Modified interleukin 2 (il-2) polypeptides, conjugates and uses thereof

Abstract

To provide modified interleukin 2 (IL-2) polypeptides, polynucleotides, e.g., DNA, RNA or viral vector, that encode the modified IL-2 polypeptides and are configured to express the modified IL-2 polypeptides in vitro and / or in vivo, conjugates comprising the modified IL-2 polypeptides, and uses thereof.SOLUTION: In one aspect, a modified interleukin 2 (IL2) polypeptide of the present invention may comprise an amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, and a substitution with a natural amino acid or an unnatural amino acid at a position selected from a group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93 and a combination thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 887,359, entitled "Modified Interleukin-2 (IL-2) Polypeptides, Conjugates, and Uses Thereof," filed August 15, 2019, and U.S. Provisional Patent Application No. 63 / 025,095, entitled "Modified Interleukin-2 (IL-2) Polypeptides, Conjugates, and Uses Thereof," filed May 14, 2020, the contents and disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0002] ASCII Text Sequence Table This patent or application file contains a sequence listing submitted in computer-readable ASCII text format (Filename: 7006-2000140_SeqList_ST25.txt, Recorded on: August 10, 2020, Size: 3,014 bytes). The contents of the sequence listing file are incorporated herein by reference in their entirety.

[0003] Technical Field The present disclosure relates to modified interleukin-2 (IL-2) polypeptides, including modified IL-2 polypeptides, polynucleotides, such as DNA, RNA or viral vectors (whether conjugated or not), that encode the modified IL-2 polypeptides and are configured to express said modified IL-2 polypeptides in vitro and / or in vivo, and uses thereof. [Background technology]

[0004] Background technology The clinical use of interleukin-2 (IL-2) for cancer treatment has been limited primarily by its toxicity and short in vivo half-life.[1,2] A marked reduction in toxicity was observed in CD25 (IL-2 receptor alpha unit, IL-2Rα)-deficient animals.[3] PEGylation, the covalent attachment of polyethylene glycol (PEG) to therapeutic agents, has been shown to overcome obstacles such as rapid body clearance, aggregation, and enzymatic degradation.[4]

[0005] WO 2019 / 028419 A1 and WO 2019 / 028425 A1 disclose interleukin (IL) conjugates (e.g., IL-2 conjugates) and their use in the treatment of one or more indications. WO 2019 / 028419 A1 and WO 2019 / 028425 A1 also describe pharmaceutical compositions and kits comprising one or more interleukin conjugates (e.g., IL-2 conjugates). There is a need in the art for improved modified interleukin-2 (IL-2) polypeptides, with or without conjugates. The present invention addresses this and other related needs in the art. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2019 / 028419 [Patent Document 2] International Publication No. 2019 / 028425 [Patent Document 3] International Publication No. 2019 / 028419 [Patent Document 4] International Publication No. 2019 / 028425 Summary of the Invention [Means for solving the problem]

[0007] Abstract The present invention is directed to modified interleukin-2 (IL-2) polypeptides, polynucleotides, e.g., DNA, RNA or viral vectors, encoding the modified IL-2 polypeptides and configured to express said modified IL-2 polypeptides in vitro and / or in vivo, conjugates comprising the modified IL-2 polypeptides, and uses thereof.

[0008] In one aspect, the invention provides a modified interleukin-2 (IL-2) polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 and a substitution with a natural or unnatural amino acid at a position selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof, wherein the modified IL-2 polypeptide is a) unconjugated or configured to be conjugated to a water soluble polymer, lipid, or polypeptide, e.g., a protein or peptide; b) reduced binding to interleukin 2 receptor alpha (IL-2Rα) compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions; c) reduced receptor signaling potency for IL-2Rαβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions; d) increased ratio of signaling potency for IL-2Rβγ over signaling potency for IL-2Rαβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions (i.e., increased ratio of signaling potency for IL-2Rβγ / signaling potency for IL-2Rαβγ); and / or e) enhanced receptor signaling potency for IL-2Rβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions, and a substitution at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and a combination thereof, with a natural or non-natural amino acid at a position within the IL-2Rα interacting region, the IL-2Rβ interacting region, and / or the IL-2Rγ interacting region, with the proviso that the modified IL-2 polypeptide comprises a substitution at amino acid residues 10 to 25,The present invention relates to a modified interleukin-2 (IL-2) polypeptide, which has at least about 80% sequence identity in the region of 80 to 100 and / or 100 to 134 with the corresponding region of a comparable IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without substitutions, and the modified IL-2 polypeptide has at least about 50% sequence identity with a comparable IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without substitutions.

[0009] In another aspect, the present invention is directed to a polynucleotide, e.g., DNA, RNA, or viral vector, encoding a modified IL-2 polypeptide and configured to express said modified IL-2 polypeptide in vitro and / or in vivo. In some embodiments, such modified IL-2 polypeptides, with or without conjugation, can be administered in the form of a protein, a fusion protein, a protein conjugate, or as part of a nanoparticle. In some embodiments, such polynucleotides, e.g., DNA, RNA, or viral vectors encoding a modified IL-2 polypeptide and configured to express said modified IL-2 polypeptide in vitro and / or in vivo, can be administered to a cell, tissue, organ, or subject, e.g., a human subject.

[0010] In yet another aspect, the present invention is directed to a modified IL-2 polypeptide conjugate comprising a modified IL-2 polypeptide as described above conjugated to a water soluble polymer, lipid, polypeptide, such as a protein or peptide.

[0011] In yet another aspect, the present invention is directed to a pharmaceutical composition comprising an effective amount of a modified IL-2 polypeptide, a polynucleotide, e.g., DNA, RNA or viral vector, or a modified IL-2 polypeptide conjugate as described above, and a pharma- ceutically acceptable carrier or excipient.

[0012] In yet another aspect, the present invention is directed to a method for treating or preventing a disease or disorder, such as a proliferative disease or disorder, an autoimmune or inflammatory disease or disorder, or an infectious disease or disorder, in a subject in need thereof comprising administering to said subject an effective amount of modified IL-2, a polynucleotide, such as DNA, RNA or viral vector, a modified IL-2 polypeptide conjugate as described above, or a pharmaceutical composition.

[0013] In yet another aspect, the invention is directed to the use of an effective amount of a modified IL-2 polypeptide, a polynucleotide, e.g., a DNA, RNA or viral vector, or a modified IL-2 polypeptide conjugate as described above for the manufacture of a medicament for treating or preventing a disease or disorder in a subject, such as a proliferative disease or disorder, an autoimmune or inflammatory disease or disorder, or an infectious disease or disorder.

[0014] In yet another aspect, the present invention provides a method for the detection of CD4 + Helper cells, CD8 + The present invention is directed to a method of expanding effector naive and memory cell, natural killer (NK) cell or natural killer T (NKT) cell populations, comprising contacting the cell population with an effective amount of a modified IL-2 polypeptide, polynucleotide, such as DNA, RNA or viral vector, or modified IL-2 polypeptide conjugate, or a pharmaceutical composition comprising a modified IL-2 polypeptide, polynucleotide, such as DNA, RNA or viral vector, or modified IL-2 polypeptide conjugate as described above, for a time sufficient to induce formation of a complex with IL-2Rβγ, thereby stimulating proliferation of the T cell, NK cell and / or NKT cell population.

[0015] In yet another aspect, the present invention provides a method for the detection of CD4 + Helper cells, CD8 +The present invention is directed to a method of expanding effector naive and memory cell, Treg cell, natural killer (NK) cell or natural killer T (NKT) cell populations, comprising contacting the cell population with an effective amount of a modified IL-2 polypeptide, polynucleotide, such as DNA, RNA or viral vector, or modified IL-2 polypeptide conjugate, or a pharmaceutical composition comprising a modified IL-2 polypeptide, polynucleotide, such as DNA, RNA or viral vector, or modified IL-2 polypeptide conjugate as described above, for a time sufficient to induce formation of a complex with IL-2Rβγ, thereby stimulating proliferation of the T cell, Treg cell, NK cell, and / or NKT cell population and reducing cell death by 10% to 100%.

[0016] In yet another aspect, the present invention provides a method for detecting CD4 + Helper cells, CD8 + The present invention relates to the use of an effective amount of a modified IL-2 polypeptide, a polynucleotide, e.g., a DNA, RNA or viral vector, or a modified IL-2 polypeptide conjugate as described above for the manufacture of a medicament for expanding effector naive and memory cells, Treg cells, natural killer (NK) cells or natural killer T (NKT) cells.

[0017] Other aspects and advantages of the present invention will become apparent from the embodiments and examples provided herein.

[0018] For the sake of brevity, the disclosures of the publications cited in this specification, including patents, are hereby incorporated by reference.

