Modified interleukin 2 receptor beta agonists

Abstract

To provide modified IL2 polypeptides and fusion proteins thereof. Methods of modulating immune responses by administering modified IL2 polypeptides or fusion proteins thereof are also provided.SOLUTION: Disclosed is a modified interleukin 2 (IL2) polypeptide comprising a modified IL2R receptor beta (IL2 beta) binding region 2 comprising X1 - X2 - X3 - D - X4 - X5 - X6 - X7 - X8 - X9 - X10 - X11 - X12 - X13 (SEQ ID NO: 1). Wherein X1, X3, X6, X8, X12, and X13 each comprise any residues; X2, X4, and X10 are uncharged residues; X5, X7, X9, and X11 each comprise uncharged apolar residues. Modified IL2 polypeptides and fusion proteins thereof exhibit increased binding to IL2R β, decreased binding to IL2R α, or both.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 886,148, filed August 13, 2019, which is incorporated by reference herein in its entirety.

[0002] Sequence Listing Description The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference. The name of the text file containing the sequence listing is 300096_401WO_SEQUENCE_LISTING.txt. The text file is 230 KB, was created on August 13, 2020, and has been submitted electronically via EFS-Web. [Background technology]

[0003] Interleukin 2 (IL2) is a cytokine that regulates lymphocyte proliferation and activation. It is 133 amino acids long and its structure contains four antiparallel amphipathic C-helices. IL2 mediates its actions by binding to the IL2 receptor (IL2R), which contains up to three distinct subunits. Association of all three subunits, namely the interleukin 2 receptor alpha chain (IL2Rα, or CD25), the interleukin 2 receptor beta chain (IL2Rβ, or CD122), and the interleukin 2 receptor gamma chain (IL2Rγ, or CD132), results in the trimeric IL2Rαβγ, a high-affinity receptor for IL2. Association of the IL2Rβ and IL2Rγ subunits results in the dimeric receptor IL2Rβγ, referred to as the intermediate-affinity IL2R. The IL2Rα subunit forms the monomeric, low-affinity IL2 receptor. Expression of IL2Rα is involved in the expansion of immunosuppressive regulatory T cells (Tregs), whereas dimeric IL2Rβγ is involved in the expansion of cytolytic CD8 in the absence of IL2Rα. + It can cause proliferation and death of T cells and NK cells. Summary of the Invention

[0004] The present disclosure provides modified IL2 polypeptides that have improved binding to IL2Rβ compared to wild-type IL2 and / or reduced binding to IL2Rα compared to wild-type IL2.

[0005] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: 10 -X 11 -X 12 -X 13 a modified interleukin 2 (IL2) polypeptide comprising a modified IL2 receptor beta (IL2Rβ) binding region 2 comprising: (SEQ ID NO: 1); In the formula, X1, X3, X6, X8, X 12 , and X 13 each containing any residue, X2, X4, and X 10 is an uncharged residue, X5, X7, X9, and X 11 each contains an uncharged apolar residue, The modified IL2 polypeptide has a K that is at least 10-fold greater than that of wild-type IL2. D and binds to IL2Rβ.

[0006] In certain embodiments, X1 is an uncharged polar residue, an uncharged apolar residue, a basic residue, or an acidic residue, and X1 is selected from C, T, G, W, I, S, E, and K, or X1 is selected from G, K, E, C, and T. In certain embodiments, X2 is an uncharged polar residue or an uncharged apolar residue, and X2 is selected from Y, P, V, W, L, A, and G, or X2 is selected from V, P, W, and A. In certain embodiments, X3 is an uncharged polar residue, an uncharged apolar residue, a basic residue, or an acidic residue, and X3 is selected from S, T, Q, G, M, E, R, and K, or X3 is selected from T, G, S, R, and E. In certain embodiments, X4 is not L, X4 is an uncharged apolar residue or an uncharged polar residue, or X4 is selected from A, V, S, and T. In certain embodiments, X5 is selected from I, L, T, and V, or X5 is selected from I and V. In certain embodiments, X6 is an uncharged polar residue, a basic residue, or an acidic residue, and X6 is selected from S, T, E, D, and R, or X6 is selected from S, D, E, and T. In certain embodiments, X7 is selected from I, A, M, and V, or X7 is selected from I, A, and M. In certain embodiments, X8 is an uncharged polar residue, an uncharged apolar residue, a basic residue, or an acidic residue, and X8 is selected from S, T, N, Q, I, G, E, K, and R, or X8 is selected from I, R, N, and T. In certain embodiments, X is selected from V, L, and I, or X is V. In certain embodiments, X 10 is an uncharged polar residue or an uncharged apolar residue, and X 10 is selected from N, T, I, and L, or X 10 is selected from I and L. In certain embodiments, X 11 is an uncharged nonpolar residue, or X 11 is selected from V, A, and I. In certain embodiments, X 12 is an uncharged polar residue, an uncharged nonpolar residue, or an acidic residue, and X 12 is selected from Q, L, G, K, and R, or X 12is selected from R, G, Q, and K. In certain embodiments, X 13 is an uncharged nonpolar or basic residue, and X 13 is selected from A, D, and E, or X 13 is selected from E and A.

[0007] In some aspects, the disclosure provides modified IL2 polypeptides comprising a substitution at least one residue selected from R81, P82, R83, L85, I86, S87, I89, N90, I92, V93, and L94. In certain aspects, the R81 substitution is selected from R81G, R81K, R81E, R81C, and R81T; the R83 substitution is selected from R83T, R83G, R83S, and R83E; the L85 substitution is selected from L85S, L85A, L85V, and L85T; the I92 substitution is I92L; and the L94 substitution is selected from L94R, L94G, L94Q, and L94K. In certain aspects, the modified IL2 polypeptide comprises substitutions at R81 and L83. In certain aspects, the modified IL2 polypeptide comprises substitutions at R81, L83, S87, N90, and N94, substitutions at R81, L83, S87, N90, and V93, substitutions at R81, L83, P82, and V93, or substitutions at R81, L83, and N90.

[0008] In one aspect, the disclosure provides a modified interleukin-2 (IL2) polypeptide comprising a modified IL2 receptor alpha (IL2Rα) binding region 1 motif comprising a substitution selected from a substitution at position K35, a substitution at R38, a substitution at F42, a substitution at Y45, or a combination thereof, wherein the modified IL2 polypeptide binds to IL2Rα with at least two-fold reduced binding kinetics compared to wild-type IL2.

[0009] In some aspects, the disclosure provides a modified IL2 polypeptide comprising a modified IL2 receptor beta (IL2Rβ) binding region 2 described above and a modified IL2 receptor alpha (IL2Rα) binding region 1 described above.

[0010] In some aspects, the present disclosure provides a modified IL2 polypeptide provided herein fused to a half-life extension.

[0011] In some aspects, the present disclosure provides a fusion polypeptide comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a modified IL2 polypeptide provided herein.

[0012] In some aspects, the disclosure provides an isolated polynucleotide encoding a modified IL2 polypeptide or a fusion polypeptide thereof, an expression vector comprising the isolated polynucleotide, or a modified cell comprising the isolated polynucleotide or expression vector.

[0013] In some aspects, the disclosure provides a pharmaceutical composition comprising a modified IL2 polypeptide or a fusion polypeptide thereof and a pharmaceutically acceptable carrier.

[0014] In some aspects, the present disclosure provides methods of modulating an immune response in a subject in need thereof, the method comprising administering a therapeutically effective amount of a modified IL2 polypeptide or a fusion polypeptide thereof, or a pharmaceutical composition thereof, to the subject. In certain aspects, the modulation of the immune response comprises at least one of enhancing effector T cell activity, enhancing NK cell activity, and suppressing regulatory T cell activity.

[0015] In some embodiments, the present disclosure provides methods of treating a disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of a modified IL2 polypeptide or a fusion polypeptide thereof, or a pharmaceutical composition thereof, to the subject. In certain embodiments, the disease is cancer. In certain embodiments, the method further comprises administering an additional therapeutic agent, such as an antigen-binding moiety, immune cells expressing a chimeric antigen receptor, immune cells expressing a modified T-cell receptor, tumor-infiltrating lymphocytes, an immune checkpoint inhibitor, an oncolytic virus, a tumor microenvironment (TME) inhibitor, or a cancer vaccine. In certain embodiments, the method comprises administering to the subject immune cells comprising a polynucleotide encoding the modified IL2 polypeptide or a fusion polypeptide thereof. [Brief explanation of the drawings]

