NEO-2 / 15 variants and their uses for preferentially stimulating T regulatory cells

Abstract

Provided herein are polypeptides comprising Neo-2 / 15 variants, polypeptides comprising Neo-2 / 15 variants and agents for targeting T regulatory cells, and methods of using them to treat diseases. Related nucleic acids, vectors, host cells, pharmaceutical compositions, and kits are also provided. Methods of making the polypeptides and treating subjects in need thereof, as well as CD25 antigen-binding proteins, are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 357,844, filed July 1, 2022, and U.S. Provisional Application No. 63 / 395,437, filed August 5, 2022, each of which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy created on June 21, 2023, is named "2023-06-21_01267-0005-00PCT_ST26" and is 95,337 bytes in size. [Background technology]

[0003] Regulatory T cells (Tregs) are important for maintaining immune tolerance, and their dysfunction contributes to inflammatory and autoimmune conditions. IL-2 is a pleiotropic cytokine that activates both immunosuppressive Tregs and inflammatory cells, including NK cells, cytotoxic T cells, and helper T cells. Low-dose IL-2 therapy effectively expands Tregs, but this intervention is limited by the co-stimulation of conventional T cells and natural killer cells. Novel therapies for expanding Treg cells without the co-stimulation of T cells and natural killer cells are needed. Summary of the Invention

[0004] Provided herein are polypeptides comprising Neo-2 / 15 variants, agents that target polypeptides comprising Neo-2 / 15 variants and T regulatory cells, and methods of using them to treat disease.

[0005] In some embodiments, the polypeptide comprises a Neo-2 / 15 variant (the Neo-2 / 15 variant comprises an amino acid sequence at least 80%, at least 85%, or at least 90% identical to SEQ ID NO:1 or SEQ ID NO:2, wherein the Neo-2 / 15 variant comprises a lysine, arginine, threonine, serine, tyrosine, glutamic acid, alanine, or histidine (Q95K or Q95R or Q95T or Q95S or Q95Y or Q95E or Q95A or Q95H) in place of the glutamine at position 95); a) serine or alanine instead of aspartic acid at position 15 (D15S or D15A); b) alanine or serine or glycine instead of asparagine at position 40 (N40A or N40S or N40G), and c) at least one additional substitution selected from alanine or serine or asparagine or threonine or tyrosine for isoleucine at position 44 (I44A or I44S or I44N or I44T or I44Y), Polypeptides are provided in which the first amino acid of SEQ ID NO:2 is designated as position 1 and the first amino acid of SEQ ID NO:1 is designated as position 4.

[0006] In some embodiments, the polypeptide comprises a Neo-2 / 15 variant (the Neo-2 / 15 variant comprises an amino acid sequence at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 2, wherein the Neo-2 / 15 variant comprises a glutamic acid, alanine, or histidine in place of glutamine at position 95 (Q95E or Q95A or Q95H)); a) serine or alanine instead of aspartic acid at position 15 (D15S or D15A); b) alanine or serine instead of asparagine at position 40 (N40A or N40S), and c) at least one additional substitution selected from alanine or serine for isoleucine at position 44 (I44A or I44S), Polypeptides are provided in which the first amino acid of SEQ ID NO:2 is designated as position 1 and the first amino acid of SEQ ID NO:1 is designated as position 4.

[0007] In some embodiments, the polypeptide comprises a Neo-2 / 15 variant (the Neo-2 / 15 variant comprises an amino acid sequence at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 2, the Neo-2 / 15 variant comprising serine or alanine instead of aspartic acid at position 15 (D15S or D15A)); a) lysine, arginine, threonine, serine, tyrosine, glutamic acid, alanine, or histidine in place of glutamine at position 95 (Q95K or Q95R or Q95T or Q95S or Q95Y or Q95E or Q95A or Q95H); b) arginine instead of histidine at position 8 (H8R); c) phenylalanine instead of histidine at position 11 (H11F); d) lysine instead of tyrosine at position 14 (Y14K); e) alanine or serine instead of asparagine at position 40 (N40A or N40S), and f) at least one additional substitution selected from alanine or serine or tyrosine for isoleucine at position 44 (I44A or I44S or I44Y), Polypeptides are provided in which the first amino acid of SEQ ID NO:1 is designated as position 4 and the first amino acid of SEQ ID NO:2 is designated as position 1.

