokinetic Endpoint Parameters• AUCO-t: Area under the concentration-time curve from the time of dosing to the time of the last observation (calculated by linear trapezoidal summation).• AUCO-co: Area under the curve from the time of dosing extrapolated to infinity (calculated by the linear trapezoidal summation and extrapolated to infinity using Clast / Xz).• +z: Terminal elimination rate constant. The value of Xz is determined from the slope of the regression line of log (concentration) vs. time with the following constraints: (i) there must be at least 3 consecutive measurable concentrations, (ii) all concentrations must be declining with time, and (iii) the correlation coefficient (r) of regression must be >0.95• Cmax: Maximum serum concentration observed postdose• Cmin: Minimum serum concentration observed postdose immediately before the second dose• tmax: Time at which Cmax occurs• tl / 2: Elimination half-life, determined as 0.693 / XzPharmacodynamic Endpoints (Biomarkers)Peripheral Biomarkers:

[0345] Biomarkers of immune activation will be measured to demonstrate that the study drug is biologically active.

[0346] Two sets of peripheral parameters will be assessed in all patients enrolled in Phase 1 (3+3 Dose Escalation and Safety / PK / PD) and in Phase lb Efficacy Expansion cohorts: Cellular parameters:• Peripheral blood mononuclear cells (PBMCs) for immunophenotyping (IPT) by flow cytometry.Soluble factors:• Cytokines and chemokines panel will include factors associated with immune activationBiomarkers Derived From Tumors:

[0347] Tissue-derived biomarkers will be evaluated on the mandatory screening and on- treatment biopsies from patients enrolled in the Phase 1 Safety / PK / PD and Phase lb Efficacy Expansion cohorts. Biomarkers may include, but may not be limited to, immunophenotyping by immunohistochemistry (IHC) and multiplex-immunofluorescence (mIF) to address immune cell activation and exhaustion.

[0348] For patients enrolled in the Phase 1 Safety / PK / PD and Phase lb Efficacy Expansion cohorts, as well as patients in the Phase 1 3+3 Dose Escalation cohorts whoconsented to the optional on-treatment biopsy, assessments will include, but may not be limited to:• Level of PD-L1 expression by IHC.• CD8+ cells density by IHC.• mIF panel.Statistical ConsiderationsSample Size:Part 1 Dose Escalation:

[0349] The sample size for the Dose Escalation part is typical of Phase 1 oncology studies in patients with no approved or established treatment options and is designed to minimize exposure to patients at subtherapeutic dosing levels. Therefore, the 3+3 design initiates near the expected therapeutic range.

[0350] Safety / PK / PD cohorts may be opened after a DL in the 3+3 Dose Escalation subpart has been cleared. Up to 40 patients may be enrolled across all Safety / PK / PD cohorts.

[0351] During the Dose Escalation part, the following number of patients may be enrolled:• Up to 42 patients in the 3+3 Dose Escalation subpart• Up to 40 patients in the Safety / PK / PD subpartPart 2 Efficacy Expansion:

[0352] Once Phase 1 (3+3 Dose Escalation and Safety / PK / PD cohorts) is completed, 20 patients with advanced melanoma who have progressed after an anti-PD-1 containing regimen are planned to be enrolled into a Phase lb Efficacy Expansion cohort.

[0353] This sample size will be sufficient to establish an initial efficacy signal within advanced melanoma patients. Among a sample size of 20 patients, there is 80% power with a one-sided type I error rate of 0.025 to exclude a null hypothesis of ORR of 15% if the true ORR among treated patients is 41.8%. The null hypothesis can be rejected if at least 7 / 20 responses are observed.Analysis Sets:Safety Population:

[0354] All patients who receive at least 1 dose of study drug. Patients will be grouped as treated.DLT-Evaluable Population:

[0355] All patients in the Safety Analysis Set that are enrolled in the 3+3 Dose Escalation subpart of the study and complete the DLT-observation period (28-days, Cycle 1) or discontinue early due to DLT or other treatment-related event.Pharmacokinetic Population:

[0356] All patients with a baseline PK measurement, who receive at least 1 dose of study drug, and have sufficient post-dose measurements for PK analysis. Patients will be grouped as treated.