[0016] [Figure 1A] The regions of IL2 involved in binding to IL2Rα (open box), IL2Rβ (dashed box), and IL2Rγ (grey box) are indicated. [Figure 1B] A graphic depiction of IL2Rα, IL2Rβ, and IL2Rγ binding to IL2 is shown. [Figure 1C] The IL2Rα binding site of IL2 is shown, highlighting four residues in IL2 (K35, R38, F42, and Y45) that are important for IL2Rα interaction. [Figure 2A] Identification of IL2Rα-reducing binding mutations by ELISA. [Figure 2B] Identification of IL2Rα-reducing binding mutations by ELISA. [Figure 3] 1 shows the characterization of IL2Rα-reduced binding mutations by surface plasmon resonance sequence alignment of IL2Rα-reduced binding mutations. [Figure 4] 1 shows the characterization of IL2Rα-reducing binding mutations by surface plasmon resonance. [Figure 5] IL2Rβ agonist mutagenic library. [Figure 6] IL2Rβ agonist expression in E. coli and binding to IL2Rβ are shown. [Figure 7] 1 shows a multiple sequence alignment of the IL2Rβ binding region 2 of IL2Rβ agonists identified by mRNA display. [Figure 8] 1 shows SDS analysis of IL2Rβ agonist clones produced in E. coli. [Figure 9A] 1 shows the sensorgram and binding kinetics of wild-type IL2 to IL2Rα in SPR. [Figure 9B] 1 shows the sensorgram and binding kinetics of wild-type IL2 to IL2Rβ in SPR. [Figure 10A] 1 shows sensorgrams of E. coli-produced wild-type IL2 and modified IL2Rβ agonists against IL2Rα in SPR. 1 shows a sensorgram of wild-type IL2. [Figure 10B] 1 shows sensorgrams of E. coli-produced wild-type IL2 and modified IL2Rβ agonists against IL2Rα in SPR. 1 shows the sensorgram of EP001. [Figure 10C] 1 shows sensorgrams of E. coli-produced wild-type IL2 and modified IL2Rβ agonists for IL2Rα in SPR. 1 shows the sensorgram of EP004. [Figure 10D] 1 shows sensorgrams of E. coli-produced wild-type IL2 and modified IL2Rβ agonists for IL2Rα in SPR. 1 shows the sensorgram of EP005. [Figure 10E] 1 shows sensorgrams of E. coli-produced wild-type IL2 and modified IL2Rβ agonists against IL2Rα in SPR. 1 shows the sensorgram of EP002. [Figure 10F] 1 shows sensorgrams of E. coli-produced wild-type IL2 and modified IL2Rβ agonists against IL2Rα in SPR. 1 shows the sensorgram of EP03. [Figure 10G] 1 shows sensorgrams of E. coli-produced wild-type IL2 and modified IL2Rβ agonists against IL2Rα in SPR. 1 shows the sensorgram of EPIM-06. [Figure 10H] 1 shows sensorgrams of E. coli-produced wild-type IL2 and modified IL2Rβ agonists for IL2Rα in SPR. 1 shows the sensorgram of EP007. [Figure 11A] 1 shows sensorgrams of E. coli-produced wild-type IL2 and modified IL2Rβ agonists against IL2Rβ in SPR. 1 shows the sensorgram of EP003. [Figure 11B] 1 shows sensorgrams of E. coli-produced wild-type IL2 and modified IL2Rβ agonists against IL2Rβ in SPR. 1 shows the sensorgram of EP005. [Figure 11C] 1 shows sensorgrams of E. coli-produced wild-type IL2 and modified IL2Rβ agonists against IL2Rβ in SPR. 1 shows the sensorgram of E002. [Figure 11D] 1 shows sensorgrams of E. coli-produced wild-type IL2 and modified IL2Rβ agonists against IL2Rβ in SPR. 1 shows the sensorgram of EP001. [Figure 11E] 1 shows sensorgrams of E. coli-produced wild-type IL2 and modified IL2Rβ agonists against IL2Rβ in SPR. 1 shows the sensorgram of EP007. [Figure 11F] 1 shows sensorgrams of E. coli-produced wild-type IL2 and modified IL2Rβ agonists against IL2Rβ in SPR. 1 shows the sensorgram of EP006. [Figure 11G] 1 shows sensorgrams of E. coli-produced wild-type IL2 and modified IL2Rβ agonists against IL2Rβ in SPR. 1 shows the sensorgram of EP004. [Figure 11H] 1 shows sensorgrams of E. coli-produced wild-type IL2 and modified IL2Rβ agonists against IL2Rβ in SPR. 1 shows a sensorgram of wild-type IL2. [Figure 12A] 1 shows a sensorgram of IL2Rβ binding to mammalian-produced wild-type IL2 in SPR. [Figure 12B]1 shows a sensorgram of IL2Rβ binding to the modified IL2Rβ agonist EP0001 in SPR. [Figure 12C] 1 shows a sensorgram of IL2Rβ binding to EP0003 in SPR. [Figure 12D] 1 shows a sensorgram of IL2Rβ binding to EP004 in SPR. [Figure 13A] 13A shows a sensorgram of IL2Rα binding to mammalian-produced wild-type IL2 (FIG. 13A) in SPR. [Figure 13B] 1 shows a sensorgram of IL2Rα binding to the modified IL2Rβ agonist EP001 in SPR. [Figure 13C] 1 shows a sensorgram of IL2Rα binding to EP003 in SPR. [Figure 13D] 1 shows a sensorgram of IL2Rα binding to EP004 in SPR. [Figure 14A] 1 shows pSTAT5 expression in CD8+ T cells measured in blood donor 1 after stimulation of human PBMCs with wild-type IL2 and modified IL2Rβ agonists. [Figure 14B] NK cell pSTAT5 expression measured from blood donor 1 after stimulation of human PBMCs with wild-type IL2 and modified IL2Rβ agonists is shown. [Figure 14C] Figure 1 shows pSTAT5 expression in Tregs measured from blood donor 1 after stimulation of human PBMCs with wild-type IL2 and modified IL2Rβ agonists. [Figure 14D] 1 shows pSTAT5 expression in CD8+ T cells measured from blood donor 2 after stimulation of human PBMCs with wild-type IL2 and modified IL2Rβ agonists. [Figure 14E] NK cell pSTAT5 expression measured from blood donor 2 after stimulation of human PBMCs with wild-type IL2 and modified IL2Rβ agonists is shown. [Figure 14F] Figure 1 shows pSTAT5 expression in Tregs measured from blood donor 2 after stimulation of human PBMCs with wild-type IL2 and modified IL2Rβ agonists. [Figure 14G] 1 shows pSTAT5 expression in CD8+ T cells measured from blood donor 3 after stimulation of human PBMCs with wild-type IL2 and modified IL2Rβ agonists. [Figure 14H] NK cell pSTAT5 expression measured from blood donor 3 after stimulation of human PBMCs with wild-type IL2 and modified IL2Rβ agonists is shown. [Figure 14I] Figure 1 shows pSTAT5 expression in Tregs measured from blood donor 3 after stimulation of human PBMCs with wild-type IL2 and modified IL2Rβ agonists. [Figure 15A] ELISA characterization of IL2Rβ agonist EP001 revertant clones for binding to IL2Rα by ELISA. [Figure 15B] ELISA characterization of IL2Rβ agonist EP001 revertant clones for binding to IL2Rβ by ELISA. [Figure 16A] 1 shows an IL2Rβ binding sensorgram of the IL2Rβ agonist EP001 revertant clone by SPR. [Figure 16B] 1 shows an IL2Rβ binding sensorgram of the IL2Rβ agonist EP001 revertant clone by SPR. [Figure 16C] 1 shows an IL2Rβ binding sensorgram of the IL2Rβ agonist EP001 revertant clone by SPR. [Figure 16D] 1 shows an IL2Rβ binding sensorgram of the IL2Rβ agonist EP001 revertant clone by SPR. [Figure 16E] 1 shows an IL2Rβ binding sensorgram of the IL2Rβ agonist EP001 revertant clone by SPR. [Figure 16F] 1 shows an IL2Rβ binding sensorgram of the IL2Rβ agonist EP001 revertant clone by SPR. [Figure 16G] 1 shows an IL2Rβ binding sensorgram of the IL2Rβ agonist EP001 revertant clone by SPR. [Figure 16H]1 shows an IL2Rβ binding sensorgram of the IL2Rβ agonist EP001 revertant clone by SPR. [Figure 17] An example of the SDS-PAGE results of purified IL2Rα / IL2Rβ clones is shown. [Figure 18A] 1 shows a binding sensorgram of modified IL2Rα / IL2Rβ clones to human IL2α by SPR. [Figure 18B] 1 shows a binding sensorgram of modified IL2Rα / IL2Rβ clones to human IL2α by SPR. [Figure 18C] 1 shows a binding sensorgram of modified IL2Rα / IL2Rβ clones to human IL2α by SPR. [Figure 18D] 1 shows a binding sensorgram of modified IL2Rα / IL2Rβ clones to human IL2α by SPR. [Figure 18E] 1 shows a binding sensorgram of modified IL2Rα / IL2Rβ clones to human IL2α by SPR. [Figure 18F] 1 shows a binding sensorgram of modified IL2Rα / IL2Rβ clones to human IL2α by SPR. [Figure 18G] 1 shows a binding sensorgram of modified IL2Rα / IL2Rβ clones to human IL2α by SPR. [Figure 18H] 1 shows a binding sensorgram of modified IL2Rα / IL2Rβ clones to human IL2α by SPR. [Figure 19A] 1 shows a binding sensorgram of modified IL2Rα / IL2Rβ clones to human IL2β by SPR. [Figure 19B] 1 shows a binding sensorgram of modified IL2Rα / IL2Rβ clones to human IL2β by SPR. [Figure 19C] 1 shows a binding sensorgram of modified IL2Rα / IL2Rβ clones to human IL2β by SPR. [Figure 19D] 1 shows a binding sensorgram of modified IL2Rα / IL2Rβ clones to human IL2β by SPR. [Figure 19E]1 shows a binding sensorgram of modified IL2Rα / IL2Rβ clones to human IL2β by SPR. [Figure 19F] 1 shows a binding sensorgram of modified IL2Rα / IL2Rβ clones to human IL2β by SPR. [Figure 19G] 1 shows a binding sensorgram of modified IL2Rα / IL2Rβ clones to human IL2β by SPR. [Figure 19H] 1 shows a binding sensorgram of modified IL2Rα / IL2Rβ clones to human IL2β by SPR. [Figure 20A] 1 shows SPR binding sensorgrams of engineered IL2Rα / IL2Rβ clones to human IL2Rα at a single concentration. [Figure 20B] 1 shows SPR binding sensorgrams of engineered IL2Rα / IL2Rβ clones to human IL2Rα at a single concentration. [Figure 20C] 1 shows SPR binding sensorgrams of engineered IL2Rα / IL2Rβ clones to human IL2Rα at a single concentration. [Figure 20D] 1 shows SPR binding sensorgrams of engineered IL2Rα / IL2Rβ clones to human IL2Rα at a single concentration. [Figure 20E] 1 shows SPR binding sensorgrams of engineered IL2Rα / IL2Rβ clones to human IL2Rα at a single concentration. [Figure 20F] 1 shows SPR binding sensorgrams of engineered IL2Rα / IL2Rβ clones to human IL2Rα at a single concentration. [Figure 20G] 1 shows SPR binding sensorgrams of engineered IL2Rα / IL2Rβ clones to human IL2Rα at a single concentration. [Figure 21A] Binding of engineered IL2Rα / IL2Rβ clones to human IL2Rβ at a single concentration by SPR is shown. [Figure 21B] Binding of engineered IL2Rα / IL2Rβ clones to human IL2Rβ at a single concentration by SPR is shown. [Figure 21C]Binding of engineered IL2Rα / IL2Rβ clones to human IL2Rβ at a single concentration by SPR is shown. [Figure 21D] Binding of engineered IL2Rα / IL2Rβ clones to human IL2Rβ at a single concentration by SPR is shown. [Figure 21E] Binding of engineered IL2Rα / IL2Rβ clones to human IL2Rβ at a single concentration by SPR is shown. [Figure 21F] Binding of engineered IL2Rα / IL2Rβ clones to human IL2Rβ at a single concentration by SPR is shown. [Figure 21G] Binding of engineered IL2Rα / IL2Rβ clones to human IL2Rβ at a single concentration by SPR is shown. [Figure 22A] Binding of engineered IL2Rα / IL2Rβ clones to human IL2Rα at multiple concentrations by SPR is shown. [Figure 22B] Binding of engineered IL2Rα / IL2Rβ clones to human IL2Rβ at multiple concentrations by SPR. [Figure 23A] Figure 1 shows ELISA binding to human IL2Rα. Figure 2 shows ELISA binding of EP252 and its IL2Rα binding-reducing mutants to human IL2Rα. [Figure 23B] Figure 1 shows ELISA binding to human IL2Rα. Figure 2 shows ELISA binding of EP253 and its IL2Rα binding-reducing mutants to human IL2Rα. [Figure 23C] Figure 1 shows ELISA binding to human IL2Rα. Figure 2 shows ELISA binding of EP258 and its IL2Rα binding-reducing mutants to human IL2Rα. [Figure 23D] Figure 1 shows ELISA binding to human IL2Rα. Figure 2 shows ELISA binding of EP260 and its IL2Rα binding-reducing mutants to human IL2Rα. [Figure 23E] Figure 1 shows ELISA binding to human IL2Rα. Figure 2 shows dose-dependent binding of selected modified IL2Rβ / IL2Rα clones. [Figure 24A] Figure 1 shows ELISA binding to human IL2Rβ. Figure 2 shows ELISA binding of EP252 and its IL2Rα binding-reducing mutants to human IL2Rβ. [Figure 24B] Figure 1 shows ELISA binding to human IL2Rβ. Figure 2 shows ELISA binding of EP253 and its IL2Rα binding-reducing mutants to human IL2Rβ. [Figure 24C] Figure 1 shows ELISA binding to human IL2Rβ. Figure 2 shows ELISA binding of EP258 and its IL2Rα binding-reducing mutants to human IL2Rβ. [Figure 24D] Figure 1 shows ELISA binding to human IL2Rβ. Figure 2 shows ELISA binding of EP260 and its IL2Rα binding-reducing mutants to human IL2Rβ. [Figure 25A] Figure 1 shows p-STAT5 activation of human CD8+ T cells from donor 656 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation of human CD8+ T cells in response to EP252 and its IL2Rα binding-reducing mutants. [Figure 25B] Figure 1 shows p-STAT5 activation of human CD8+ T cells from donor 656 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation of human CD8+ T cells in response to EP253 and its IL2Rα binding-reducing mutants. [Figure 25C] Figure 1 shows p-STAT5 activation of human CD8+ T cells from donor 656 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation of human CD8+ T cells in response to EP258 and its IL2Rα binding-reducing mutants. [Figure 25D] Figure 1 shows p-STAT5 activation of human CD8+ T cells from donor 656 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation of human CD8+ T cells in response to EP260 and its IL2Rα binding-reducing mutants. [Figure 26A] Figure 1 shows p-STAT5 activation of human CD8+ T cells from donor 648 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation of human CD8+ T cells in response to EP252 and its IL2Rα binding-reducing mutants. [Figure 26B]Figure 1 shows p-STAT5 activation of human CD8+ T cells from donor 648 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation of human CD8+ T cells in response to EP253 and its IL2Rα binding-reducing mutants. [Figure 26C] Figure 1 shows p-STAT5 activation of human CD8+ T cells from donor 648 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation of human CD8+ T cells in response to EP258 and its IL2Rα binding-reducing mutants. [Figure 26D] Figure 1 shows p-STAT5 activation of human CD8+ T cells from donor 648 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation of human CD8+ T cells in response to EP260 and its IL2Rα binding-reducing mutants. [Figure 27A] Figure 1 shows p-STAT5 activation in human NK cells from donor 656 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation in human NK cells in response to EP252 and its IL2Rα binding-reducing mutants. [Figure 27B] Figure 1 shows p-STAT5 activation in human NK cells from donor 656 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation in human NK cells in response to EP253 and its IL2Rα binding-reducing mutants. [Figure 27C] Figure 1 shows p-STAT5 activation in human NK cells from donor 656 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation in human NK cells in response to EP258 and its IL2Rα binding-reducing mutants. [Figure 27D] Figure 1 shows p-STAT5 activation in human NK cells from donor 656 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation in human NK cells in response to EP260 and its IL2Rα binding-reducing mutants. [Figure 28A] Figure 1 shows p-STAT5 activation in human NK cells from donor 648 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation in human NK cells in response to EP252 and its IL2Rα binding-reducing mutants. [Figure 28B]Figure 1 shows p-STAT5 activation in human NK cells from donor 648 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation in human NK cells in response to EP253 and its IL2Rα binding-reducing mutants. [Figure 28C] Figure 1 shows p-STAT5 activation of human NK cells from donor 648 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation of human NK cells in response to EP258 and its IL2Rα binding-reducing mutants. [Figure 28D] Figure 1 shows p-STAT5 activation in human NK cells from donor 648 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation in human NK cells in response to EP260 and its IL2Rα binding-reducing mutants. [Figure 29A] Figure 1 shows p-STAT5 activation of human Treg cells from donor 656 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation of human Treg cells in response to EP252 and its IL2Rα binding-reducing mutants. [Figure 29B] Figure 1 shows p-STAT5 activation of human Treg cells from donor 656 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation of human Treg cells in response to EP253 and its IL2Rα binding-reducing mutants. [Figure 29C] Figure 1 shows p-STAT5 activation of human Treg cells from donor 656 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation of human Treg cells in response to EP258 and its IL2Rα binding-reducing mutants. [Figure 29D] Figure 1 shows p-STAT5 activation of human Treg cells from donor 656 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation of human Treg cells in response to EP260 and its IL2Rα binding-reducing mutants. [Figure 30A] Figure 1 shows p-STAT5 activation of human Treg cells from donor 648 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation of human Treg cells in response to EP252 and its IL2Rα binding-reducing mutants. [Figure 30B] Figure 1 shows p-STAT5 activation of human Treg cells from donor 648 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation of human Treg cells in response to EP253 and its IL2Rα binding-reducing mutants. [Figure 30C] Figure 1 shows p-STAT5 activation of human Treg cells from donor 648 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation of human Treg cells in response to EP258 and its IL2Rα binding-reducing mutants. [Figure 30D] Figure 1 shows p-STAT5 activation of human Treg cells from donor 648 by modified IL2Rα / IL2Rβ clones. Figure 2 shows p-STAT5 activation of human Treg cells in response to EP260 and its IL2Rα binding-reducing mutants. [Figure 31A] 1 shows p-STAT5 activation in mouse CD8+ T cells. 1 shows p-STAT5 activation in mouse CD8+ T cells in response to EP252 and its IL2Rα binding-reducing mutants. [Figure 31B] 1 shows p-STAT5 activation in mouse CD8+ T cells. 1 shows p-STAT5 activation in mouse CD8+ T cells in response to EP253 and its IL2Rα binding-reducing mutants. [Figure 31C] 1 shows p-STAT5 activation in mouse CD8+ T cells. 1 shows p-STAT5 activation in mouse CD8+ T cells in response to EP258 and its IL2Rα binding-reducing mutants. [Figure 31D] 1 shows p-STAT5 activation in mouse CD8+ T cells. 1 shows p-STAT5 activation in mouse CD8+ T cells in response to EP260 and its IL2Rα binding-reducing mutants. [Figure 32A] 1 shows p-STAT5 activation in mouse NK cells. 2 shows p-STAT5 activation in mouse NK cells in response to EP252 and its IL2Rα binding-reducing mutants. [Figure 32B] 1 shows p-STAT5 activation in mouse NK cells. 2 shows p-STAT5 activation in mouse NK cells in response to EP253 and its IL2Rα binding-reducing mutants. [Figure 32C]1 shows p-STAT5 activation in mouse NK cells. 2 shows p-STAT5 activation in mouse NK cells in response to EP258 and its IL2Rα binding-reducing mutants. [Figure 32D] 1 shows p-STAT5 activation in mouse NK cells. 2 shows p-STAT5 activation in mouse NK cells in response to EP260 and its IL2Rα binding-reducing mutants. [Figure 33A] 1 shows p-STAT5 activation in mouse regulatory T cells. 2 shows p-STAT5 activation in mouse regulatory T cells of EP252 and its IL2Rα binding-reducing mutant. [Figure 33B] 1 shows p-STAT5 activation in mouse regulatory T cells. 2 shows p-STAT5 activation in mouse regulatory T cells of EP253 and its IL2Rα binding-reducing mutant. [Figure 33C] 1 shows p-STAT5 activation in mouse regulatory T cells. 2 shows p-STAT5 activation in mouse regulatory T cells of EP258 and its IL2Rα binding-reducing mutants. [Figure 33D] 1 shows p-STAT5 activation in mouse regulatory T cells. 2 shows p-STAT5 activation in mouse regulatory T cells of EP260 and its IL2Rα binding-reducing mutant. [Figure 34] Schematic representation of p-STAT5 activation in mouse CD8+ T cells, NK cells, and Tregs. [Figure 35A] 1 shows structural diagrams of monovalent and bivalent IL2Rβ agonist Fc fusion proteins. [Figure 35B] 1 shows structural diagrams of monovalent and bivalent IL2Rβ agonist Fc fusion proteins. [Figure 35C] 1 shows structural diagrams of monovalent and bivalent IL2Rβ agonist Fc fusion proteins. [Figure 36A] 1 shows SDS-PAGE analysis of purified IL2Rβ agonist Fc fusion proteins. [Figure 36B] 1 shows SDS-PAGE analysis of purified IL2Rβ agonist Fc fusion proteins. [Figure 37A] 1 shows receptor binding analysis by ELISA of bivalent IL2Rβ agonist Fc fusion proteins. [Figure 37B] 1 shows receptor binding analysis by ELISA of bivalent IL2Rβ agonist Fc fusion proteins. [Figure 37C] 1 shows receptor binding analysis by ELISA of bivalent IL2Rβ agonist Fc fusion proteins. [Figure 37D] 1 shows receptor binding analysis by ELISA of bivalent IL2Rβ agonist Fc fusion proteins. [Figure 37E] 1 shows receptor binding analysis by ELISA of bivalent IL2Rβ agonist Fc fusion proteins. [Figure 37F] 1 shows receptor binding analysis by ELISA of bivalent IL2Rβ agonist Fc fusion proteins. [Figure 37G] 1 shows receptor binding analysis by ELISA of bivalent IL2Rβ agonist Fc fusion proteins. [Figure 38A] 1 shows receptor binding analysis by ELISA of monovalent IL2Rβ agonist Fc fusion proteins. [Figure 38B] 1 shows receptor binding analysis by ELISA of monovalent IL2Rβ agonist Fc fusion proteins. [Figure 39A] 1 shows receptor binding analysis of monovalent IL2RβFc ​​fusion proteins by SPR. [Figure 39B] 1 shows receptor binding analysis of monovalent IL2RβFc ​​fusion proteins by SPR. [Figure 39C] 1 shows receptor binding analysis of monovalent IL2RβFc ​​fusion proteins by SPR. [Figure 39D] 1 shows receptor binding analysis of monovalent IL2RβFc ​​fusion proteins by SPR. [Figure 40A] 1 shows p-STAT5 activation in human PBMCs by bivalent IL2Rβ agonist Fc fusion proteins. [Figure 40B] 1 shows p-STAT5 activation in human PBMCs by bivalent IL2Rβ agonist Fc fusion proteins. [Figure 40C] 1 shows p-STAT5 activation in human PBMCs by bivalent IL2Rβ agonist Fc fusion proteins. [Figure 41A] 1 shows p-STAT5 activation in human PBMCs by monovalent IL2Rβ agonist Fc fusion proteins. [Figure 41B] 1 shows p-STAT5 activation in human PBMCs by monovalent IL2Rβ agonist Fc fusion proteins. [Figure 41C] 1 shows p-STAT5 activation in human PBMCs by monovalent IL2Rβ agonist Fc fusion proteins. [Figure 42A] Pharmacokinetics of an IL2Rβ agonist following iv administration to mice. [Figure 42B] Pharmacokinetics of an IL2Rβ agonist following ip administration to mice. [Figure 43A] Normalized numbers of tumor-infiltrating immune cells after administration of an IL2Rβ agonist are shown. [Figure 43B] Normalized numbers of tumor-infiltrating immune cells after administration of an IL2Rβ agonist are shown. [Figure 43C] Normalized numbers of tumor-infiltrating immune cells after administration of an IL2Rβ agonist are shown. [Figure 43D] Normalized numbers of tumor-infiltrating immune cells after administration of an IL2Rβ agonist are shown. [Figure 44A] The ratio of effector cells to regulatory T cells in the tumor is shown. [Figure 44B] The ratio of effector cells to regulatory T cells in the tumor is shown. [Figure 45A] The percentages of effector and memory T cells are shown. [Figure 45B] The percentages of effector and memory T cells are shown. [Figure 45C] The percentages of effector and memory T cells are shown. DETAILED DESCRIPTION OF THE INVENTION

[0017] Although IL2 has become a promising new immunotherapy, therapies based on wild-type human IL2 can activate regulatory T cells in addition to activating effector T cells and NK cells. Activation of regulatory T cells by IL2 can prevent the anti-cancer responses that IL2 can induce in the absence of regulatory T cell activation. Therefore, there is a need for IL2-based therapies that have reduced activation of regulatory T cells and / or preferential activation of T effector cells, NK cells, or a combination thereof.

[0018] Presented herein are rationally designed IL2Rβ agonists, which are modified IL2 polypeptides with amino acid substitutions in IL2Rβ binding region 2 that enhance binding to IL2Rβ. The modified IL2Rβ agonists offer the advantage of increased stimulation of NK cells and T effector cells, but not regulatory T cells, compared to wild-type IL2. Thus, the modified IL2Rβ agonists are useful for modulating or activating immune responses, for example, for the treatment of cancer.

[0019] As used herein, the term "interleukin 2" or "IL2" refers to IL2 from any vertebrate source, including mammals such as humans or mice, unless otherwise indicated. The term encompasses precursor or unprocessed IL2, as well as any form of IL2 resulting from cellular processing. The term also encompasses naturally occurring variants of IL2, such as splice variants or allelic variants. An exemplary amino acid sequence of mature human IL2 is set forth in SEQ ID NO: 65. Precursor or unprocessed human IL2 is set forth in SEQ ID NO: 66 and includes a 20-residue signal peptide that is not present in the mature IL2 polypeptide. "Wild-type" or "native" when used with respect to IL2 is intended to refer to the mature IL2 molecule (e.g., SEQ ID NO: 65). The term "modified IL2" or "modified IL2 polypeptide" as used herein encompasses IL2 having at least one residue that differs from native or wild-type IL2, including full-length IL2, truncated forms of IL2, and forms in which IL2 is linked or fused to another molecule, such as another polypeptide. The various forms of modified IL2 are characterized by having at least one amino acid substitution that affects the interaction of IL2 with IL2Rβ and / or IL2Rα. The various modified forms of IL2 described herein are identified with respect to the sequence shown, for example, in SEQ ID NO: 22. Various identifiers may be used herein to refer to the same residue substitution. For example, a substitution of arginine to threonine at position 81 may be indicated as R81T or 81T.

[0020] IL2Rβ-binding region 1 and IL2Rβ-binding region 2 are involved in the binding of IL2 to IL2Rβ. As used herein, "IL2Rβ-binding region 1" refers to residues 11-23 of wild-type or naturally occurring human IL2. The amino acid sequence of IL2Rβ-binding region 1 is provided in SEQ ID NO: 67. As used herein, "IL2Rβ-binding region 2" refers to residues 81-95 of wild-type or naturally occurring human IL2. The amino acid sequence of IL2Rβ-binding region 2 is provided in SEQ ID NO: 68.

[0021] IL2Rα-binding region 1 and IL2Rα-binding region 2 are involved in the binding of IL2 to IL2Rα. As used herein, "IL2Rα-binding region 1" refers to residues 34-45 of wild-type or native human IL2. The amino acid sequence of IL2Rα-binding region 1 is provided in SEQ ID NO:223.

[0022] As used herein, the term "substitution" or "residue substitution" refers to the replacement of a native or wild-type residue with a different residue.

[0023] As used herein, "any residue" refers to an amino acid residue having one of the 20 standard amino acid side chains: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0024] As used herein, "uncharged residue" refers to an amino acid residue having a side chain that does not carry a charge at physiological pH (pH = 7). Uncharged residues are as follows: alanine (Ala, A), asparagine (Asn, N), cysteine ​​(Cys, C), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0025] As used herein, "uncharged polar residue" refers to an amino acid residue having a side chain that does not carry a charge and is hydrophilic at physiological pH (pH=7). Uncharged polar residues are as follows: asparagine (Asn, N), cysteine ​​(Cys, C), glutamine (Gln, Q), serine (Ser, S), threonine (Thr, T), and tyrosine (Tyr, Y).

[0026] As used herein, "uncharged nonpolar residue" refers to an amino acid residue having a side chain that does not carry a charge at physiological pH (pH = 7) and is hydrophobic. Uncharged nonpolar residues are as follows: alanine (Ala, A), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), tryptophan (Trp, W), and valine (Val, V).

[0027] As used herein, a "basic residue" refers to an amino acid residue having a side chain that carries a positive charge at physiological pH (pH=7). Basic residues are lysine (Lys, K) and arginine (Arg, R).

[0028] As used herein, "acidic residue" refers to an amino acid residue having a side chain that carries a negative charge at physiological pH (pH=7). Acidic residues are aspartic acid (Asp, D) and glutamic acid (Glu, E).

[0029] A "fusion polypeptide" or "fusion protein" refers to a polypeptide that is encoded by at least two different DNA sequences corresponding to genes or fragments thereof that are not naturally expressed from the same gene. An example of a fusion polypeptide is a modified IL2-Fc fusion polypeptide that contains the amino acid sequence of a modified IL2 polypeptide and the amino acid sequence of an Fc domain.

[0030] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., a receptor and a ligand). The affinity of molecule X for partner Y is generally determined by the dissociation constant (K D ), which can be expressed as the dissociation and association rate constants (k off and k on ) is the ratio of the rate constants. Thus, equivalent affinities may involve different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by methods known to those of skill in the art, including those described herein.

[0031] As used herein, a "half-life extender" refers to a molecule that, when attached (e.g., covalently) to a second molecule, extends the half-life of the second molecule. Examples of half-life extenders include Fc domains, human serum albumin (HSA), HSA-binding molecules, polyethylene glycol (PEG), and polypropylene glycol (PPG).

[0032] As used herein, "Fc domain" or "Fc region" refers to a polypeptide derived from the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes polypeptides having a native sequence Fc region, or variants thereof. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is usually defined to stretch from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Examples of Fc regions are disclosed in U.S. Patent Nos. 7,317,091, 8,735,545, 7,371,826, 7,670,600, and 9,803,023, all of which are incorporated by reference in their entireties.

[0033] "Human serum albumin" or "HSA" refers to serum albumin found in human blood. A commonly used form of HSA has a molecular mass of 66.5 kDa and a half-life of approximately 20 days. Examples of HSA molecules are disclosed in U.S. Patent No. 8,143,026 and U.S. Patent No. 7,189,690, which are incorporated by reference in their entireties.

[0034] "HSA binding molecule" refers to a molecule that specifically binds to human serum albumin (HSA), such as an antigen-binding moiety having an HSA binding domain.

[0035] "Polyethylene glycol" or "PEG," also known as polyethylene oxide or polyoxyethylene, is a polyether polymer that can be used to extend half-life.

[0036] "Polypropylene glycol" or "PPG," also known as polypropylene oxide, is a polymer of propylene glycol that can be used to extend half-life.

[0037] "Antigen-binding portion" refers to the site (i.e., amino acid residues) of an antigen-binding molecule (e.g., an antibody) that interacts with an antigen epitope. The antigen-binding portion may comprise one or more antibody variable domains (also referred to as antibody variable regions). Preferably, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of antigen-binding portions include immunoglobulins, Fab molecules, scFvs, bispecific antibodies, diabodies, bispecific T-cell engagers, and nanobodies. Specific examples of antigen-binding portions include nivolumab, pembrolizumab, pidilizumab, atezolizumab, ipilimumab, tremelimumab, rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, ublituximab, and bevacizumab.

[0038] "Immunoglobulin" refers to a protein having the structure of a naturally occurring antibody. As an example, an IgG class immunoglobulin is a heterotetrameric glycoprotein consisting of two light chains and two heavy chains disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain, also called a light chain constant region. Immunoglobulin heavy chains may be assigned to one of five classes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which may be further divided into subclasses, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the sequence of their constant domains. Immunoglobulins contain two Fab molecules and an Fc domain linked via an immunoglobulin hinge region.

[0039] A "Fab molecule" or "antigen-binding fragment" is an antigen-binding fragment of an antibody that contains the variable and constant domains of the light chain and the variable and CH1 domains of the heavy chain.

[0040] "Single-chain variable domain" or "scFv" refers to an antigen-binding portion comprising the variable regions of the heavy and light chains connected by a linker peptide.

[0041] A "bispecific antibody" refers to an artificial antibody that has two different antigen-binding sites. A bispecific antibody may refer to an intact immunoglobulin protein that has two different antigen-binding sites, or to other molecules that have two antigen-binding moieties, such as a fusion protein containing two Fabs or two scFvs.