[0008] In some embodiments, the polypeptide comprises a Neo-2 / 15 variant (the Neo-2 / 15 variant comprises an amino acid sequence at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 2, the Neo-2 / 15 variant comprising serine or alanine instead of aspartic acid at position 15 (D15S or D15A)); a) glutamic acid, alanine, or histidine in place of glutamine at position 95 (Q95E, Q95A, or Q95H); b) arginine instead of histidine at position 8 (H8R); c) phenylalanine instead of histidine at position 11 (H11F); d) lysine instead of tyrosine at position 14 (Y14K); e) alanine or serine instead of asparagine at position 40 (N40A or N40S), and f) at least one additional substitution selected from alanine or serine for isoleucine at position 44 (I44A or I44S), Polypeptides are provided in which the first amino acid of SEQ ID NO:1 is designated as position 4 and the first amino acid of SEQ ID NO:2 is designated as position 1.

[0009] In some embodiments, a polypeptide is provided comprising a Neo-2 / 15 variant, wherein the Neo-2 / 15 variant comprises an amino acid sequence at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 2, wherein the Neo-2 / 15 variant comprises: a) phenylalanine instead of histidine at position 11 (H11F); b) alanine or serine instead of asparagine at position 40 (N40A or N40S), and c) containing alanine or serine or tyrosine instead of isoleucine at position 44 (I44A or I44S or I44Y), Polypeptides are provided in which the first amino acid of SEQ ID NO:1 is designated as position 4 and the first amino acid of SEQ ID NO:2 is designated as position 1.

[0010] In some embodiments, a polypeptide is provided comprising a Neo-2 / 15 variant, wherein the Neo-2 / 15 variant comprises an amino acid sequence at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 2, wherein the Neo-2 / 15 variant comprises: a) phenylalanine instead of histidine at position 11 (H11F); b) alanine or serine instead of asparagine at position 40 (N40A or N40S), and c) containing an alanine or serine instead of the isoleucine at position 44 (I44A or I44S); Polypeptides are provided in which the first amino acid of SEQ ID NO:1 is designated as position 4 and the first amino acid of SEQ ID NO:2 is designated as position 1.

[0011] In some embodiments, a polypeptide is provided comprising a Neo-2 / 15 variant, the Neo-2 / 15 variant comprising domains D1, D2, D3, and D4; (a) D1 has the amino acid sequence: KIQLHAEHALYX 15 ALMILNI (SEQ ID NO: 61), and D3 comprises the amino acid sequence LEDYAFNFELILEEIARLFESG (SEQ ID NO: 64), or (b) D1 comprises the amino acid sequence: KIQLHAEHALYDALMILNI (SEQ ID NO: 62) and D3 comprises the amino acid sequence LEDYAFX 40 FELX 44 LEEIARLFESG (SEQ ID NO: 65), D2 comprises an amino acid sequence at least 8 amino acids in length, D4 has the amino acid sequence EDEQEEMANAIITILX 95 SWIFS (SEQ ID NO: 66), wherein (i) D1, D2, D3, and D4 can be in any order in the Neo-2 / 15 mutant; (ii) an amino acid linker may be present between any of the domains (a "domain linker"); (iii)X 15 is serine or alanine; X 95 is glutamic acid, alanine, or histidine; X 40 is serine or alanine; X 44 is serine or alanine, or X 15 is serine or alanine; X 95is glutamic acid, lysine, arginine, threonine, serine, tyrosine, alanine, or histidine; X 40 is serine, alanine, or glycine; X 44 is serine or alanine or asparagine or threonine or tyrosine, (iv) The Neo-2 / 15 variant contains a total of no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, no more than 1, or no substitutions at amino acid positions not designated as X. [Brief explanation of the drawings]