[0357] Additional analysis sets may be defined within a separate Statistical Analysis Plan (SAP).Statistical Methods:

[0358] All collected data will be reported using summary tables and figures, as appropriate. Data summaries will be presented by dose level and part of study. Tabulations will be produced for appropriate disposition, demographic, baseline, safety, efficacy, and PK / PD parameters. Categorical variables will be summarized by frequency distributions (number and percentage of patients) and continuous variables will be summarized by descriptive statistics (mean, standard deviation, median, minimum, and maximum). For time- to-event variables, percentages of patients experiencing that event will be presented and median time to event will be estimated using Kaplan-Meier methodology. As appropriate, a 95% confidence interval (CI) will be presented.

[0359] All data will be provided in by-subject data listings.

[0360] A detailed statistical analysis plan (SAP) will provide additional information surrounding statistical considerations.Safety Analysis

[0361] Safety analyses, including reporting of DLTs, adverse events, clinical laboratory assessments, ECGs, vital signs, ECOG, and concomitant medications, will be summarized using descriptive statistics.Efficacy Analysis

[0362] RECIST 1.1 will be used for determination of tumor response and establishment of initial evidence of efficacy. In determining the objective response rate (ORR), a minimum 4-week metric will be used to qualify stable disease and to confirm CR, PR, or progressive disease.

[0363] ORR will be defined by confirmed complete or partial responses and will be summarized as the percentage of patients achieving CR or PR and presented with a Clopper- Pearson exact 95% CI.

[0364] DOR will be defined as the time from first response (CR or PR) to the date of the first documented tumor progression as determined according to RECIST 1.1 or death due to any cause, whichever occurs first.

[0365] PFS will be defined as the time from first administration of study drug until first observation of progressive disease or death, when death occurs within 12 weeks of the last tumor assessment or last administration of study drug (whichever is later).

[0366] OS will be defined as the time from first administration of study drug to death.

[0367] Median PFS and OS will be presented in patient listings and analyzed using theKaplan -Meier method. In addition, the number and percentage of patients achieving 12- month PFS and 12-month OS will be reported.Pharmacokinetics, Pharmacodynamics, and Biomarkers

[0368] Pharmacokinetic concentration parameters will be estimated using noncompartmental analysis (NCA) and summarized using descriptive statistics.

[0369] Descriptive summaries of PD parameters and biomarkers will also be presented. Assessment of EfficacyCT or MRI Scans for Tumor Assessment during Screening

[0370] A CT scan or MRI (if MRI is used, CT of chest is mandatory) of the chest, abdomen, and pelvis (at a minimum and other established assessments of tumor burden if CT / MRI imaging is not sufficient for the individual patient; other regions as specifically required for specific tumor indications) will be performed within 28 days prior to study drug start in order to document the baseline status of the tumor disease using RECIST 1.1 target and non-target lesions. However, if the results of a CT scan or MRI performed within 4 weeks prior to first treatment are available, the Screening CT / MRI does not need to be performed.

[0371] A brain CT / MRI scan (contrast preferred) during Screening and on study is only required if clinically indicated.Tumor Imaging and Disease Response Assessment

[0372] For all patients in all cohorts, tumor response assessment will be performed by CT scan or MRI (if MRI is used, CT of chest is mandatory) imaging of the chest / abdomen / pelvis (plus other regions as specifically required for specific tumor types) and other established assessments of tumor burden if CT / MRI imaging is insufficient for the individual patient. All the scans performed at baseline and other imaging performed as clinically required (other supportive imaging) need to be repeated at subsequent visits. In general, lesions detected atbaseline need to be followed using the same imaging methodology and preferably the same imaging equipment at subsequent tumor evaluation visits.

[0373] Skin metastasis can be used as target lesions according to RECIST 1.1 using measurements by caliper, if they fulfill RECIST 1.1 for target lesions as described below. The presence of new cutaneous lesions will be considered diagnostic of progression for RECIST 1.1, even if not imaged.

[0374] For each patient, the Investigator will designate 1 or more of the following measures of tumor status to follow for determining response: CT or MRI images of primary and / or metastatic tumor masses, physical examination (PE) findings, and the results of other assessments. All available images collected during the study period will be considered. The most appropriate measures to evaluate the tumor status of a patient should be used. The measure(s) to be chosen for sequential evaluation during the study have to correspond to the measures used to document the progressive tumor status that qualifies the patient for enrollment. The tumor response assessment will be performed at the timepoints designated in the SoA.