[0042] "Diabodies" refer to a class of antigen-binding molecules that are bivalent and bispecific. The fragments comprise a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain to form two antigen-binding sites.

[0043] "Bispecific T cell engagers" refer to a class of bispecific antibodies that have a first antigen-binding portion that binds to T cells (e.g., by binding to CD3) and a second antigen-binding portion that binds to a different antigen (e.g., a tumor antigen).

[0044] "Nanobody" or "single domain antibody" refers to an antigen-binding moiety that consists of a single monomeric variable antibody domain.

[0045] "Transferrin" is an iron transport protein that can be used in fusion proteins to extend its half-life. Human transferrin has a serum half-life of 12 days.

[0046] As used herein, "cytokine" refers to a class of small (<25 kDa) proteins involved in cell signaling and immune regulation. Cytokines include, for example, IL2, interleukin 10 (IL-10), interleukin 1 (IL-1), interleukin 17 (IL-17), interleukin 18 (IL-18), interferon α, interferon β, interferon γ, TGF-β1, TGF-β2, and TGF-β3, chemokine (C-C motif) ligand 2 (CCL2), and chemokine (C-C motif) ligand 19 (CCL19).

[0047] A "subject" according to any of the above embodiments is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the subject is a human.

[0048] "Modulating the immune response" may include one or more of a general increase, an increase in T effector cell responses (e.g., cytotoxicity against tumor cells and virally infected cells), an increase in B cell activation, restoration of lymphocyte activation and proliferation, an increase in IL2 receptor expression, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, a decrease in regulatory T cell responses against other T cells, and the like.

[0049] "Regulatory T cells" or "Treg cells" refer to a special type of CD4+ T cell that can function to suppress the responses of other T cells. Treg cells express the IL2 receptor (CD25) and the α subunit of the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)) and are involved in the induction and maintenance of peripheral self-tolerance to antigens, including those expressed by tumors. Treg cells require IL2 for their function and for the development and induction of their suppressive properties.

[0050] "T effector cells" refers to a population of T cells that respond to a stimulus such as IL2. T effector cells include CD8+ cytotoxic T cells and CD4+ helper T cells. As used herein, T effector cells do not include regulatory T cells.

[0051] "Natural killer cells" or "NK cells" are cytotoxic lymphocytes that are components of the innate immune system and play an important role in the rejection of tumor and virus-infected cells.

[0052] "Treatment," "treating," or "ameliorating" refers to the medical management of a subject's (e.g., patient's) condition, disease, or disorder, which may be curative, prophylactic / preventative, or a combination thereof.

[0053] An "effective amount" or "therapeutically effective amount" may refer to the amount of a therapeutic agent (e.g., a modified IL2 polypeptide or modified IL2 fusion polypeptide described herein) that provides a desired physiological change, such as an anti-cancer effect. The desired physiological change may be, for example, a reduction in disease symptoms or a reduction in disease severity, or a reduction in disease progression. With respect to cancer, the desired physiological change may include, for example, tumor regression, a reduction in the rate of tumor progression, a reduction in the level of a cancer biomarker, a reduction in symptoms associated with cancer, prevention or delay of metastasis, or clinical remission.

[0054] A "checkpoint inhibitor" refers to an agent that reduces the activity of an immune checkpoint protein. A checkpoint inhibitor can be an antigen-binding moiety that binds to and reduces the activity of an immune checkpoint protein. Immune checkpoint proteins include, for example, programmed cell death protein 1 (PD-1 or CD279), programmed death-ligand 1 (PD-L1 or CD274), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4 or CD152), T-cell immunoglobulin mucin 3 (TIM3), lymphocyte activation 3 (LAG3 or CD223), B7-H2 (ICOSL or CD275), and B7-H3 (CD276). Examples of checkpoint inhibitors include ipilimumab (anti-CTLA-4 antibody), nivolumab (anti-PD-1 antibody), and pembrolizumab (anti-PD-1 antibody).

[0055] "Cancer antigen" refers to a molecule preferentially expressed by cancer cells. Examples of cancer antigens include CD19, CD20, ROR1, fibroblast activation protein alpha, and carcinoembryonic antigen (CEA).

[0056] "Oncolytic viruses" refer to viruses that preferentially infect and kill cancer cells. For example, oncolytic herpes viruses have been engineered to be deficient in ICP34.5, resulting in viruses that replicate only in cancer (and not healthy cells). An example of an oncolytic virus is talimogene laherparepvec, which is used to treat melanoma.

[0057] A "cancer vaccine" refers to a vaccine that presents cancer epitopes to the immune system to elicit an anti-cancer response from the immune system. For example, sipuleucel-T is a vaccine for metastatic prostate cancer that targets the immune response to the prostate cancer antigen prostatic acid phosphatase (PAP).

[0058] A "chimeric antigen receptor" or "CAR" is an engineered antigen-binding receptor that, when expressed in certain types of immune cells, activates the immune cell upon antigen binding. CARs typically contain an extracellular domain that includes an antigen-binding portion (e.g., an scFv), a transmembrane domain, and an intracellular immune signaling domain (e.g., containing signaling domains from CD3ζ, 4-1BB, and / or CD28). CARs can be expressed, for example, by T cells or NK cells and can contain an antigen-binding portion that targets a cancer antigen, such as CD19 or ROR1.

[0059] "Tumor infiltrating lymphocytes" or "TILs" refer to lymphocytes that are isolated from tumor tissue, manipulated in vitro (e.g., stimulated using cytokines such as interleukin-2), and then infused back into the patient so that the activated TILs home to the tumor site and induce tumor regression.

[0060] "Tumor microenvironment inhibitors" refer to agents that suppress one or more conditions or cell types present in the local environment surrounding a tumor that promote tumor growth. For example, bevacizumab can suppress the tumor microenvironment by reducing angiogenesis in the tumor microenvironment.

[0061] As used herein, the term "about" means ±20% of the stated range, value, or structure, unless otherwise indicated. The term "consisting essentially of" limits the scope of a claim to certain materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. As used herein, the terms "a" and "an" should be understood to refer to "one or more" of the listed components. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof. As used herein, the terms "include" and "have" are used interchangeably, and these terms and variations thereof are intended to be open-ended. The term "comprise" refers to the presence of the stated features, details, steps, or components recited in the claim, but does not exclude the presence or addition of one or more other features, details, steps, components, or groups thereof.

[0062] Recombinant DNA, molecular cloning, and gene expression techniques used in this disclosure are known in the art and are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd Ed., Cold Spring Harbor Laboratory, New York, 2001, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD, 1999.

[0063] Modified Interleukin-2 Polypeptides As noted above, the IL2 polypeptides of the present disclosure include IL2 receptor β (IL2Rβ) agonists having a modified IL2Rβ binding region 2. In some embodiments, binding region 2 comprises: X1-X2-X3-D-X4-X5-X6-N-X7-X8-X9-X 10 -X11 -X 12 -X 13 (SEQ ID NO: 1) In the formula, X1, X3, X6, X8, X 12 , and X 13 each containing any residue, X2, X4, and X 10 is an uncharged residue, X5, X7, X9, and X 11 Each contains an uncharged apolar residue.

[0064] For example, in some embodiments, the modified IL2 polypeptide has the following amino acid sequence: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLX1X2X3DX4X5X6NX7X8X9X 10 X 11 X 12 X 13 LKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 22) In the formula, X1, X3, X6, X8, X 12 , and X 13 each containing any residue, X2, X4, and X 10 is an uncharged residue, X5, X7, X9, and X 11 Each contains an uncharged apolar residue.

[0065] In certain embodiments, X1 is an uncharged polar residue, an uncharged nonpolar residue, a basic residue, or an acidic residue. In some embodiments, X1 is selected from C, T, G, W, I, S, E, and K. In some embodiments, X1 is selected from G, K, E, C, and T. In certain embodiments, X2 is an uncharged polar residue or an uncharged nonpolar residue. In some embodiments, X2 is selected from Y, P, V, W, L, A, and G. In some embodiments, X2 is selected from V, P, W, and A. In certain embodiments, X3 is an uncharged polar residue, an uncharged nonpolar residue, a basic residue, or an acidic residue. In some embodiments, X3 is selected from S, T, Q, G, M, E, R, and K. In some embodiments, X3 is selected from T, G, S, R, and E. In certain embodiments, X4 is not L. In some embodiments, X4 is an uncharged apolar residue or an uncharged polar residue. In some embodiments, X4 is selected from A, V, S, and T. In certain embodiments, X5 is selected from I, L, T, and V. In some embodiments, X5 is selected from I and V. In certain embodiments, X6 is an uncharged polar residue, a basic residue, or an acidic residue. In some embodiments, X6 is selected from S, T, E, D, and R. In some embodiments, X6 is selected from S, D, E, and T. In certain embodiments, X7 is selected from I, A, M, and V. In some embodiments, X7 is selected from I, A, and M. In certain embodiments, X8 is an uncharged polar residue, an uncharged apolar residue, a basic residue, or an acidic residue. In some embodiments, X8 is selected from S, T, N, Q, I, G, E, K, and R. In some embodiments, X8 is selected from I, R, N, and T. In certain embodiments, X is selected from V, L, and I. In some embodiments, X is V. In certain embodiments, X 10 is an uncharged polar residue or an uncharged apolar residue. In some embodiments, X 10 is selected from N, T, I, and L. In some embodiments, X 10is selected from I and L. In certain embodiments, X 11 is selected from V, A, and I. In certain embodiments, X 12 is an uncharged polar residue, an uncharged nonpolar residue, or an acidic residue. 12 is selected from Q, L, G, K, and R. In some embodiments, X 12 is selected from R, G, Q, and K. In certain embodiments, X 13 is an uncharged nonpolar residue or a basic residue. 13 is selected from A, D, and E. In some embodiments, X 13 is selected from E and A.

[0066] In some embodiments, IL2Rβ binding region 2 is selected from the group consisting of GVTDSISNAIVLARE (SEQ ID NO: 2), KWGDAVSNARVLAGE (SEQ ID NO: 3), KWGDAVSNARVLAGA (SEQ ID NO: 4), TLMDTTDNIGVLVRE (SEQ ID NO: 5), EPSDVISNINVLVQE (SEQ ID NO: 6), SPQDSIENISVLVRE (SEQ ID NO: 7), WASDSIENITLLIQE (SEQ ID NO: 8), CPTDTIENITVLIQE (SEQ ID NO: 9), RYKDSLENMQIIIQE (SEQ ID NO: 10), TARDAVDNMRVIIQE (SEQ ID NO: 11), TPRDVVENMNVLVLE (SEQ ID NO: 12), TPSDVIENMEVLILD (SEQ ID NO: 13), TPSDAIENINVLIRE ​​(SEQ ID NO: 14), TPSDVIENITVLVQE (SEQ ID NO: 15), G and selected from VGDTIDNINVLVKE (SEQ ID NO: 16), IGRDSIDNIKVIVQE (SEQ ID NO: 17), WATDTIRNVEVLVQE (SEQ ID NO: 18), TAEDVVTNITVLVQE (SEQ ID NO: 19), TAEDVISNIRVNVQE (SEQ ID NO: 20), TPSDVIDNVSITVQE (SEQ ID NO: 21), TARDAISNIRVIVQE (SEQ ID NO: 210), RARDAIDNIRVIVQE (SEQ ID NO: 211), TPRDAIDNINVIIQE (SEQ ID NO: 212), TPRDAIDNIRVIVQE (SEQ ID NO: 213), TPRDAIDNIRVIILE (SEQ ID NO: 214), TARDAISNINVIIQE (SEQ ID NO: 215), and TARDAIDNINVIVQE (SEQ ID NO: 216), and TARDAIDNIRVIVLE (SEQ ID NO: 217).

[0067] In some embodiments, the modified IL2Rβ binding region 2 is selected from TPRDAIDNIRVIVQE (SEQ ID NO: 213), TPRDAIDNIRVIILE (SEQ ID NO: 214), TARDAISNINVIIQE (SEQ ID NO: 215), and TARDAIDNINVIVQE (SEQ ID NO: 216).

[0068] In some embodiments, the modified IL2Rβ binding region 2 is selected from GVTDSISNAIVLARE (SEQ ID NO: 2), KWGDAVSNARVLAGA (SEQ ID NO: 4), EPSDVISNINVLVQE (SEQ ID NO: 6), CPTDTIENITVLIQE (SEQ ID NO: 9), TARDAVDNMRVIIQE (SEQ ID NO: 11), GVGDTIDNINVLVKE (SEQ ID NO: 16), TAEDVVTNITVLVQE (SEQ ID NO: 19).

[0069] In some embodiments, the modified IL2Rβ binding region 2 is selected from GVTDSISNAIVLARE (SEQ ID NO: 2), CPTDTIENITVLIQE (SEQ ID NO: 9), and TARDAVDNMRVIIQE (SEQ ID NO: 11).

[0070] In some embodiments, the modified IL2 polypeptide has the amino acid sequence of SEQ ID NO: 22. In some embodiments, the modified IL2 polypeptide has the amino acid sequence of any one of SEQ ID NOs: 23-42.

[0071] In some embodiments, an IL2Rβ agonist of the present disclosure comprises a modified IL2 polypeptide comprising a substitution of at least one residue selected from R81, P82, R83, L85, 186, S87, 189, N90, 192, V93, and L94 relative to SEQ ID NO: 65. In certain embodiments, the at least one substitution is to residue L85. In certain embodiments, the IL2 polypeptide comprises substitutions of at least two residues selected from R81, P82, R83, L85, 186, S87, 189, N90, 192, V93, and L94. In some embodiments, the at least two residues are selected from R81, R83, L85, 192, and L94. In some embodiments, the IL2 polypeptide comprises substitutions of at least three residues selected from R81, R83, L85, 192, and L94. In some embodiments, the modified IL2 polypeptide comprises substitutions at R81, R83, L85, I92, and L94. In some embodiments, the R81 substitution is selected from R81G, R81K, R81E, R81C, and R81T. In some embodiments, the R83 substitution is selected from R83T, R83G, R83S, and R83E. In some embodiments, the L85 substitution is selected from L85S, L85A, L85V, and L85T. In some embodiments, the I92 substitution is I92L. In some embodiments, the L94 substitution is selected from L94R, L94G, L94Q, and L94K. In some embodiments, the modified IL2 polypeptide comprising a substitution with at least one residue selected from R81, P82, R83, L85, I86, S87, I89, N90, I92, V93, and L94 has an IL2Rβ binding region 2 of any of SEQ ID NOs: 2-24.

[0072] In some embodiments, the modified IL2 polypeptide has increased affinity for IL2Rβ compared to wild-type IL2. In certain embodiments, the K Dis at least 10-fold greater, at least 15-fold greater, at least 20-fold greater, at least 25-fold greater, or at least 30-fold greater than the binding of wild-type IL2 to IL2Rβ. In some embodiments, the modified IL2 polypeptide has a K that is at least 30-fold greater than wild-type IL2. D In some embodiments, the affinity of the modified IL2 polypeptide for IL2Rβ is increased by at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 30-fold compared to wild-type IL2.

[0073] In some embodiments, the affinity of the modified IL2 polypeptide for IL2Rα is reduced compared to wild-type IL2, hi certain embodiments, the affinity of the modified IL2 polypeptide for IL2Rα is reduced by at least 5%, at least 10%, at least 15%, or at least 20% compared to wild-type IL2.

[0074] In some embodiments, the affinity of the modified IL2 polypeptide for IL2Rα is similar compared to wild-type IL2. In certain embodiments, the modified IL2 polypeptide has an affinity for IL2Rα that differs by no more than ±20%, no more than ±15%, no more than ±10%, or no more than ±5% from the affinity of wild-type IL2 for IL2Rα.

[0075] Some embodiments of the present disclosure provide a modified IL2 polypeptide comprising a modified IL2 receptor alpha (IL2Rα) binding region 1. The modified IL2Rα binding region 1 may comprise a substitution selected from a substitution at position K35, a substitution at R38, a substitution at F42, a substitution at Y45, or a combination thereof. In some embodiments, the modified IL2 polypeptide binds to IL2Rα with at least two-fold reduced binding kinetics compared to wild-type IL2.

[0076] In some embodiments, the modified IL2 polypeptide may comprise a substitution at position K35. In some embodiments, the substitution at position K35 comprises a non-basic residue. In some embodiments, the substitution at position K35 comprises an uncharged residue or an acidic residue. In some embodiments, the substitution at position K35 is selected from K35G, K35L, K35S, K35V, K35D, K35E, and K35C.

[0077] In some embodiments, the modified IL2 polypeptide comprises a substitution at position R38. In some embodiments, the substitution at position R38 comprises a non-basic residue. In some embodiments, the substitution at position R38 comprises an uncharged residue or an acidic residue. In some embodiments, the substitution at position R38 is selected from R38V, R38D, R38E, R38S, R38I, R38A, R38Y, R38G, R38C, and R38N.

[0078] In some embodiments, the modified IL2 polypeptide may comprise a substitution at position F42. In some embodiments, the substitution at position F42 comprises an uncharged residue. In some embodiments, the substitution at position F42 comprises a basic residue. In some embodiments, the substitution at position F42 is selected from F42A, F42R, F42G, F42I, F42L, F42P, and F42H.

[0079] In some embodiments, the modified IL2 polypeptide may comprise a substitution at position Y45. In some embodiments, the substitution at position Y45 comprises an uncharged residue. In some embodiments, the substitution at position Y45 comprises an uncharged polar residue or an uncharged nonpolar residue. In some embodiments, the Y45 substitution is Y45S, Y45P, Y45A, Y45V, Y45C, Y45T, and Y45F.

[0080] In some embodiments, the modified IL2 polypeptide may comprise a substitution at position K35 and a substitution at position R38. In some embodiments, the modified IL2 polypeptide comprises a K35G substitution and an R38E substitution.

[0081] In some embodiments, the modified IL2 polypeptide may comprise a substitution at position K35 and a substitution at position F42. In some embodiments, the modified IL2 polypeptide comprises a K35S substitution and a F42G substitution.

[0082] In some embodiments, the modified IL2 polypeptide may comprise a substitution at position K35, a substitution at position R38, and a substitution at position F42. In some embodiments, the modified IL2 polypeptide comprises a K35L substitution, an R38D substitution, and an F42R substitution.

[0083] In some embodiments, the modified IL2 polypeptide may comprise a substitution at position R38 and a substitution at position Y45S. In some embodiments, the modified IL2 polypeptide comprises an R38D substitution and a Y45S substitution. In some embodiments, the modified IL2 polypeptide comprises an R38V substitution and a Y45S substitution.

[0084] In some embodiments, the modified IL2 polypeptide binds to IL2Rα with binding kinetics that are at least 10-fold reduced compared to wild-type IL2.

[0085] In some embodiments, IL2Rα binding region 1 is selected from the group consisting of PVLTRMLTIKFY (SEQ ID NO: 183), PKLTRMLTLKFP (SEQ ID NO: 184), PDLTSMLAFKFY (SEQ ID NO: 185), PGLTEMLTFKFY (SEQ ID NO: 186), PSLTMLTGKFY (SEQ ID NO: 187), PELTIMLTPKFY (SEQ ID NO: 188), PCLTAMLTLKFA (SEQ ID NO: 189), PCLTAMLTLKFA (SEQ ID NO: 190), PKLTRMLTHKFV (SEQ ID NO: 191), PCLTDMLTFKFY (SEQ ID NO: 192), PLLTDMLTRKFY (SEQ ID NO: 193), PLLTDMLTFKFY (SEQ ID NO: 194), PKLTDMLTFKFS (SEQ ID NO: 195), PKL TYMLTRKFY (SEQ ID NO: 196), PKLTRMLTFKFC (SEQ ID NO: 197), PKLTSMLTFKFS (SEQ ID NO: 198), PKLTSMLTFKFS (SEQ ID NO: 199), PKLTYMLTFKFS (SEQ ID NO: 200), PKLTYMLTFKFS (SEQ ID NO: 201), PKLTGMLTFKFS (SEQ ID NO: 202), PKLTVMLTFKFT (SEQ ID NO: 203), PKLTVMLTFKFS (SEQ ID NO: 204), PKLTVMLTFKFP (SEQ ID NO: 205), PKLTVMLTFKFF (SEQ ID NO: 206), PKLTCMLTFKFA (SEQ ID NO: 207), PKLTNMLTFKFA (SEQ ID NO: 208), and PKLTNMLTFKFS (SEQ ID NO: 209).