[0012] [Figure 1] Graphs of the attenuation of STAT5 signaling for the Neo-2 / 15 mutants, Neo-2 / 15_D15S_Q95E and Neo-2 / 15_D15A_Q95A, are provided. In A, the percentages of CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) showing STAT5 phosphorylation after stimulation with Neo-2 / 15_D15S_Q95E (solid line, filled shapes) and Neo-2 / 15 (dotted line, open shapes) are shown, and in B, the percentages of CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) showing STAT5 phosphorylation after stimulation with Neo-2 / 15_D15A_Q95A (solid line, filled shapes) and Neo-2 / 15 (dotted line, open shapes) are shown. [Figure 2] Representations of fusion protein formats are provided. In A, the Neo-2 / 15 variant and the scFv are both positioned at opposite ends of the Fc. In B, the Neo-2 / 15 variant is linked to the N-terminus of the antibody heavy chain. In C, the Neo-2 / 15 variant is linked to the C-terminus of the antibody heavy chain. [Figure 3A]Graphs of STAT5 signaling for Neo-2 / 15 and Neo-2 / 15 variants fused to anti-CD25 scFv are provided. The percentages of total T cells (squares), CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) showing STAT5 phosphorylation are provided after stimulation with titrations of scFv fusion proteins (solid line, filled shapes) and human IL-2 (dotted line, open shapes). Neo-2 / 15 fused to anti-CD25 ScFv is provided. [Figure 3B] Graphs of STAT5 signaling for Neo-2 / 15 and Neo-2 / 15 variants fused to anti-CD25 scFv are provided. Percentages of total T cells (squares), CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) exhibiting STAT5 phosphorylation after stimulation with titrations of scFv fusion proteins (solid lines, filled shapes) and human IL-2 (dotted lines, open shapes) are provided. Neo-2 / 15_D15S_Q95E fused to anti-CD25 ScFv is provided. [Figure 3C] Graphs of STAT5 signaling for Neo-2 / 15 and Neo-2 / 15 variants fused to anti-CD25 scFv are provided. Percentages of total T cells (squares), CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) exhibiting STAT5 phosphorylation after stimulation with titrations of scFv fusion proteins (solid lines, filled shapes) and human IL-2 (dotted lines, open shapes) are provided. Neo-2 / 15_D15A_Q95A fused to anti-CD25 ScFv is provided. [Figure 3D] Graphs of STAT5 signaling for Neo-2 / 15 and Neo-2 / 15 variants fused to anti-CD25 scFv are provided. The percentages of total T cells (squares), CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) showing STAT5 phosphorylation are provided after stimulation with titrations of scFv fusion proteins (solid lines, filled shapes) and human IL-2 (dotted lines, open shapes). Neo-2 / 15_D15A_Q95H fused to anti-CD25 ScFv is provided. [Figure 3E] Graphs of STAT5 signaling for Neo-2 / 15 and Neo-2 / 15 variants fused to anti-CD25 scFv are provided. Percentages of total T cells (squares), CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) exhibiting STAT5 phosphorylation after stimulation with titrations of scFv fusion proteins (solid lines, filled shapes) and human IL-2 (dotted lines, open shapes) are provided. Neo-2 / 15_L13R_L17E_Q95H fused to anti-CD25 ScFv is provided. [Figure 3F] Graphs of STAT5 signaling for Neo-2 / 15 and Neo-2 / 15 variants fused to anti-CD25 scFv are provided. Percentages of total T cells (squares), CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) exhibiting STAT5 phosphorylation after stimulation with titrations of scFv fusion proteins (solid lines, filled shapes) and human IL-2 (dotted lines, open shapes) are provided. Neo-2 / 15_L13R_L17E_N40A_Q95H fused to anti-CD25 ScFv is provided. [Figure 4] Graphs of STAT5 signaling for Neo-2 / 15 fused to anti-CD25 ScFV and Neo-2 / 15_D15S_Q95E fused to anti-CD25 ScFV are provided for a second patient sample. The percentages of total T cells (circles), CD8+ cells (upright triangles), CD4+ cells (delta triangles), and Treg cells (squares) showing STAT5 phosphorylation after stimulation with titrated scFv fusion proteins (solid lines, filled shapes) and human IL-2 control at 1 nM (dotted line) are provided. A provides Neo-2 / 15 fused to anti-CD25 ScFV, and B provides Neo-2 / 15_D15S_Q95E fused to anti-CD25 ScFv. [Figure 5]Graphs of STAT5 signaling for Neo-2 / 15 mutant D15S_Q95E unfused (A) and fused (B) to anti-CD25 ScFv are provided. The percentages of CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) showing STAT5 phosphorylation after stimulation with a titration of test samples are provided. [Figure 6A] 1 provides a graph of STAT5 signaling for Neo-2 / 15 mutant D15S_Q95E fused to anti-CD25 ScFv. The percentage of total T cells (diamonds), CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) showing STAT5 phosphorylation after stimulation with a titration of test samples is provided. [Figure 6B] 1 provides a graph of STAT5 signaling for the Neo-2 / 15 mutant D15S_Q95E fused at the C-terminus to a full-length antibody. The percentage of total T cells (diamonds), CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) showing STAT5 phosphorylation after stimulation with a titration of test samples is provided. [Figure 