[0019] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. In certain embodiments, for example, the following items are provided: (Item 1) A modified interleukin 2 (IL-2) polypeptide, comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, and Q13, L19, N29, N30, Y31, K32, N33 , P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof, with natural or non-natural amino acids, wherein the modified IL-2 polypeptide comprises: a) is unconjugated or is configured to be conjugated to a water-soluble polymer, lipid, or polypeptide, such as a protein or peptide; and / or b) has reduced binding to interleukin-2 receptor alpha (IL-2Rα) compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without said substitutions; and / or c) has a reduced receptor signaling potency for IL-2Rαβγ compared to an IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without said substitutions; and / or d) an increased ratio of signaling potency for IL-2Rβγ to signaling potency for IL-2Rαβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without said substitutions (i.e., an increased ratio of signaling potency for IL-2Rβγ / signaling potency for IL-2Rαβγ); and / or e) has enhanced receptor signaling potency towards IL-2Rβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without said substitutions; and / or with the proviso that when the modified IL-2 polypeptide comprises a substitution with a non-natural amino acid, the modified IL-2 polypeptide comprises a substitution at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof, and a substitution with a natural or non-natural amino acid at a position within the IL-2Rα interacting region, IL-2Rβ interacting region, and / or IL-2Rγ interacting region; provided that the modified IL-2 polypeptide has at least about 80% sequence identity in the regions of amino acid residues 10 to 25, 80 to 100, and / or 100 to 134 to the corresponding regions of a comparable IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, and that the modified IL-2 polypeptide has at least about 50% sequence identity to a comparable IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution. (Item 2) 2. The modified IL-2 polypeptide of item 1, comprising a substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at a position selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof. (Item 3) a) comprising substitutions with natural amino acids at positions selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof, and a water-soluble polysaccharide at said positions selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof; and / or configured to be conjugated to a mer, lipid, protein or peptide; b) The modified IL-2 polypeptide according to item 1 or 2, comprising substitutions with natural amino acids at positions selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76 and combinations thereof, and configured for conjugation to a water soluble polymer, lipid, protein or peptide at the N-terminus and / or C-terminus of the polypeptide. (Item 4) a) comprising a substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine or tyrosine at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76 and combinations thereof; and / or b) The modified IL-2 polypeptide according to item 3, comprising a substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine or tyrosine at positions selected from the group consisting of N30, Y31, N33, P34, K35, R38, T41, K43, K48, K49, K64, P65, N71, Q74, K76 and combinations thereof. (Item 5) a) comprising a substitution with cysteine ​​at a position selected from the group consisting of N29, N30, Y31, N33, P34, K35, R38, T41, K43, K48, K49, K64, P65, N71, Q74, K76, and combinations thereof; b) comprising a substitution with cysteine ​​at a position selected from the group consisting of N29, Y31, K35, P65, N71, Q74, and combinations thereof; c) at positions Y31, N29, or a combination thereof, containing a substitution with any amino acid; d) comprising a substitution at position Y31, N29, or a combination thereof with cysteine, serine, or alanine; e) at position Y31, containing a substitution by cysteine, f) at position N29, containing a substitution by cysteine; and / or g) A modified IL-2 polypeptide according to item 4, comprising a substitution at position P65 with cysteine. (Item 6) 6. The modified IL-2 polypeptide according to any one of items 3 to 5, further comprising a substitution with a natural or non-natural amino acid at a position within the IL-2Rα interacting region, the IL-2Rβ interacting region and / or the IL-2Rγ interacting region. (Item 7) 7. The modified IL-2 polypeptide according to item 6, further comprising a substitution with a natural amino acid at a position within the IL-2Rα interacting region. (Item 8) 8. The modified IL-2 polypeptide according to item 7, comprising a substitution with a natural amino acid at a position selected from the group consisting of R38, F42, Y45, E62, P65 and combinations thereof. (Item 9) 9. The modified IL-2 polypeptide of item 8, comprising a substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at positions selected from the group consisting of R38, F42, Y45, E62, P65, and combinations thereof. (Item 10) a) comprising a substitution with cysteine ​​at a position selected from the group consisting of R38, F42, Y45, E62, P65, and combinations thereof; b) comprising a substitution at position F42 with alanine, lysine or serine; c) at position F42, comprising a substitution by alanine, d) comprising a substitution at position F42 with serine; e) at position F42, containing a substitution by lysine, f) comprising a substitution at position Y45 with alanine, histidine or serine; g) comprising a substitution at position Y45 with alanine, h) comprising a substitution at position Y45 with histidine, i) comprising a substitution at position R38 with alanine, aspartic acid or serine; j) containing a substitution at position R38 with aspartic acid, k) at position P65, comprising a substitution by alanine, l) at position P65, containing a substitution by serine, m) containing a substitution at position E62 by alanine; and / or n) The modified IL-2 polypeptide according to item 6 or 9, comprising a substitution at position F42 with lysine, at position Y31 with cysteine, or a combination thereof. (Item 11) 7. The modified IL-2 polypeptide of item 6, further comprising a substitution with a natural amino acid at a position within the IL-2Rβ interacting region. (Item 12) 12. The modified IL-2 polypeptide of item 11, comprising a substitution with a natural amino acid at a position selected from the group consisting of Q13, L19, R81, L85, S87, V91, I92, V93 and combinations thereof. (Item 13) 13. The modified IL-2 polypeptide of item 12, comprising a substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at positions selected from the group consisting of Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof. (Item 14) 14. The modified IL-2 polypeptide of item 13, comprising a substitution with cysteine ​​at a position selected from the group consisting of Q13, L19, R81, L85, S87, V91, I92, V93 and combinations thereof. (Item 15) a) substitutions with natural amino acids at positions within the IL-2Rα interacting region and substitutions with natural amino acids at positions within the IL-2Rβ interacting region; b) a substitution with a natural amino acid at a position within the IL-2Rα interacting region and a substitution with a natural amino acid at a position within the IL-2Rγ interacting region, or c) The modified IL-2 polypeptide according to any one of items 6 to 14, further comprising a substitution with a natural amino acid at a position within the IL-2Rα interacting region, a substitution with a natural amino acid at a position within the IL-2Rβ interacting region and a substitution with a natural amino acid at a position within the IL-2Rγ interacting region. (Item 16) 16. The modified IL-2 polypeptide according to any one of items 1 to 15, which has reduced binding to IL-2Rα as compared to an IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution. (Item 17) The binding affinity to IL-2Rα is reduced by about 10% to about 100%, or by about 17. The modified IL-2 polypeptide of item 16, wherein the expression level of the polypeptide is reduced by about 100,000-fold or more. (Item 18) 17. The modified IL-2 polypeptide of item 16, which has no detectable binding to IL-2Rα. (Item 19) 19. The modified IL-2 polypeptide according to any one of items 1 to 18, which has a reduced receptor signaling potency for IL-2Rαβγ, as compared to an IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution. (Item 20) 20. The modified IL-2 polypeptide according to item 19, wherein the ratio of the signaling potency for IL-2Rαβγ to the signaling potency for IL-2Rαβγ of the comparable IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution is about 1 / 2 to about 1 / 100,000. (Item 21) 20. The modified IL-2 polypeptide of item 19, having no detectable receptor signaling effect on IL-2Rαβγ. (Item 22) 22. The modified IL-2 polypeptide according to any one of items 1 to 21, which has reduced binding to IL-2Rα as compared to an IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution, and has reduced receptor signaling potency for IL-2Rαβγ as compared to an IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution. (Item 23) 23. The modified IL-2 polypeptide of item 22, having no detectable binding to IL-2Rα and no detectable receptor signaling efficacy on IL-2Rαβγ. (Item 24) 24. The modified IL-2 polypeptide according to any one of items 1 to 23, which retains a substantial level of binding to interleukin-2 receptor beta (IL-2Rβ) or interleukin-2 receptor gamma (IL-2Rγ) or has a higher level of binding compared to an IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitutions, and / or retains a substantial receptor signaling potency for IL-2Rβγ or has a higher receptor signaling potency compared to an IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitutions. (Item 25) 26. The modified IL-2 polypeptide according to claim 24, which retains a substantial level of binding to IL-2Rβ or IL-2Rγ or has a higher level of binding compared to an IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution. 25. The modified IL-2 polypeptide of item 24, which retains substantial receptor signaling potency for IL-2Rβγ or has greater receptor signaling potency compared to an IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution. (Item 27) 25. The modified IL-2 polypeptide of item 24, which retains substantial or has a higher level of binding to IL-2Rβ or IL-2Rγ compared to an IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitutions, and retains substantial or has a higher receptor signaling potency for IL-2Rβγ compared to an IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitutions. (Item 28) 28. The modified IL-2 polypeptide according to items 1 to 27, which has an increased ratio of signaling potency for IL-2Rβγ to signaling potency for IL-2Rαβγ (i.e., an increase in the ratio of signaling potency for IL-2Rβγ / signaling potency for IL-2Rαβγ) compared to an IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution. (Item 29) 29. The modified IL-2 polypeptide according to any one of items 1 to 28, having an N-terminal deletion, such as an N-terminal deletion of amino acid residues 1 to 30, and / or a C-terminal deletion, such as a C-terminal deletion of amino acid residues 114 to 134. (Item 30) 30. The modified IL-2 polypeptide according to any one of items 1 to 29, having an N-terminal deletion and a C-terminal deletion. (Item 31) 31. The modified IL-2 polypeptide according to any one of items 1 to 30, which is part of a fusion polypeptide, e.g. a recombinant fusion protein, comprising the modified IL-2 polypeptide and a further amino acid sequence. (Item 32) 32. The modified IL-2 polypeptide according to claim 31, wherein the N-terminus or C-terminus of the modified IL-2 polypeptide is fused to the additional amino acid sequence. (Item 33) 33. The modified IL-2 polypeptide according to item 32, wherein the further amino acid sequence comprises an antibody sequence or a part or fragment thereof, such as the Fc portion of an antibody. (Item 34) 34. The modified IL-2 polypeptide according to any one of items 1 to 33, which is in isolated form. (Item 35) 35. A polynucleotide, such as a DNA, RNA or viral vector, encoding the modified IL-2 polypeptide according to any one of items 1 to 34 and configured to express said modified IL-2 polypeptide in vitro and / or in vivo. (Item 36) 36. A modified IL-2 polypeptide conjugate comprising a modified IL-2 polypeptide according to any one of items 1 to 34 or 1 to 35 conjugated to a water soluble polymer, lipid, polypeptide, such as a protein or peptide. (Item 37) 37. The modified IL-2 polypeptide conjugate according to item 36, wherein the modified IL-2 polypeptide is covalently conjugated to a water-soluble polymer, lipid, protein or peptide. (Item 38) 37. The modified IL-2 polypeptide conjugate according to item 36, wherein the modified IL-2 polypeptide conjugate is non-covalently conjugated to a water-soluble polymer, lipid, protein or peptide. (Item 39) 39. The modified IL-2 polypeptide conjugate according to any one of items 36 to 38, wherein the modified IL-2 polypeptide is conjugated to a water soluble polymer, lipid, protein or peptide via a substituted natural or unnatural amino acid at a position selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93 and combinations thereof. (Item 40) 40. The modified IL-2 polypeptide conjugate of item 39, wherein the modified IL-2 polypeptide is conjugated to a water soluble polymer, lipid, protein or peptide via a substituted natural amino acid at a position selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93 and combinations thereof. (Item 41) 40. The modified IL-2 polypeptide conjugate of item 39, wherein the modified IL-2 polypeptide is conjugated to a water soluble polymer, lipid, protein or peptide via a substituted lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine or tyrosine at a position selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93 and combinations thereof. (Item 42) 40. The modified IL-2 polypeptide conjugate of item 39, wherein the modified IL-2 polypeptide is conjugated to a water soluble polymer, lipid, protein or peptide via a substituted cysteine ​​at a position selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93 and combinations thereof. (Item 43) 40. The modified IL-2 polypeptide conjugate of item 39, wherein the modified IL-2 polypeptide is conjugated to a water soluble polymer, lipid, protein or peptide via a substituted natural or unnatural amino acid at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76 and combinations thereof. (Item 44) 44. The modified IL-2 polypeptide conjugate according to item 43, wherein the modified IL-2 polypeptide is conjugated to a water soluble polymer, lipid, protein or peptide via a substituted natural amino acid at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76 and combinations thereof. (Item 45) 45. The modified IL-2 polypeptide conjugate according to item 44, wherein the modified IL-2 polypeptide is conjugated to a water soluble polymer, lipid, protein or peptide via a substituted lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine or tyrosine at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76 and combinations thereof. (Item 46) 46. ​​The modified IL-2 polypeptide conjugate according to item 45, wherein the modified IL-2 polypeptide is conjugated to a water soluble polymer, lipid, protein or peptide via a substituted cysteine ​​at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76 and combinations thereof. (Item 47) 47. The modified IL-2 polypeptide conjugate according to any one of items 36 to 46, wherein the modified IL-2 polypeptide is conjugated to a water soluble polymer, lipid, protein or peptide via a single amino acid residue of the modified IL-2 polypeptide. (Item 48) The modified IL-2 polypeptide comprises: i) the alpha amino group of the N-terminal amino acid residue of the modified IL-2 polypeptide; ii) the epsilon amino group of a lysine amino acid residue of the modified IL-2 polypeptide, or iii) via the N- or O-glycosylation site of the modified IL-2 polypeptide, 48. The modified IL-2 polypeptide conjugate according to item 47, conjugated to a water-soluble polymer, lipid, protein or peptide. (Item 49) 49. The modified IL-2 polypeptide conjugate according to any one of items 36 to 48, wherein the modified IL-2 polypeptide is covalently conjugated to a water soluble polymer, lipid, protein or peptide via a linker. (Item 50) 47. The modified IL-2 polypeptide conjugate according to any one of items 36 to 46, wherein the modified IL-2 polypeptide is conjugated to a water-soluble polymer, lipid, protein or peptide via a single amino acid residue in a fusion polypeptide comprising the modified IL-2 polypeptide and a further amino acid sequence. (Item 51) 51. The modified IL-2 polypeptide conjugate of claim 50, wherein the single amino acid residue is located within the modified IL-2 polypeptide. (Item 52) 51. The modified IL-2 polypeptide conjugate according to claim 50, wherein the single amino acid residue is located within the further amino acid sequence. (Item 53) 53. The modified IL-2 polypeptide conjugate according to any one of items 50 to 52, wherein the further amino acid sequence comprises an antibody sequence or a part or fragment thereof. (Item 54) 54. The modified IL-2 polypeptide conjugate according to claim 53, wherein the further amino acid sequence comprises an Fc portion of an antibody. (Item 55) The modified IL-2 polypeptide comprises: i) the alpha amino group of the N-terminal amino acid residue of said fusion polypeptide; ii) the epsilon amino group of a lysine amino acid residue of said fusion polypeptide, or iii) via an N- or O-glycosylation site of the fusion polypeptide, 55. The modified IL-2 polypeptide conjugate according to any one of items 50 to 54, conjugated to a water-soluble polymer, lipid, protein or peptide. (Item 56) 56. The modified IL-2 polypeptide conjugate according to item 55, wherein the fusion polypeptide is covalently conjugated via a linker to a water-soluble polymer, lipid, protein or peptide. (Item 57) 57. The modified IL-2 polypeptide conjugate according to any one of items 36 to 56, wherein the modified IL-2 polypeptide is conjugated to a water soluble polymer. (Item 58) 48. The modified IL-2 polypeptide conjugate of item 47, wherein the water soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), a copolymer of ethylene glycol and propylene glycol, a poly(oxyethylated polyol), a poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or a combination thereof. (Item 59) 59. The modified IL-2 polypeptide conjugate of claim 58, wherein the water soluble polymer comprises a PEG molecule. (Item 60) 60. The modified IL-2 polypeptide conjugate according to item 59, wherein the PEG molecule is a linear PEG. (Item 61) 60. The modified IL-2 polypeptide conjugate according to claim 59, wherein the PEG molecule is a branched PEG. (Item 62) 62. The modified IL-2 polypeptide conjugate of item 61, wherein the branched PEG has about 3 to about 10 PEG chains emanating from a central core group. (Item 63) 62. The modified IL-2 polypeptide conjugate of claim 61, wherein the branched PEG is a star-shaped PEG comprising about 10 to about 100 PEG chains emanating from a central core group. (Item 64) 62. The modified IL-2 polypeptide conjugate of claim 61, wherein the branched PEG is a comb PEG comprising multiple PEG chains grafted onto a polymer backbone. (Item 65) 65. The modified IL-2 polypeptide conjugate according to any one of items 59 to 64, wherein the PEG molecule has a molecular weight range of about 300 g / mol to about 10,000,000 g / mol. (Item 66) 65. The modified IL-2 polypeptide conjugate according to any one of items 59 to 64, wherein the PEG molecule has an average molecular weight of from about 5,000 daltons to about 1,000,000 daltons. (Item 67) 67. The modified IL-2 polypeptide conjugate according to item 66, wherein the PEG molecule has an average molecular weight of about 20,000 daltons to about 30,000 daltons. (Item 68) 68. The modified IL-2 polypeptide conjugate according to any one of items 59 to 67, wherein the PEG molecules are monodisperse, uniform or discrete PEG molecules. (Item 69) 58. The modified IL-2 polypeptide conjugate of claim 57, wherein the water soluble polymer comprises a polysaccharide. (Item 70) 57. The modified IL-2 polypeptide conjugate according to any one of items 36 to 56, wherein the modified IL-2 polypeptide is conjugated to a lipid. (Item 71) 71. The modified IL-2 polypeptide conjugate of claim 70, wherein the lipid comprises a fatty acid. (Item 72) 57. The modified IL-2 polypeptide conjugate according to any one of items 36 to 56, wherein the modified IL-2 polypeptide is conjugated to a protein. (Item 73) 73. The modified IL-2 polypeptide conjugate of claim 72, wherein the protein comprises an antibody or a binding fragment thereof. (Item 74) 74. The modified IL-2 polypeptide conjugate according to item 73, wherein the antibody or binding fragment thereof comprises an Fc portion of an antibody. (Item 75) 75. The modified IL-2 polypeptide conjugate according to any one of items 36 to 74, wherein a water soluble polymer, lipid, protein or peptide is indirectly attached to the substituted natural or unnatural amino acid of the modified IL-2 polypeptide via a linker. (Item 76) 75. The modified IL-2 polypeptide conjugate according to any one of items 36 to 74, wherein a water soluble polymer, lipid, protein or peptide is directly attached to the substituted natural or unnatural amino acid of the modified IL-2 polypeptide. (Item 77) 77. The modified IL-2 polypeptide conjugate according to any one of items 36 to 76, having an in vivo half-life of from about 5 minutes to about 10 days. (Item 78) A pharmaceutical composition comprising an effective amount of a modified IL-2 polypeptide according to any one of items 1 to 34, a polynucleotide according to item 35 or a modified IL-2 polypeptide conjugate according to any one of items 36 to 77, and a pharma- ceutically acceptable carrier or excipient. (Item 79) 79. The pharmaceutical composition according to item 78, further comprising another active ingredient. (Item 80) 80. The pharmaceutical composition according to item 78 or 79, adapted to treat or prevent a proliferation disorder in a subject. (Item 81) 80. The pharmaceutical composition according to item 79, wherein the further active ingredient is an antitumor agent. (Item 82) A method for treating or preventing a disease or disorder, such as a proliferative disease or disorder, an autoimmune or inflammatory disease or disorder, or an infectious disease or disorder, in a subject in need thereof, comprising administering to said subject an effective amount of a modified IL-2 polypeptide according to any one of items 1 to 34, a polynucleotide according to item 35, a modified IL-2 polypeptide conjugate according to any one of items 36 to 77, or a pharmaceutical composition according to any one of items 78 to 81. (Item 83) 83. The method of claim 82, wherein the subject is a human. (Item 84) 83. The method of claim 82, wherein the subject is a non-human mammal. (Item 85) The method of any one of items 82 to 84, which is used to treat a proliferation disorder in a subject. (Item 86) 85. The method according to any one of items 82 to 84, which is used to prevent a proliferation disorder in a subject. (Item 87) 87. The method of any one of items 82 to 86, wherein the proliferation disorder is a tumor. (Item 88) 87. The method of any one of items 82 to 86, wherein the proliferation disorder is cancer. (Item 89) 89. The method according to item 87 or 88, wherein the tumor or cancer is a solid tumor or cancer. (Item 90) The solid tumor or cancer is selected from the group consisting of chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma of bone / osteosarcoma, osteosarcoma, rhabdomyosarcoma, cardiac cancer, astrocytoma, brain stem glioma, pilocytic astrocytoma, ependymoma, primitive neuroectodermal tumor, cerebellar astrocytoma, cerebral astrocytoma, glioma, medulloblastoma, neuroblastoma, oligodendroglioma, pineal astrocytoma, pituitary adenoma, visual pathway and hypothalamic glioma, breast cancer, invasion lobular carcinoma, tubular carcinoma, invasive breast cancer, medullary carcinoma, male breast cancer, phyllodes tumor, inflammatory breast cancer, adrenal cortical carcinoma, islet cell carcinoma (endocrine pancreas), multiple endocrine neoplasia syndrome, parathyroid carcinoma, pheochromocytoma, thyroid carcinoma, Merkel cell carcinoma, uveal melanoma, retinoblastoma, anal cancer, appendix cancer, bile duct cancer, carcinoid tumor, gastrointestinal, colon cancer, extrahepatic bile duct cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal interstitial GIST, hepatocellular carcinoma, pancreatic islet cell cancer, rectal cancer, bladder cancer, cervical cancer, endometrial cancer, extragonadal germ cell tumor, ovarian cancer, ovarian epithelial cancer (surface epithelial stromal tumor), ovarian germ cell tumor, penile cancer, renal cell carcinoma, renal pelvis and ureter, transitional cell carcinoma, prostate cancer, testicular cancer, gestational trophoblastic tumor, ureter and renal pelvis, transitional cell carcinoma, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms' tumor, esophageal cancer, head and neck cancer, nasopharyngeal 90. The method of item 89, wherein the cancer is selected from the group consisting of head cancer, oral cavity cancer, oropharynx cancer, paranasal sinus and nasal cavity cancer, pharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, basal cell carcinoma, melanoma, skin cancer (non-melanoma), bronchial adenoma / carcinoma, small cell lung cancer, mesothelioma, non-small cell lung cancer, pleuropulmonary blastoma, laryngeal cancer, thymoma and thymic carcinoma, AIDS-related cancer, Kaposi's sarcoma, epithelioid hemangioendothelioma (EHE), desmoplastic small round cell tumor, and liposarcoma. (Item 91) 89. The method of item 87 or 88, wherein the tumor or cancer is a hematological malignancy. (Item 92) The hematological malignancies are myeloid neoplasms, leukemias, lymphomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, hepatosplenic T-cell lymphoma, B-cell lymphoma reticuloendotheliosis, reticulosis, microglioma, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, primary mediastinal B-cell lymphoma, Waldenstrom's macroglobulinemia, nodal follicular zone B-cell lymphoma, splenic marginal zone lymphoma, Intravascular large B-cell lymphoma, Primary effusion lymphoma, Lymphomatoid granulomatosis, Nodular lymphocyte-predominant Hodgkin lymphoma, Plasma cell leukemia, Acute dysentery and erythroleukemia, Acute erythroblastic myelopathy, Acute erythroblastic leukemia, Heilmeyer-Schoener disease, Acute megakaryoblastic leukemia, Mast cell leukemia, Panmyelosis, Acute panmyelosis with myelofibrosis, Lymphosarcoma cell leukemia, Acute leukemia of unspecified cell type, Blast phase chronic myeloid leukemia, Stem cell leukemia, Chronic leukemia of unspecified cell type, Subacute leukemia of unspecified cell type, Accelerated phase chronic myelogenous leukemia Myeloid leukemia, acute myeloid leukemia, polycythemia vera, acute promyelocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, adult T-cell leukemia / lymphoma, aggressive NK cell leukemia, B-cell prolymphocytic leukemia, B-cell chronic lymphocytic leukemia, B-cell leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, chronic neutrophilic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, chronic idiopathic myelofibrosis, multiple myeloma, Köhler's disease , myelomatosis, solitary myeloma, plasma cell leukemia, plasmacytoma, extramedullary, malignant plasma cell neoplasm NOS, plasmacytoma NOS, monoclonal gammopathy, multiple myeloma, angiocentric immunoproliferative lesions, lymphocytic granulomatosis, angioimmunoblastic lymphadenopathy, T-gamma lymphoproliferative disorders, Waldenstrom's macroglobulinemia, alpha heavy chain disorder, gamma heavy chain disorder, Franklin's disease, immunoproliferative small intestinal disease, Mediterranean disease, malignant immunoproliferative disorder, unspecified, and immunoproliferative disorder NOS. (Item 93) 83. The method of claim 82, wherein the disease or disorder is an immunodeficiency disease or disorder. (Item 94) The immunodeficiency disease or disorder may be agammaglobulinemia: X-linked and autosomal recessive, ataxia telangiectasia, chronic granulomatous disease and other phagocytic cell disorders, common variable immunodeficiency, complement deficiencies, DiGeorge syndrome, hemophagocytic lymphohistiocytosis (HLH), hyper-IgE syndrome, hyper-IgM syndrome, IgG subclass deficiencies, innate immunity deficiencies, NEMO deficiency syndrome, selective IgA deficiency, selective IgM deficiency, severe combined immunodeficiency, deficiency and combined immunodeficiency, specific antibody deficiency, transient hypogammopathy of infancy, 94. The method of claim 93, wherein the immunodeficiency is selected from the group consisting of: hypogammaglobulinemia, WHIM syndrome (warts, hypogammaglobulinemia, infections, and myelocatechusis), Wiskott-Aldrich syndrome, other antibody deficiencies, other primary cellular immunodeficiencies, severe combined immunodeficiency (SCID), common variable immunodeficiency (CVID), human immunodeficiency virus / acquired immune deficiency syndrome (HIV / AIDS), drug-induced immunodeficiency, graft versus host syndrome, primary immunodeficiency disorder (PIDD), and lymphopenia. (Item 95) 85. The method according to any one of items 82 to 84, which is used to treat or prevent an autoimmune or inflammatory disease or disorder in a subject. (Item 96) The inflammatory or autoimmune disease or disorder is inflammation, an autoimmune disease, a tumor-associated autoimmune disease, inflammation of cartilage, a fibrotic disease and / or bone deterioration, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Letters syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reter's syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), dermatomyositis, psoriatic arthritis inflammation, scleroderma, systemic lupus erythematosus, vasculitis, myositis, polymyositis, dermatomyositis, osteoarthritis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, scleroderma, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis , lupus, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, Guillain-Barre disease, type I diabetes, thyroiditis (including Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, and graft rejection. (Item 97) 85. The method according to any one of items 82 to 84, which is used to treat or prevent an infectious disease or disorder in a subject. (Item 98) The infectious disease is selected from the group consisting of Acinetobacter infection, Actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (Acquired Immune Deficiency Syndrome), amebiasis, anaplasmosis, Angiostrongylus cantonensis anisakiasis, anthrax, hemolytic Arcanobacterium infection, Argentine hemorrhagic fever, ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacillus cereus infection, bacterial meningitis, bacterial pneumonia, bacterial vaginosis, Bacteroides infection, Balantidiosis, Bartonellosis, Baylisascari s infection, BK virus infection, black sand lichen, cystic cystosis, blastomycosis, Bolivian hemorrhagic fever, botulism (and infant botulism), Brazilian hemorrhagic fever, brucellosis, bubonic plague, Burkholderia infection, Buruli ulcer, Calicivirus infection (Norovirus and Sapovirus), Campylobacteriosis, Candidiasis (Moniliasis; Thrush), Capillariasis, Carrion disease, Cat scratch disease, Cellulitis, Chagas disease (American trypanosomiasis), Chancroid, Chickenpox, Chikungunya Fever, Chlamydia, Chlamydophila pneumoniae infection (Taiwan Acute Respiratory Pathogen or TWAR), Cholera, Chromoblastomycosis, Frog chytridiomycosis, Clonorchiasis, Clostridium difficile colitis, Coccidioidomycosis, Colorado Tick Fever (CTF), Common Cold (Acute Viral Rhanopharyngitis; Acute Coryza), Coronavirus Disease 2019 (COVID-19), Creutzfeldt-Jakob Disease (CJD), Crimean-Congo Hemorrhagic Fever (CCHF), Cryptococcosis, Cryptosporidiosis, Cutaneous Larva Migration (CLM), Cyclosporosis, Cysticercosis, Cytoplasmic Leukemia (CLM), ... Megalovirus infection, Dengue fever, Desmodesmus infection, Dinula amebiasis, Diphtheria, Diphyllobothriasis, Dracunculiasis, Ebola hemorrhagic fever, Echinococcosis, Ehrlichiosis, Enterobiasis (helminth infection), Enterococcus infection, Enterovirus infection, Typhus, Erythema infectiosum (fifth disease), Exanthema subitum (sixth disease), Fascioliasis, Fascioliasis, Fatal familial insomnia (FFI), Filariasis, Food poisoning due to Clostridium perfringens, Free-living ameba infection, Fusobacterium infection, Gas gangrene (Clostridial myonecrosis), Geotrichum, Gerstmann-Straussler Scheinker's syndrome (GSS), giardiasis, glanders, gnathostomiasis, gonorrhea, granulomatosis inguinale (donovanosis), group A streptococcal infection, group B streptococcal infection, Haemophilus influenzae infection, foot and mouth disease (HFMD), Hantavirus pulmonary syndrome (HPS), Heartland virus disease, Helicobacter pylori infection, hemolytic uraemic syndrome (HUS), hemorrhagic fever with renal syndrome (HFRS), Hendra virus infection, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex, histoplasmosis, hookworm infection, human bocavirus infection, human ewi ngii ehrlichiosis, human granulocytic anaplasmosis (HGA), human metapneumovirus infection, human monocytic ehrlichiosis, human papillomavirus (HPV) infection, human parainfluenza virus infection, membranous tapeworm disease, Epstein-Barr virus infectious mononucleosis (mono), influenza (influenza), isosporosis, Kawasaki disease, keratitis, kingella kingae infection, kuru, Lassa fever, legionnaires' disease (legionnaires' disease), Pontiac fever, leishmaniasis, leprosy, leptospirosis, listeriosis, Lyme disease (Lyme borreliosis),Lymphatic filariasis (elephantiasis), lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever (MHF), measles, Middle East respiratory syndrome (MERS), melioidosis (Whitmore's disease), meningitis, meningococcal disease, schizophrenia, microsporidiosis, leukemia (MC), monkeypox, mumps, typhus (endemic typhoid), mycoplasma pneumonia, mycoplasma genitalium infection, mycetoma, myiasis, neonatal conjunctivitis (ophthalmia neonatorum), Nipah virus infection, norovirus (children and infants), (new) variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Nocardiosis, Onchocerciasis (river blindness), Opisthorchiasis, Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis, Pasteurellosis, Pediculosis capitis (head lice), Pediculosis corporis (body lice), Pediculosis pubis (pubic lice, pubic lice), Pelvic inflammatory disease (PID), Whooping cough (pertussis), Plague, Pneumococcal infection, Pneumocystis jirovecii pneumonia (PCP), Pneumonia, Acute poliomyelitis, Prevotella infection, Primary amebic meningoencephalitis (PAM), Progressive multifocal leukoencephalopathy, Psittacosis, Q fever, Rabies, Relapsing fever, Respiratory syncytial virus Infectious diseases, rhinosporidiosis, rhinovirus infections, rickettsial infections, rickettsial smallpox, Rift Valley fever (RVF), Rocky Mountain spotted fever (RMSF), rotavirus infections, rubella, salmonellosis, SARS (Severe Acute Respiratory Syndrome), scabies, scarlet fever, schistosomiasis, septicemia, shigellosis (bacterial dysentery), shingles (shingles), smallpox (variola), sporotrichosis, staphylococcal food poisoning, staphylococcal infections, bacillariasis, subacute sclerosing panencephalitis, bejel, syphilis, and yaws, taeniasis, tetanus (lockjaw), tinea barbae (razor burn), tinea capitis (scalp ringworm), tinea corporis (ringworm of the body), tinea cruris (jock itch), tinea manubrium (ringworm of the hand), tinea nigricans, athlete's foot (athlete's foot), tinea unguium (onychomycosis), tinea versicolor (tinea versicolor), toxocariasis (ocular larva migrans (OLM)), toxocariasis (visceral larva migrans (VLM)), toxoplasmosis, trachoma, trichinosis, trichomoniasis, trichuriasis (whipworm infection), tuberculosis, tularemia, typhoid fever, typhoid fever, Ureaplasma urealyticum infection, valley fever, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Vibrio vulnificus infection, Vibrio parahaemolyticus enteritis, viral pneumonia, West Nile fever,White sand hair disease (tinea blanca), E, 98. The method of item 97, wherein the infection is selected from the group consisting of Yersinia pseudotuberculosis, yersiniosis, yellow fever, Zeaspora, Zika, and zygomycosis. (Item 99) 99. The method of any one of items 82-99, further comprising administering an effective amount of a second therapeutic agent to treat or prevent the proliferation disorder in the subject. (Item 100) Use of an effective amount of a modified IL-2 polypeptide according to any one of items 1 to 34, a polynucleotide according to item 35 or a modified IL-2 polypeptide conjugate according to any one of items 36 to 77 for the manufacture of a medicament for treating or preventing a disease or disorder, such as a proliferative disease or disorder, an autoimmune or inflammatory disease or disorder or an infectious disease or disorder, in a subject. (Item 101) CD4 + Helper cells, CD8 + 81. A method for expanding effector naive and memory cell, natural killer (NK) cell or natural killer T (NKT) cell populations, comprising contacting the cell population with an effective amount of a modified IL-2 polypeptide according to any one of items 1 to 34, a polynucleotide according to item 35, a modified IL-2 polypeptide conjugate according to any one of items 36 to 77 or a pharmaceutical composition according to any one of items 78 to 81 for a time sufficient to induce formation of a complex with IL-2Rβγ, thereby stimulating proliferation of said T cell, NK cell and / or NKT cell population. (Item 102) CD4 + Helper cells, CD8 +81. A method for expanding effector naive and memory cell, Treg cell, natural killer (NK) cell or natural killer T (NKT) cell populations, comprising contacting the cell population with an effective amount of a modified IL-2 polypeptide according to any one of items 1 to 34, a polynucleotide according to item 35, a modified IL-2 polypeptide conjugate according to any one of items 36 to 77 or a pharmaceutical composition according to any one of items 78 to 81 for a time sufficient to induce formation of a complex with IL-2Rβγ, thereby stimulating proliferation of said T cell, Treg cell, NK cell and / or NKT cell population and reducing cell death by 10% to 100%. (Item 103) The modified IL-2 polypeptide according to any one of items 1 to 34, the polynucleotide according to item 35, the modified IL-2 polypeptide conjugate according to any one of items 36 to 77, or the pharmaceutical composition according to any one of items 78 to 81 is administered to a subject in need of treatment with CD4 + T regulatory (Treg) cells were contacted with a comparable IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitutions. + 103. The method of claim 101 or 102, wherein 20%, 15%, 10%, 5%, 1% or less of the CD3+ cell population is expanded relative to the expansion of Treg cells. (Item 104) The modified IL-2 polypeptide according to any one of items 1 to 34, the polynucleotide according to item 35, the modified IL-2 polypeptide conjugate according to any one of items 36 to 77, or the pharmaceutical composition according to any one of items 78 to 81 is capable of inhibiting CD4+ in the cell population. + The method according to item 101 or 102, which does not expand Treg cells. (Item 105) 103. The method according to item 101 or 102, wherein the ratio of Teff cells to Treg cells in the cell population after incubation with the modified IL-2 polypeptide according to any one of items 1 to 34, the polynucleotide according to item 35, the modified IL-2 polypeptide conjugate according to any one of items 36 to 77, or the pharmaceutical composition according to any one of items 78 to 81 is about or at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, 100:1 or more. (Item 106) The method according to any one of items 101 to 105, which is carried out in vivo. (Item 107) 106. The method according to any one of items 101 to 105, which is carried out in vitro. (Item 108) 106. The method according to any one of items 101 to 105, which is carried out ex vivo. (Item 109) CD4 in cell populations + Helper cells, CD8 + Use of an effective amount of a modified IL-2 polypeptide according to any one of items 1 to 34, a polynucleotide according to item 35 or a modified IL-2 polypeptide conjugate according to any one of items 36 to 77 for the manufacture of a medicament for expanding effector naive and memory cells, Treg cells, natural killer (NK) cells or natural killer T (NKT) cell populations. (Item 110) CD4 in subjects + Helper cells, CD8 + 110. The use according to item 109, configured to expand effector naive and memory cell, Treg cell, natural killer (NK) cell or natural killer T (NKT) cell populations. [Brief description of the drawings]