[0086] In some embodiments, the modified IL2 polypeptide shares at least 80%, e.g., at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with residues outside of IL2Rβ binding region 2 (i.e., residues 1-80 and 96-133) of SEQ ID NO: 22 and binds to IL2Rβ. In some embodiments, the modified IL2 polypeptide shares at least 80%, e.g., at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with residues outside of IL2Rα binding region 1 (i.e., residues 1-33 and 46-133) of SEQ ID NO: 223, has reduced binding to IL2Rα, and binds to IL2Rβ. In some embodiments, the modified IL2 polypeptide shares at least 80%, e.g., at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with residues outside of IL2Rα binding region 1 and IL2Rβ binding region 2 (i.e., residues 1-33, 46-80, 96-133) of SEQ ID NO: 22 or SEQ ID NO: 223, has reduced binding to IL2Rα, and binds to IL2Rβ. In some embodiments, the disclosure provides a modified IL2 polypeptide comprising the modified IL2Rβ binding region 2 described above and the modified IL2Rα binding region 1 described above. In certain embodiments, the modified IL2 polypeptide is selected from the group consisting of PVLTRMLTIKFY (SEQ ID NO: 183), PKLTRMLTLKFP (SEQ ID NO: 184), PDLTSMLAFKFY (SEQ ID NO: 185), PGLTEMLTFKFY (SEQ ID NO: 186), PSLTMLTGKFY (SEQ ID NO: 187), PELTIMLTPKFY (SEQ ID NO: 188), PCLTAMLTLKFA (SEQ ID NO: 189), PCLTAMLTLKFA (SEQ ID NO: 190), PKLTRMLTHKFV (SEQ ID NO: 191), PCLTDMLTFKFY (SEQ ID NO: 192), PLLTDMLTRKFY (SEQ ID NO: 193),PLLTDMLTFKFY (SEQ ID NO: 194), PKLTDMLTFKFS (SEQ ID NO: 195), PKLTYMLTRKFY (SEQ ID NO: 196), PKLTRMLTFKFC (SEQ ID NO: 197), PKLTSMLTFKFS (SEQ ID NO: 198), PKLTSMLTFKFS (SEQ ID NO: 199), PKLTYMLTFKFS (SEQ ID NO: 200), PKLTYMLTFKFS (SEQ ID NO: 201), PKLTGMLTFKFS (SEQ ID NO: 202), PKLTVMLTFKFT (SEQ ID NO: 203), PKLTVMLTFKFS (SEQ ID NO: 204), PKLTVML TFKFP (SEQ ID NO: 205), PKLTVMLTFKFF (SEQ ID NO: 206), PKLTCMLTFKFA (SEQ ID NO: 207), PKLTNMLTFKFA (SEQ ID NO: 208), and PKLTNMLTFKFS (SEQ ID NO: 209), and ISVLVRE (SEQ ID NO: 7), WASDSIENITLLIQE (SEQ ID NO: 8), CPTDTIENITVLIQE (SEQ ID NO: 9), RYKDSLENMQIIIQE (SEQ ID NO: 10), TARDAVDNMRVIIQE (SEQ ID NO: 11), TPRDVVENMNVLVLE (SEQ ID NO: 12), TPSDVIENMEVLILD (SEQ ID NO: 13), TPSDAIENINVLIRE ​​(SEQ ID NO: 14), TPSDVIENITVLVQE (SEQ ID NO: 15), GVGDTIDNINVLVKE (SEQ ID NO: 16), IGRDSIDNIKVIVQE (SEQ ID NO: 17). 17), WATDTIRNVEVLVQE (SEQ ID NO: 18), TAEDVVTNITVLVQE (SEQ ID NO: 19), TAEDVISNIRVNVQE (SEQ ID NO: 20), TPSDVIDNVSITVQE (SEQ ID NO: 21), TARDAISNIRVIVQE (SEQ ID NO: 210), RARDAIDNIRVIVQE (SEQ ID NO: 211), TPRDAIDNINVIIQE (SEQ ID NO: 212), TPRDAIDNIRVIVQE (SEQ ID NO: 213), TPRDAIDNIRVIILE (SEQ ID NO: 214), TARDAISNINVIIQE (SEQ ID NO: 215),and a modified IL2Rβ binding region 2 selected from TARDAIDNINVIVQE (SEQ ID NO: 216), and TARDAIDNIRVIVLE (SEQ ID NO: 217).

[0087] In certain embodiments, the modified IL2 polypeptide comprises a modified IL2Rβ binding region 2 selected from GVTDSISNAIVLARE (SEQ ID NO: 2), TARDAVDNMRVIIQE (SEQ ID NO: 11), TPRDAIDNIRVIVQE (SEQ ID NO: 213), TPRDAIDNIRVIILE (SEQ ID NO: 214), TARDAISNINVIIQE (SEQ ID NO: 215), and TARDAIDNINVIVQE (SEQ ID NO: 216), and a modified IL2Rα binding region 1 described above.

[0088] In some embodiments, the modified IL2 polypeptide is selected from any one of SEQ ID NOs: 147-170, optionally including (or excluding) a C-terminal histidine tag. In some embodiments, the C-terminal histidine tag is replaced with another linker, such as a gly-ser linker.

[0089] Modified IL2 fusion polypeptides Some embodiments of the present disclosure provide modified IL2 fusion polypeptides. The modified IL2 fusion polypeptide may comprise a modified IL2 polypeptide as described herein above and at least one additional molecule covalently linked to the modified IL2 polypeptide via a peptide bond or other chemical bond. In some embodiments, the at least one additional molecule of the fusion polypeptide is a half-life extending molecule. In some embodiments, the half-life extending molecule comprises a half-life extending polypeptide. In some embodiments, the half-life extending polypeptide comprises an Fc domain, human serum albumin (HSA), an HSA binding molecule, or transferrin.

[0090] In certain embodiments, the IL2 fusion polypeptide comprises an Fc domain. In some embodiments, the Fc domain is derived from an IgG antibody. Human IgG antibodies have several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the Fc domain is derived from an IgG1 antibody or an IgG4 antibody. In some embodiments, the Fc domain has one or more substitutions that reduce the effector function of the Fc domain. Examples of substitutions that reduce Fc effector function include L34A, L235A, and P329G. "LALAPG" may refer to a modified Fc domain comprising L34A, L235A, and P329G, respectively. In some embodiments, the Fc domain comprises a modification of at least one amino acid residue that increases serum half-life. Exemplary modifications to the Fc domain are described in U.S. Patent Nos. 7,317,091, 8,735,545, 7,371,826, 7,670,600, and 9,803,023. In some embodiments, the Fc domain is SEQ ID NO: 64. In some embodiments, the modified IL2-Fc fusion polypeptide comprises a sequence selected from SEQ ID NOs: 39-49.

[0091] In some embodiments, at least one additional molecule of the fusion polypeptide is an antigen-binding moiety. In some embodiments, the antigen-binding moiety comprises an immunoglobulin, a Fab molecule, an scFv, a bispecific T cell engager, a diabody, a single domain antibody, or a nanobody. The antigen-binding moiety may bind to, for example, carcinoembryonic antigen (CEA), GD2, or CD20. An example of a CEA antigen moiety is CH1A1A-2F. An example of a GD-2 antigen-binding moiety is dinutuximab, and an example of a CD20 antigen-binding moiety is rituximab.

[0092] In some embodiments, at least one additional molecule of the fusion polypeptide is a cytokine. In some embodiments, the cytokine is selected from interleukin 2, interleukin 15, interleukin 7, interleukin 10, and CC motif chemokine ligand 19 (CCL19). In some embodiments, the additional molecule of the fusion polypeptide is a second modified IL2 polypeptide described herein.

[0093] In some embodiments, the half-life extending molecule comprises polyethylene glycol (PEG) or polypropylene glycol (PPG).

[0094] In some embodiments, the fusion polypeptide is a monovalent fusion polypeptide. A monovalent fusion polypeptide refers to a fusion polypeptide having one copy of a modified IL2 polypeptide.

[0095] In certain embodiments, a monovalent fusion polypeptide comprises a modified IL2 polypeptide linked to a fusion partner, such as an Fc region. A variety of linkers are known in the art and may be used to covalently link the modified IL2 described herein to a fusion partner, such as an Fc region. As used herein, the terms "linker" and "linker sequence" refer to a molecule or group of molecules (e.g., a monomer or polymer) that connects two molecules and often serves to position the two molecules in a preferred configuration. A linker may contain amino acid residues that provide flexibility. Thus, a linker peptide may primarily comprise the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide must be of sufficient length to link the two molecules in a suitable conformation relative to each other so as to retain the desired activity. A suitable length for this purpose includes at least one and at most 30 amino acid residues. Preferably, the linker is about 1 to 30 amino acids in length, with linkers of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acids in length being preferred. Useful linkers include glycine-serine polymers (e.g., (GS)n, (GSGGS)n (SEQ ID NO: 218), (GGGGS)n (SEQ ID NO: 219), and (GGGS)n (SEQ ID NO: 220), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. In some embodiments, the fusion polypeptide is a bivalent fusion polypeptide. A bivalent fusion protein may refer to a molecular complex containing two copies of a modified IL2 polypeptide, which may have the same or different sequences. The molecular complex may be non-covalently bound. For example, a bivalent fusion protein may comprise two Fc regions non-covalently linked together, e.g., by one or more disulfide bridges or by knobs-into-hole chemistry.

[0096] Method for producing modified IL2 polypeptide Modified IL2 polypeptides or modified IL2 fusion polypeptides can be prepared by genetic or chemical methods well known in the art and by methods disclosed in the Examples below. Genetic methods can include, for example, site-directed mutagenesis of the DNA sequence encoding the polypeptide, PCR, and gene synthesis. The intended nucleotide changes can be confirmed by sequencing. The nucleotide sequence of native IL2 is described by Taniguchi et al. (Nature 302, 305-10 (1983)), and nucleic acids encoding native human IL2 are available, for example, from the American Type Culture Collection (Rockville, MD).

[0097] The modified IL2 polypeptide or modified IL2 fusion polypeptide can be obtained, for example, by recombinant production or solid-phase peptide synthesis. For recombinant production, a polynucleotide encoding the modified IL2 polypeptide or modified IL2 fusion polypeptide can be isolated and inserted into one or more vectors for cloning and / or expression in a host cell. Such polynucleotides can be readily isolated and sequenced by conventional procedures. In certain embodiments, vectors, such as expression vectors, are provided that contain one or more of the polynucleotides of the present disclosure. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the modified IL2 polypeptide or modified IL2 fusion polypeptide, along with appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL (FOURTH EDITION), Cold Spring Harbor Laboratory, NY (2012), and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, NY (1993). Expression vectors can be part of a plasmid, a virus, or can be nucleic acid fragments. Expression vectors contain an expression cassette into which a polynucleotide encoding a modified IL2 polypeptide or modified IL2 fusion polypeptide (i.e., a coding region) is cloned in operable association with a promoter and / or other transcriptional or translational control elements. As used herein, a "coding region" is a portion of a nucleic acid consisting of codons translated into amino acids.A "stop codon" (TAG, TGA, or TAA), although not translated into amino acids, may be considered part of a coding region when present; however, any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, etc., are not part of a coding region. Two or more coding regions may be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any vector may contain a single coding region or may include two or more coding regions; for example, the vectors disclosed herein may encode one or more polyproteins, which are post-translationally or co-translationally cleaved into final proteins via proteolytic cleavage. The vectors, polynucleotides, or nucleic acids of the present disclosure may also encode heterologous coding regions, fused or unfused to a first or second polynucleotide encoding a polypeptide disclosed herein or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. Operable association occurs when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences such that expression of the gene product is under the influence or control of the regulatory sequence(s). Two DNA fragments (e.g., a polypeptide coding region and its associated promoter) are "operably associated" if induction of promoter function results in transcription of mRNA encoding the desired gene product, and if the nature of the binding between the two DNA fragments does not interfere with the ability of the expression control sequences to induce expression of the gene product or the ability of the DNA template to be transcribed. Thus, a promoter region is operably associated with a nucleic acid encoding a polypeptide if the promoter is capable of affecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that induces substantial transcription of DNA only in predetermined cells.In addition to promoters, other transcription control elements, such as enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to induce cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as promoters and enhancer segments derived from cytomegalovirus (e.g., the immediate early promoter in combination with intron A), Simian Virus 40 (e.g., the early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers, as well as inducible promoters (e.g., tetracycline-inducible promoters). Similarly, a variety of translation control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly internal ribosome entry sites or IRES, also called CITE sequences). Expression cassettes may also include other features such as origins of replication and / or chromosomal integration elements such as retroviral long terminal repeats (LTRs) or adeno-associated viral (AAV) inverted terminal repeats (ITRs).

[0098] The polynucleotide and nucleic acid coding regions of the present disclosure may be associated with additional coding regions encoding secretory or signal peptides that direct the secretion of a polypeptide encoded by a polynucleotide of the present disclosure. For example, if secretion of a modified IL2 polypeptide or a modified IL2 fusion polypeptide is desired, DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding the mature amino acids of the modified IL2 polypeptide or modified IL2 fusion polypeptide. Those skilled in the art will recognize that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce the secreted or "mature" form of the polypeptide. For example, native human IL2 is translated with a 20-amino acid signal sequence at the N-terminus of the polypeptide, which is subsequently cleaved to yield the mature 133-amino acid human IL2. In some embodiments, a native signal peptide, e.g., the IL2 signal peptide or an immunoglobulin heavy or light chain signal peptide, is used, or a functional derivative of that sequence that retains the ability to direct the secretion of an operably associated polypeptide is used.

[0099] In some embodiments, the polynucleotide encoding the modified IL2 polypeptide or modified IL2 fusion polypeptide further comprises a DNA sequence encoding a sequence (e.g., a histidine tag) to facilitate purification or to label the modified IL2 polypeptide or modified IL2 fusion polypeptide within or at the end of the polynucleotide encoding the modified IL2 polypeptide or modified IL2 fusion polypeptide.

[0100] In certain embodiments, host cells are provided that contain one or more polynucleotides encoding modified IL2 polypeptides or modified IL2 fusion polypeptides. In certain embodiments, the host cells contain one or more vectors encoding the modified IL2 polypeptides or modified IL2 fusions. The host cells can be any type of cell line that can be used to produce modified IL2 polypeptides or modified IL2 fusion polypeptides. Such cells can be transfected or transduced with a particular expression vector encoding the modified IL2 polypeptide or modified IL2 fusion, as needed, and large quantities of the vector-containing cells can be grown for inoculation into large-scale fermenters to obtain sufficient amounts of the modified IL2 polypeptide or modified IL2 fusion for clinical use. Suitable host cells include prokaryotic microorganisms, such as E. coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, and the like. For example, polypeptides can be produced in bacteria, particularly if glycosylation is not required. After expression, the polypeptide can be isolated from the bacterial cells in a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce polypeptides with partially or completely human glycosylation patterns. Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plants and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include the SV40 (COS-7) transformed monkey kidney CV1 line; human embryonic kidney lines (e.g., 293 or 293T cells described in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells (MMT060562), TRI cells (e.g., Mather et al., Annals of NY Acad Sci 383, 44-68 (1982), MRC5 cells, and FS4 cells. Other useful mammalian host cell lines include dhfr. - Examples include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines, such as YO, NS0, P3X63, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as cultured mammalian cells, yeast cells, insect cells, bacterial cells, and plant cells, to name just a few, including cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell).

[0101] Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing a modified IL2 polypeptide fused to either the heavy or light chain of an antigen-binding moiety, such as an antibody, may be modified to also express the remainder of the antibody chains, such that the expressed modified IL2 fusion polypeptide comprises an antibody having both a heavy and a light chain.

[0102] In some embodiments, methods for producing a modified IL2 polypeptide or a modified IL2 fusion polypeptide are provided. In some embodiments, the methods comprise culturing a host cell comprising a polynucleotide encoding a modified IL2 polypeptide or a modified IL2 fusion polypeptide provided herein under conditions suitable for expression of the modified IL2 polypeptide or the modified IL2 fusion polypeptide, and, optionally, recovering and / or purifying the modified IL2 polypeptide or the modified IL2 fusion polypeptide from the host cell (or from the culture medium of the host cell, e.g., if the host cell secretes the polypeptide).

[0103] Pharmaceutical Composition Provided herein are pharmaceutical compositions comprising a modified IL2 polypeptide or modified IL2 fusion polypeptide described herein and a pharmaceutically acceptable diluent(s), excipient(s), or carrier(s). In some embodiments, the pharmaceutical composition comprises a modified IL2 polypeptide or modified IL2 fusion polypeptide disclosed herein and an additional therapeutic agent (e.g., a combination therapy). Non-limiting examples of such therapeutic agents are described herein below. Pharmaceutical compositions may be formulated in a conventional manner using pharmaceutically acceptable carriers, including excipients and auxiliary agents that facilitate processing of the modified IL2 or IL2 fusion polypeptide into a pharmaceutically usable preparation. Appropriate formulations will depend on the selected route of administration. Any pharmaceutically acceptable means, carriers, and excipients may be used as suitable for formulating the pharmaceutical compositions described herein, including, but not limited to, Remington: The Science and Practice of Pharmacy, Nineteenth Ed. (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999). Examples of IL-2 compositions are described in U.S. Patent Nos. 4,604,377 and 4,766,106, which are incorporated herein by reference.

[0104] As used herein, "pharmaceutically acceptable carrier" and "physiologically acceptable carrier" are used interchangeably and include any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungals), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like materials, and combinations thereof, known to those of skill in the art, and generally are molecular entities and compositions that are non-toxic to recipients, i.e., do not produce adverse, allergic, or other untoward reactions when administered to an animal, e.g., a human, as appropriate, at the dosages and concentrations employed (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0105] A pharmaceutical composition may contain different types of carriers depending on whether it is to be administered in solid, liquid, or aerosol form, and whether it needs to be sterile for the route of administration, such as injection. The modified IL2 polypeptides or modified IL2 fusion polypeptides described herein (and any additional therapeutic agents) can be administered intravenously, intradermally, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravascularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, infusion, continuous infusion, by local perfusion bathing directly at target cells, via catheter, via lavage, in a cream, in a lipid composition (e.g., liposomes), or by other methods or combinations of any of the above as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, incorporated herein by reference). Parenteral administration, particularly intravenous injection, is most commonly used to administer polypeptide molecules such as the modified IL2 polypeptides or modified IL2 fusion polypeptides described herein.

[0106] Parenteral compositions include those designed for administration by injection, e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection. For injection, the modified IL2 polypeptides or modified IL2 fusion polypeptides described herein can be formulated in aqueous solutions, preferably physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. The solutions may contain formulating agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the modified IL2 polypeptides or modified IL2 fusion polypeptides may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the modified IL2 polypeptides or modified IL2 fusion polypeptides in the required amount in the appropriate solvent, with various other ingredients as listed below, as needed. Sterilization can be readily accomplished, for example, by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and / or other ingredients. In the case of sterile powders for preparing sterile injectable solutions, suspensions, or emulsions, preparation methods include vacuum drying or freeze-drying techniques to produce a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered liquid medium. The liquid medium should be appropriately buffered, if necessary, and the liquid diluent is first rendered isotonic with sufficient saline or glucose prior to injection. Pharmaceutical compositions are preferably stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. It will be understood that endotoxin contamination should be kept to a minimum, for example, at a safe level, e.g., less than 0.5 ng / mg protein.Suitable pharmaceutically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polymers, Examples of suitable suspensions include peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. In some embodiments, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound, allowing for the preparation of highly concentrated solutions. Additionally, suspensions of the active compound may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl acetate or triglycerides, or liposomes.

[0107] In some embodiments, the aqueous suspension contains one or more polymers as a suspending agent. Exemplary polymers include water-soluble polymers, such as cellulose-based polymers, such as hydroxypropylmethylcellulose, and water-insoluble polymers, such as cross-linked carboxyl-containing polymers. Certain pharmaceutical compositions described herein include a mucoadhesive polymer selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran.

[0108] In some embodiments, the pharmaceutical composition includes a solubilizing agent that aids in the solubility of the modified IL2 polypeptide or modified IL2 fusion polypeptide. The term "solubilizing agent" generally includes agents that result in the formation of a micellar or true solution of the drug. Certain acceptable non-ionic surfactants, such as polysorbate 80, are useful as solubilizing agents. Examples include glycols, polyglycols, such as polyethylene glycol 400, and glycol ethers.

[0109] In some embodiments, the pharmaceutical compositions comprise one or more pH adjusting or buffering agents, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trishydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0110] In some embodiments, the pharmaceutical composition comprises one or more salts in an amount necessary to render the composition tolerable in osmolality. Such salts include those having sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0111] In some embodiments, the pharmaceutical composition includes one or more preservatives that inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merphen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0112] In some embodiments, the pharmaceutical composition contains one or more surfactants to enhance physical stability or for other purposes. Suitable nonionic surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, such as octylphenol 10 and octylphenol 40.

[0113] In some embodiments, pharmaceutical compositions contain one or more antioxidants where required to enhance chemical stability. Suitable antioxidants include, by way of example only, ascorbic acid and sodium metabisulfite.

[0114] In certain embodiments, aqueous suspension compositions are packaged in single-dose non-reclosable containers. Alternatively, multi-dose reclosable containers are used, in which case the compositions typically contain a preservative.