[0375] Tumor responses to treatment will be assigned based on the evaluation of the response of target, non-target, and new lesions according to RECIST 1.1.

[0376] To assess ORR, the tumor burden at baseline will be estimated and used for comparison with subsequent measurements. At baseline, tumor lesions will be categorized in target and non-target lesions.

[0377] Results for these evaluations will be recorded with as much specificity as possible so that pre- and post-treatment results will provide the best opportunity for evaluating tumor response.

[0378] Any CR or PR should be confirmed. In the case of a PR or CR, a confirmatory CT or MRI scan must be done no sooner than 28 days after the initial scan resulting in a reading of PR or CR (preferably at the scheduled 8-week interval). Confirmation of PR can be confirmed at an assessment later than the next assessment after the initial documentation of PR.

[0379] Patients who discontinue DF IL-2-Fc before disease progression will have a computed tomography (CT) / magnetic resonance imaging (MRI) performed every 12 weeks (±2 weeks) until progressive disease is declared / confirmed or the patient starts treatment with a new anti-cancer therapy.

[0380] The Investigator may perform scans in addition to a scheduled study scan for medical reasons or if the Investigator suspects progressive disease.

[0381] Collect any additional imaging that may demonstrate tumor response or progression (including scans performed at unscheduled timepoints and / or at an outside institution) for RECIST 1.1 tumor assessment.Assessment of Safety

[0382] The safety profile of DF IL-2-Fc will be assessed through the recording, reporting, and analyzing of baseline medical conditions, AEs, physical examination findings, vital signs, ECG, and safety laboratory tests. In addition, patients will be using wearable cardiac / temperature and blood pressure monitoring devices. The data collected by the wearable device will be available to the site study team for all days (cumulative) prior to each patient visit.INCORPORATION BY REFERENCE

[0383] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.EQUIVALENTS

[0384] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A variant human IL-2 protein comprising a substitution of the threonine at position 3 to an alanine, a substitution of the glycine at position 27 to a methionine, a substitution of the isoleucine at position 28 to a leucine, a substitution of the lysine at position 32 to an aspartic acid, and a substitution of the cysteine at position 125 to a serine; wherein the substitutions are relative to the amino acid sequence of wild-type human IL-2 protein represented by SEQ ID NO: 1.

2. The variant human IL-2 protein of claim 1 further comprising a substitution of the leucine at position 72 to a glutamine and a substitution of the arginine at position 81 to an aspartic acid.

3. The variant human IL-2 protein of claim 1 further comprising a substitution of the methionine at position 39 to a leucine, a substitution of the glutamic acid at position 52 to a serine, a substitution of the leucine at position 72 to an alanine, a substitution of the glutamine at position 74 to a glycine, a substitution of the serine at position 75 to an aspartic acid, a substitution of the lysine at position 76 to an aspartic acid, a substitution of the asparagine at position 77 to a proline, a substitution of the phenylalanine at position 78 to a lysine, a substitution of the histidine at position 79 to a threonine, a substitution of the leucine at position 80 to an isoleucine, a deletion of the arginine at position 81, a deletion of the proline at position 82, a substitution of the leucine at position 85 to a valine, a substitution of the isoleucine at position 86 to a valine, and a substitution of the isoleucine at position 92 to a phenylalanine.

4. A variant human IL-2 protein comprising a substitution of the threonine at position 3 to an alanine, a substitution of the glycine at position 27 to a methionine, a substitution of the isoleucine at position 28 to a leucine, a substitution of the lysine at position 32 to an aspartic acid, a substitution of the leucine at position 72 to a glutamine, a substitution of the arginine at position 81 to an aspartic acid, and a substitution of the cysteine at position 125 to a serine; wherein the substitutions are relative to the amino acid sequence of wild-type human IL-2 protein represented by SEQ ID NO: 1.