[0020] [Figure 1]Figure 1A shows the sequence of an exemplary recombinant human IL-2 (rhIL-2) mutated from cysteine ​​to serine at position 125 [5]. The amino acid sites selected to be PEGylated via individual cysteine ​​substitutions and / or sites selected by mutation to disrupt IL-2Rα interaction and / or enhance IL-2Rβγ interaction are labeled with superscript numbers. Figure 1B shows the 3D structure of IL-2 and the receptor IL-2Rαβγ complex from PDB structure 2b5i. See, for example, The Protein Data Bank HM Berman, J. Westbrook, Z. Feng, G. Gilliland, TNBhat, H. Weissig, IN Shindyalov, PE Bourne (2000) Nucleic Acids Research, 28:235-242. doi:10.1093 / nar / 28.1.235. The sites described in Figure 1A are shown as red spheres.

[0021] [Diagram 2] FIG. 2 shows that expression of exemplary functional IL-2 mutants with individual cysteine ​​substitutions was determined by HEK-blue assay in cell culture supernatants diluted 1:10000.

[0022] [Figure 3-1] Figure 3 shows exemplary or typical profiles of chromatography and SDS-PAGE analysis for exemplary IL-2 muteins and PEG-conjugates. Figure 3A shows chromatography of N29C on a Superdex 75 Increase column. Figure 3B shows chromatography of N29C-PEG30 conjugate on a SP Sepharose FF column. Figure 3C shows chromatography of N29C-PEG30 conjugate on a Superdex 75 Increase column. Figure 3D shows SDS-PAGE analysis of N29C-PEG30 fractions eluted from the SP Sepharose FF column and the subsequent Superdex 75 Increase column. [Figure 3-2] Same as above.

[0023] [Figure 4-1] FIG. 4 shows an exemplary or representative sensorgram of an exemplary IL-2 mutein and PEG-conjugate binding to IL-2Rα obtained by an Octet Qke (ForteBio, San Jose, Calif.). [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above. [Figure 4-5] Same as above. [Figure 4-6] Same as above. [Diagram 4-7] Same as above. [Figure 4-8] Same as above. [Figure 4-9] Same as above.

[0024] [Diagram 5] FIG. 5 shows that the Y31C mutation and PEGylation did not affect cytokine binding to IL2Rα as demonstrated by Octet Qke (ForteBio, San Jose, Calif.).

[0025] [Figure 6-1] FIG. 6 shows that the Y31C mutein has enhanced binding to IL2Rαβγ expressing cells such as CTLL2 cells and CD25+ human T cells. [Figure 6-2] Same as above.

[0026] [Figure 7-1] FIG. 7 shows activation of pSTAT5 by exemplary IL-2 muteins and PEG-conjugates in human T cell subpopulations. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above.

[0027] [Figure 8]FIG. 8 shows plots of concentration-time curves following a single injection of rhIL-2, P65C-PEG20 conjugate, and Y31C-PEG20+F42K conjugate in mice.

[0028] [Figure 9-1] FIG. 9 shows that rhIL-2, the P65C-PEG20 conjugate and the Y31C-PEG20+F42K conjugate stimulate ex vivo proliferation of T cells and NK cells. [Figure 9-2] Same as above.

[0029] [Figure 10] FIG. 10 shows that rhIL-2, P65C-PEG20 conjugate and Y31C-PEG20+F42K conjugate expanded LAK cells (NK cells) have enhanced cytotoxicity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Detailed Description A. General Technology The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, immunology, and pharmacology, which are within the skill of one of ordinary skill in the art. Such techniques may be practiced in accordance with the teachings of Molecular Cloning: A Laboratory Manual, 2 nded. (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F. M. Usubel et al., eds., 1987, and periodic revisions); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Remington, The Science and Practice of Pharmacy, 20 th This is fully explained in such sources as (Lippincott, Williams & Wilkins 2003).

[0031] B. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. All patents, patent applications (published or unpublished), and other publications mentioned herein are incorporated by reference in their entirety. If the definitions set forth in this section are contrary to or inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth in this section shall take precedence over the definitions incorporated herein by reference.

[0032] As used herein, "a" or "an" means "at least one" or "one or more."

[0033] The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length, e.g., at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1,000 or more amino acids. The polymer may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified, either naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art, are also included in the definition.

[0034] As used herein, the term "variant" is used in reference to a polypeptide having a degree of amino acid sequence identity with a parent polypeptide sequence. A variant is similar to the parent sequence but has at least one substitution, deletion or insertion in the amino acid sequence that makes it different in sequence from the parent polypeptide. In addition, a variant may retain functional characteristics of the parent polypeptide, e.g., it may retain at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the biological activity of the parent polypeptide.

[0035] An "antibody" is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule, and may be any class of immunoglobulin, such as IgG, IgM, IgA, IgD, and IgE. IgY, the major antibody type in avian species such as chickens, is also included within the definition. As used herein, the term encompasses intact polyclonal or monoclonal antibodies, as well as fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain (ScFv), variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion having an antigen recognition site of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site of the required specificity.

[0036] As used herein, the term "antigen" refers to a target molecule that is specifically bound by an antibody through its antigen recognition site. An antigen can be monovalent or multivalent, i.e., it can have one or more epitopes recognized by one or more antibodies. Types of antigens that can be recognized by antibodies include polypeptides, oligosaccharides, glycoproteins, polynucleotides, lipids, etc.

[0037] As used herein, the term "epitope" refers to a portion of an antigen, e.g., a peptide sequence of at least about 3-5, preferably about 5-10 or 15, and up to about 1,000 amino acids (or any integer in between), that by itself or as part of a larger sequence defines a sequence that binds to an antibody generated in response to such sequence. There is no critical upper limit to the length of the fragment, which may, for example, comprise nearly the entire length of the antigen sequence, or even a fusion protein that contains two or more epitopes from the target antigen. Epitopes for use in the present invention are not limited to peptides having the exact sequence of the portion of the parent protein from which they are derived, but also include sequences identical to the native sequence, as well as modifications to the native sequence, such as deletions, additions and substitutions (essentially conservative).

[0038] As used herein, the term "specifically binds" refers to the binding specificity of a specific binding pair. Recognition by an antibody of a specific target in the presence of other potential targets is one characteristic of such binding. Specific binding involves two different molecules, one of which specifically binds to a second molecule by chemical or physical means. The two molecules are related in the sense that their binding is such that their binding partners can be distinguished from other assay components with similar characteristics. Members of a pair of binding components are referred to as ligand and receptor (antiligand), specific binding pair (SBP) members and SBP partners, etc. Molecules can also be SBP members for aggregation of molecules. For example, an antibody raised against an immune complex of a second antibody and its corresponding antigen can be considered to be an SBP member of the immune complex.

[0039] "Polynucleotide" or "nucleic acid," as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps", substitution of one or more analogs of naturally occurring nucleotides, internucleotide modifications such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. Additionally, any hydroxyl groups normally present in the sugar may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or conjugated to a solid support. The 5' and 3' terminal OH may be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxo sugars, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced with P(O)S ("thioate"), P(S)S ("dithioate"), "(O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or a substituted or unsubstituted alkyl (1-20C) optionally containing an ether (--O--) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

[0040] As used herein, "oligonucleotide" generally refers to a short, generally single-stranded, generally synthetic polynucleotide that is generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotide is equally fully applicable to oligonucleotide.

[0041] As used herein, the term "homolog" is used to refer to a nucleic acid that differs from a naturally occurring nucleic acid (e.g., a "prototype" or "wild-type" nucleic acid) by minor modifications to the naturally occurring nucleic acid, but maintains the basic nucleotide structure of the naturally occurring form. Such changes include, but are not limited to, changes of one or a few nucleotides, including deletions (e.g., truncated versions of the nucleic acid) insertions and / or substitutions. Homologs may have enhanced, decreased, or substantially similar properties compared to the naturally occurring nucleic acid. Homologs may be complementary or identical to the naturally occurring nucleic acid. Homologs can be produced using techniques known in the art for the production of nucleic acids, including, but not limited to, recombinant DNA technology, chemical synthesis, and the like.