[0115] In some embodiments, the modified IL2 polypeptides or modified IL2 fusion polypeptides described herein are delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing a therapeutic agent. A variety of sustained-release materials are useful herein. In some embodiments, sustained-release capsules release the modified IL2 polypeptides or modified IL2 fusion polypeptides for several weeks to over 100 days. Depending on the chemical nature and biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed. Examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films or microcapsules. In some embodiments, sustained absorption of injectable compositions can be achieved by the use in the compositions of agents that delay absorption, such as, for example, aluminum monostearate, gelatin, or combinations thereof.

[0116] In some embodiments, the modified IL2 polypeptides or modified IL2 fusion polypeptides described herein may be encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methsylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed., Mack Printing Company, 1990).

[0117] In some embodiments, the modified IL2 polypeptides or modified IL2 fusion polypeptides described herein may also be formulated as depot preparations. Such long-acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or intramuscular injection. Thus, for example, the modified IL2 polypeptides or modified IL2 fusion polypeptides may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.

[0118] Pharmaceutical compositions containing the modified IL2 polypeptides or modified IL2 fusion polypeptides described herein may be produced by conventional mixing, dissolving, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or auxiliaries that facilitate processing of the protein into a pharmaceutically usable preparation. The appropriate formulation will depend on the selected route of administration.

[0119] In some embodiments, modified IL2 polypeptides or modified IL2 fusion polypeptides may be formulated into compositions in a free acid or base, neutral, or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include acid addition salts, e.g., those formed with free amino groups of a proteinaceous composition, or those formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other aprotic solvents than the corresponding free base forms.

[0120] In some embodiments, the modified IL2 polypeptides or modified IL2 fusion polypeptides described herein are formulated for oral administration. In various embodiments, the modified IL2 polypeptides or modified IL2 fusion polypeptides described herein are formulated in oral dosage forms including, by way of example only, tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions, and the like.

[0121] In certain embodiments, pharmaceutical preparations for oral use are obtained by mixing one or more solid excipients with one or more modified IL2 polypeptides or modified IL2 fusion polypeptides described herein, optionally grinding the resulting mixture, and optionally processing the granulated mixture after adding suitable excipients to obtain tablets or dragee cores. Suitable excipients include, in particular, fillers, such as sugars including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. In certain embodiments, disintegrants are optionally added. Disintegrants include, by way of example only, cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.

[0122] In some embodiments, dosage forms such as dragee cores and tablets are provided with one or more suitable coatings. In certain embodiments, a concentrated sugar solution is used to coat the dosage form. The sugar solution optionally contains additional ingredients, such as, by way of example only, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, a lacquer solution, and a suitable organic solvent or solvent mixture. Dyes and / or pigments are also optionally added to the coating for identification purposes. Furthermore, dyes and / or pigments are optionally used to characterize different combinations of active drug doses.

[0123] In certain embodiments, a therapeutically effective amount of at least one of the modified IL2 polypeptides or modified IL2 fusion polypeptides described herein is formulated into other oral dosage forms. Oral dosage forms include push-fit capsules made of gelatin and sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. In certain embodiments, push-fit capsules contain the active ingredient mixed with one or more fillers. The fillers include binders, such as lactose or starch, and / or lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In some embodiments, soft capsules contain one or more active agents dissolved or suspended in a suitable liquid. Suitable liquids may include one or more fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers are optionally added.

[0124] In some embodiments, a therapeutically effective amount of at least one of the modified IL2 polypeptides or modified IL2 fusion polypeptides described herein is formulated for buccal or sublingual administration. Formulations suitable for buccal or sublingual administration include, by way of example only, tablets, lozenges, or gels.

[0125] In some embodiments, the modified IL2 polypeptide or modified IL2 fusion polypeptide is administered topically. The modified IL2 polypeptide or modified IL2 fusion polypeptide described herein is formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams, or ointments. Such pharmaceutical compositions optionally contain solubilizers, stabilizers, tonicity enhancers, buffers, and preservatives.

[0126] In some embodiments, the modified IL2 polypeptide or modified IL2 fusion polypeptide is formulated for transdermal administration. In certain embodiments, transdermal formulations use transdermal delivery devices and transdermal delivery patches, and may be lipophilic emulsions or buffered aqueous solutions, dissolved and / or dispersed in polymers or adhesives. In various embodiments, such patches are constructed for continuous, pulsatile, or on-demand delivery of pharmaceutical agents. In additional embodiments, transdermal delivery of the modified IL2 polypeptide or modified IL2 fusion polypeptide is achieved by means of an iontophoretic patch or the like. In certain embodiments, the transdermal patch provides controlled delivery of the modified IL2 polypeptide or modified IL2 fusion polypeptide. In certain embodiments, the absorption rate is slowed by using rate-controlling membranes or by trapping the modified IL2 polypeptide or modified IL2 fusion polypeptide within a polymer matrix or gel. In alternative embodiments, absorption enhancers are used to increase absorption. The absorption enhancer or carrier comprises absorbable pharmaceutically acceptable solvents that aid in passage through the skin. For example, in one embodiment, the transdermal device is in the form of a bandage comprising a backing, optionally a reservoir containing the modified IL2 polypeptide or modified IL2 fusion polypeptide together with a carrier, optionally a rate-controlling barrier for delivering the modified IL2 polypeptide or modified IL2 fusion polypeptide to the host's skin at a controlled, predetermined rate over an extended period of time, and a means for securing the device to the skin.

[0127] In some embodiments, the modified IL2 polypeptide or modified IL2 fusion polypeptide is formulated for administration by inhalation. Various forms suitable for administration by inhalation include, but are not limited to, aerosols, mists, or powders. The pharmaceutical composition of the modified IL2 polypeptide or modified IL2 fusion polypeptide can conveniently be delivered in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). In certain embodiments, the dosage unit of the pressurized aerosol is determined by providing a valve to deliver a metered amount. In certain embodiments, gelatin capsules and cartridges for use in an inhaler or insufflator are formulated, by way of example only, containing a powder mix of the modified IL2 polypeptide or modified IL2 fusion polypeptide and a suitable powder base, such as lactose or starch.

[0128] In some embodiments, the modified IL2 polypeptide or modified IL2 fusion polypeptide is formulated into a rectal composition, e.g., an enema, rectal gel, rectal foam, rectal aerosol, suppository, jelly suppository, or retention enema, containing a conventional suppository base such as cocoa butter or other glycerides, and a synthetic polymer such as polyvinylpyrrolidone or PEG. In a suppository form of composition, a low melting wax, such as, but not limited to, a mixture of fatty acid glycerides, optionally combined with cocoa butter, is first melted.

[0129] In certain embodiments, the formulations described herein include one or more antioxidants, metal chelators, thiol-containing compounds, and / or other general stabilizers. Examples of such stabilizers include, but are not limited to, (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w / v ascorbic acid, (f) 0.003 to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.05% w / v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrin, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc, or (n) combinations thereof.

[0130] In some embodiments, the concentration of modified IL2 polypeptide or modified IL2 fusion polypeptide provided in a pharmaceutical composition of the present disclosure is 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08% , 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or less than 0.0001% w / w, w / v, or v / v.

[0131] In some embodiments, the concentration of modified IL2 polypeptide or modified IL2 fusion polypeptide provided in the pharmaceutical composition of the present disclosure is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50%, 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8% ,7.75%,7.50%,7.25%,7%,6.75%,6.50%,6.25%,6%,5.75%,5.50%,5.25%,5%,4.75%,4.50%,4.25%,4%,3.75%,3.50%,3.25%,3%,2.75%,2.50%,2.25%,2%,1.75%,1.50%,125%,1%,0.5%,0.4%,0.3%,0.2%,0.1%,0.09%,0.08%,0.07%,0 Greater than 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w / w, w / v, or v / v.

[0132] In some embodiments, the concentration of modified IL2 polypeptide or modified IL2 fusion polypeptide provided in the pharmaceutical composition of the present disclosure is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, or from about 0. The range is from about 0.07% to about 24%, from about 0.08% to about 23%, from about 0.09% to about 22%, from about 0.1% to about 21%, from about 0.2% to about 20%, from about 0.3% to about 19%, from about 0.4% to about 18%, from about 0.5% to about 17%, from about 0.6% to about 16%, from about 0.7% to about 15%, from about 0.8% to about 14%, from about 0.9% to about 12%, or from about 1% to about 10% w / w, w / v, or v / v.

[0133] In some embodiments, the concentration of modified IL2 polypeptide or modified IL2 fusion polypeptide provided in the pharmaceutical composition of the present disclosure is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, or about 0.1% to about 0.9% w / w, w / v, or v / v.

[0134] In some embodiments, the amount of modified IL2 polypeptide or modified IL2 fusion polypeptide provided in a pharmaceutical composition of the present disclosure is 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3g, 0.25g, 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g, or 0.0001g.

[0135] In some embodiments, the amount of modified IL2 polypeptide or modified IL2 fusion polypeptide provided in the pharmaceutical composition of the present disclosure is 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008g, 0.0009g, 0.001g, 0.0 015g, 0.002g, 0.0025g, 0.003g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006 g, 0.0065g, 0.007g, 0.0075g, 0.008g, 0.0085g, 0.009g, 0.0095g, 0.01g, 0.015g, 0.0 2g, 0.025g, 0.03g, 0.035g, 0.04g, 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0.07g, 0. 075g, 0.08g, 0.085g, 0.09g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0. Greater than 45g, 0.5g, 0.55g, 0.6g, 0.65g, 0.7g, 0.75g, 0.8g, 0.85g, 0.9g, 0.95g, 1g, 1.5g, 2g, 2.5, 3g, 3.5, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g, or 10g.

[0136] In some embodiments, the amount of modified IL2 polypeptide or modified IL2 fusion polypeptide provided in a pharmaceutical composition of the present disclosure ranges from 0.0001 to 10 g, 0.0005 to 9 g, 0.001 to 8 g, 0.005 to 7 g, 0.01 to 6 g, 0.05 to 5 g, 0.1 to 4 g, 0.5 to 4 g, or 1 to 3 g.

[0137] Methods of Treatment and Use In some embodiments of the present disclosure, provided herein are methods for modulating an immune response in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a modified IL2 polypeptide, a modified IL2 fusion polypeptide, or a pharmaceutical composition thereof, as described hereinabove. In certain embodiments, the modulation of the immune response comprises at least one of enhancing effector T cell activity, enhancing NK cell activity, and suppressing regulatory T cell activity. In some embodiments of the present disclosure, provided herein are the above-described modified IL2 polypeptides, the above-described fusion polypeptides, and / or the above-described pharmaceutical compositions for use in the methods for modulating an immune response in a subject in need thereof. In some embodiments, the modulation of the immune response comprises increasing STAT5 phosphorylation compared to WT IL2.

[0138] In some embodiments of the present disclosure, there is a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a modified IL2 polypeptide, a modified IL2 fusion polypeptide, or a pharmaceutical composition thereof, as described hereinabove. In some embodiments of the present disclosure, there is provided herein the modified IL2 polypeptide, the fusion polypeptide, and / or the pharmaceutical composition described above, for use in the method of treating a subject with a disease. Non-limiting examples of diseases or conditions contemplated by the method include proliferative disorders such as cancer, and immunosuppression.

[0139] In some embodiments, there are methods for treating a proliferative disorder, comprising administering to a subject a therapeutically effective amount of a modified IL2 polypeptide, modified IL2 fusion polypeptide, or a pharmaceutical composition thereof, as described hereinabove. In some embodiments, the proliferative disorder is cancer. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, glioblastoma, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, skin cancer, melanoma, bone cancer, renal cell carcinoma, and kidney cancer. Precancerous conditions or lesions and cancer metastasis are also included. Other cell proliferative disorders include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid gland), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thoracic region, and genitourinary system. Similarly, other cell proliferative disorders, such as hypergammaglobulinemia, lymphoproliferative disorders, dysproteinemia, purpura, sarcoidosis, Sézary syndrome, Waldenstrom's macroglobulinemia, Gaucher's disease, histiocytosis, and any other cell proliferative disease other than neoplasms located in the above organ systems, can also be treated.

[0140] In some embodiments, the method of treating or modulating an immune response further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent (e.g., a combination therapy). In certain embodiments, the additional therapeutic agent is an anti-cancer agent. Examples of anti-cancer agents include checkpoint inhibitors (e.g., anti-PD1 antibodies), chemotherapeutic agents, agents that suppress the tumor microenvironment, cancer vaccines (e.g., sipuleucel-T), oncolytic viruses (e.g., talimogene laherparepvec), immune cells expressing chimeric antigen receptors, and tumor-infiltrating lymphocytes. In certain embodiments, the additional therapeutic agent is a molecule comprising an antigen-binding portion. In certain specific embodiments, the antigen-binding portion is selected from a single-domain antibody, a Fab molecule, an scFv, a diabody, a nanobody, a bispecific T-cell engager, or an immunoglobulin. In certain embodiments, the antigen-binding portion is specific for a tumor antigen (e.g., carcinoembryonic antigen, fibroblast activation protein-α, CD20) or a checkpoint protein (e.g., CTLA-4, PD-1, or PD-L1). In some embodiments, the additional therapeutic agent comprises a chimeric antigen receptor-expressing immune cell, a modified T cell receptor-expressing immune cell, or a tumor-infiltrating lymphocyte. In certain embodiments, the modified IL2 polypeptide or modified IL2 fusion polypeptide may be encoded by a polynucleotide that is transfected, transvected, or otherwise introduced into a chimeric antigen receptor-expressing immune cell, a modified T cell receptor-expressing immune cell, or a tumor-infiltrating lymphocyte. In such embodiments, the immune cell may be an enhanced chimeric antigen receptor-expressing cell. The polynucleotide may further encode a secretion signal (e.g., the native IL2 signal sequence or a signal sequence from another protein) immediately upstream of the modified IL2 polypeptide coding sequence to enable the cell to secrete the modified IL2 polypeptide or modified IL2 fusion polypeptide.

[0141] In some embodiments, a method of treating or modulating an immune response comprises administering to a subject a modified IL2 polypeptide having an IL2Rβ binding region 2 of SEQ ID NO: 1. In certain embodiments, a method of treating or modulating an immune response comprises administering to a subject a modified IL2 polypeptide having an IL2Rβ binding region 2 of any one of SEQ ID NOs: 2-21. In certain embodiments, a method comprises administering to a subject a modified IL2 polypeptide (optionally including a C-terminal histidine tag) of any one of SEQ ID NOs: 23-42, any one of SEQ ID NOs: 44-63, any one of SEQ ID NOs: 147-170, or an Fc fusion polypeptide of SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 144, or SEQ ID NO: 145. In certain embodiments, a method of treating or modulating an immune response comprises administering to a subject a modified IL2 polypeptide of SEQ ID NO: 22 or any one of SEQ ID NOs: 23-42. In certain embodiments, a method of treating or modulating an immune response comprises administering to a subject a modified IL2 fusion polypeptide of SEQ ID NO: 51. In certain embodiments, a method of treating or modulating an immune response comprises administering to a subject a modified IL2 fusion polypeptide of SEQ ID NO: 43, or of any one of SEQ ID NOs: 44-63.

[0142] Suitable routes of administration include, but are not limited to, intravenous, parenteral, transdermal, oral, rectal, aerosol, intraocular, pulmonary, transmucosal, intravaginal, otic, nasal, and topical administration. Additionally, by way of example only, parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injection, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injection.

[0143] In certain embodiments, the modified IL2 polypeptide or IL2 fusion polypeptide is administered systemically. In certain embodiments, the modified IL2 polypeptide or modified IL2 fusion polypeptide described herein is administered locally rather than systemically, for example, by injecting the modified IL2 polypeptide or modified IL2 fusion polypeptide directly into an organ, tissue, or tumor. In some embodiments, the long-acting formulation is administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Further, in some embodiments, the drug is a targeted drug delivery system, for example, in a liposome coated with an organ- or cell-specific antibody. In such embodiments, the liposome is targeted and taken up selectively by the organ. In some embodiments, the modified IL2 polypeptide or modified IL2 fusion polypeptide described herein is provided in the form of an immediate-release formulation, a sustained- or extended-release formulation, an intermediate-release formulation, or a depot preparation. In some embodiments, the modified IL2 polypeptide or modified IL2 fusion polypeptide described herein is administered locally.

[0144] The appropriate dosage of modified IL2 polypeptide or modified IL2 fusion polypeptide (used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease or condition, the route of administration, the subject's weight, the severity and progression of the disease, whether the polypeptide is administered for prophylactic or therapeutic purposes, previous or concurrent therapeutic interventions, the subject's medical history and response to the modified IL2 polypeptide or modified IL2 fusion polypeptide, and the judgment of the attending physician. The physician responsible for administration will be able to determine the concentration of the active ingredient(s) in the composition and appropriate dose for the subject to be treated. Various administration schedules are contemplated herein, including, but not limited to, single or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0145] A single dose of modified IL2 polypeptide can range from about 50,000 IU / kg to about 1,000,000 IU / kg, or more. This can be repeated several times daily (e.g., 2-4 times daily) for several days (e.g., 3-5 consecutive days), followed by one or more doses after a rest period (e.g., 7-14 days). Thus, a therapeutically effective amount can include only a single dose, or multiple doses over a period of time (e.g., about 10-30 separate doses of about 600,000 IU / kg of IL2, each given over a period of about 5-20 days). When administered in the form of a fusion polypeptide, the therapeutically effective amount of modified IL2 fusion polypeptide can be lower than that of a non-fused modified IL2 polypeptide (e.g., 10,000 IU / kg to about 600,000 IU / kg). Similarly, modified IL2 fusion polypeptide can be administered to a patient all at once or over a series of treatments as described above.

[0146] In certain embodiments, the daily dosage of the modified IL2 polypeptide or modified IL2 fusion polypeptide ranges from about 1 μg / kg to about 100 mg / kg, or more. For repeated administration over several days or more, depending on the condition, treatment may be sustained until a desired suppression of disease symptoms (e.g., tumor shrinkage) occurs. In some embodiments, a single dose of the modified IL2 polypeptide or modified IL2 fusion polypeptide ranges from about 0.005 mg / kg to about 10 mg / kg. In some embodiments, the dose may be about 1 μg / kg / body weight, about 5 μg / kg / body weight, about 10 μg / kg / body weight, about 50 μg / kg / body weight, about 100 μg / kg / body weight, about 200 μg / kg / body weight, about 350 μg / kg / body weight, about 500 μg / kg / body weight, about 1 mg / kg / body weight, about 5 mg / kg / body weight, about 10 mg / kg / body weight, about 50 mg / kg / body weight, about 100 mg / kg / body weight, about 200 mg / kg / body weight, about 350 mg / kg / body weight, about 500 mg / kg / body weight to about 1000 mg / kg / body weight, and any range derivable therein. Non-limiting examples of ranges derivable from the values ​​described herein include about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight, etc., which can be administered based on the above values. Such doses may be administered intermittently, for example, two to three times daily, weekly, or every three weeks. An initial higher loading dose, followed by one or more lower doses, may also be administered. However, other dosing regimens may be useful.

[0147] The modified IL2 polypeptides and modified IL2 fusion polypeptides described herein may be used in an amount effective to achieve the intended purpose. When used to treat or prevent a condition, the modified IL2 polypeptide or modified IL2 fusion polypeptide, or a pharmaceutical composition thereof, is administered in a therapeutically effective amount. Determining a therapeutically effective amount is within the capabilities of those skilled in the art, especially in light of the details provided herein.

[0148] For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. 50 Doses can be formulated in animal models to achieve a circulating concentration range that includes the IL2 polypeptide and IL2 fusion polypeptide (as determined in cell culture). Such information can be used to more accurately determine useful doses in humans. Initial dosages can also be extrapolated from in vivo data, e.g., animal models, using techniques well known in the art. Administration to humans can be readily optimized by those of skill in the art based on animal data. Dosage amounts and intervals can be adjusted to obtain plasma levels of the modified IL2 polypeptide and modified IL2 fusion polypeptide, respectively, that are sufficient to maintain therapeutic effect. Plasma levels can be measured, for example, by HPLC. [Example]

[0149] Example 1 A library strategy to identify IL2Rα-reducing binding mutations An IL2 mutation library to identify IL2Rα-reducing binders was rationally designed based on structural modeling of IL2 interaction with IL2Rα (Figures 1A and 1B). In summary, K35, R38, F42, and Y45 residues of IL2 were identified as key residues interacting with IL2Rα (Figure 1C). Mutagenic oligos with two, three, and four randomized mutations at these residues were used for library construction (Table 1). [Table 1] [Table 2]

[0150] The uppercase nucleotide trimers were mixed at 50% WT and 50% NNS or 70% WT and 30% NNS, where "N" refers to any nucleotide and "S" refers to G or C. Three libraries were constructed by multistep PCR and overlap PCR of the mutagenic oligos described above using the WT IL2 sequence as a template. Library 1 contained mutagenic oligos 1–6, Library 2 contained mutagenic oligos 7–9, and Library 3 contained mutagenic oligos 10–11 (Table 2). These mutagenic libraries were further modified to contain in vitro transcription and translation signals at the N-terminus. A Flag tag sequence was also added to the C-terminus for selection and purification purposes.