5. A variant human IL-2 protein comprising a substitution of the threonine at position 3 to an alanine, a substitution of the glycine at position 27 to a methionine, a substitution of the isoleucine at position 28 to a leucine, a substitution of the lysine at position 32 to an aspartic acid, a substitution of the methionine at position 39 to a leucine, a substitution of the glutamic acid at position 52 to a serine, a substitution of the leucine at position 72 to an alanine, a substitution of the glutamine at position 74 to a glycine, a substitution of the serine at position 75 to an aspartic acid, a substitution of the lysine at position 76 to an aspartic acid, a substitution of the asparagine at position 77 to a proline, a substitution of the phenylalanine at position 78 to a lysine, a substitution of the histidine at position 79 to a threonine, a substitution of the leucine at position 80 to an isoleucine, a deletion of the arginine at position 81, a deletion of the proline at position 82, a substitution of the leucine at position 85 to a valine, a substitution of the isoleucine at position 86 to a valine, a substitution of the isoleucine at position 92 to a phenylalanine, and a substitution of the cysteine at position 125 to a serine; wherein the substitutions are relative to the amino acid sequence of wild-type human IL-2 protein represented by SEQ ID NO: 1.

6. The variant human IL-2 protein of any one of claims 1, 2, and 4, wherein the protein comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2.

7. The variant human IL-2 protein of claim 6, wherein the protein comprises an amino acid sequence of SEQ ID NO: 2.

8. The variant human IL-2 protein of any one of claims 1, 3, and 5, wherein the protein comprises an amino acid sequence at least 95% identical to SEQ ID NO: 3.

9. The variant human IL-2 protein of claim 8, wherein the protein comprises an amino acid sequence of SEQ ID NO: 3.

10. A heterodimeric Fc-fusion protein comprising: a variant human IL-2 protein of any one of claims 1 to 9, and an IgG Fc domain.

11. The heterodimeric Fc-fusion protein of claim 10, wherein the IgG Fc domain comprises a first Fc domain polypeptide.

12. The heterodimeric Fc-fusion protein of claim 10 or 11, wherein the IgG Fc domain comprises a first Fc domain polypeptide and a second Fc domain polypeptide.

13. The heterodimeric Fc-fusion protein of any one of claims 10-12, wherein the IgG Fc domain comprises a first Fc domain polypeptide and a second Fc domain polypeptide, and wherein the first and second Fc domain polypeptides each comprise one or more different mutations promoting heterodimerization, and wherein the Fc domain first polypeptide and the second Fc domain polypeptide are bound to each other.

14. The heterodimeric Fc-fusion protein of claim 12 or 13, wherein the IgG Fc domain is derived from an IgGl, IgG2, IgG3, or IgG4 Fc domain.

15. The heterodimeric Fc-fusion protein of claim 14, wherein the IgG Fc domain is derived from a human IgGl Fc domain.

16. The heterodimeric Fc-fusion protein of any one of claims 13-15, wherein the one or more different mutations for promoting heterodimerization are selected from the group consisting of: Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, D356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, F405L, Y407A, Y407I, Y407V, K409F, K409W, K409D, K409R, T41 ID, T41 IE, K439D, and K439E, numbered according to the EU numbering system.

17. The heterodimeric Fc-fusion protein of claim 16, wherein the one or more mutations for promoting heterodimerization comprise K360E and K409W, numbered according to the EU numbering system.

18. The heterodimeric Fc-fusion protein of claim 16, wherein the one or more mutations for promoting heterodimerization comprise Q347R, D399V, and F405T, numbered according to the EU numbering system.

19. The heterodimeric Fc-fusion protein of claim 17 or 18, wherein the first Fc domain polypeptide and the second Fc domain polypeptide each comprise one or more mutations thatpromote disulfide bond formation between the first Fc domain polypeptide and the second Fc domain polypeptide.

20. The heterodimeric Fc-fusion protein of claim 19, wherein the first or second Fc domain polypeptide comprises heterodimerization mutations Q347R, D399V, and F405T, and the one or more mutations promoting disulfide bond formation comprise S354C, numbered according to the EU numbering system.

21. The heterodimeric Fc-fusion protein of claim 19, wherein the first or second Fc domain polypeptide comprises heterodimerization mutations K360E and K409W, and the one or more mutations promoting disulfide bond formation comprise Y349C, numbered according to the EU numbering system.

22. The heterodimeric Fc-fusion protein of claim 20, wherein the first or second Fc domain polypeptide comprises an amino acid sequence of SEQ ID NO: 4.

23. The heterodimeric Fc-fusion protein of claim 21, wherein the first or second Fc domain polypeptide comprises an amino acid sequence of SEQ ID NO: 5.

24. The heterodimeric Fc-fusion protein of any one of claims 12 to 23, wherein the first Fc domain polypeptide is fused to the variant human IL-2 protein, and the second Fc domain polypeptide is not fused to the variant human IL-2 protein.