[0042] As used herein, "substantially complementary or substantially identical" means that two nucleic acid sequences have at least 90% sequence identity. Preferably, two nucleic acid sequences have at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity. Alternatively, "substantially complementary or substantially identical" means that two nucleic acid sequences can hybridize under high stringency conditions.

[0043] Generally, the stability of hybrids is a function of ion concentration and temperature. Typically, hybridization reactions are performed under conditions of lower stringency, followed by washing of various but higher stringency. Moderately stringent hybridization refers to conditions that allow a nucleic acid molecule, such as a probe, to bind to a complementary nucleic acid molecule. Hybridized nucleic acid molecules generally have at least 60% identity, and include, for example, at least 70%, 75%, 80%, 85%, 90%, or 95% identity. Moderately stringent conditions are equivalent to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS at 42°C. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.1x SSPE and 0.1% SDS at 65°C. Low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5x Denhardt's solution, 6x SSPE, 0.2% SDS at 22°C, followed by washing in 1x SSPE, 0.2% SDS at 37°C. Denhardt's solution contains 1% Ficoll, 1% polyvinylpyrrolidone, and 1% bovine serum albumin (BSA). 20x SSPE (sodium chloride, sodium phosphate, ethylenediamidetetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025M (EDTA). Other suitable medium stringency and high stringency hybridization buffers and conditions are well known to those of skill in the art.

[0044] As used herein, "vector (or plasmid)" refers to a discrete element used to introduce heterologous DNA into cells for either its expression or replication. The selection and use of such vehicles is well within the skill of one of ordinary skill in the art. Expression vectors include vectors capable of expressing DNA operably linked with regulatory sequences, such as promoter regions, capable of effecting expression of such DNA fragments. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, that upon introduction into an appropriate host cell results in expression of the cloned DNA. Suitable expression vectors are well known to those of ordinary skill in the art and include those capable of replicating in eukaryotic and / or prokaryotic cells, those that remain episomal, or those that integrate into the host cell genome.

[0045] As used herein, "promoter region or promoter element" refers to a segment of DNA or RNA that controls transcription of the DNA or RNA to which it is operably linked. The promoter region contains specific sequences sufficient for recognition, binding, and transcription initiation of RNA polymerase. This portion of the promoter region is called the promoter. In addition, the promoter region contains sequences that regulate this recognition, binding, and transcription initiation activity of RNA polymerase. These sequences may be cis-acting or responsive to trans-acting factors. Depending on the nature of the regulation, promoters may be constitutive or regulated. Exemplary promoters contemplated for use in prokaryotes include bacteriophage T7 and T3 promoters, etc.

[0046] As used herein, "operably linked or operably associated" refers to the functional relationship of DNA to nucleotide regulatory and effector sequences, such as promoters, enhancers, transcription and translation termination sites, and other signal sequences. For example, operably linked DNA to a promoter refers to a physical and functional relationship between the DNA and the promoter such that transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA. To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' untranslated portion of the clone to eliminate extra potential inappropriate alternative translation initiation (i.e., start) codons or other sequences that may disrupt or reduce expression at the transcription or translation level. Alternatively, a consensus site can be inserted immediately 5' to the start codon, which can enhance expression. See, e.g., Kozak (1991) J. Biol. Chem. 266:19867-19870. The desirability (or need for) such modifications can be determined empirically.

[0047] "Treating" or "treatment" or "palliation" refers to a therapeutic treatment that is a slowing (relief) of the targeted pathological condition or disorder, if not curing it or preventing the recurrence of the condition. A subject is successfully "treated" if, after receiving a therapeutic amount of a therapeutic agent or treatment, the subject shows an observable and / or measurable reduction in one or more signs and symptoms of a particular disease. The reduction in the signs and symptoms of the disease may also be felt by the patient. A patient is also considered to be treated if the patient experiences stable disease. In some embodiments, treatment with a therapeutic agent is effective to cause the patient to be disease-free at 3 months after treatment, preferably 6 months after treatment, more preferably 1 year, and even more preferably 2 years or more after treatment. These parameters for evaluating successful treatment and improvement of a disease are easily measurable by routine procedures well known to those skilled in the art. In some embodiments, "treatment" refers to any method in which the symptoms, disorders, or signs of a disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein. In some embodiments, "amelioration" of symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any permanent or temporary, sustained or transient, relief that may result from or be associated with administration of the composition.

[0048] The term "prediction" or "prognosis" is often used herein to refer to the likelihood that a patient will respond favorably or unfavorably to a drug or set of drugs, or the likely outcome of a disease. In one embodiment, the prediction relates to the extent of those responses or outcomes. In one embodiment, the prediction relates to whether and / or the likelihood that a patient will survive or improve for a period of time without disease recurrence after treatment, e.g., treatment with a particular therapeutic agent. The predictive method of the present invention can be used clinically to make treatment decisions by selecting the most appropriate treatment modality for any particular patient. The predictive method of the present invention is a valuable tool in predicting whether a patient is likely to respond favorably to a treatment regimen, e.g., a given treatment regimen including administration of a given therapeutic agent or combination, surgical intervention, steroid treatment, etc.

[0049] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharma- ceutically active substances is well known in the art. See, for example, The Science and Practice of Pharmacy, 20 th ed., (Lippincott, Williams & Wilkins 2003). Except insofar as any conventional media or agent is incompatible with the active compound, use of such in the compositions is contemplated.

[0050] "Pharmaceutically acceptable salt" is intended to mean a salt of a free acid or free base of a compound represented herein that is non-toxic, biologically tolerable, or biologically suitable for administration to a subject. See generally, Berge et al., J.Pharm.Sci., 1977, 66, 1-19. Preferred pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of a subject without undue toxicity, irritation, or allergic response. The modified interleukin-2 (IL-2) polypeptides or conjugates thereof described herein have sufficiently acidic groups, sufficiently basic groups, both types of functional groups, or more than one of each type, and thus can react with several inorganic or organic bases, as well as inorganic and organic acids, to form pharmaceutically acceptable salts.

[0051] Examples of pharma- ceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, and hexyne-1,6-dioate. , benzoate, chlorobenzoate, methyl benzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propylsulfonate, besylate, xylenesulfonate, oxysulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate and mandelate.

[0052] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of a therapeutic agent that is effective when administered to a cell, tissue, or subject, alone or in combination with an additional therapeutic agent, to prevent or ameliorate a disease or disorder, a proliferative disease or disorder of the subject. A therapeutically effective dose further refers to an amount of a therapeutic agent sufficient to cause amelioration of symptoms, such as treatment, cure, prevention, or amelioration of an associated medical condition, or an increase in the rate of treatment, cure, prevention, or amelioration of such a condition. When individual active ingredients are administered alone, the therapeutically effective dose refers to that ingredient alone. When a combination is administered, the therapeutically effective dose refers to the combined amount of active ingredients that results in a therapeutic effect, regardless of whether they are administered in combination, sequentially, or simultaneously. In some embodiments, an "effective amount of a compound for treating a particular disease" is an amount sufficient to ameliorate or in some way reduce symptoms associated with the disease. Such an amount may be administered as a single dose or according to a regimen, whereby it is effective. The amount may cure the disease, but is typically administered to ameliorate the symptoms of the disease. Repeated administration may be necessary to achieve the desired amelioration of symptoms.

[0053] The term "combination" refers to either a fixed combination in one dosage unit form, or a kit-of-parts for co-administration, in which the modified interleukin-2 (IL-2) polypeptide or conjugate thereof and a combination partner (e.g., another drug, also referred to as a "therapeutic agent" or "adjunct") can be administered separately, either simultaneously or within time intervals, particularly those time intervals that allow the combination partners to exhibit a cooperative, e.g., synergistic, effect. Terms such as "co-administration" or "co-administration" as used herein are meant to encompass administration of the selected combination partners to a single subject (e.g., patient) in need thereof, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term "pharmaceutical combination" as used herein refers to a product resulting from the mixing or combination of more than one active ingredient, and includes both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that both active ingredients, e.g., the modified interleukin-2 (IL-2) polypeptide or conjugate thereof and the combination partner, are administered to the patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that both active ingredients, e.g., modified interleukin-2 (IL-2) polypeptide or conjugate thereof and combination partner, are administered to a patient as separate entities simultaneously, simultaneously or sequentially without specific time limitations, such administration providing therapeutically effective levels of the two substances in the patient's body. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0054] As used herein, "biological sample" refers to any sample obtained from a living or viral source or other source of macromolecules and biological molecules, including any cell type or tissue of a subject from which nucleic acids or proteins or other macromolecules can be obtained. A biological sample can be a sample obtained directly from a living source or a processed sample. For example, amplified isolated nucleic acids constitute a biological sample. Biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples from animals and plants, and processed samples derived therefrom.

[0055] The term "level" or "level" is used to refer to the presence and / or amount of a target, e.g., a substance or organism that is part of the pathogenesis of a disease or disorder, and can be determined qualitatively or quantitatively. A "qualitative" change in target level refers to the appearance or disappearance of a target that is undetectable or present in a sample obtained from a normal control. A "quantitative" change in the level of one or more targets refers to a measurable increase or decrease in the target level when compared to a healthy control.

[0056] A "healthy control" or "normal control" is a biological sample taken from an individual not afflicted with a disease or disorder, such as a proliferative disease or disorder. A "negative control" is a sample that does not contain any of the particular analyte that the assay is designed to detect, thus providing a reference baseline for the assay.

[0057] As used herein, "mammal" refers to any species of the mammalian class. Often, as used herein, the term "mammal" refers to a human, a human subject, or a human patient. "Mammal" also refers to any species of the non-human mammalian class, e.g., experimental, companion, or economic non-human mammals. Exemplary non-human mammals include mice, rats, rabbits, cats, dogs, pigs, cows, sheep, goats, horses, monkeys, gorillas, and chimpanzees.

[0058] As used herein, "production by recombinant means" refers to a production method that uses recombinant nucleic acid methods that rely on well-known methods of molecular biology to express a polypeptide or protein encoded by a cloned nucleic acid.

[0059] As used herein, the term "subject" is not limited to a particular species or sample type. For example, the term "subject" may refer to a patient, often a human patient. However, the term is not limited to humans, and thus encompasses a variety of non-human animal or mammalian species.

[0060] As used herein, a "prodrug" is a substance that is metabolized or converted into the biologically, pharma- ceutical or therapeutically active form of the substance upon in vivo administration. To produce a prodrug, a pharma- ceutical active substance is modified so that the active substance is regenerated by metabolic processes. Prodrugs can be designed to alter the metabolic stability or transport properties of a drug, mask side effects or toxicity, improve the taste of a drug, or change other properties or characteristics of a drug. Knowledge of in vivo pharmacodynamic processes and drug metabolism allows one skilled in the art to design a prodrug of a compound once a pharma- ceutical active compound is known (see, for example, Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392).

[0061] It is understood that aspects and embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" aspects and embodiments.

[0062] Throughout this disclosure, various aspects of the invention are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0063] Other objects, advantages and features of the present invention will become apparent from the following specification taken in conjunction with the accompanying drawings.

[0064] C. Modified Interleukin 2 (IL-2) Polypeptides and Polynucleotides Encoding and Expressing Same In one aspect, the invention provides a modified IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 and substitutions of natural or unnatural amino acids at positions Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93 or a combination thereof, wherein the modified IL-2 polypeptide comprises: a) a water soluble polymer, lipid or polypeptide, e.g. and b) has reduced binding to interleukin 2 receptor alpha (IL-2Rα) compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions, and / or c) has reduced receptor signaling potency towards IL-2Rαβγ compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions, provided that the modified IL-2 polypeptide does not comprise a substitution with a non-natural amino acid. wherein the modified IL-2 polypeptide comprises a substitution at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof, and a substitution with a natural or non-natural amino acid at a position within the IL-2Rα, IL-2Rβ, and / or IL-2Rγ interacting region, and with the proviso that the modified IL-2 polypeptide comprises The present invention relates to a modified interleukin-2 (IL-2) polypeptide, which has at least about 80% sequence identity in the regions of amino acid residues 10 to 25, 80 to 100, and / or 100 to 134 with the corresponding regions of a comparable IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without substitutions, and which has at least about 50% sequence identity with a comparable IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without substitutions.

[0065] The amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 is shown below. [ka]

[0066] In one embodiment, the modified IL-2 polypeptide has at least about 80% sequence identity, e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, in the region of amino acid residues 10-25, to the corresponding region of a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions.

[0067] In another embodiment, the modified IL-2 polypeptide has at least about 80% sequence identity, e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, in the region of amino acid residues 80-100, to the corresponding region of a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitutions.

[0068] In yet another embodiment, the modified IL-2 polypeptide has at least about 80% sequence identity, e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, in the region of amino acid residues 100-134 to the corresponding region of a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitutions.

[0069] In yet another embodiment, the modified IL-2 polypeptide has at least about 80% sequence identity to the corresponding region of a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitutions, e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity in the regions of amino acid residues 10-25 and 80-100.

[0070] In yet another embodiment, the modified IL-2 polypeptide has at least about 80% sequence identity to the corresponding region of a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitutions, e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity in the regions of amino acid residues 10-25 and 100-134.

[0071] In yet another embodiment, the modified IL-2 polypeptide has at least about 80% sequence identity, e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, in the regions of amino acid residues 80-100 and 100-134 to the corresponding regions of a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitutions.

[0072] In yet another embodiment, the modified IL-2 polypeptide has at least about 80% sequence identity to the corresponding region of a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitutions, e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity in the regions of amino acid residues 10-25, 80-100 and 100-134.

[0073] In one embodiment, the modified IL-2 polypeptide has at least about 50% sequence identity, e.g., at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, to a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions.

[0074] The modified IL-2 polypeptides can include any suitable substitution with a natural amino acid. For example, the modified IL-2 polypeptides can include a substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at positions Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, or a combination thereof.

[0075] In one embodiment, the modified IL-2 polypeptide comprises: a) a substitution with a natural amino acid at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and a combination thereof; and and / or b) comprises substitutions with natural amino acids at positions selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof, and is configured to be conjugated to a water soluble polymer, lipid, protein or peptide at the N-terminus and / or C-terminus of the polypeptide.

[0076] In another embodiment, the modified IL-2 polypeptide comprises: a) a lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalan ... and / or b) comprising a substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine or tyrosine at positions selected from the group consisting of N29, N30, Y31, N33, P34, K35, R38, T41, K43, K48, K49, K64, P65, N71, Q74, K76 and combinations thereof.

[0077] In yet another embodiment, the modified IL-2 polypeptide comprises a) a substitution with a cysteine ​​at a position selected from the group consisting of N29, N30, Y31, N33, P34, K35, R38, T41, K43, K48, K49, K64, P65, N71, Q74, K76, and combinations thereof; b) a substitution with a cysteine ​​at a position selected from the group consisting of N29, Y31, K35, P65, N71, Q74, and combinations thereof; c) a substitution with a cysteine ​​at position Y31; and / or d) a substitution with a cysteine ​​at position P65.

[0078] In yet another embodiment, the modified IL-2 polypeptide comprises a substitution at position Y31 with any amino acid. For example, the modified IL-2 polypeptide may comprise a substitution at position Y31 with serine or alanine.

[0079] The modified IL-2 polypeptides can further comprise substitutions of natural or non-natural amino acids at positions within the IL-2Rα, IL-2Rβ and / or IL-2Rγ interacting regions.

[0080] The modified IL-2 polypeptide can further comprise a substitution with a natural amino acid at a position within the IL-2Rα interacting region. The modified IL-2 polypeptide can further comprise a substitution with a natural amino acid at any suitable position within the IL-2Rα interacting region. For example, the modified IL-2 polypeptide can further comprise a substitution with a natural amino acid at a position selected from the group consisting of R38, F42, Y45, E62, P65, and combinations thereof.

[0081] The modified IL-2 polypeptides can include any suitable substitution with a natural amino acid at a position within the IL-2Rα interacting region. For example, the modified IL-2 polypeptides can include substitutions with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at a position selected from the group consisting of R38, F42, Y45, E62, P65, and combinations thereof.

[0082] In one embodiment, the modified IL-2 polypeptide comprises: a) a substitution with cysteine ​​at a position selected from the group consisting of R38, F42, Y45, E62, P65, and combinations thereof; b) a substitution with alanine, lysine, or serine at position F42; c) a substitution with alanine at position F42; d) a substitution with serine at position F42; e) a substitution with lysine at position F42; f) a substitution with alanine, histidine, or serine at position Y45; and g) a substitution with alanine, histidine, or serine at position Y45. , alanine; h) a substitution at position Y45 with histidine; i) a substitution at position R38 with alanine, aspartic acid or serine; j) a substitution at position R38 with aspartic acid; k) a substitution at position P65 with alanine; l) a substitution at position P65 with serine; m) a substitution at position E62 with alanine; and / or n) a substitution at position F42 with lysine, at position Y31 with cysteine, or a combination thereof.

[0083] The modified IL-2 polypeptide can further comprise a substitution with a natural amino acid at a position within the IL-2Rβ interacting region. The modified IL-2 polypeptide can further comprise a substitution with a natural amino acid at any suitable position within the IL-2Rβ interacting region. For example, the modified IL-2 polypeptide can further comprise a substitution with a natural amino acid at a position selected from the group consisting of Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof.

[0084] The modified IL-2 polypeptides can include any suitable substitution with a natural amino acid at a position within the IL-2Rβ interacting region. For example, the modified IL-2 polypeptides can include a substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at a position selected from the group consisting of Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof. In one embodiment, the modified IL-2 polypeptides can include a substitution with cysteine ​​at a position selected from the group consisting of Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof.

[0085] In one embodiment, the modified IL-2 polypeptide can further comprise a) substitutions with natural amino acids at positions within the IL-2Rα interacting region and substitutions with natural amino acids at positions within the IL-2Rβ interacting region; b) substitutions with natural amino acids at positions within the IL-2Rα interacting region and substitutions with natural amino acids at positions within the IL-2Rγ interacting region; or c) substitutions with natural amino acids at positions within the IL-2Rα interacting region, substitutions with natural amino acids at positions within the IL-2Rβ interacting region and substitutions with natural amino acids at positions within the IL-2Rγ interacting region.