[0151] Example 2 Selection and identification of IL2Rα reduced binding clones Using mRNA display technology, IL2 mutants with reduced IL2Rα binding were selected from three IL2 mutagenic libraries. Briefly, the DNA library was first transcribed into an mRNA library and then translated into an mRNA-IL2 mutant fusion library by covalent conjugation via a puromycin linker. The library was purified and converted into an mRNA / cDNA fusion library. The fusion library was counterselected with human and mouse IgG (negative proteins) to remove nonspecific binders, and then counterselected three times against IL2Rα. The library flow-through (non-binding molecules) was collected, and PCR was performed to recover non-IL2Rα binding molecules, which were then gel-purified. The recovered pool was subcloned into the pET22b vector and expressed in E. coli Rosetta II strains. Supernatants from individual clones were tested in an IL2Rα binding ELISA. Figure 2A shows the ELISA results for the supernatants of IL2 clones selected against IL2Rα. Figure 2B shows clone expression plotted against IL2Rα binding to the supernatant. Figure 3 shows a sequence alignment of clones identified with reduced IL2Rα binding.

[0152] Example 3 Binding kinetics analysis of IL2Rα-reduced binding clones The binding kinetics of IL2Rα-reduced binding clones to IL2Rα were evaluated using SPR technology with a Biacore T200 and software version 2.0. In each cycle, 1 μg / mL of human IL2Rα was captured on flow cell 2 in 1x HBSP buffer on a protein A sensor chip at a flow rate of 10 μL / min for 60 seconds. 100 nM of each HIS- and Flag-tagged purified IL2 variant was injected onto both reference flow cell 1 and IL2Rα-captured flow cell 2 at a flow rate of 30 μL / min for 150 seconds, followed by a 300-second wash. The flow cells were then regenerated with glycine, pH 2.0, at a flow rate of 30 μL / min for 60 seconds. HBSP+buffer was included with each sample as a starting control. The assay was configured in a 96-well format. Kinetic data were analyzed using Biacore T200 evaluation software 3.0. Specific binding response units were derived from binding to IL2Rα flow cell 2 minus binding to reference flow cell 1 and subtraction of the buffer control. WT IL2 was included as a control. For each IL2Rα reduced binding clone, the relative response (RU) was determined (Figure 4, Table 3). [Table 3]

[0153] Example 4 Library strategies for generating IL2Rβ agonists We generated engineered IL2Rβ-binding agonists through rational IL2 mutagenesis library design and subsequent selection using an mRNA display technology platform. Briefly, two wild-type IL2-binding regions for IL2Rβ were identified through structural analysis: IL2Rβ-binding region 1 "QLQLEHLLLDLQM" (SEQ ID NO: 67) and IL2Rβ-binding region 2 "RPRDLISNINVIVLE" (SEQ ID NO: 68). To generate mutagenesis libraries containing mutations in IL2Rβ-binding region 1, IL2Rβ-binding region 2, or IL2Rβ-binding region 1 and IL2Rβ-binding region 2, we designed two mutagenic oligomers (Oligo 1 and Oligo 2) encoding the sequences of these two regions (Table 4). In the Oligo 1 and Oligo 2 sequences, each codon trimer, with nucleotides indicated in lowercase, was a mixture of 50% WT and 50% NNS ("N" refers to any nucleotide, and "S" refers to G or C). Additional oligomers (oligos 3 through 12) encoding the WT IL2 sequence were designed from the WT region for mutagenic library assembly (Table 4). These oligos were used to construct three mutagenic libraries (Figure 5). Library 4 was constructed using an overlapping PCR strategy with mutagenic oligo 1 and oligos 3 through 12. Library 5 was constructed using an overlapping PCR strategy with mutagenic oligo 2 and oligos 3 through 9, 11, and 12. Library 6 was constructed using an overlapping PCR strategy with mutagenic oligos 1 and 2 and oligos 3 through 8, 11, and 12. Furthermore, the three mutagenic libraries were modified to contain an in vitro transcription and translation signal at the N-terminus and a FLAG tag at the C-terminus for selection using mRNA display. [Table 4]

[0154] Example 5 Selection and identification of IL2Rβ agonist clones To identify IL2Rβ agonists from three IL2 mutagenic libraries, we used an mRNA display technology platform. The DNA library was first transcribed into an mRNA library and then translated into an mRNA-IL2 mutant fusion library by covalent linkage via a puromycin linker. The library was then purified and converted into an mRNA / cDNA fusion library (see, e.g., U.S. Patent No. 6,258,558, incorporated herein by reference). The fusion library was first counterselected with human IgG (a negative protein) to remove nonspecific binders, then counterselected to remove IL2Rα binders, followed by selection against recombinant IL2Rβ / Fc protein captured on Protein G magnetic beads. IL2Rβ binders were recovered and enriched by PCR amplification. A total of five rounds of selection were performed to obtain highly enriched engineered IL2 mutants binding to IL2Rβ.

[0155] After five rounds of selection, the enriched library was cloned into the bacterial periplasmic expression vector pET22b and transformed into TOP10-competent E. coli cells. Each modified IL2 molecule was modified to contain a C-terminal Flag and 6xHIS tag for purification and assay detection. Clones from TOP10 cells were pooled, miniprep DNA was prepared, and then transformed into E. coli Rosetta II strain for expression. Single clones were picked, grown, and induced with 0.25 mM IPTG in 96-well plates for expression. Supernatants were collected after 16–24 h of induction at 30°C for assays to identify binders.

[0156] Supernatants containing the modified IL2 variants were evaluated by a sandwich ELISA assay to screen for expression. Briefly, anti-HIS tag antibodies (R&D Systems) were immobilized in 96-well plates at a final concentration of 2 μg / mL in a total volume of 50 μL per well of 1× PBS. The plates were incubated overnight at 4°C and subsequently blocked with 200 μL of Superblock per well for 1 hour. Supernatants diluted 1:10 in 1× PBST (100 μL) were added to each well and incubated with shaking for 1 hour. The expression levels of the modified IL2 variants were detected by adding 50 μL of anti-Flag HRP diluted 1:5000 in 1× PBST for 1 hour. Between each step, the plates were washed three times with 1× PBST using a plate washer. The plates were then developed with 50 μL of TMB substrate for 5 minutes and stopped by adding 50 μL of 2N sulfuric acid. The plates were analyzed using a Biotek plate reader at OD . 450 The readings were taken at nm and the data were analyzed using Prism 8.1 software.

[0157] Next, single clones were screened for IL2Rβ binding. To identify individual modified IL2 variants, an IL2Rβ binding screening ELISA was developed. Briefly, 96-well plates were immobilized with human Fc and human IL2Rβ at a final concentration of 2 μg / mL in a total volume of 50 μL per well of 1× PBS. The plates were incubated overnight at 4°C and subsequently blocked with 200 μL of Superblock per well for 1 hour. 100 μL of supernatant was added to both the Fc-immobilized wells and the IL2Rβ-immobilized wells and incubated with shaking for 1 hour. Binding of the modified IL2 variants was detected by adding 50 μL of anti-Flag HRP diluted 1:5000 in 1× PBST. Between each step, the plates were washed three times with 1× PBST using a plate washer. The plates were then developed with 50 μL of TMB substrate for 5 minutes and stopped by adding 50 μL of 2N sulfuric acid. Plates were read at OD 450 nm using a Biotek plate reader and binding and selectivity were analyzed. The correlation between expression and IL2Rβ binding was plotted using Prism 8.1 software.

[0158] High IL2Rβ-binding clones (indicated to the right of the vertical OD450 nm cutoff) were identified from library 5 for further characterization as engineered IL2Rβ agonists (Figure 6). The IL2Rβ-binding activity of the clones generally correlated with their expression level. Multiple sequence alignment of the IL2Rβ-binding region 2 revealed both highly conserved and highly divergent amino acids compared to IL2 WT, as well as clone sequences that were independently identified multiple times (Figure 7). No specific IL2Rβ-binding clones were identified from libraries 4 and 6.

[0159] Example 6 Production of IL2Rβ agonist clones in E. coli and mammalian cells To produce IL2Rβ agonists in E. coli, glycerol stocks of each modified agonist clone were inoculated into TB medium for overnight growth. The next day, cells from the overnight cultures were inoculated into TB medium to an OD of 0.6–0.8. 600 The cells were grown to a cell density of 1000 kJ / ml. Expression was induced overnight at 30°C by adding IPTG to a final concentration of 1 mM. The supernatant was collected by centrifugation. The modified agonists were purified using a Ni-Sepharose (GE Healthcare) affinity column according to the manufacturer's protocol. The purity of the modified agonists was further improved by Flag-tag affinity column purification (Sigma). For size-exclusion chromatography column purification, the agonists were individually concentrated and loaded onto an AKTA Sephadex 200 Increase 10 / 300GL column. Highly homogeneous monomer peak fractions of the agonists were pooled and concentrated. Endotoxin was further removed using endotoxin removal resin (Pierce) according to standard protocols. Final endotoxin levels were less than 10 EU / mg. Protein purity was confirmed by LC-MS spectroscopy and SDS gel analysis (Figure 8). Proteins were stored in 1× PBS buffer for binding and functional assays, respectively.

[0160] To produce IL2Rβ agonists in mammalian cells, DNA sequences corresponding to the amino acid sequences were codon-optimized, synthesized, and subcloned into pCDNA3.4 (Invitrogen). Each modified IL2 polypeptide was transiently expressed in ExpiHEK293-F cells using the Freestyle system (Invitrogen) according to standard protocols. Cells were grown under the above conditions for 7 days before harvesting. The supernatant was collected by centrifugation and filtered through a 0.2 μm PES membrane. The agonists were first purified on a Ni Sepharose Excel resin column (GE Healthcare) and buffer exchanged into PBS pH 7.4 + 300 mM NaCl (total) on a 7 kDa Zeba column. Each polypeptide was then concentrated to 1 mL and purified to homogeneity on a Superdex 200 Increase 10 / 300 GL column (GE Healthcare). The monomer peak fractions were pooled and concentrated. The final purified protein contained less than 10 EU / mg of endotoxin. The identity of the IL2 polypeptide was confirmed by LC-MS spectroscopy, and purity was analyzed by SDS gel analysis. The protein was stored in 1x PBS / 300 nM NaCl buffer for binding, function, and mechanistic analyses.

[0161] Example 7 Binding kinetics analysis of IL2Rβ agonist clones using surface plasmon resonance The binding kinetics of modified IL2 polypeptides produced in E. coli cells and mammalian cells was assessed using surface plasmon resonance technology on a Biacore T200 instrument. The assay was performed using Biacore T200 control software version 2.0. In each cycle, 1 μg / mL of human IL2Rβ or IL2Rα was captured at 10 μL / min on flow cell 2 of a Protein A sensor chip in 1x HBST buffer for 60 seconds. Two-fold serial dilutions of purified HIS-tagged modified IL2 variants were injected at a flow rate of 30 μL / min for 150 seconds on both reference flow cell 1 and flow cell 2 loaded with IL2Rβ or IL2Rα, followed by a 300-second wash. The flow cells were then regenerated for 40 seconds with glycine pH 2 at a flow rate of 30 μL / min. Eight concentration points from 0 to 100 nM were assayed for each IL2Rβ agonist clone in a 96-well plate format. Kinetic data were analyzed using Biacore T200 Evaluation Software 3000. Specific binding response units were derived from the subtraction of binding to reference flow cell 1 from binding to target flow cell 2.

[0162] Wild-type IL2 was used to validate the binding protocol and was included in each experiment as a control (Figures 9A and 9B). Representative sensorgrams of the binding kinetics of E. coli- and mammalian-produced IL2Rβ agonists to IL2Rα (E. coli-produced, Figures 10A-10H; mammalian-produced, Figures 12A-12D) and IL2Rβ (E. coli, Figures 11A-11H; mammalian-produced, Figures 13A-13D) are shown. The binding kinetics of E. coli-produced IL2 (Table 5) and mammalian-produced IL2 (Table 6) are summarized.

[0163] The IL2Rβ agonists EP001, EP006, and EP007 either showed no detectable IL2Rα binding or a significant reduction (less than 20-fold) in IL2Rα binding, but a significant increase in IL2Rβ binding compared to WT IL2 (Tables 5 and 6). In contrast, the IL2Rβ agonists EP002, EP003, EP004, and EP005 did not show a significant reduction (less than 20-fold) in IL2Rα binding compared to wild-type IL2, but showed a significant increase in IL2Rβ binding compared to WT IL2 (Tables 5 and 6). [Table 5] [Table 6]

[0164] Example 8 IL2Rβ agonist P-STAT5 activation in human PBMCs Human PBMCs were isolated from peripheral blood from three separate donors and plated at 250,000 cells / well in 75 μL of medium in 96-well plates. Cells were incubated at 37°C for 1 hour. Cells were stimulated with human wild-type IL2 or modified His-Flag-tagged IL2 at a concentration of 4x in 25 μL for 20 minutes at 37°C. Stimulated PBMCs were immediately fixed, permeabilized, stained for lineage markers (CD3, CD56, CD4, CD8, FOXP3) and p-STAT5, and visualized using an Attune flow cytometer. CD8+ T cells were defined as CD3+CD56-CD4-CD8+. NK cells were defined as CD3-CD56+. Regulatory T cells were defined as CD3+CD56-CD4+CD8-FOXP3+. The % of cells that were p-STAT5+ was determined and graphed for each IL2 titration (Figures 14A-14C for blood donor 1, Figures 14D-14F for blood donor 2, and Figures 14G-14I for blood donor 3). EC50 values ​​for p-STAT5 activation were determined using Prism software (Table 7). [Table 7]

[0165] Example 9 Rational generation of IL2Rβ agonist revertant clones A rationally designed IL2Rβ agonist backmutation strategy was implemented to generate a series of IL2Rβ agonist candidate mutations. EP001 contains the following mutations: R81T, P82A, L85A, I86V, S87D, I89M, N90R, V93I, and L94Q. Four backmutations to WT IL2 were designed for each candidate. I86 and I89 were backmutated to WT IL2 residues 86I and 89I for all mutations. Global backmutations were then applied to the other two residues in combination with 86I and 89I. A total of 21 backmutation combinations were designed and generated by site-directed mutagenesis using EP001 as a template (Table 8). IL2Rβ agonist backmutation clones were sequence confirmed after mutagenesis. [Table 8]

[0166] Example 10 Characterization of IL2Rβ agonist revertant clones The EP001 revertant clones were characterized for their binding activity to the IL2Rβ and IL2Rα receptors by ELISA. Briefly, 384-well plates were immobilized with human IL2Rα and IL2Rβ Fc fusion proteins at a final concentration of 2 μg / mL in 1x PBS in a total volume of 25 μL per well. The plates were incubated overnight at 4°C and blocked with 80 μL of Superblock per well for 1 hour. The purified EP001 revertant clones were serially diluted from 100 nM to 0 nM. Each dilution was added to IL2Rα or IL2Rβ wells in duplicate in parallel. Binding of the IL2 mutants was detected by adding 25 μL of anti-Flag HRP diluted 1:5000 in 1x PBST. Between each step, the plate was washed three times with 1x PBST using a plate washer. Plates were then developed with 25 μL of TMB substrate for 5 minutes and stopped by adding 25 μL of 2N sulfuric acid. Plates were read at OD450 nm using a Biotek plate reader and analyzed with Prism 8.1 software to obtain EC50 values ​​(Figure 15A for IL2Rα and Figure 15B for IL2Rβ, summarized in Table 9). [Table 9]

[0167] Example 11 Binding kinetics of revertant clones of IL2Rβ agonists using surface plasmon resonance The binding kinetics of the EP001 revertant clones were evaluated using SPR technology on a Biacore T200. The assay was performed using Biacore T200 control software version 2.0. In each cycle, 1 μg / mL of human IL2Rβ was captured on flow cell 2 in 1x HBSP buffer on a protein A sensor chip at a flow rate of 10 μL / min for 60 seconds. 100 nM of each purified IL2 variant with HIS and Flag tags was serially diluted 2-fold and injected onto both reference flow cell 1 and IL2Rβ capture flow cell 2 at a flow rate of 30 μL / min for 150 seconds, followed by a 300-second wash. The flow cells were then regenerated with glycine pH 2 at a flow rate of 30 μL / min for 60 seconds. The assay was configured in a 96-well format with eight serial dilution concentration points. Kinetic data were analyzed using Biacore T200 evaluation software 3.0. Specific binding response units were derived from the subtraction of binding to reference flow cell 1 from binding to target flow cell 2 (Figures 16A-16F, Table 10). [Table 10]

[0168] Example 12 Modified IL2Rβ agonists with reduced IL2Rα activity IL2 mutants with potentially reduced or eliminated IL2Rα binding, generated through mRNA library selection and screening, were first expressed in E. coli and purified using Ni-Sepharose (GE Healthcare) affinity columns and Flag-tag affinity column purification (Sigma) according to the manufacturer's protocol. Next, IL2 mutants with significantly reduced IL2Rα binding activity, as confirmed by both Biacore SPR binding and ELISA binding, were selected for the generation of IL2Rβ agonists with reduced IL2Rα binding activity. Briefly, mutations were introduced into the IL2Rβ agonist construct in the pCDNA3.4 mammalian expression vector using site-directed mutagenesis techniques and confirmed by DNA sequence analysis (Table 11). Each modified IL2 polypeptide was transiently expressed in ExpiHEK293-F cells using the FreeStyle system (Invitrogen) according to standard protocols. Cells were grown under the above conditions for 5 days before harvest. The supernatant was collected by centrifugation and filtered through a 0.2 μm PES membrane. The agonist was first purified on a Ni Sepharose Excel resin column (GE Healthcare) and further purified to greater than 95% homogeneity on a Superdex 200 Increase 10 / 300 GL column (GE Healthcare) (Figure 17). The final purified protein has less than 10 EU / mg endotoxin. The protein was stored in 1x PBS buffer for binding, functional, and mechanistic analyses. [Table 11]

[0169] Example 13 Binding kinetics analysis of engineered IL2Rα / IL2Rβ clones using surface plasmon resonance Kinetic analysis of the receptor binding activity of IL2Rβ agonists with IL2Rα-reducing mutations was evaluated using SPR technology on a Biacore T200. The assay was performed using Biacore T200 control software version 2.0. In each cycle, 1 μg / mL of human IL2Rβ was captured on flow cell 2 in 1x HBSP buffer on a protein A sensor chip at a flow rate of 10 μl / min for 60 seconds. 100 nM of each purified IL2 mutant with HIS and Flag tags was serially diluted 2-fold and injected onto both reference flow cell 1 and IL2Rβ-capture flow cell 2 at a flow rate of 30 μl / min for 150 seconds, followed by a 300-second wash. The flow cells were then regenerated with glycine pH 2 at a flow rate of 30 μl / min for 60 seconds. The assay was configured in a 96-well format with eight serial dilution concentration points. Kinetic data were analyzed using Biacore T200 evaluation software 3.0. Specific binding response units were derived from subtracting binding to reference flow cell 1 from binding to target flow cell 2. Figures 18A-18H show titrations of binding of engineered IL2Rβ / α clones to IL2Rα, and Figures 19A-19H show titrations of binding of engineered IL2Rβ / α clones to IL2Rβ. A description of the clones and a summary of the kinetic data are shown in Table 12. [Table 12]

[0170] Figures 20A-20G show the binding of IL2Rα at a single concentration, Figures 21A-21G show the binding of IL2Rβ at a single concentration, and Figures 22A and 22B show the binding of IL2Rα at multiple concentrations, which are summarized in Table 13. [Table 13]

[0171] Example 14 ELISA binding analysis of engineered IL2Rα / IL2Rβ clones Recombinant Fc-tagged human IL2Rα and IL2Rβ were added to 25 μL of 1× PBS in a 384-well plate and incubated overnight at 4°C to coat the plate. The plate was washed three times with 0.05% Tween 20 / 1× PBS. The plate was blocked with 100 μL of SuperBlock at room temperature for 1 hour and then washed three times with 0.05% Tween 20 / 1× PBS. IL2 variants were diluted from 1000 nM to 0 nM in 0.05% Tween 20 / 1× PBS and added to the plate for 2 hours at room temperature. The plate was then washed six times with 0.05% Tween 20 / 1× PBS. Anti-His tag-HRP was diluted 1:5000 in 0.05% Tween 20 / 1× PBS and added to the plate for 1 hour at room temperature. Plates were then washed six times with 0.05% Tween 20 / 1x PBS, and TMB was added to develop a blue color. The reaction was stopped with 2N hydrogen sulfide, and the absorbance at 450 nm was read on a BioTek plate reader. Single-point absorbance for human IL2Rα and titrations for human IL2Rα and IL2Rβ are graphed (Figures 23A-23E for IL2Rα, Figures 24A-24D for IL2Rβ). A summary of ELISA binding EC50 values ​​is shown (Table 14). [Table 14]

[0172] Example 15 P-STAT5 activation in human PBMCs by engineered IL2Rα / IL2Rβ clones Human PBMCs were isolated from peripheral blood from two donors and plated at 250,000 cells / well in 75 μL of medium in 96-well plates. Cells were incubated at 37°C for 1 hour. Cells were stimulated with 4x concentrations of human IL2 WT and His-Flag-tagged IL2 in 25 μL for 20 minutes at 37°C. Stimulated PBMCs were immediately fixed, permeabilized, stained for lineage markers (CD3, CD56, CD4, CD8, FOXP3) and p-STAT5, and visualized using an Attune flow cytometer. CD8+ T cells were defined as CD3+CD56-CD4-CD8+. NK cells were defined as CD3-CD56+. Regulatory T cells were defined as CD3+CD56-CD4+CD8-FOXP3+. The percent of cells that were p-STAT5+ were determined and graphed for each IL2 titration (Figures 25A-25D for CD8+ T cells from donor 656; Figures 26A-26D for CD8=+ T cells from donor 648; Figures 27A-27D for NK cells from donor 656; Figures 28A-28D for NK cells from donor 648; Figures 29A-29D for regulatory T cells from donor 656; and Figures 30A-30D for regulatory T cells from donor 648). A summary of the EC50 values ​​for p-STAT5 activation in each cell type is shown in Table 15 for blood donor 1 and Table 16 for blood donor 2. [Table 15] [Table 16]

[0173] Example 16 P-STAT5 activation in mouse cells by engineered IL2Rα / IL2Rβ clones Mouse splenocytes were plated at 250,000 cells / well in 75 μL of medium in a 96-well plate. Cells were incubated at 37°C for 1 hour. Cells were stimulated with 4x concentrations of human IL2 WT and His-Flag-tagged IL2 in 25 μL for 20 minutes at 37°C. Stimulated mouse splenocytes were immediately fixed, permeabilized, stained for lineage markers (CD3, CD56, CD4, CD8, FOXP3) and p-STAT5, and visualized using an Attune flow cytometer. CD8+ T cells were defined as CD3+CD56-CD4-CD8+. The percentage of cells that were p-STAT5+ was determined and graphed for each IL2 titration (Figures 31A-31D). A summary of the EC50 values ​​for p-STAT5 activation in each cell type is shown in Figure 34.