25. The heterodimeric Fc-fusion protein of any one of claims 12 to 23, wherein the second Fc domain polypeptide is fused to the variant human IL-2 protein, and the first Fc domain polypeptide is not fused to the variant human IL-2 protein.

26. The heterodimeric Fc-fusion protein of any one of claims 12 to 25, wherein the first Fc domain polypeptide and the second Fc domain polypeptide each comprise one or more mutations that reduce effector functions.

27. The heterodimeric Fc-fusion protein of claim 26, wherein the first Fc domain polypeptide and the second Fc domain polypeptide each comprise one or more mutations at positions 234, 235, 237, 329, 330, and / or 331, according to the EU numbering system.

28. The heterodimeric Fc-fused protein of claim 27, wherein the first Fc domain polypeptide and the second Fc domain polypeptide each comprise one or more mutationsselected from the group consisting of L234A, L235A, L235E, G237A, P329A, A330S, and P331S.

29. The heterodimeric Fc-fusion protein of claim 27 or 28, wherein the first Fc domain polypeptide and the second Fc domain polypeptide each comprise mutations L234A, L235A, and P329A.

30. The heterodimeric Fc-fusion protein of any one of claims 10 to 29, wherein the variant human IL-2 protein is fused to the IgG Fc domain via a linker polypeptide.

31. The heterodimeric Fc-fusion protein of claim 30, wherein the linker polypeptide comprises the amino acid sequence (GGGGS)s (SEQ ID NO: 6).

32. The heterodimeric Fc-fusion protein of any one of claims 10 to 31, wherein the human IL-2 protein is fused to the C-terminus of the IgG Fc domain.

33. A heterodimeric Fc-fusion protein comprising: a first polypeptide comprising an amino acid sequence of SEQ ID NO: 7; and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 8.

34. The variant human IL-2 protein of any one of claims 1 to 9, or the heterodimeric Fc- fusion protein of any one of claims 10 to 34, wherein the variant human IL-2 protein has decreased capacity to induce pro-inflammatory cytokine production; as compared to a wildtype human IL-2 protein under comparable assay conditions, wherein the pro-inflammatory cytokine production is assayed using a cytokine release assay.

35. An isolated nucleic acid encoding a variant human IL-2 protein of any one of claims 1 to 9 and 34, or the heterodimeric Fc-fusion protein of any one of claims 10 to 34.

36. An expression vector comprising a nucleic acid according to claim 35.

37. A pharmaceutical composition comprising a variant human IL-2 protein of any one of claims 1 to 9 and 34, or the heterodimeric Fc-fusion protein of any one of claims 10 to 34, and a pharmaceutically acceptable carrier.

38. A recombinant cell comprising one or more nucleic acids according to claim 35 or one or more vectors according to claim 36.

39. A method of expanding T cells, comprising contacting the T cells with an effective amount of a variant human IL-2 protein according to any one of claims 1 to 9 and 34, the heterodimeric Fc-fusion protein of any one of claims 10 to 34, or the pharmaceutical composition of claim 37.

40. A method of treating a disease in a subject in need thereof comprising administering to the subject a variant human IL-2 protein according to any one of claims 1 to 9 and 34, the heterodimeric Fc-fusion protein of any one of claims 10 to 34, or the pharmaceutical composition of claim 37.

41. The method of claim 40, wherein the disease is a hyperproliferative disease.

42. The method of claim 40 or 41, wherein the disease is a cancer.

43. The method of any one of claims 40-42, wherein the subject has received treatment with a checkpoint inhibitor.

44. The method of claim 43, wherein the subject has progressed following treatment with the checkpoint inhibitor.

45. The method of claim 43 or claim 44, wherein the checkpoint inhibitor comprises an anti-PD-1 therapy.

46. A variant human IL-2 protein according to any one of claims 1 to 9 and 34, or the heterodimeric Fc-fusion protein of any one of claims 10 to 34, wherein the variant human IL- 2 protein or heterodimeric Fc-fusion protein is a purified variant human IL-2 protein or purified heterodimeric Fc-fusion protein.

47. The variant human IL-2 protein or heterodimeric Fc-fusion protein of claim 46, wherein the variant human IL-2 protein or heterodimeric Fc-fusion protein is purified using a method selected from the group consisting of: centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification, gel filtration, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

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