[0086] The modified IL-2 polypeptide can include any suitable substitution with a non-natural amino acid. For example, the non-natural amino acids disclosed in WO2019 / 028425A1 and WO2019 / 028419A1 can be used. In one embodiment, the non-natural amino acid can be a lysine analog, a cysteine ​​analog, or a histidine analog, and includes an aromatic side chain, includes an azide group, includes an alkyne group, or includes an aldehyde group or a ketone group. In another embodiment, the non-natural amino acid does not include an aromatic side chain. In yet another embodiment, the unnatural amino acid is N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxonoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl Examples of suitable tyrosine include p-allyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic acid, or selenocysteine.

[0087] Unnatural amino acids can be incorporated into modified IL-2 polypeptides by any suitable means or method. For example, unnatural amino acids can be incorporated into modified IL-2 polypeptides by an orthogonal tRNA synthetase / tRNA pair. Any suitable orthogonal tRNA can be used. For example, the orthogonal tRNA of the orthogonal synthetase / tRNA pair can include at least one unnatural nucleobase.

[0088] The modified IL-2 polypeptide may have reduced or no detectable binding to IL-2Rα compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitutions. In one embodiment, the binding affinity of the modified IL-2 polypeptide to IL-2Rα may be reduced by about 10% to about 100%, such as by about 10%, 20%, 30%, 40%, 50%, 60%, 17%, 80%, 90%, 100%, or subranges thereof. In another embodiment, the binding affinity of the modified IL-2 polypeptide to IL-2Rα may be reduced by about 10% to about 100%, or by about 1-fold to about 100,000-fold or more, such as by about 1-fold, 10-fold, 100-fold, 1,000-fold, 10,000-fold, 100,000-fold or more, or subranges thereof. In yet another embodiment, the modified IL-2 polypeptide has no detectable binding to IL-2Rα.

[0089] The modified IL-2 polypeptide has reduced or no detectable receptor signaling potency for IL-2Rαβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions. In one embodiment, the ratio of the signaling potency for IL-2Rαβγ of the modified IL-2 polypeptide to the signaling potency for IL-2Rαβγ of a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions can be about 1 / 2 to about 1 / 100,000, e.g., about 1 / 2, 1 / 5, 1 / 10, 1 / 100, 1 / 1,000, 1 / 10,000, 1 / 100,000 or more, or any subrange thereof. In another embodiment, the modified IL-2 polypeptide has no detectable receptor signaling potency for IL-2Rαβγ.

[0090] In one embodiment, the modified IL-2 polypeptide has reduced binding to IL-2Rα compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions, and reduced receptor signaling potency for IL-2Rαβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions. In another embodiment, the modified IL-2 polypeptide has no detectable binding to IL-2Rα and no detectable receptor signaling potency for IL-2Rαβγ.

[0091] The modified IL-2 polypeptides may substantially retain or have a higher level of binding to interleukin 2 receptor beta (IL-2Rβ) or interleukin 2 receptor gamma (IL-2Rγ) compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions, and / or may substantially retain or have a higher receptor signaling potency for IL-2Rβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions. In one embodiment, the modified IL-2 polypeptides retain substantial binding levels or have a higher level of binding to IL-2Rβ or IL-2Rγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions. In another embodiment, the modified IL-2 polypeptides retain substantial receptor signaling potency or have a higher receptor signaling potency for IL-2Rβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions. In yet another embodiment, the modified IL-2 polypeptide retains substantial or has higher binding levels to IL-2Rβ or IL-2Rγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions, and retains substantial or has higher receptor signaling potency for IL-2Rβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without substitutions.

[0092] The modified IL-2 polypeptide can comprise a deletion at any suitable position. In one embodiment, the modified IL-2 polypeptide has an N-terminal deletion, such as an N-terminal deletion of amino acid residues 1-30, or a subrange thereof. In another embodiment, the modified IL-2 polypeptide has a C-terminal deletion, such as a C-terminal deletion of amino acid residues 114-134, or a subrange thereof. In yet another embodiment, the modified IL-2 polypeptide has an N-terminal deletion and a C-terminal deletion.

[0093] The modified IL-2 polypeptide may be part of a fusion polypeptide, e.g., a recombinant fusion protein, comprising the modified IL-2 polypeptide and an additional amino acid sequence. The modified IL-2 polypeptide may be fused to the additional amino acid sequence in any suitable manner. For example, the N-terminus or C-terminus of the modified IL-2 polypeptide may be fused to the additional amino acid sequence. The additional amino acid sequence may comprise any suitable sequence or content. For example, the additional amino acid sequence may comprise an antibody sequence or a portion or fragment thereof. In another example, the additional amino acid sequence may comprise the Fc portion of an antibody.

[0094] The modified IL-2 polypeptide may be in any suitable form, for example, the modified IL-2 polypeptide may be in isolated or purified form.

[0095] The modified IL-2 polypeptides can be prepared using any suitable technique or process. For example, the modified IL-2 polypeptides of the present invention can be prepared by recombinant production, chemical synthesis, or a combination thereof.

[0096] In another aspect, the present invention is directed to a polynucleotide, e.g., a DNA, RNA or viral vector, encoding a modified IL-2 polypeptide as described above and configured to express said modified IL-2 polypeptide in vitro and / or in vivo.

[0097] The modified IL-2 polypeptide may be administered in any suitable form. For example, the modified IL-2 polypeptide may be administered in the form of a protein, a fusion protein, a protein conjugate, with or without conjugation, or as part of a nanoparticle. In some embodiments, a polynucleotide, e.g., a DNA, RNA, or viral vector, encoding a modified IL-2 polypeptide and configured to express said modified IL-2 polypeptide in vitro and / or in vivo, may be administered to a cell, tissue, organ, or subject, e.g., a human subject.

[0098] D. Modified Interleukin 2 (IL-2) Polypeptide Conjugates In another aspect, the present invention is directed to a modified IL-2 polypeptide conjugate comprising a modified IL-2 polypeptide as described above conjugated to another moiety, such as a water soluble polymer, a lipid, a polypeptide, such as a protein or a peptide.

[0099] The modified IL-2 polypeptide may be conjugated to another moiety, such as a water soluble polymer, lipid, protein, or peptide, in any suitable manner. For example, the modified IL-2 polypeptide may be covalently conjugated to a water soluble polymer, lipid, protein, or peptide. In another example, the modified IL-2 polypeptide may be non-covalently conjugated to a water soluble polymer, lipid, protein, or peptide. In yet another example, the modified IL-2 polypeptide may be conjugated to a water soluble polymer, lipid, protein, or peptide via a substituted natural or unnatural amino acid at any suitable position.

[0100] In one embodiment, the modified IL-2 polypeptide is conjugated to another moiety, e.g., a water soluble polymer, lipid, protein, or peptide, via a substituted natural or unnatural amino acid at a position selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof. In another embodiment, the modified IL-2 polypeptide is conjugated to another moiety, e.g., a water soluble polymer, lipid, protein, or peptide, via a substituted native amino acid at a position selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof. In yet another embodiment, the modified IL-2 polypeptide is conjugated to another moiety, such as a water soluble polymer, lipid, protein, or peptide, via a substituted lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at a position selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof. In yet another embodiment, the modified IL-2 polypeptide is conjugated to another moiety, e.g., a water soluble polymer, lipid, protein, or peptide, via a substituted cysteine ​​at a position selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof.

[0101] The modified IL-2 polypeptide may be conjugated to another moiety, e.g., a water soluble polymer, lipid, protein or peptide, via a substituted natural or unnatural amino acid at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76 and combinations thereof. In one embodiment, the modified IL-2 polypeptide is conjugated to another moiety, e.g., a water soluble polymer, lipid, protein or peptide, via a substituted native amino acid at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76 and combinations thereof. In another embodiment, the modified IL-2 polypeptide is conjugated to another moiety, e.g., a water soluble polymer, lipid, protein, or peptide, via a substituted lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof. In yet another embodiment, the modified IL-2 polypeptide is conjugated to another moiety, e.g., a water soluble polymer, lipid, protein, or peptide, via a substituted cysteine ​​at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof.

[0102] The modified IL-2 polypeptide may be conjugated to another moiety, such as a water soluble polymer, lipid, protein or peptide, through a single amino acid residue or multiple amino acid residues of the modified IL-2 polypeptide. In one embodiment, the modified IL-2 polypeptide may be conjugated to another moiety, such as a water soluble polymer, lipid, protein or peptide, through i) the alpha amino group of the N-terminal amino acid residue of the modified IL-2 polypeptide, ii) the epsilon amino group of a lysine amino acid residue of the modified IL-2 polypeptide, or iii) an N-glycosylation site or an O-glycosylation site of the modified IL-2 polypeptide.

[0103] The modified IL-2 polypeptide may be covalently conjugated to another moiety, such as a water-soluble polymer, lipid, protein, or peptide, via a linker. The modified IL-2 polypeptide may also be covalently conjugated directly to another moiety, such as a water-soluble polymer, lipid, protein, or peptide, without a linker.

[0104] The modified IL-2 polypeptide may be conjugated to another moiety, such as a water-soluble polymer, lipid, protein, or peptide, via a single amino acid residue in a fusion polypeptide comprising the modified IL-2 polypeptide and an additional amino acid sequence. The single amino acid residue may be located at any suitable position. For example, the single amino acid residue may be located within the modified IL-2 polypeptide. In another example, the single amino acid residue may be located within the additional amino acid sequence.

[0105] The additional amino acid sequence in the modified IL-2 polypeptide conjugate may comprise any suitable sequence or content. For example, the additional amino acid sequence in the modified IL-2 polypeptide conjugate may comprise an antibody sequence or a portion or fragment thereof. In another example, the additional amino acid sequence in the modified IL-2 polypeptide conjugate may comprise the Fc portion of an antibody.

[0106] The modified IL-2 polypeptide may be conjugated to another moiety, such as a water-soluble polymer, lipid, protein or peptide in a fusion polypeptide in any suitable manner. For example, the modified IL-2 polypeptide may be conjugated to another moiety, such as a water-soluble polymer, lipid, protein or peptide, via i) the alpha amino group of the N-terminal amino acid residue of the fusion polypeptide, ii) the epsilon amino group of a lysine amino acid residue of the fusion polypeptide, or iii) an N-glycosylation site or an O-glycosylation site of the fusion polypeptide. In another example, the fusion polypeptide may be covalently conjugated to a water-soluble polymer, lipid, protein or peptide, either directly or via a linker.

[0107] The modified IL-2 polypeptides of the present invention can be conjugated to any suitable water-soluble polymer. For example, the water-soluble polymer can include polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamides), poly(hydroxyalkyl methacrylates), poly(saccharides), poly(a-hydroxy acids), poly(vinyl alcohols), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholines), or combinations thereof. See, for example, WO 2019 / 028425 A1 and WO 2019 / 028419 A1.

[0108] In the modified IL-2 polypeptide conjugates of the present invention, the water-soluble polymer can comprise a PEG molecule. The PEG molecule can be a linear PEG or a branched PEG. The branched PEG can have any suitable configuration and / or any suitable number of PEG chains. For example, the branched PEG can have about 3 to about 10 PEG chains emanating from a central core group. In another example, the branched PEG can be a star PEG comprising about 10 to about 100 PEG chains emanating from a central core group. In yet another example, the branched PEG can be a comb PEG comprising multiple PEG chains grafted to a polymer backbone.

[0109] The PEG molecule in the modified IL-2 polypeptide conjugate can have any suitable molecular weight. For example, the PEG molecule can have a molecular weight range of about 300 g / mol to about 10,000,000 g / mol, such as about 300 g / mol, 500 g / mol, 1,000 g / mol, 10,000 g / mol, 100,000 g / mol, 1,000,000 g / mol, 10,000,000 g / mol or any subrange thereof. In another example, the PEG molecule can have an average molecular weight of about 5,000 daltons to about 1,000,000 daltons, such as about 5,000 daltons, 10,000 daltons, 100,000 daltons, 1,000,000 daltons or any subrange thereof. In yet another example, the PEG molecules can have an average molecular weight of about 20,000 daltons to about 30,000 daltons, e.g., about 20,000 daltons, 21,000 daltons, 22,000 daltons, 23,000 daltons, 24,000 daltons, 25,000 daltons, 26,000 daltons, 27,000 daltons, 28,000 daltons, 29,000 daltons, 30,000 daltons, or any subrange thereof.

[0110] The PEG molecules in the modified IL-2 polypeptide conjugates can be in any suitable form, for example, the PEG molecules can be monodisperse, uniform, or individual PEG molecules.

[0111] The water soluble polymer in the modified IL-2 polypeptide conjugate can comprise a polysaccharide.

[0112] The modified IL-2 polypeptide in the modified IL-2 polypeptide conjugate can be conjugated to any suitable lipid. For example, the lipid in the modified IL-2 polypeptide conjugate can comprise a fatty acid.

[0113] The modified IL-2 polypeptide in the modified IL-2 polypeptide conjugate may be conjugated to any suitable protein. For example, the protein in the modified IL-2 polypeptide conjugate may comprise an antibody or a binding fragment thereof. The antibody or a binding fragment thereof may comprise the Fc portion of an antibody.

[0114] In the modified IL-2 polypeptide conjugates of the invention, the other moiety, e.g., a water-soluble polymer, lipid, protein, or peptide, can be attached to the modified IL-2 polypeptide using any suitable method. For example, the other moiety, e.g., a water-soluble polymer, lipid, protein, or peptide, can be indirectly attached to the substituted natural or unnatural amino acid of the modified IL-2 polypeptide via a linker. In another example, the other moiety, e.g., a water-soluble polymer, lipid, protein, or peptide, can be directly attached to the substituted natural or unnatural amino acid of the modified IL-2 polypeptide.

[0115] The modified IL-2 polypeptide conjugates of the invention can have any suitable in vivo half-life, for example, the modified IL-2 polypeptide conjugates can have an in vivo half-life of about 5 minutes to about 10 days, e.g., about 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or any subrange thereof.

[0116] E. Pharmaceutical Compositions In another aspect, the present invention is directed to a pharmaceutical composition comprising an effective amount of a modified IL-2 polypeptide, a polynucleotide, e.g., DNA, RNA or viral vector, or a modified IL-2 polypeptide conjugate as described above, and a pharma- ceutically acceptable carrier or excipient.

[0117] The pharmaceutical composition can be adapted to treat or prevent any suitable disease, disorder or condition. For example, the pharmaceutical composition can be adapted to treat or prevent a proliferation disorder in a subject.

[0118] In one embodiment, the pharmaceutical composition is adapted to treat or prevent a solid tumor or cancer in a subject, such as chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma of bone / osteosarcoma, osteosarcoma, rhabdomyosarcoma, cardiac cancer, astrocytoma, brain stem glioma, pilocytic astrocytoma, ependymoma, primitive neuroectodermal tumor, cerebellar astrocytoma, cerebral astrocytoma, glioma, medulloblastoma, neuroblastoma, oligodendroglioma, pineal astrocytoma, pituitary adenoma, visual pathway and hypothalamic glioma, breast cancer, Invasive lobular carcinoma, tubular carcinoma, invasive breast cancer, medullary carcinoma, male breast cancer, phyllodes tumor, inflammatory breast cancer, adrenal cortical carcinoma, islet cell carcinoma (endocrine pancreas), multiple endocrine neoplasia syndrome, parathyroid carcinoma, pheochromocytoma, thyroid carcinoma, Merkel cell carcinoma, uveal melanoma, retinoblastoma, anal cancer, appendix cancer, bile duct cancer, carcinoid tumor, gastrointestinal, colon cancer, extrahepatic bile duct cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor tumor, gastrointestinal stromal tumor (GIST), hepatocellular carcinoma, pancreatic islet cell cancer, rectal cancer, bladder cancer, cervical cancer, endometrial cancer, extragonadal germ cell tumor, ovarian cancer, ovarian epithelial cancer (surface epithelial stromal tumor), ovarian germ cell tumor, penile cancer, renal cell carcinoma, renal pelvis and ureter, transitional cell carcinoma, prostate cancer, testicular cancer, gestational trophoblastic tumor, ureter and renal pelvis, transitional cell carcinoma, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms' tumor, edema The cancer may be of the following types: esophageal cancer, head and neck cancer, nasopharyngeal cancer, oral cancer, oropharynx cancer, paranasal sinus and nasal cavity cancer, pharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, basal cell carcinoma, melanoma, skin cancer (non-melanoma), bronchial adenoma / carcinoma, small cell lung cancer, mesothelioma, non-small cell lung cancer, pleuropulmonary blastoma, laryngeal cancer, thymoma and thymic carcinoma, AIDS-related cancer, Kaposi's sarcoma, epithelioid hemangioendothelioma (EHE), desmoplastic small round cell tumor, or liposarcoma.