[0174] Isolated NK cells or murine regulatory T cells were plated at 20,000 cells / well in 75 μL of medium in a 96-well plate. Cells were incubated at 37°C for 1 hour. Cells were stimulated with 4x concentrations of human IL2 WT and His-Flag-tagged IL2 in 25 μL for 20 minutes at 37°C. Stimulated murine NK cells or murine regulatory T cells were immediately fixed, permeabilized, stained for p-STAT5, and visualized on an Attune flow cytometer. The percentage of cells that were p-STAT5+ was determined and graphed for each IL2 titration (Figures 32A-32D, 33A-33D).

[0175] A summary of the EC50 values ​​for P-STAT5 activation in each cell type is shown in FIG.

[0176] Example 17 Design of IL2Rβ agonist Fc fusion proteins To generate bivalent IL2Rβ agonist Fc fusion proteins, the protein sequences encoding the modified IL2 polypeptides EP003 (SEQ ID NO: 2), EP007 (SEQ ID NO: 4), EP002 (SEQ ID NO: 6), EP004 (SEQ ID NO: 09), EP001 (SEQ ID NO: 11), EP006 (SEQ ID NO: 16), EP009 (SEQ ID NO: 18), and EP005 (SEQ ID NO: 19) were fused to the N-terminal site of the constant frame sequence of human IgG1 isoform to produce the modified agonist Fc fusion proteins (SEQ ID NOs: 44, 46, 48, 51, 53, 58, and 61). To eliminate complement fixation and Fc-γ-dependent antibody-dependent cellular cytotoxicity (ADCC) effects, the L234A, L235A, and P329G mutations of human IgG1 were introduced (Lo et al., JBC 2017) (Figure 35A).

[0177] To generate monovalent IL2Rβ agonist Fc fusion proteins, protein sequences encoding the modified IL2 polypeptides EP003 (SEQ ID NO: 2), EP007 (SEQ ID NO: 4), EP002 (SEQ ID NO: 6), EP004 (SEQ ID NO: 09), EP001 (SEQ ID NO: 11), EP006 (SEQ ID NO: 16), EP009 (SEQ ID NO: 18), and EP005 (SEQ ID NO: 19) were fused to the N-terminal site of the constant frame sequence of the respective human IgG1 and IgG4 isoforms to produce modified agonist Fc fusion proteins (SEQ ID NOs: 44, 46, 48, 51, 53, 58, and 61). Knob mutations of S354C, T366W, and K409A were introduced into the constructs. Hole mutations of Y349C, T366S, L368A, F405K, and Y407V were introduced into the CH2 and CH3 fragments of IgG1 and IgG4, respectively. To eliminate complement fixation and Fc-γ-dependent antibody-dependent cellular cytotoxicity (ADCC) effects, we introduced the L234A, L235A, and P329G mutations of human IgG1 (Lo et al., JBC 2017) (Figures 35B and 35C). Next, DNA encoding the entire Fc-fusion agonist protein was synthesized with codons optimized for mammalian cell expression and subcloned into pCDNA3.4 (Invitrogen).

[0178] Example 18 Production of IL2Rβ agonist Fc fusion proteins To produce bivalent IL2-Fc fusion proteins, agonists were transiently expressed in ExpiHEK293-F cells using the Freestyle system (Invitrogen) according to standard protocols. Cells were grown under the above conditions for 7 days before harvesting. The supernatant was collected by centrifugation and filtered through a 0.2 μm PES membrane. First, the Fc fusion agonist was purified using MabSelect PrismA Protein A resin (GE Health). The protein was eluted with 100 mM Gly (pH 2.5) + 150 mM NaCl and quickly neutralized with 20 mM citric acid (pH 5.0) + 300 mM NaCl. The agonist protein was then concentrated to 1 mL and further purified on a Superdex 200 Increase 10 / 300GL column. The monomer peak fractions were pooled and concentrated. The final purified protein has less than 10 EU / mg endotoxin and is maintained in 20 mM citrate (pH 5.0) + 300 mM NaCl. The purified IL2-Fc fusion agonist was run on an SDS gel (4-12% Bis-Tris Bolt gel with MES running buffer) to compare samples treated under reducing vs. non-reducing conditions (Figure 36A).

[0179] To produce monovalent IL2-Fc fusion proteins, the "knob" and "hole" constructs of the respective IgG1 and IgG4 backbone formats were transfected into ExpiHEK293-F cells at a 1:1 ratio. Cells were grown under the conditions described above for 5 days before harvest. The supernatant was collected by centrifugation and filtered through a 0.2 μm PES membrane. First, the Fc fusion agonist was purified using MabSelect PrismA Protein A resin (GE Health). The protein was eluted with 100 mM Gly (pH 2.5) + 150 mM NaCl and quickly neutralized with 20 mM citric acid (pH 5.0) + 300 mM NaCl. The agonist protein was then concentrated to 1 mL and further purified on a Superdex 200 Increase 10 / 300GL column. The monomer peak fractions were pooled and concentrated. The final purified protein has less than 10 EU / mg endotoxin and is maintained in 20 mM citrate (pH 5.0) + 300 mM NaCl. The purified monovalent IL2-Fc fusion agonist was run on an SDS gel (4-12% Bis-Tris Bolt gel with MES running buffer) to compare samples treated under reducing vs. non-reducing conditions (Figure 36B).

[0180] Example 19 ELISA binding analysis of IL2Rβ agonist Fc fusion proteins For bivalent Fc fusion proteins, human IL2Rα and human IL2Rβ were immobilized in a 384-well plate at a final concentration of 2 μg / mL in a total volume of 25 μL of 1x PBS per well. The plate was incubated overnight at 4°C and then blocked with 80 μL of Superblock per well for 1 hour. 100 nM purified modified IL2 mutant Fc fusion protein was serially diluted 3-fold 12 times. Each dilution was added to IL2Rα and IL2Rβ wells in parallel. Modified IL2 mutant binding was detected by adding 50 μL of anti-human Fc HRP diluted 1:5000 in 1x PBST. Between each step, the plate was washed three times with 1x PBST using a plate washer. The plate was then developed with 25 μL of TMB substrate for 5 minutes and stopped by adding 25 μL of 2N sulfuric acid. Plates were read at OD 450 nm using a Biotek plate reader and analyzed for EC50 using Prism 8.1 software. Absorbance versus IL-2 concentration is graphed for human IL2Rα and IL2Rβ (Figures 37A-37G). A summary of ELISA binding EC50 values ​​is shown (Table 17). [Table 17]

[0181] For monovalent Fc fusion proteins, recombinant His-tagged human IL2Rα and IL2Rβ were added to 25 μL of 1× PBS in a 384-well plate and incubated overnight at 4°C to coat the plate. The plate was washed three times with 0.05% Tween 20 / 1× PBS. The plate was blocked with 100 μL of SuperBlock at room temperature for 1 hour and then washed three times with 0.05% Tween 20 / 1× PBS. IL2 variants were diluted from 1000 nM to 0 nM in 0.05% Tween 20 / 1× PBS and added to the plate for 2 hours at room temperature. The plate was then washed six times with 0.05% Tween 20 / 1× PBS. Anti-His tag-HRP was diluted 1:5000 in 0.05% Tween 20 / 1× PBS and added to the plate for 1 hour at room temperature. Plates were then washed six times with 0.05% Tween 20 / 1x PBS, and TMB was added to develop a blue color. The reaction was stopped with 2N hydrogen sulfide, and the absorbance at 450 nm was read on a BioTek plate reader. Absorbance versus IL2 concentration is graphed for human IL2Rα and IL2Rβ (Figures 38A-38B). A summary of ELISA binding EC50 values ​​is shown (Table 18). [Table 18]

[0182] Example 20 Binding kinetics of monovalent IL2Rβ Fc fusion proteins The binding kinetics of monovalent IL2Rβ Fc fusion proteins were analyzed using SPR technology with a Biacore T200. Briefly, anti-hFc antibodies were immobilized on flow cells 1 and 2. In each cycle, 1 μg / mL of IL2 Fc fusion protein was captured on flow cell 2 in 1X HBSP buffer on an anti-hFc-immobilized chip at a flow rate of 10 μl / min for 60 s. 100 nM of IL2Rα-His-tagged or IL2Rβ-His-tagged antibodies were serially diluted two-fold and injected into both reference flow cell 1, and the IL2 Fc fusion protein was captured on flow cell 2 at a flow rate of 30 μl / min for 150 s. A 300-s wash was performed after the final injection. The assay was configured in a 96-well format with eight serial dilution concentration points. Kinetic data were analyzed using Biacore T200 Evaluation Software 3.0. Specific binding response units were derived from the subtraction of binding to reference flow cell 1 from binding to target flow cell 2 (Figures 39A-39D).

[0183] Example 21 P-STAT5 activation in human PBMCs by IL2Rβ agonist Fc fusion proteins Human PBMCs were isolated from peripheral blood and plated at 250,000 cells / well in 75 μl of medium in 96-well plates. Cells were incubated at 37°C for 1 hour. Cells were stimulated with human IL2 WT and IL2Rβ agonist Fc fusion proteins at 4x concentrations in 25 μl for 20 minutes at 37°C. Stimulated PBMCs were immediately fixed, permeabilized, stained for lineage markers (CD3, CD56, CD4, CD8, FOXP3) and p-STAT5, and visualized using an Attune flow cytometer. CD8+ T cells were defined as CD3+CD56-CD4-CD8+. NK cells were defined as CD3-CD56+. Regulatory T cells were defined as CD3+CD56-CD4+CD8-FOXP3+. The % of cells that were P-STAT5+ was determined and graphed for each IL2 titration (for bivalent fusion proteins, Figures 40A-40C; for bivalent fusion proteins, Figures 41A-41C; see Table 19 for summary). [Table 19]

[0184] Example 22 In vivo pharmacokinetic analysis of IL2Rβ agonist mice C57BL / 6 mice were injected intravenously or intravenously with 10 μg of IL2-WT, EP001, or EP003 in 200 μL of saline. Blood was collected at 0 min, 10 min, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h, 24 h, and 48 h and immediately centrifuged to separate the plasma. To determine the plasma concentrations of IL2-WT and EP001, the plasma was serially diluted and analyzed using a Duoset IL2 ELISA kit (R&D Systems) according to the manufacturer's instructions. IL2-WT, EP001, and EP001 concentrations were determined by comparing plasma absorbance values ​​with spiked controls prepared with equivalently diluted untreated C57BL / 6 mouse plasma. IL2 concentrations are plotted against time on a logarithmic scale (Figures 42A-42B; Table 20). [Table 20]

[0185] Example 23 In vivo tumor cell invasion in IL2Rβ agonist mice Seven-week-old female C57BL / 6 mice were injected subcutaneously into the dorsal flank with 100,000 MC38 cells in 50% Matrigel. Tumors were measured with calipers. The average volume was 100 mm. 3At the time of tumor growth, mice were treated with 32 μg of WT IL2, EP001, EP003, or EP004 BID for 5 days. On day 6, mice were sacrificed and tumor-infiltrating immune cells were analyzed by flow cytometry. Tumor sections used for flow cytometry were weighed to obtain normalized cell counts. CD4+ T cells were defined as CD45+CD3+CD49b-CD4+CD8-. CD8+ T cells were defined as CD45+CD3+CD49b-CD4-CD8+. NK cells were defined as CD45+CD3-CD49b+. Regulatory T cells (NK cells) were defined as CD45+CD3+CD49b-CD4+CD8-FOXP3+. Naive T cells were defined as CD44 低 CD62L 高 Effector T cells are defined as CD44 高 CD62L 低 Central memory T cells are defined as CD44 高 CD62L 高 The normalized number of tumor-infiltrating immune cells (Figures 43A-43D), the effector to regulatory cell ratio (Figures 44A and 44B), and T cell subtypes (Figures 45A-45C) are graphed for the IL2 clone-treated groups.

Claims

1. 1. A modified interleukin 2 (IL2) polypeptide comprising: X １ -X ２ -X ３ -D-X ４ -X- ５ -X ６ -N-X ７ -X ８ -X ９ -X １０ -X １１ -X １２ -X １３ (SEQ ID NO: 1), In the formula, X １ , X ３ , X ６ , X ８ , X １２ , and X １３ each containing any residue, X ２ , X ４ , and X １０ is an uncharged residue, X ５ , X ７ , X ９ , and X １１ each containing an uncharged apolar residue, The modified IL2 polypeptide has a K that is at least 10-fold greater than wild-type IL2. Ｄ and binds to IL2Rβ. The modified interleukin 2 (IL2) polypeptide.

2. X １ 2. The modified IL2 polypeptide of claim 1, wherein is an uncharged polar residue, an uncharged nonpolar residue, a basic residue, or an acidic residue.

3. X １ The modified IL2 polypeptide of claim 1 or 2, wherein is selected from C, T, G, W, I, S, E, and K.

4. X １ The modified IL2 polypeptide of any one of claims 1 to 3, wherein is selected from G, K, E, C, and T.

5. X ２ The modified IL2 polypeptide of any one of claims 1 to 4, wherein is an uncharged polar residue or an uncharged apolar residue.

6. X ２ The modified IL2 polypeptide of any one of claims 1 to 5, wherein is selected from Y, P, V, W, L, A, and G.

7. X ２ The modified IL2 polypeptide of any one of claims 1 to 6, wherein is selected from V, P, W, and A.

8. X ３ The modified IL2 polypeptide of any one of claims 1 to 7, wherein is an uncharged polar residue, an uncharged nonpolar residue, a basic residue, or an acidic residue.

9. X ３ The modified IL2 polypeptide of any one of claims 1 to 8, wherein is selected from S, T, Q, G, M, E, R, and K.

10. X ３ The modified IL2 polypeptide of any one of claims 1 to 9, wherein is selected from T, G, S, R, and E.

11. X ４ The modified IL2 polypeptide of any one of claims 1 to 10, wherein:

12. X ４ The modified IL2 polypeptide of any one of claims 1 to 11, wherein is an uncharged nonpolar residue or an uncharged polar residue.

13. X ４ The modified IL2 polypeptide of any one of claims 1 to 12, wherein is selected from A, V, S, and T.

14. X ５ The modified IL2 polypeptide of any one of claims 1 to 13, wherein is selected from I, L, T, and V.

15. X ５ The modified IL2 polypeptide of any one of claims 1 to 14, wherein is selected from I and V.

16. X ６ The modified IL2 polypeptide of any one of claims 1 to 15, wherein is an uncharged polar residue, a basic residue, or an acidic residue.

17. X ６ The modified IL2 polypeptide of any one of claims 1 to 16, wherein is selected from S, T, E, D, and R.

18. X ６ The modified IL2 polypeptide of any one of claims 1 to 17, wherein is selected from S, D, E, and T.

19. X ７ The modified IL2 polypeptide of any one of claims 1 to 18, wherein is selected from I, A, M, and V.

20. X ７ The modified IL2 polypeptide of any one of claims 1 to 19, wherein is selected from I, A, and M.

21. X ８ 21. The modified IL2 polypeptide of any one of claims 1 to 20, wherein is an uncharged polar residue, an uncharged nonpolar residue, a basic residue, or an acidic residue.

22. X ８ is selected from S, T, N, Q, I, G, E, K, and R.

23. X ８ The modified IL2 polypeptide of any one of claims 1 to 22, wherein is selected from I, R, N, and T.

24. X ９ The modified IL2 polypeptide of any one of claims 1 to 23, wherein is selected from V, L, and I.

25. X ９ The modified IL2 polypeptide of any one of claims 1 to 24, wherein is V.

26. X １０ The modified IL2 polypeptide of any one of claims 1 to 25, wherein is an uncharged polar residue or an uncharged apolar residue.

27. X １０ The modified IL2 polypeptide of any one of claims 1 to 26, wherein is selected from N, T, I, and L.

28. X １０ The modified IL2 polypeptide of any one of claims 1 to 27, wherein is selected from I and L.

29. X １１ The modified IL2 polypeptide of any one of claims 1 to 28, wherein is selected from V, A, and I.

30. X １２ 30. The modified IL2 polypeptide of any one of claims 1 to 29, wherein is an uncharged polar residue, an uncharged nonpolar residue, or an acidic residue.

31. X １２ The modified IL2 polypeptide of any one of claims 1 to 30, wherein is selected from Q, L, G, K, and R.

32. X １２ The modified IL2 polypeptide of any one of claims 1 to 31, wherein is selected from R, G, Q, and K.

33. X １３ The modified IL2 polypeptide of any one of claims 1 to 32, wherein is an uncharged apolar residue or a basic residue.

34. X １３ The modified IL2 polypeptide of any one of claims 1 to 33, wherein is selected from A, D, and E.

35. X １３ The modified IL2 polypeptide of any one of claims 1 to 33, wherein is selected from E and A.

36. The modified IL2Rβ binding region 2 is selected from the group consisting of GVTDSISNAIVLARE (SEQ ID NO: 2), KWGDAVSNARVLAGE (SEQ ID NO: 3), KWGDAVSNARVLAGA (SEQ ID NO: 4), TLMDTTDNIGVLVRE (SEQ ID NO: 5), EPSDVISNINVLVQE (SEQ ID NO: 6), SPQDSIENISVLVRE (SEQ ID NO: 7), WASDSIENITLLIQE (SEQ ID NO: 8), CPT DTIENITVLIQE (SEQ ID NO: 9), RYKDSLENMQIIIQE (SEQ ID NO: 10), TARDAVDNMRVIIQE (SEQ ID NO: 11), TPRDVVENMNVLVLE (SEQ ID NO: 12), TPSDVIENMEVLILD (SEQ ID NO: 13), TPSDAIENINVLIRE ​​(SEQ ID NO: 14), TPSDVIENITVLVQE (SEQ ID NO: 15), GVGDTIDNINVLVKE ( SEQ ID NO: 16), IGRDSIDNIKVIVQE (SEQ ID NO: 17), WATDTIRNVEVLVQE (SEQ ID NO: 18), TAEDVVTNITVLVQE (SEQ ID NO: 19), TAEDVISNIRVNVQE (SEQ ID NO: 20), and TPSDVIDNVSITVQE (SEQ ID NO: 21), TARDAISMIRVIVQE (SEQ ID NO: 210), RARDAIDNIRVIVQE (SEQ ID NO: 211), 2. The modified IL2 polypeptide of claim 1, selected from TPRDAIDNINVIIQE (SEQ ID NO: 212), TPRDAIDNIRVIVQE (SEQ ID NO: 213), TPRDAIDNIRVIILE (SEQ ID NO: 214), TARDAISNINVIQE (SEQ ID NO: 215), and TARDAIDNIRVIVQE (SEQ ID NO: 216), and TARDAIDNIRVIVLE (SEQ ID NO: 217).