[0119] In another embodiment, the pharmaceutical composition is configured to treat or prevent a hematological malignancy in a subject, including myeloid neoplasms, leukemia, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, hepatosplenic T-cell lymphoma, B-cell lymphoma reticuloendotheliosis, reticulosis, microglioma, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, primary mediastinal B-cell lymphoma, Waldenstrom's macroglobulinemia, nodal follicular zone B-cell lymphoma, splenic marginal zone lymphoma, lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, nodular lymphocyte-predominant Hodgkin lymphoma, plasma cell leukemia, acute dysentery and erythroleukemia, acute erythroblastic myelopathy, acute erythroblastic leukemia, Heilmeyer-Schoener disease, acute megakaryoblastic leukemia, mast cell leukemia, panmyelosis, acute panmyelosis with myelofibrosis, lymphosarcoma cell leukemia, acute leukemia of unspecified cell type, blast phase chronic myeloid leukemia, stem cell leukemia, chronic leukemia of unspecified cell type, subacute leukemia of unspecified cell type, adenocarcinoma Rapid-phase chronic myeloid leukemia, acute myeloid leukemia, polycythemia vera, acute promyelocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, adult T-cell leukemia / lymphoma, aggressive NK cell leukemia, B-cell prolymphocytic leukemia, B-cell chronic lymphocytic leukemia, B-cell leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, chronic neutrophilic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, chronic idiopathic myelofibrosis, multiple myelofibrosis The disease may be myeloma, Köhler's disease, myelomatosis, solitary myeloma, plasma cell leukemia, plasmacytoma, extramedullary, malignant plasma cell neoplasm NOS, plasmacytoma NOS, monoclonal gammopathy, multiple myeloma, angiocentric immunoproliferative lesion, lymphocytic granulomatosis, angioimmunoblastic lymphadenopathy, T-gamma lymphoproliferative disorder, Waldenstrom's macroglobulinemia, alpha heavy chain disorder, gamma heavy chain disorder, Franklin's disease, immunoproliferative small enteropathy, Mediterranean disease, malignant immunoproliferative disorder, unspecified, or immunoproliferative disorder NOS.

[0120] In yet another embodiment, the pharmaceutical composition is configured to treat or prevent an immunodeficiency disease or disorder in a subject, the immunodeficiency disease or disorder being selected from the group consisting of agammaglobulinemia: X-linked and autosomal recessive, ataxia telangiectasia, chronic granulomatous disease and other phagocytic cell disorders, common variable immunodeficiency, complement deficiencies, DiGeorge syndrome, hemophagocytic lymphohistiocytosis (HLH), hyper-IgE syndrome, hyper-IgM syndrome, IgG subclass deficiencies, innate immunity deficiencies, NEMO deficiency syndrome, selective IgA deficiency, selective IgM deficiency, severe combined immunodeficiency, deficiency and combined immunodeficiency, specific antibody deficiencies, infantile immunodeficiencies, and combination immunodeficiencies. The cause may be transient hypogammaglobulinemia of early stage, WHIM syndrome (warts, hypogammaglobulinemia, infection, and bone marrow cathexis), Wiskott-Aldrich syndrome, other antibody deficiencies, other primary cellular immunodeficiencies, severe combined immunodeficiency (SCID), common variable immunodeficiency (CVID), human immunodeficiency virus / acquired immune deficiency syndrome (HIV / AIDS), drug-induced immunodeficiency, graft-versus-host syndrome, primary immunodeficiency disorder (PIDD), or lymphopenia.

[0121] The pharmaceutical composition may further comprise another active ingredient. The other active ingredient may be any suitable active ingredient for treating or preventing any suitable disease, disorder or condition. For example, the other active ingredient may be an anti-tumor agent.

[0122] The additional active ingredients may be formulated in a separate pharmaceutical composition from at least one exemplary modified IL-2 polypeptide or modified IL-2 polypeptide conjugate of the present disclosure, or may be included in a single pharmaceutical composition together with at least one exemplary modified IL-2 polypeptide or modified IL-2 polypeptide conjugate of the present disclosure.

[0123] The pharmaceutical compositions of the present invention may be formulated to be administered orally, parenterally, by inhalation, topically, rectally, nasally, buccally, vaginally, via an implanted reservoir, or by other methods of drug administration. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0124] Sterile injectable compositions, such as sterile injectable aqueous or oily suspensions, can be formulated according to techniques known in the art using suitable dispersants or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents. Acceptable vehicles and solvents that can be used include mannitol, water, Ringer's solution and isotonic sodium chloride solution. Suitable carriers and other pharmaceutical composition ingredients are typically sterile.

[0125] In addition, sterile fixed oils are conventionally used as a solvent or suspending medium (e.g., synthetic mono- or diglycerides). Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are pharma-ceutically acceptable oils such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, or carboxymethylcellulose or similar dispersants. Various emulsifiers or bioavailability enhancers commonly used in the manufacture of pharma-ceutically acceptable solid, liquid, or other dosage forms can also be used for formulation purposes.

[0126] Compositions for oral administration may be any orally acceptable dosage form, including, but not limited to, tablets, capsules, emulsions, and aqueous suspensions, dispersions, and solutions. For tablets for oral administration, commonly used carriers include lactose and cornstarch. Lubricants such as magnesium stearate may also be added. For oral administration in capsule form, useful diluents include lactose and dry cornstarch. For oral administration of aqueous suspensions or emulsions, the active ingredient may be suspended or dissolved in the oil phase in combination with emulsifying or suspending agents. If necessary, certain sweetening, flavoring, or coloring agents may be added. Nasal aerosol or inhalation compositions may be prepared according to techniques well known in the art of pharmaceutical formulation, and may be prepared, for example, as a solution in saline, using suitable preservatives (e.g., benzyl alcohol), absorption enhancers to enhance bioavailability, and / or other solubilizing or dispersing agents known in the art.

[0127] Any suitable formulation of the compounds described herein can be prepared. See generally Remington's Pharmaceutical Sciences, (2000) Hoover, JE editor, 20th edition, Lippincott Williams and Wilkins Publishing Company, Easton, Pa., pages 780-857. Formulations are selected to suit the appropriate route of administration. If the compound is sufficiently basic or acidic to form a stable non-toxic acid or base salt, it may be appropriate to administer the compound as a salt. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form physiologically acceptable anions, such as tosylates, methanesulfonates, acetates, citrates, malonates, tartrates, succinates, benzoates, ascorbates, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts can also be formed, including hydrochlorides, sulfates, nitrates, bicarbonates, and carbonates. Pharmaceutically acceptable salts are obtained using standard procedures well known in the art, for example, by incorporating sufficiently basic compounds, such as amines, with a suitable acid to provide a physiologically acceptable anion. Alkali metal (e.g., sodium, potassium or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids are also prepared.

[0128] When the contemplated compound or substance is administered in a pharmacological composition, it is expected that the compound or substance can be formulated in admixture with a pharma- ceutically acceptable excipient and / or carrier. For example, the contemplated compound or substance can be administered intravenously as a neutral compound or substance, or as a pharma- cetically acceptable salt, or in saline. Conventional buffers such as phosphate, bicarbonate, or citrate can be used for this purpose. Of course, one skilled in the art can modify the formulation within the teachings of this specification to provide multiple formulations for a particular route of administration. In particular, the contemplated compounds or substances can be modified to make them more soluble in water or other vehicles, which can be easily achieved, for example, with minor modifications (salt formulations, esterification, etc.) that are well within the scope of one skilled in the art. It is also well within the scope of one skilled in the art to modify the administration route and dosing regimen of a particular compound or substance to manage the pharmacokinetics of the compound or substance of the present invention, for example, the modified IL-2 polypeptide or modified IL-2 polypeptide conjugate of the present invention, for maximum beneficial effect in the patient.

[0129] The modified IL-2 polypeptide or modified IL-2 polypeptide conjugate may be soluble in organic solvents such as chloroform, dichloromethane, ethyl acetate, ethanol, methanol, isopropanol, acetonitrile, glycerol, N,N-dimethylformamide, N,N-dimethylacetoamide, dimethylsulfoxide, and the like. In one embodiment, the invention provides a formulation prepared by mixing the modified IL-2 polypeptide or modified IL-2 polypeptide conjugate of the invention with a pharma- ceutically acceptable carrier. In one aspect, the formulation may be prepared using a method comprising: a) dissolving the described compound or substance in a water-soluble organic solvent, a non-ionic solvent, a water-soluble lipid, a cyclodextrin, a vitamin such as tocopherol, a fatty acid, a fatty acid ester, a phospholipid, or a combination thereof to provide a solution; and b) adding saline or a buffer containing 1-10% carbohydrate solution. In one example, the carbohydrate comprises dextrose. The pharmaceutical compositions obtained using the method are stable and useful for veterinary and clinical applications.

[0130] Illustrative examples of water-soluble organic solvents for use in the pharmaceutical compositions of the present invention include, but are not limited to, polyethylene glycol (PEG), alcohol, acetonitrile, N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide, or combinations thereof. Examples of alcohols include, but are not limited to, methanol, ethanol, isopropanol, glycerol, or propylene glycol.

[0131] Illustrative examples of water-soluble non-ionic surfactants for use in the pharmaceutical compositions of the present invention include, but are not limited to, CREMOPHOR®. EL, polyethylene glycol modified CREMOPHOR® (polyoxyethylene glycerol triricinol 35), hydrogenated CREMOPHOR®. RH40, hydrogenated CREMOPHOR®. RH60, PEG-succinate, polysorbate 20, polysorbate 80, SOLUTOL®. HS (polyethylene glycol 660 12-hydroxystearate), sorbitan monooleate, poloxamer, LABRAFIL® (ethoxylated persic oil), LABRASOL® (capryl-caproyl macrogol-8-glyceride), GELUCIRE® (glycerol esters), SOFTIGEN® (PEG 6 caprylic acid glyceride), glycerin, glycol-polysorbate, or combinations thereof.

[0132] Illustrative examples of water-soluble lipids for use in the pharmaceutical compositions of the present invention include, but are not limited to, vegetable oils, triglycerides, plant oils, or combinations thereof.Examples of lipid oils include, but are not limited to, castor oil, polyoxyl castor oil, corn oil, olive oil, cottonseed oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oil, hydrogenated soybean oil, coconut triglyceride, palm seed oil, and hydrogenated forms thereof, or combinations thereof.

[0133] Illustrative examples of fatty acids and fatty acid esters for use in the pharmaceutical compositions of the present invention include, but are not limited to, oleic acid, monoglycerides, diglycerides, mono- or di-fatty acid esters of PEG, or combinations thereof.

[0134] Illustrative examples of cyclodextrins for use in the pharmaceutical compositions of the invention include, but are not limited to, alpha-cyclodextrin, beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, or sulfobutylether-beta-cyclodextrin.

[0135] Illustrative examples of phospholipids for use in the pharmaceutical compositions of the present invention include, but are not limited to, soybean phosphatidylcholine, or distearoylphosphatidylglycerol, and hydrogenated forms thereof, or combinations thereof.

[0136] Those skilled in the art may modify the formulation within the teachings of this specification to provide multiple formulations for specific administration routes. In particular, compounds or substances may be modified to make them more soluble in water or other vehicles. It is also well within the scope of those skilled in the art to modify the administration route and administration regimen of a particular compound or substance to manage the pharmacokinetics of the compound or substance for maximum beneficial effect in patients.

[0137] F. Methods for Treating or Preventing a Disease or Disorder In yet another aspect, the present invention is directed to a method for treating or preventing a disease or disorder, such as a proliferative disease or disorder, an autoimmune or inflammatory disease or disorder, or an infectious disease or disorder, in a subject in need thereof, comprising administering to said subject an effective amount of a modified IL-2 polypeptide, polynucleotide, such as a DNA, RNA or viral vector, a modified IL-2 polypeptide conjugate, or a pharmaceutical composition, as described above.

[0138] The method can be used to treat or prevent a disease or disorder, such as a proliferative disease or disorder, in any suitable subject. For example, the method can be used to treat or prevent a disease or disorder, such as a proliferative disease or disorder, in a human. In another example, the method can be used to treat or prevent a disease or disorder, such as a proliferative disease or disorder, in a non-human mammal.

[0139] In one embodiment, the method can be used to treat a proliferation disorder in a subject. In another embodiment, the method can be used to prevent a proliferation disorder in a subject.

[0140] The method can be used to treat or prevent any suitable proliferative disease or disorder in a subject. For example, the method can be used to treat or prevent a tumor in a subject. In another example, the method can be used to treat or prevent a cancer in a subject.

[0141] In one embodiment, the method can be used to treat or prevent a solid tumor or cancer in a subject. The method can be used to treat or prevent any suitable solid tumor or cancer in a subject. For example, the solid tumor or cancer can be chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma of bone / osteosarcoma, osteosarcoma, rhabdomyosarcoma, cardiac cancer, astrocytoma, brain stem glioma, pilocytic astrocytoma, ependymoma, primitive neuroectodermal tumor, cerebellar astrocytoma, cerebral astrocytoma, glioma, medulloblastoma, neuroblastoma, oligodendroglioma, pineal astrocytoma, pituitary adenoma, visual pathway and hypothalamic glioma, Breast cancer, invasive lobular carcinoma, tubular carcinoma, invasive breast cancer, medullary carcinoma, male breast cancer, phyllodes tumor, inflammatory breast cancer, adrenal cortical carcinoma, islet cell carcinoma (endocrine pancreas), multiple endocrine neoplasia syndrome, parathyroid carcinoma, pheochromocytoma, thyroid cancer, Merkel cell carcinoma, uveal melanoma, retinoblastoma, anal cancer, appendix cancer, bile duct cancer, carcinoid tumor, gastrointestinal, colon cancer, extrahepatic bile duct cancer, gallbladder cancer, stomach (gastric) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), hepatocellular carcinoma, pancreatic islet cell cancer, rectal cancer, bladder cancer, cervical cancer, endometrial cancer, extragonadal germ cell tumor, ovarian cancer, ovarian epithelial cancer (surface epithelial stromal tumor), ovarian germ cell tumor, penile cancer, renal cell carcinoma, renal pelvis and ureter, transitional cell carcinoma, prostate cancer, testicular cancer, gestational trophoblastic tumor, ureter and renal pelvis, transitional cell carcinoma, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms' tumor, The cancer may be esophageal cancer, head and neck cancer, nasopharyngeal cancer, oral cancer, oropharynx cancer, paranasal sinus and nasal cavity cancer, pharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, basal cell carcinoma, melanoma, skin cancer (non-melanoma), bronchial adenoma / carcinoma, small cell lung cancer, mesothelioma, non-small cell lung cancer, pleuropulmonary blastoma, laryngeal cancer, thymoma and thymic carcinoma, AIDS-related cancer, Kaposi's sarcoma, epithelioid hemangioendothelioma (EHE), desmoplastic small round cell tumor, or liposarcoma.

[0142] In another embodiment, the method can be used to treat or prevent a hematological malignancy in a subject. The method can be used to treat or prevent any suitable hematological malignancy in a subject.For example, hematological malignancies include myeloid neoplasms, leukemias, lymphomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, hepatosplenic T-cell lymphoma, B-cell lymphoma reticuloendotheliosis, reticulosis, microglioma, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, primary mediastinal B-cell lymphoma, Waldenström's macroglobulinemia, nodal follicular zone B-cell lymphoma, splenic marginal zone Lymphoma, Intravascular large B-cell lymphoma, Primary effusion lymphoma, Lymphomatoid granulomatosis, Nodular lymphocyte-predominant Hodgkin lymphoma, Plasma cell leukemia, Acute dysentery and erythroleukemia, Acute erythroblastic myelopathy, Acute erythroblastic leukemia, Heilmeyer-Schoener disease, Acute megakaryoblastic leukemia, Mast cell leukemia, Panmyelosis, Acute panmyelosis with myelofibrosis, Lymphosarcoma cell leukemia, Acute leukemia of unspecified cell type, Blast phase chronic myeloid leukemia, Stem cell leukemia, Chronic leukemia of unspecified cell type, Subacute leukemia of unspecified cell type , accelerated phase chronic myeloid leukemia, acute myeloid leukemia, polycythemia vera, acute promyelocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, adult T cell leukemia / lymphoma, aggressive NK cell leukemia, B cell prolymphocytic leukemia, B cell chronic lymphocytic leukemia, B cell leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, chronic neutrophilic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, chronic idiopathic myelofibrosis, polycythemia vera, acute promyelocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia, acute ... The disease may be myeloma, Köhler's disease, myelomatosis, solitary myeloma, plasma cell leukemia, plasmacytoma, extramedullary, malignant plasma cell neoplasm NOS, plasmacytoma NOS, monoclonal gammopathy, multiple myeloma, angiocentric immunoproliferative lesions, lymphocytic granulomatosis, angioimmunoblastic lymphadenopathy, T-gamma lymphoproliferative disorder, Waldenstrom's macroglobulinemia, alpha heavy chain disorder, gamma heavy chain disorder, Franklin's disease, immunoproliferative small enteropathy, Mediterranean disease, malignant immunoproliferative disorder, unspecified, or immunoproliferative disorder NOS.

[0143] In yet another embodiment, the method can be used to treat or prevent an immunodeficiency disease or disorder in a subject. The method can be used to treat or prevent any suitable immunodeficiency disease or disorder in a subject. For example, immunodeficiency diseases or disorders include agammaglobulinemia: X-linked and autosomal recessive, ataxia telangiectasia, chronic granulomatous disease and other phagocytic cell disorders, common variable immunodeficiency, complement deficiencies, DiGeorge syndrome, hemophagocytic lymphohistiocytosis (HLH), hyper-IgE syndrome, hyper-IgM syndrome, IgG subclass deficiencies, innate immunity deficiencies, NEMO deficiency syndrome, selective IgA deficiency, selective IgM deficiency, severe combined immunodeficiency, deficiencies and combined immunodeficiencies, specific antibody deficiencies, It may be transient hypogammaglobulinemia of infancy, WHIM syndrome (warts, hypogammaglobulinemia, infection, and bone marrow cathexis), Wiskott-Aldrich syndrome, other antibody deficiencies, other primary cellular immunodeficiencies, severe combined immunodeficiency (SCID), common variable immunodeficiency (CVID), human immunodeficiency virus / acquired immunodeficiency syndrome (HIV / AIDS), drug-induced immunodeficiency, graft-versus-host syndrome, primary immunodeficiency disorder (PIDD), or lymphopenia.

[0144] In yet another embodiment, the method can be used to treat or prevent an autoimmune disease or disorder. For example, the method can be used to treat or prevent inflammation, autoimmune diseases, paraneoplastic autoimmune diseases, cartilage inflammation, fibrotic diseases and / or bone deterioration, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Lett. syndrome, SEA syndrome (serum negative). , enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reter's syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), dermatomyositis, psoriatic arthritis, scleroderma, systemic Lupus erythematosus, vasculitis, myositis, polymyositis, dermatomyositis, osteoarthritis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, scleroderma, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, steatohepatitis, The present invention can be used to treat or prevent various autoimmune diseases, including idiopathic leukemia, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, Guillain-Barre disease, type I diabetes, thyroiditis (such as Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, and / or transplant rejection. However, autoimmune diseases or disorders are a very active area of ​​research, and additional diseases or disorders can be identified as the present invention can be obtained by treatment. In some embodiments, an autoimmune disease or disorder refers to a disease or disorder in which the immune system attacks its own proteins, cells, tissues, organs, and the like. For example, in some human autoimmune diseases or disorders, the human immune system attacks its own proteins, cells, tissues, organs, and the like, including diseased proteins, cells, tissues, and organs.A review of several autoimmune diseases or disorders and a list of them can be found in Autoimmune Diseases (Rose and Mackay, 6. th Edition, 2019, Academic Press). The method can further include administering an effective amount of a second therapeutic agent to treat or prevent the proliferation disorder in the subject. For example, the method can be used to treat or prevent a proliferation disease or proliferation disorder, such as a tumor or cancer, in a subject, and can further include administering an anti-tumor agent to the subject.

[0145] To practice the methods of the invention, the modified IL-2 polypeptide, polynucleotide, e.g., DNA, RNA or viral vector, modified IL-2 polypeptide conjugate or pharmaceutical composition as described above can be administered via any suitable route. For example, the modified IL-2 polypeptide, polynucleotide, e.g., DNA, RNA or viral vector, modified IL-2 polypeptide conjugate or pharmaceutical composition as described above can be administered orally, parenterally, by inhalation, topically, rectally, nasally, buccally, vaginally, via an implanted reservoir, or by other drug administration methods. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0146] Sterile injectable compositions, such as sterile injectable aqueous or oily suspensions, can be formulated according to techniques known in the art using suitable dispersants or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents. Acceptable vehicles and solvents that can be used include mannitol, water, Ringer's solution and isotonic sodium chloride solution. Suitable carriers and other pharmaceutical composition ingredients are typically sterile.

[0147] In addition, sterile fixed oils are conventionally used as a solvent or suspending medium (e.g., synthetic mono- or diglycerides). Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are pharma-ceutically acceptable oils such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, or carboxymethylcellulose or similar dispersants. Various emulsifiers or bioavailability enhancers commonly used in the manufacture of pharma-ceutically acceptable solid, liquid, or other dosage forms can also be used for formulation purposes.

[0148] Compositions for oral administration may be any orally acceptable dosage form, including, but not limited to, tablets, capsules, emulsions, and aqueous suspensions, dispersions, and solutions. For tablets for oral administration, commonly used carriers include lactose and cornstarch. Lubricants such as magnesium stearate may also be added. For oral administration in capsule form, useful diluents include lactose and dry cornstarch. For oral administration of aqueous suspensions or emulsions, the active ingredient may be suspended or dissolved in the oil phase in combination with emulsifying or suspending agents. If necessary, certain sweetening, flavoring, or coloring agents may be added. Nasal aerosol or inhalation compositions may be prepared according to techniques well known in the art of pharmaceutical formulation, and may be prepared, for example, as a solution in saline, using suitable preservatives (e.g., benzyl alcohol), absorption enhancers to enhance bioavailability, and / or other solubilizing or dispersing agents known in the art.

[0149] In yet another aspect, the present invention is directed to the use of an effective amount of a modified IL-2 polypeptide, a polynucleotide, such as a DNA, RNA or viral vector, or a modified IL-2 polypeptide conjugate as described above for the manufacture of a medicament for treating or preventing a disease or disorder, such as a proliferative disease or disorder, in a subject.

[0150] G. Methods for Proliferating Various Immune Cells In yet another aspect, the present invention provides a method for the detection of CD4 + Helper cells, CD8 + The present invention is directed to a method of expanding effector naive and memory cell, natural killer (NK) cell or natural killer T (NKT) cell populations, comprising contacting the cell population with an effective amount of a modified IL-2 polypeptide, polynucleotide, e.g., DNA, RNA or viral vector, modified IL-2 polypeptide conjugate or pharmaceutical composition as described above for a time sufficient to induce formation of a complex with IL-2Rβγ, thereby stimulating proliferation of the T cell, NK cell and / or NKT cell population.

[0151] In yet another aspect, the present invention provides a method for the detection of CD4 + Helper cells, CD8 + The present invention is directed to a method of expanding effector naive and memory cell, Treg cell, natural killer (NK) cell or natural killer T (NKT) cell population, comprising contacting the cell population with an effective amount of a modified IL-2 polypeptide, a polynucleotide, e.g., DNA, RNA or viral vector, a modified IL-2 polypeptide conjugate as described above, or a pharmaceutical composition, for a time sufficient to induce formation of a complex with IL-2Rβγ, thereby stimulating proliferation of the T cell, Treg cell, NK cell and / or NKT cell population and reducing cell death by 10% to 100%, e.g., reducing cell death by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any subrange thereof.

[0152] In one embodiment, the modified IL-2 polypeptide, polynucleotide, e.g., DNA, RNA or viral vector, modified IL-2 polypeptide conjugate or pharmaceutical composition as described above is administered to a CD3+ cell line contacted with a comparable IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without substitutions. + CD4 in cell population + Compared with the proliferation of Treg cells, CD4 +T regulatory (Treg) cells are classified as CD3 + In another embodiment, the modified IL-2 polypeptide, polynucleotide, e.g., DNA, RNA or viral vector, modified IL-2 polypeptide conjugate or pharmaceutical composition as described above expands the CD4 + In yet another embodiment, the ratio of Teff cells to Treg cells in the cell population after incubation with a modified IL-2 polypeptide, polynucleotide, such as DNA, RNA or viral vector, modified IL-2 polypeptide conjugate or pharmaceutical composition as described above is about or at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, 100:1 or more.

[0153] The method can be performed in any suitable manner. In one embodiment, the method is performed in vivo. In another embodiment, the method is performed in vitro. In yet another embodiment, the method is performed ex vivo.

[0154] In yet another aspect, the present invention provides a method for detecting CD4 + Helper cells, CD8 + The present invention is directed to the use of an effective amount of a modified IL-2 polypeptide, polynucleotide, e.g., DNA, RNA or viral vector, or modified IL-2 polypeptide conjugate as described above for the manufacture of a medicament for expanding effector naive and memory cells, Treg cells, natural killer (NK) cells or natural killer T (NKT) cell populations. In one embodiment, the use relates to the manufacture of a medicament for expanding CD4 + Helper cells, CD8 + It is configured to expand effector naive and memory cell, Treg cell, natural killer (NK) cell or natural killer T (NKT) cell populations. EXAMPLES

[0155] H. Working Example Example 1. Design of PEG-modified IL2 muteins Selection of PEG attachment sites on IL-2 From the human IL-2 peptide sequence, muteins can be conjugated with maleimide-activated PEG reagents by selecting one of the amino acids from the list of "site 1" (Table 1) and replacing it with a cysteine. The PEG-conjugated muteins are expected to have a longer half-life compared to the native IL-2 molecule. These PEGylations may also disrupt binding to the alpha unit of the IL-2 receptor (IL-2Rα), while binding to the beta and gamma units (IL-2Rβ, IL-2Rγ) remains unchanged (Figure 1, Table 1). All constructs are made with the substitution C125S, which removes this unpaired cysteine ​​residue in IL-2 on a background of wild-type human IL-2 (herein referred to as rhIL-2). Table 1. IL-2 mutation design [Table 1] The final design of the PEGylated IL-2 mutein molecule is expected to have reduced affinity for IL-2Rα, strong binding to IL-2Rβγ, and extended half-life in humans and other animals.

[0156] Selection of additional mutation sites that disrupt IL-2Rα or enhance IL-2Rβ interactions In addition to the PEGylation modifications described in step 1, further modifications may be introduced. The modifications have at least one mutation substituting an amino acid from the list of "site 2" (Table 1) with any other amino acid. Mutations at these sites reduced the binding to IL-2Rα, while keeping the binding of IL-2Rβ and IL-2Rγ substantially unchanged (Table 1). The modifications may also have at least one mutation substituting an amino acid from the list of "site 3" (Table 1) with any other amino acid to enhance its binding to IL-2Rβ.

[0157] Example 2. Production and purification of IL-2 muteins cDNAs encoding IL-2 muteins were synthesized and cloned into the pcDNA3.1(-) vector. HEK293F cells were transiently transfected with PEI MAX (Polysciences) and cultured for 96 hours. Supernatants were collected by centrifugation of the cultures at 4000 x g for 20 minutes.

[0158] IL-2 expression levels were measured using HEK-Blue™ IL-2 reporter cells (InvivoGen, hkb-il2). Upon IL-2 stimulation, HEK-Blue™ IL-2 cells trigger the activation of STAT5 and subsequent secretion of SEAP. The level of STAT5-induced SEAP can be easily monitored using QUANTI-Blue™. Cells were seeded at 100,000 cells / well in 100 μl, and then 100 μl of rhIL-2 or IL-2 mutein was added to the wells. After 20-24 hours, 180 μl of supernatant was collected and mixed with 20 μl of Quantiblue in a flat-bottom plate. After 90 minutes of incubation at 37°C, absorbance was read at 620 nm. Figure 2 showed that the IL-2 mutants had different detectable expression levels using 10,000 diluted culture supernatants.

[0159] The protein of interest was isolated from the supernatant using standard protein purification techniques. Briefly, the protein of interest was captured by a cOmplete® His-Tag Purification column (Roche) and polished by a Superdex 75 Increase column (GE Healthcare). The purified protein was eluted in a buffer containing 0.1M MES and 150mM NaCl, pH 6.0, and stored at -80°C for further use.

[0160] Example 3. PEGylation of IL-2 muteins Purified IL-2 muteins (1 mg / ml) were reduced with 5 mM TCEP (Thermo Fisher) for 15 min at room temperature, followed by addition of a 50-fold molar excess of maleimide-PEG. The maleimide-PEG 20k was reacted with L-cysteine ​​(Sigma) at room temperature for 30 min. The reaction was stopped by adding a 2-fold molar excess of L-cysteine ​​(Sigma) over maleimide-PEG 20k. The PEG conjugate was further purified by SP Sepharose FF column followed by a Superdex 75 Increase column (GE Healthcare). A representative chromatogram and SDS-PAGE analysis of the purification process are shown in Figure 3.

[0161] Example 4. Binding of IL-2 muteins and PEG conjugates to the IL-2 receptor Binding of purified IL-2 muteins or PEG conjugates to the IL-2 receptor was determined by Octet QKe (ForteBio). IL-2Rα or IL-2Rβ in human Fc fusion protein format (ACROBiosystems) was captured with an anti-human IgG Fc Capture (AHC) sensor. After establishing a baseline in 1× kinetic buffer, the sensor was immersed in wells containing serially diluted rhIL-2, muteins or PEG conjugates to measure association constants. Dissociation was detected after transferring the sensor to wells containing buffer only. Data were collected and analyzed by Octet User Software. A 1:1 curve fitting model was used for analysis of kinetic constants. Table 2 shows the kinetic parameters of IL-2 mutants binding to individual IL-2 receptor subunits. Typical sensorgrams of binding are shown in Figure 4. Most PEGylated muteins showed reduced or abolished binding to IL-2Rα. Table 2. Rate constants of IL-2 mutants interacting with IL-2Rα. [Table 2] Note: ND = Not detectable

[0162] Example 5. Surface binding of IL2 muteins on IL2Rαβγ expressing cells For this analysis, two different IL2Rαβγ expressing cells were tested: 1. CTLL2 cells; 2. IL2Rα+ T cells generated by anti-CD3 / CD28 Dynabeads reactivated human T cells from PBMCs (at least 90% of cells were positive for IL2Rα). Either CTLL2 or IL2Rα+ T cells were harvested and resuspended at 2-4 million cells / ml in cold binding buffer (FBB, 5% FBS in DPBS). His-tagged IL-2 and variants were added to the cell suspension, mixed and kept at 4°C for 40 minutes. Cells were washed once with wash buffer (FWB, 1% FBS in DPBS). Cell pellets were resuspended in FBB and reacted with 1:100 anti-His-APC (BioLegend 362605) for 15 minutes at room temperature. Cells were washed with 120uL of FWB and then resuspended for flow cytometry analysis. Both Y31C and Y31C-PEG20 showed enhanced binding to CTLL2 cells and IL2Rα positive human T cells. (See FIG. 6.)

[0163] Example 6. T cell activity of PEGylated IL-2 muteins Frozen PBMCs were thawed in serum-free AIM-V medium (ThermoFisher) and kept at 37°C for 2–4 h before the experiment. 5Cells / well were seeded in 96-well plates. To avoid phosphorylation of STAT-5 at different time points, different IL-2 muteins were added on top of the cells at 4C. Cells were mixed with the muteins and incubated for 15 min at 37°C. The rest of the protocol was performed at room temperature. After centrifugation, cell pellets were stained for extracellular markers (1:300-anti-human CD4 FITC, CD8 APC, CD25 BV650, R45RA BV421, BioLegend) and Fixable Viability Dye (1:1000-eFluor 780, ThermoFisher) in 50 μL staining buffer (PBS+1% FBS+2mM EDTA) for 15 min. Cells were washed with 200 μL washing buffer (PBS+1% FBS) and centrifuged. The cell pellet was fixed with 200 μL of 1× fixation buffer (FoxP3 / transcription factor staining buffer set, eBioscience) for 30 minutes in the dark. The cells were spun and permeabilized with 100 μL of cold 100% methanol overnight at 4°C. After this period, 100 μL of wash buffer was added, the cells were spun and stained with 50 μL of anti-human P STAT5-PE (1:80, BioLegend) for 30 minutes at room temperature in the dark. 250 μL of wash buffer was added, the cells were centrifuged and resuspended in 110 μL of staining buffer. CD8 + CD45RA + CD25 low Naive (IL-2Rβγ expressing T cells) and CD4 + CD45RA - CD25 high (IL-2Rαβγ-expressing T cells) Phosphorylation of the indicated surface markers and STAT-5 from T cells was assessed by flow cytometry (NovoCyte, ACEA Biosciences).

[0164] Figure 7 shows the dose-response phosphorylation of STAT5 in different IL-2 muteins and the respective PEGylated proteins. When compared to rhIL-2 protein, the PEGylated IL-2 muteins showed dramatically reduced activity against cells expressing IL-2Rαβγ, whereas their activity against T cells expressing IL-2Rβγ was almost unchanged (Table 3). The favorable bioselectivity of the PEGylated muteins for T cells expressing IL-2Rβγ over IL-2Rαβγ was also demonstrated by the ratio of EC50 for the two different cell populations. Table 3. T cell activity of PEGylated IL2 muteins [Table 3]

[0165] Example 7. PK study in C57BL / 6 mice Pharmacokinetic studies of P65C-PEG20 or Y31C-PEG20+F42K were performed in C57BL / 6 mice. In the following, P65C-PEG20 was used as an example. Three mice were used for blood collection at each time point. Each mouse was administered a single IV dose of 0.56 mg / kg P65C-PEG20. Blood samples were collected at 0.033, 0.083, 0.17, 0.5, 1, 4, 24, 48, 72, and 96 hours after administration. Blood was allowed to clot at room temperature and then processed by centrifugation at 5000 rpm for 10 minutes. Serum was collected, frozen in dry ice, and kept at -80°C until ELISA analysis.

[0166] ELISA was performed in two stages. In the first phase, most samples were diluted 10-fold, except for samples from the early time points 2 min to 30 min after administration. They were diluted 100-1,000-fold. The diluted samples were added to ELISA plates coated with rabbit anti-IL-2 antibody P600 (ThermoFisher) and detected with biotin-conjugated monoclonal IL-2 antibody M600B (ThermoFisher). For samples taken after 1 h, the samples were further tested with a High Sensitivity IL-2 Human ELISA Kit (ThermoFisher) to detect low levels of IL-2. All tests were performed in duplicate. ELISA readings were converted to concentrations using the standard curve of the corresponding IL-2 construct and the Pade(1,1) fitting model (Prism).

[0167] PK parameters were calculated by noncompartmental methods using Phenix WinNonLin version 8.1 software (Certara USA, Inc., Princeton, NJ, USA) (Figure 8). The serum concentration-time profile of P65C-PEG20 in mice was similar to that reported for aldesleukin [REF-1]. The terminal half-life (t 1 / 2 ) and mean residence time (MRT inf The half-lives of rhIL-2 were 4.0 and 0.20 hours, respectively (REF-1). The area under the concentration-time curve (AUC last) was 8051h*ng / mL at a 0.56mg / kg dose, whereas the AUC of aldesleukin was 1380h*ng / mL at a 0.8mg / kg dose [REF-1]. P65C-PEG20 showed longer terminal half-life (5.8-fold) and residence time (14.2-fold) and higher exposure (8.3-fold, dose-normalized) than aldesleukin. REF-1:Charych D,Khalili S,Dixit V,Kirk P,Chang T,Langowski J et al. (2017)Modeling the receptor pharmacology,pharmacokinetics,and pharmacodynamics of NKTR-214,a kinetically-controlled interleukin-2 (IL2) receptor agonist for cancer immunotherapy. PLoS ONE 12(7):e0179431. Table 4. PK parameters of IL2 P65C-PEG20 [Table 4]

[0168] Example 8. Ex vivo expansion of T cells and NK cells T cell expansion PBMCs were thawed and expanded at 5 million cells / ml in AIM V, 5% FBS, 5ng / ml OKT3 and IL-2 or muteins at the indicated concentrations. Starting on day 5, cells were split every 3-4 days with medium and IL-2 refresher. Starting on day 7, cells were stained and counted for total cells and lymphocyte subtypes every 2-3 days. Ex vivo expanded T cells have significantly reduced Treg and enhanced CD8 T / Treg ratios in the presence of P65C-PEG20 or Y31C-PEG20+F42K compared to rhIL2 (Figure 9).