37. 2. The modified IL2 polypeptide of claim 1, wherein the modified IL2Rβ binding region 2 is selected from TPRDAIDNIRVIVQE (SEQ ID NO: 213), TPRDAIDNIRVIILE (SEQ ID NO: 214), TARDAISNINVIIQE (SEQ ID NO: 215), and TARDAIDNINVIVQE (SEQ ID NO: 216).

38. 2. The modified IL2 polypeptide of claim 1, wherein the modified IL2Rβ binding region 2 is selected from GVTDSISNAIVLARE (SEQ ID NO: 2), KWGDAVSNARVLAGA (SEQ ID NO: 4), EPSDVISNINVLVQE (SEQ ID NO: 6), CPTDTIENITVLIQE (SEQ ID NO: 9), TARDAVDNMRVIIQE (SEQ ID NO: 11), GVGDTIDNINVLVKE (SEQ ID NO: 16), and TAEDVVTNITVLVQE (SEQ ID NO: 19).

39. 2. The modified IL2 polypeptide of claim 1, wherein the modified IL2Rβ binding region 2 is selected from GVTDSISNAIVLARE (SEQ ID NO: 2), CPTDTIENITVLIQE (SEQ ID NO: 9), and TARDAVDNMRVIIQE (SEQ ID NO: 11).

40. A modified IL2 polypeptide comprising a substitution of at least one residue selected from R81, P82, R83, L85, I86, S87, I89, N90, I92, V93, and L94.

41. 41. The modified IL2 polypeptide of claim 40, wherein the at least one residue is L85.

42. 41. The modified IL2 polypeptide of claim 40, comprising substitutions of at least two residues selected from R81, P82, R83, L85, I86, S87, I89, N90, I92, V93, and L94.

43. 43. The modified IL2 polypeptide of claim 42, comprising substitutions at R81 and L85.

44. 44. The modified IL2 polypeptide of claim 43, further comprising substitutions at S87, N90, and L94.

45. 44. The modified IL2 polypeptide of claim 43, further comprising substitutions at S87, N90, and V93.

46. 44. The modified IL2 polypeptide of claim 43, further comprising substitutions at P82 and V93.

47. 47. The modified IL2 polypeptide of claim 46, further comprising a substitution at N90.

48. 43. The modified IL2 polypeptide of claim 42, wherein the at least two residues are selected from R81, R83, L85, I92, and L94.

49. 41. The modified IL2 polypeptide of claim 40, comprising substitutions of at least three residues selected from R81, R83, L85, I92, and L94.

50. 41. The modified IL2 polypeptide of claim 40, comprising substitutions at R81, R83, L85, I92, and L94.

51. (a) the R81 substitution is selected from R81G, R81K, R81E, R81C, and R81T; (b) the R83 substitution is selected from R83T, R83G, R83S, and R83E; (c) the L85 substitution is selected from L85S, L85A, L85V, and L85T; (d) the I92 substitution is I92L; (e) the L94 substitution is selected from L94R, L94G, L94Q, and L94K; 41. The modified IL2 polypeptide of claim 40.

52. 52. The modified IL2 polypeptide of any one of claims 1 to 51, wherein the affinity of the modified IL2 polypeptide for IL2Rβ is increased compared to the wild-type IL2.

53. 53. The modified IL2 polypeptide of claim 52, wherein the affinity of the modified IL2 polypeptide for IL2Rβ is increased by at least 10-fold compared to the wild-type IL2.

54. 54. The modified IL2 polypeptide of any one of claims 1 to 53, wherein the affinity of the modified IL2 polypeptide for IL2Rα is reduced compared to the wild-type IL2.

55. 55. The modified IL2 polypeptide of any one of claims 1 to 54, wherein the affinity of the modified IL2 polypeptide for IL2Rα is similar compared to wild-type IL2.

56. A modified interleukin-2 (IL2) polypeptide, comprising a modified IL2 receptor alpha (IL2Rα) binding region 1 comprising a substitution selected from a substitution at position K35, a substitution at R38, a substitution at F42, a substitution at Y45, or any combination thereof, wherein the modified IL2 polypeptide binds to IL2Rα with binding kinetics that are reduced by at least two-fold compared to wild-type IL2.

57. 57. The modified IL2 polypeptide of claim 56, comprising a substitution at position K35.

58. 58. The modified IL2 polypeptide of claim 57, wherein the substitution at position K35 comprises a non-basic residue.

59. 58. The modified IL2 polypeptide of claim 57, wherein the substitution at position K35 comprises an uncharged or acidic residue.

60. 58. The modified IL2 polypeptide of claim 57, wherein the substitution at position K35 is selected from K35G, K35L, K35S, K35V, K35D, K35E, and K35C.

61. 61. The modified IL2 polypeptide of any one of claims 56 to 60, comprising a substitution at position R38.

62. 62. The modified IL2 polypeptide of claim 61, wherein the substitution at position R38 comprises a non-basic charged residue.

63. 62. The modified IL2 polypeptide of claim 61, wherein the substitution at position R38 comprises an uncharged or acidic residue.

64. 62. The modified IL2 polypeptide of claim 61, wherein the substitution at position R38 is selected from R38V, R38D, R38E, R38S, R38I, R38A, R38Y, R38G, R38C, or R38N.

65. 65. The modified IL2 polypeptide of any of claims 56 to 64, comprising a substitution at position F42.

66. 66. The modified IL2 polypeptide of claim 65, wherein the substitution at position F42 comprises an uncharged residue.

67. 66. The modified IL2 polypeptide of claim 65, wherein the substitution at position F42 comprises a positively charged residue.

68. 66. The modified IL2 polypeptide of claim 65, wherein the substitution at position F42 is selected from F42A, F42R, F42G, F42I, F42L, F42P, and F42H.

69. 69. The modified IL2 polypeptide of any of claims 56 to 68, comprising a substitution at position Y45.

70. 70. The modified IL2 polypeptide of claim 69, wherein the substitution at position Y45 comprises an uncharged residue.

71. 70. The modified IL2 polypeptide of claim 69, wherein the substitution at position Y45 comprises an uncharged polar residue or an uncharged apolar residue.

72. 70. The modified IL2 polypeptide of claim 69, wherein the substitutions at position Y45 are Y45S, Y45P, Y45A, Y45V, Y45C, Y45T, and Y45F.

73. 73. The modified IL2 polypeptide of any one of claims 56 to 72, comprising a substitution at position K35 and a substitution at position R38.

74. 74. The modified IL2 polypeptide of claim 73, comprising a K35G substitution and a R38E substitution.

75. 75. The modified IL2 polypeptide of any one of claims 56 to 74, comprising a substitution at position K35 and a substitution at position F42.

76. 76. The modified IL2 polypeptide of claim 75, comprising a K35S substitution and a F42G substitution.

77. 77. The modified IL2 polypeptide of any one of claims 56 to 76, comprising a substitution at position K35, a substitution at position R38, and a substitution at position F42.

78. 78. The modified IL2 polypeptide of any of claims 77, comprising a K35L substitution, a R38D substitution, and a F42R substitution.

79. 79. The modified IL2 polypeptide of any one of claims 56 to 78, comprising a substitution at position R38 and a substitution at position Y45S.

80. 80. The modified IL2 polypeptide of any of claims 79, comprising an R38D substitution and a Y45S substitution.

81. 80. The modified IL2 polypeptide of any of claims 79, comprising an R38V substitution and a Y45S substitution.

82. containing a substitution at least one of positions K35, R38, F42, and Y45; i) the substitution at position K35 is selected from K35G, K35L, K35S, K35V, K35D, K35E, and K35C; ii) said substitution at position R38 is selected from R38V, R38D, R38E, R38S, R38I, R38A, R38Y, R38G, R38C, or R38N; iii) the substitution at position F42 is selected from F42A, F42R, F42G, F42I, F42L, F42P, and F42H; iv) the substitutions at position Y45 are Y45S, Y45P, Y45A, Y45V, Y45C, Y45T, and Y45F; A modified IL2 polypeptide according to any one of claims 56 to 81.

83. 83. The modified IL2 polypeptide of claim 82, wherein the substitutions are at least two, at least three, or all four of positions K35, R38, F42, and Y45.

84. 84. The modified IL2 polypeptide of any of claims 56 to 83, wherein the modified IL2 polypeptide binds to IL2Rα with binding kinetics that are at least 10-fold reduced compared to wild-type IL2.

85. The modified IL2Rα binding region 1 is selected from the group consisting of PVLTRMLTIKFY (SEQ ID NO: 183), PKLTRMLTLKFP (SEQ ID NO: 184), PDLTSMLAFKFY (SEQ ID NO: 185), PGLTEMLTFKFY (SEQ ID NO: 186), PSLTRMLTGKFY (SEQ ID NO: 187), PELTIMLTPKFY (SEQ ID NO: 188), PCLTAMLTLKFA (SEQ ID NO: 189), and the like. Sequence number 189), PCLTAMLTLKFA (SEQ ID NO: 190), PKLTRMLTHKFV (SEQ ID NO: 191), PCLTDMLTFKFY (SEQ ID NO: 192), PLLTDMLTRKFY (SEQ ID NO: 193), PLLTDMLTFKFY (SEQ ID NO: 194), PKLTDMLTFKFS (SEQ ID NO: 195), PKLTYMLTRKFY (SEQ ID NO: 196), PKLTRMLTFKFC (SEQ ID NO: 197), PKLTSMLTFKFS (SEQ ID NO: 198), PKLTSMLTFKFS (SEQ ID NO: 199), PKLTYMLTFKFS (SEQ ID NO: 200), PKLTYMLTFKFS (SEQ ID NO: 201), PKLTGMLTFKFS (SEQ ID NO: 202), PKLTVMLTFKFT (SEQ ID NO: 203), PKLTVMLT 85. The modified IL2 polypeptide of any of claims 56-84, selected from PKLTVMLTFKFS (SEQ ID NO: 204), PKLTVMLTFKFP (SEQ ID NO: 205), PKLTVMLTFKFF (SEQ ID NO: 206), PKLTCMLTFKFA (SEQ ID NO: 207), PKLTNMLTFKFA (SEQ ID NO: 208), and PKLTNMLTFKFS (SEQ ID NO: 209).

86. A modified IL2 polypeptide comprising a modified IL2 receptor beta (IL2Rβ) binding region 2 described in any one of claims 1 to 55 and a modified IL2 receptor alpha (IL2Rα) binding region 1 described in any one of claims 56 to 85.

87. The modified IL2Rα binding region 1 is selected from the group consisting of PVLTRMLTIKFY (SEQ ID NO: 183), PKLTRMLTLKFP (SEQ ID NO: 184), PDLTSMLAFKFY (SEQ ID NO: 185), PGLTEMLTFKFY (SEQ ID NO: 186), PSLTRMLTGKFY (SEQ ID NO: 187), PELTIMLTPKFY (SEQ ID NO: 188), PCLTAMLTLKFA (SEQ ID NO: 189), PCLTAMLTLKFA (SEQ ID NO: 190), PKLTRMLTHKFV (SEQ ID NO: 191), PCLTDMLTFKFY (SEQ ID NO: 192), and PLLTDMLTRK FY (SEQ ID NO: 193), PLLTDMLTFKFY (SEQ ID NO: 194), PKLTDMLTFKFS (SEQ ID NO: 195), PKLTYMLTRKFY (SEQ ID NO: 196), PKLTRMLTFKFC (SEQ ID NO: 197), PKLTSMLTFKFS (SEQ ID NO: 198), PKLTSMLTFKFS (SEQ ID NO: 199), PKLTYMLTFKFS (SEQ ID NO: 200), PKLTYMLTFKFS (SEQ ID NO: 201), PKLTGMLTFKFS (SEQ ID NO: 202), PKLTVMLTFKFT (SEQ ID NO: 203), PKLTVMLTFKFS (SEQ ID NO: 204), SEQ ID NO: 204), PKLTVMLTFKFP (SEQ ID NO: 205), PKLTVMLTFKFF (SEQ ID NO: 206), PKLTCMLTFKFA (SEQ ID NO: 207), PKLTNMLTFKFA (SEQ ID NO: 208), and PKLTNMLTFKFS (SEQ ID NO: 209), and the modified IL2Rβ binding region 2 is selected from GVTDSISNAIVLARE (SEQ ID NO: 2), KWGDAVSNARVLAGE (SEQ ID NO: 3), KWGDAVSNARVLAGA (SEQ ID NO: 4), TLMDTTDNIGVLVRE (SEQ ID NO: 5), EPSDVISNINVLV QE (SEQ ID NO: 6), SPQDSIENISVLVRE (SEQ ID NO: 7), WASDSIENITLLIQE (SEQ ID NO: 8), CPTDTIENITVLIQE (SEQ ID NO: 9), RYKDSLENMQIIIQE (SEQ ID NO: 10), TARDAVDNMRVIIQE (SEQ ID NO: 11), TPRDVVENMNVLVLE (SEQ ID NO: 12), TPSDVIENMEVLILD (SEQ ID NO: 13), TPSDAIENINVLIRE ​​(SEQ ID NO: 14), TPSDVIENITVLVQE (SEQ ID NO: 15), GVGDTIDNINVLVKE (SEQ ID NO: 16),IGRDSIDNIKVIVQE (SEQ ID NO: 17), WATDTIRNVEVLVQE (SEQ ID NO: 18), TAEDVVTNITVLVQE (SEQ ID NO: 19), TAEDVISNIRVNVQE (SEQ ID NO: 20), and TPSDVIDNVSITVQE (SEQ ID NO: 21), TARDAISNIRVIVQE (SEQ ID NO: 210), RARDAIDNIRVIVQE (SEQ ID NO: 211), TPRDA 87. The modified IL2 polypeptide of claim 86, selected from IDNINVIIQE (SEQ ID NO: 212), TPRDAIDNIRVIVQE (SEQ ID NO: 213), TPRDAIDNIRVIILE (SEQ ID NO: 214), TARDAISNINVIQE (SEQ ID NO: 215), and TARDAIDNINVIVQE (SEQ ID NO: 216), and TARDAIDNIRVIVLE (SEQ ID NO: 217).

88. A fusion polypeptide comprising a modified IL2 polypeptide of any one of claims 1 to 87 fused to a half-life extension molecule.

89. 89. The fusion polypeptide of claim 88, wherein the half-life extending molecule comprises a half-life extending polypeptide.

90. 89. The fusion polypeptide of claim 88, wherein the half-life extending polypeptide comprises an Fc domain, human serum albumin (HSA), an HSA binding molecule, or transferrin.

91. 89. The fusion polypeptide of claim 88, wherein the half-life extending polypeptide comprises an Fc domain.

92. 89. The fusion polypeptide of claim 88, wherein the half-life extension molecule comprises polyethylene glycol (PEG) or polypropylene glycol (PPG).

93. 88. A fusion polypeptide comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a modified IL2 polypeptide of any one of claims 1 to 87.

94. 94. The fusion polypeptide of claim 93, wherein the second polypeptide comprises an antigen-binding portion.

95. 95. The fusion polypeptide of claim 94, wherein the antigen-binding portion comprises an immunoglobulin.

96. 96. The fusion polypeptide of claim 95, wherein the antigen-binding portion comprises a Fab molecule, an scFv, a bispecific T cell engager, a diabody, a single domain antibody, or a nanobody.

97. 94. The fusion polypeptide of claim 93, wherein the second polypeptide comprises a cytokine.

98. 98. The fusion polypeptide of claim 97, wherein the second polypeptide comprises interleukin 2, interleukin 15, interleukin 7, interleukin 10, or C-C motif chemokine ligand 19 (CCL19).

99. 94. The fusion polypeptide of claim 93, wherein the second polypeptide comprises a second modified IL2 polypeptide of any one of claims 1 to 87.

100. An isolated polynucleotide encoding at least one polypeptide according to any one of claims 1 to 99.

101. An expression vector comprising the polynucleotide of claim 100.

102. 102. A modified cell comprising the isolated polynucleotide of claim 100 or the expression vector of claim 101.

103. 103. The modified cell of claim 102, further comprising a modified T cell receptor or a chimeric antigen receptor.

104. A pharmaceutical composition comprising a modified IL2 polypeptide according to any one of claims 1 to 87 or a fusion polypeptide according to any one of claims 88 to 99 and a pharmaceutically acceptable carrier.

105. 104. The modified IL2 polypeptide of any one of claims 1 to 87, the fusion polypeptide of any one of claims 88 to 99, or the pharmaceutical composition of claim 104 for use in a method of modulating an immune response in a subject in need thereof.

106. 106. The method of claim 105, wherein the modulation of the immune response comprises at least one of enhancing effector T cell activity, enhancing NK cell activity, and suppressing regulatory T cell activity.

107. 104. The modified IL2 polypeptide of any one of claims 1 to 87, the fusion polypeptide of any one of claims 88 to 99, or the pharmaceutical composition of claim 104 for use in a method of treating a disease in a subject in need thereof.

108. 108. The modified IL2 polypeptide of any one of claims 1 to 87, the fusion polypeptide of any one of claims 88 to 99, or the pharmaceutical composition of claim 104, used according to claim 107, wherein the disease comprises cancer or immunosuppression.

109. 108. The modified IL2 polypeptide of any one of claims 1 to 87, the fusion polypeptide of any one of claims 88 to 99, or the pharmaceutical composition of claim 104, used according to claim 108, wherein the cancer comprises breast cancer, pancreatic cancer, lung cancer, glioblastoma, renal cell carcinoma, or melanoma.

110. 108. The modified IL2 polypeptide of any one of claims 1 to 87, the fusion polypeptide of any one of claims 88 to 99, or the pharmaceutical composition of claim 104, for use according to any one of claims 107 to 109, wherein the subject is treated with an additional therapeutic agent.

111. 110. The modified IL2 polypeptide of any one of claims 1 to 87, the fusion polypeptide of any one of claims 88 to 99, or the pharmaceutical composition of claim 104, used according to claim 110, wherein the additional therapeutic agent comprises an antigen-binding portion.

112. 112. The modified IL2 polypeptide of any one of claims 1 to 87, the fusion polypeptide of any one of claims 88 to 99, or the pharmaceutical composition of claim 104, used in accordance with claim 111, wherein the antigen-binding portion comprises a single domain antibody, a Fab molecule, an scFv, a diabody, a nanobody, a bispecific T cell engager, or an immunoglobulin.

113. 113. The modified IL2 polypeptide of any one of claims 1 to 87, the fusion polypeptide of any one of claims 88 to 99, or the pharmaceutical composition of claim 104, used according to claim 111 or 112, wherein the antigen-binding portion is directed against a tumor antigen.

114. 110. The modified IL2 polypeptide of any one of claims 1 to 87, the fusion polypeptide of any one of claims 88 to 99, or the pharmaceutical composition of claim 104, used in accordance with claim 110, wherein the additional therapeutic agent comprises an immune cell expressing a chimeric antigen receptor, an immune cell expressing a modified T-cell receptor, or a tumor-infiltrating lymphocyte.

115. The modified IL2 polypeptide of any one of claims 1 to 87, the fusion polypeptide of any one of claims 88 to 99, or the pharmaceutical composition of claim 104, used according to claim 114, wherein said immune cells comprise a polynucleotide encoding the modified IL2 polypeptide of any one of claims 1 to 87, or the fusion polypeptide of any one of claims 88 to 99.

116. 110. The modified IL2 polypeptide of any one of claims 1 to 87, the fusion polypeptide of any one of claims 88 to 99, or the pharmaceutical composition of claim 104, used in accordance with claim 110, wherein the additional therapeutic agent comprises an immune checkpoint inhibitor.

117. 117. The modified IL2 polypeptide of any one of claims 1 to 87, the fusion polypeptide of any one of claims 88 to 99, or the pharmaceutical composition of claim 104, used in accordance with claim 116, wherein the checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor, a LAG3 inhibitor, a B7-H2 inhibitor, or a B7-H3 inhibitor.

118. 110. The modified IL2 polypeptide of any one of claims 1 to 87, the fusion polypeptide of any one of claims 88 to 99, or the pharmaceutical composition of claim 104, used according to claim 110, wherein the additional therapeutic agent comprises an oncolytic virus.

119. 110. The modified IL2 polypeptide of any one of claims 1 to 87, the fusion polypeptide of any one of claims 88 to 99, or the pharmaceutical composition of claim 104, used in accordance with claim 110, wherein the additional therapeutic agent comprises a tumor microenvironment (TME) inhibitor.

120. 110. The modified IL2 polypeptide of any one of claims 1 to 87, the fusion polypeptide of any one of claims 88 to 99, or the pharmaceutical composition of claim 104, used according to claim 110, wherein the additional therapeutic agent comprises a cancer vaccine.

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