[0169] NK cell proliferation PBMCs were thawed and expanded at 5 million cells / ml in 24-well plates at 0.5 ml / well in AIM V, 5% FBS, IL-2 or muteins at the indicated concentrations. Cells were split every 2-3 days with medium and IL-2 refresher. From day 7, cells were stained and counted for total cells and lymphocyte subtypes every 3-6 days. Compared to rhIL2 or rhIL5, P65C-PEG20 or Y31C-PEG20+F42K promoted better proliferation of NK cells (Figure 9).

[0170] LAK cell cytotoxicity PBMC-derived LAK cells cultured for 2–4 weeks were used as effector cells. K562 stained with CFSE and grown overnight were used as target cells. 30,000 K562 are mixed with different amounts of LAK cells in wells of a 96-well U-bottom plate. At different time points after co-culture, K562 cell viability is measured by staining with Annexin V 7-AAD and the CFSE+Annexin V+ population is counted. P65C-PEG20 or Y31C-PEG20+F42K-activated LAK cells showed enhanced proliferation and enhanced cytotoxicity against target K562 cells compared to rhIL2 (Figure 10). I. References

[0171] The cited references are listed below. [ka] [ka]

Claims

1. 1. A modified interleukin-2 (IL-2) polypeptide comprising an amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 and a substitution at a position selected from the group consisting of Y31, N29, N30, and a combination thereof with a natural or non-natural amino acid, wherein the modified IL-2 polypeptide has at least about 90% sequence identity in the region of amino acid residues 10-25, 80-100, and / or 100-134 with the corresponding region of an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, and wherein the modified IL-2 polypeptide has at least about 90% sequence identity with an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitution.

2. 2. The modified IL-2 polypeptide of claim 1, comprising a substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at a position selected from the group consisting of Y31, N29, N30, and combinations thereof.

3. a) comprising a substitution with a natural amino acid at a position selected from the group consisting of Y31, N29, N30, and combinations thereof, and configured to be conjugated to a water soluble polymer, lipid, protein, or peptide at said position selected from the group consisting of Y31, N29, N30, and combinations thereof; and / or 3. The modified IL-2 polypeptide of claim 1 or 2, wherein said modified IL-2 polypeptide comprises a natural amino acid substitution at a position selected from the group consisting of Y31, N29, N30 and combinations thereof, and is configured for conjugation to a water soluble polymer, lipid, protein or peptide at the N-terminus and / or C-terminus of said modified IL-2 polypeptide.

4. a) comprising a substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine or tyrosine at a position selected from the group consisting of Y31, N29, N30 and combinations thereof; and / or b) comprising a substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine or tyrosine at a position selected from the group consisting of Y31, N30 and combinations thereof; and / or c) comprising a substitution with cysteine ​​at a position selected from the group consisting of Y31, N29, N30 and combinations thereof; and / or d) comprising a substitution with cysteine ​​at a position selected from the group consisting of Y31, N29, and combinations thereof; and / or e) containing a substitution with any amino acid at positions Y31, N29 or a combination thereof; and / or f) comprising a substitution with cysteine, serine or alanine at positions Y31, N29 or a combination thereof; and / or g) containing a substitution at position Y31 by cysteine; and / or h) containing a substitution at position N29 with a cysteine; and / or i) further comprising a substitution at position F42 with a cysteine; and / or The modified IL-2 polypeptide of claim 1 or 3, further comprising j) a substitution at position F42 with a natural amino acid.

5. A modified IL-2 polypeptide according to any one of claims 1 to 4, comprising a substitution at position F42 with lysine and at position Y31 with cysteine.

6. has reduced binding to IL-2Rα compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without said substitutions; and / or retains or has a higher level of binding to interleukin-2 receptor beta (IL-2Rβ) or interleukin-2 receptor gamma (IL-2Rγ) compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without said substitutions; and / or retains or has a higher receptor signaling potency for IL-2Rβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without said substitutions; The modified IL-2 polypeptide of any one of claims 1 to 5.

7. A polynucleotide encoding a modified IL-2 polypeptide according to any one of claims 1 to 6 and adapted to express said modified IL-2 polypeptide in vitro and / or in vivo, a) DNA; and / or b) RNA; and / or c) a viral vector; and / or A polynucleotide comprising a combination of a) to c).

8. A modified IL-2 polypeptide conjugate comprising a modified IL-2 polypeptide according to any one of claims 1 to 6 conjugated to a water soluble polymer, lipid, polypeptide, protein or peptide.

9. The modified IL-2 polypeptide comprises: via a linker; and / or 9. The modified IL-2 polypeptide conjugate of claim 8, covalently conjugated to a water soluble polymer, lipid, protein or peptide via a single amino acid residue in a fusion polypeptide comprising the amino acid sequences of the modified IL-2 polypeptide and the protein or peptide.

10. 10. The modified IL-2 polypeptide conjugate of claim 9, wherein said amino acid sequence of said protein or peptide comprises an antibody sequence or an antibody portion having an antigen recognition site of the required specificity.

11. 11. The modified IL-2 polypeptide conjugate of any one of claims 8-10, wherein the water soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), a copolymer of ethylene glycol and propylene glycol, a poly(oxyethylated polyol), a poly(olefinic alcohol), a poly(vinylpyrrolidone), a poly(hydroxyalkyl methacrylamide), a poly(hydroxyalkyl methacrylate), a poly(saccharide), a poly(a-hydroxy acid), a poly(vinyl alcohol), a polyphosphazene, a polyoxazoline (POZ), a poly(N-acryloylmorpholine), or a combination thereof.

12. 12. A pharmaceutical composition comprising an effective amount of a modified IL-2 polypeptide according to any one of claims 1 to 6, a polynucleotide according to claim 7, or a modified IL-2 polypeptide conjugate according to any one of claims 8 to 11, and a pharma- ceutically acceptable carrier or excipient.

13. 13. The pharmaceutical composition of claim 12 for treating or preventing a disease or disorder, wherein the disorder comprises a proliferative disease or disorder, an autoimmune disease or disorder, an immunodeficiency disease or disorder, or an inflammatory disease or disorder, or an infectious disease or disorder in a subject having the disease or disorder.

14. The proliferative disease or disorder is selected from the group consisting of chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma of bone / osteosarcoma, osteosarcoma, rhabdomyosarcoma, cardiac cancer, astrocytoma, brain stem glioma, pilocytic astrocytoma, ependymoma, primitive neuroectodermal tumor, cerebellar astrocytoma, cerebral astrocytoma, glioma, medulloblastoma, neuroblastoma, oligodendroglioma, pineal astrocytoma, pituitary adenoma, visual pathway and hypothalamic glioma, breast cancer, invasive lobular carcinoma, tubular carcinoma, invasive breast cancer, medullary carcinoma, male breast cancer, phyllodes tumor, inflammatory breast cancer, adrenal cortical carcinoma, islet cell carcinoma, multiple endocrine neoplasia syndrome, parathyroid carcinoma, pheochromocytoma, thyroid carcinoma, Merkel cell carcinoma, alveolar carcinoma, uveal melanoma, retinoblastoma, anal cancer, appendix cancer, bile duct cancer, carcinoid tumor, colon cancer, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, hepatocellular carcinoma, pancreatic cancer, rectal cancer, bladder cancer, cervical cancer, endometrial cancer, extragonadal germ cell tumor, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, penile cancer, renal cell carcinoma, transitional cell carcinoma, prostate cancer, testicular cancer, gestational trophoblastic tumor, transitional cell carcinoma, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms' tumor, esophageal cancer, head and neck cancer, nasopharyngeal cancer, oral cancer, oropharynx cancer, paranasal sinuses and nasal cavity cancer, pharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, basal cell carcinoma, melanoma, skin cancer, bronchial adenoma / Carcinoma, small cell lung cancer, mesothelioma, non-small cell lung cancer, pleuropulmonary blastoma, laryngeal cancer, thymoma and thymic carcinoma, AIDS-related cancer, Kaposi's sarcoma, epithelioid hemangioendothelioma, desmoplastic small round cell tumor and liposarcoma, myeloid tumors, leukemia, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, hepatosplenic T-cell lymphoma, B-cell lymphoma reticuloendotheliosis, reticulosis, microglioma, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, Primary mediastinal B-cell lymphoma, Waldenstrom's macroglobulinemia, nodal follicular zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, nodular lymphocyte-predominant Hodgkin's lymphoma, plasma cell leukemia, acute dysentery and erythroleukemia, acute erythroblastic myelopathy, acute erythroblastic leukemia, Heilmeyer-Schoener's disease, acute megakaryoblastic leukemia, mast cell leukemia, panmyelosis, acute panmyelosis with myelofibrosis, lymphosarcoma cell leukemia, acute leukemia, blast phase chronic myelogenous leukemia, stem cell leukemia, chronic leukemia,Subacute leukemia, Accelerated phase chronic myeloid leukemia, Acute myeloid leukemia, Polycythemia vera, Acute promyelocytic leukemia, Acute basophilic leukemia, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Adult T-cell leukemia / lymphoma, Aggressive NK cell leukemia, B-cell prolymphocytic leukemia, B-cell chronic lymphocytic leukemia, B-cell leukemia, Chronic myelogenous leukemia, Chronic myelomonocytic leukemia, Chronic neutrophilic leukemia, Chronic lymphocytic leukemia, Hairy cell leukemia, Chronic idiopathic myeloblastoma fibrosis, multiple myeloma, Köhler's disease, myelomatosis, solitary myeloma, plasma cell leukemia, plasmacytoma, malignant plasma cell neoplasm NOS, plasmacytoma NOS, monoclonal gammopathy, multiple myeloma, angiocentric immunoproliferative lesions, lymphocytic granulomatosis, angioimmunoblastic lymphadenopathy, T-gamma lymphoproliferative disorders, Waldenstrom's macroglobulinemia, alpha heavy chain disorders, gamma heavy chain disorders, Franklin's disease, immunoproliferative small intestinal disorders, Mediterranean disease, malignant immunoproliferative disorders, and immunoproliferative disorders NOS; The immunodeficiency disease or disorder is selected from the group consisting of X-linked and autosomal recessive agammaglobulinemia, ataxia telangiectasia, chronic granulomatous disease and phagocytic disorders, common variable immunodeficiency, complement deficiencies, DiGeorge syndrome, hemophagocytic lymphohistiocytosis, hyper-IgE syndrome, hyper-IgM syndrome, IgG subclass deficiencies, innate immunity deficiencies, NEMO deficiency syndrome, selective IgA deficiency, selective IgM deficiency, severe combined immune deficiency, combined immune deficiency, specific antibody deficiency, transient hypogammaglobulinemia of infancy, WHIM syndrome, Wiskott-Aldrich syndrome, antibody deficiency, primary cellular immune deficiency, severe combined immune deficiency, human immunodeficiency virus / acquired immune deficiency syndrome, drug-induced immune deficiency, graft versus host syndrome, primary immune deficiency, and lymphopenia. The inflammatory disease or disorder or autoimmune disease or disorder is selected from the group consisting of inflammation, autoimmune disease, tumor-associated autoimmune disease, cartilage inflammation, fibrotic disease and / or bone deterioration, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, SEA syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome, dermatomyositis, psoriatic arthritis, scleroderma, psoriasis, systemic lupus erythematosus, vasculitis, myositis, polymyositis, dermatomyositis, osteoarthritis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, scleroderma, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, systemic lupus erythematosus, myasthenia gravis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis, asthma, COPD, Guillain-Barre disease, type I diabetes mellitus, thyroiditis, Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, and graft rejection, The infectious disease is Acinetobacter infection, actinomycosis, African sleeping sickness, AIDS (acquired immune deficiency syndrome), amebiasis, anaplasmosis, Angiostrongylus cantonensis anisakiasis, anthrax, hemolytic Arcanobacterium infection, Argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, Bacillus cereus infection, bacterial meningitis, bacterial pneumonia, bacterial vaginosis, Bacteroides infection, balantidiosis, bartonellosis, Baylisascaris infection, BK virus infection, black sand lichen, cysticercosis, blastomycosis, Bolivian hemorrhagic fever. , botulism, Brazilian hemorrhagic fever, brucellosis, bubonic plague, Burkholderia infection, Buruli ulcer, calicivirus infection, campylobacteriosis, candidiasis, capitis, carrion disease, cat scratch disease, cellulitis, Chagas disease, chancroid, chickenpox, chikungunya fever, chlamydia, chlamydophila pneumonia infection, cholera, chromoblastomycosis, chytridiomycosis, liver fluke, Clostridium difficile colitis, coccidioidomycosis, Colorado tick fever, common cold, coronavirus disease 2019, Creutzfeldt-Jakob disease, Crimean-Congo exacerbation Blood fever, cryptococcosis, cryptosporidiosis, cutaneous larval migration, cyclosporosis, cysticercosis, cytomegalovirus infection, dengue fever, desmodesmus infection, diamoebiasis, diphtheria, diphyllobothriasis, dracunculiasis, Ebola hemorrhagic fever, echinococcosis, ehrlichiosis, enterobiasis, enterococcal infection, enterovirus infection, typhus, erythema infectiosum, exanthema subitum, fascioliasis, fascioliasis, fatal familial insomnia, filariasis, food poisoning with Clostridium perfringens, free-living ameba infection, Fusobacterium infection, gas gangrene, geotrichum, genomic DNA, Lustmann-Straussler-Scheinker syndrome, giardiasis, glanders, gnathostomiasis, gonorrhea, inguinal granuloma, group A streptococcal infection, group B streptococcal infection, Haemophilus influenzae infection, hand and mouth disease, hantavirus pulmonary syndrome, heartland virus disease, Helicobacter pylori infection, hemolytic uraemic syndrome, hemorrhagic fever with renal syndrome, Hendra virus infection, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex, histoplasmosis, hookworm infection, human bocavirus infection, human ewingii ehrlichiosis, human granulocytic anaplasmosis,Human metapneumovirus infection, human monocytic ehrlichiosis, human papillomavirus infection, human parainfluenza virus infection, membranous tapeworm disease, Epstein-Barr virus infectious mononucleosis, influenza, isosporosis, Kawasaki disease, keratitis, Kingella kingae infection, kuru, Lassa fever, Legionnaires' disease, Pontiac fever, leishmaniasis, leprosy, leptospirosis, listeriosis, Lyme disease, lymphatic filariasis, lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever, measles, Middle East respiratory syndrome, melioidosis, meningeal Infection, meningococcal disease, schizophrenia, microsporidiosis, molluscum contagiosum, monkeypox, mumps, typhus, mycoplasma pneumonia, mycoplasma genitalium infection, mycetoma, myiasis, neonatal conjunctivitis, Nipah virus infection, norovirus, variant Creutzfeldt-Jakob disease, nocardiosis, onchocerciasis, opisthorchiasis, paracoccidioidomycosis, paragonimiasis, pasteurellosis, head lice infestation, body lice infestation, pelvic inflammatory disease, whooping cough, plague, pneumococcal infection, Pneumocystis pneumonia, pneumonia, acute ash Leukomyelitis, Prevotella infections, primary amebic meningoencephalitis, progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, relapsing fever, respiratory syncytial virus infections, rhinosporidiosis, rhinovirus infections, rickettsial infections, rickettsial smallpox, Rift Valley fever, Rocky Mountain spotted fever, rotavirus infections, rubella, salmonellosis, SARS, scabies, scarlet fever, schistosomiasis, septicemia, shigellosis, shingles, smallpox, sporotrichosis, staphylococcal food poisoning, staphylococcal infections, bacillariasis, subacute sclerosing panencephalitis, bejel, syphilis, and yaws, taeniasis, tetanus tinea barbae, tinea capitis, tinea corporis, tinea cruris, tinea manus, tinea nigra, athlete's foot, tinea unguium, tinea versicolor, toxocariasis, toxoplasmosis, trachoma, trichinosis, trichomoniasis, trichuriasis, tuberculosis, tularemia, typhoid fever, typhoid fever, Ureaplasma urealyticum infection, valley fever, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Vibrio vulnificus infection, Vibrio parahaemolyticus enteritis, viral pneumonia, West Nile fever, white sand lichen, Yersinia pseudotuberculosis infection, yersiniosis, yellow fever, Zika fever, and zygomycosis; The pharmaceutical composition of claim 13.

15. 15. The pharmaceutical composition of claim 13 or claim 14, further comprising an effective amount of a second therapeutic agent.

16. CD4 ＋ Helper cells, CD8 ＋ 13. A method of expanding effector naive and memory cell, natural killer (NK) cell or natural killer T (NKT) cell populations in vitro or ex vivo, comprising contacting the cell population with an effective amount of a modified IL-2 polypeptide according to any one of claims 1 to 6, a polynucleotide according to claim 7, a modified IL-2 polypeptide conjugate according to any one of claims 8 to 10, or a pharmaceutical composition according to claim 12 comprising said modified IL-2 polypeptide, said polynucleotide or said modified IL-2 polypeptide conjugate, for a time sufficient to induce formation of a complex with IL-2Rβγ, thereby stimulating proliferation of said CD4+ helper cell, CD8+ effector naive and memory cell, NK cell and / or NKT cell population.

17. CD4 ＋ Helper cells, CD8 ＋ 13. A modified IL-2 polypeptide according to any one of claims 1 to 6, a polynucleotide according to claim 7, a modified IL-2 polypeptide conjugate according to any one of claims 8 to 10 or a pharmaceutical composition according to claim 12 for use in a method for expanding in vivo a population of effector naive and memory cells, natural killer (NK) cells or natural killer T (NKT) cells, said method comprising contacting a population of cells with an effective amount of said modified IL-2 polypeptide, said polynucleotide or said modified IL-2 polypeptide conjugate for a time sufficient to induce the formation of a complex with IL-2Rβγ, thereby increasing the CD4 ＋ Helper cells, CD8 ＋ A modified IL-2 polypeptide, polynucleotide, modified IL-2 polypeptide conjugate, or pharmaceutical composition comprising stimulating the proliferation of effector naive and memory cell, NK cell, and / or NKT cell populations.

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