6C] 1 provides a graph of STAT5 signaling for the Neo-2 / 15 mutant D15S_Q95E fused at the N-terminus to a full-length antibody. The percentage of total T cells (diamonds), CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) showing STAT5 phosphorylation after stimulation with a titration of test samples is provided. [Figure 7A] Graphs of STAT5 signaling of Neo-2 / 15 variants fused to the C-terminus of the heavy chain of an anti-CD25 antibody (shown in FIG. 2C) are provided. Percentages of CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) showing STAT5 phosphorylation after stimulation with titrations of scFv fusion proteins (solid lines, filled shapes) and human IL-2 (dotted lines, open shapes) are provided. Fusion proteins with Neo-2 / 15_H11F_N40S_I44S are provided. [Figure 7B]Graphs of STAT5 signaling of Neo-2 / 15 mutants fused to the C-terminus of the heavy chain of an anti-CD25 antibody (shown in FIG. 2C) are provided. The percentages of CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) showing STAT5 phosphorylation after stimulation with titrations of scFv fusion proteins (solid lines, filled shapes) and human IL-2 (dotted lines, open shapes) are provided. Fusion proteins with Neo-2 / 15_Y14K_D15S are provided. [Figure 7C] Graphs of STAT5 signaling of Neo-2 / 15 variants fused to the C-terminus of the heavy chain of an anti-CD25 antibody (shown in FIG. 2C) are provided. The percentages of CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) showing STAT5 phosphorylation after stimulation with titrations of scFv fusion proteins (solid lines, filled shapes) and human IL-2 (dotted lines, open shapes) are provided. Fusion proteins with Neo-2 / 15_N40S_Q95E are provided. [Figure 7D] Graphs of STAT5 signaling of Neo-2 / 15 variants fused to the C-terminus of the heavy chain of an anti-CD25 antibody (shown in FIG. 2C) are provided. Percentages of CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) showing STAT5 phosphorylation after stimulation with titrations of scFv fusion proteins (solid lines, filled shapes) and human IL-2 (dotted lines, open shapes) are provided. Fusion proteins with Neo-2 / 15_D15S_Q95E are provided. [Figure 8A]Figure 2C provides graphs of STAT5 signaling of Neo-2 / 15 variants fused to the C-terminus of the heavy chain of an anti-CD25 antibody (shown in Figure 2C). The percentages of CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) showing STAT5 phosphorylation after stimulation with a titration of scFv fusion protein (solid line, filled shapes) and human IL-2 (dotted line, open shapes) are provided. This experiment repeats the experiment of Figure 7 using a different patient sample. Figure 7A provides fusion proteins with Neo-2 / 15_H11F_N40S_I44S, 7B provides fusion proteins with Neo-2 / 15_Y14K_D15S, 7C provides fusion proteins with Neo-2 / 15_N40S_Q95E, and 7D provides fusion proteins with Neo-2 / 15_D15S_Q95E. [Figure 8B] Figure 2C provides graphs of STAT5 signaling of Neo-2 / 15 variants fused to the C-terminus of the heavy chain of an anti-CD25 antibody (shown in Figure 2C). The percentages of CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) showing STAT5 phosphorylation after stimulation with a titration of scFv fusion protein (solid line, filled shapes) and human IL-2 (dotted line, open shapes) are provided. This experiment repeats the experiment of Figure 7 using a different patient sample. Figure 7A provides fusion proteins with Neo-2 / 15_H11F_N40S_I44S, 7B provides fusion proteins with Neo-2 / 15_Y14K_D15S, 7C provides fusion proteins with Neo-2 / 15_N40S_Q95E, and 7D provides fusion proteins with Neo-2 / 15_D15S_Q95E. [Figure 8C]Figure 2C provides graphs of STAT5 signaling of Neo-2 / 15 variants fused to the C-terminus of the heavy chain of an anti-CD25 antibody (shown in Figure 2C). The percentages of CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) showing STAT5 phosphorylation after stimulation with a titration of scFv fusion protein (solid line, filled shapes) and human IL-2 (dotted line, open shapes) are provided. This experiment repeats the experiment of Figure 7 using a different patient sample. Figure 7A provides fusion proteins with Neo-2 / 15_H11F_N40S_I44S, 7B provides fusion proteins with Neo-2 / 15_Y14K_D15S, 7C provides fusion proteins with Neo-2 / 15_N40S_Q95E, and 7D provides fusion proteins with Neo-2 / 15_D15S_Q95E. [Figure 8D] Figure 2C provides graphs of STAT5 signaling of Neo-2 / 15 variants fused to the C-terminus of the heavy chain of an anti-CD25 antibody (shown in Figure 2C). The percentages of CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) showing STAT5 phosphorylation after stimulation with a titration of scFv fusion protein (solid line, filled shapes) and human IL-2 (dotted line, open shapes) are provided. This experiment repeats the experiment of Figure 7 using a different patient sample. Figure 7A provides fusion proteins with Neo-2 / 15_H11F_N40S_I44S, 7B provides fusion proteins with Neo-2 / 15_Y14K_D15S, 7C provides fusion proteins with Neo-2 / 15_N40S_Q95E, and 7D provides fusion proteins with Neo-2 / 15_D15S_Q95E. [Figure 9] Graphs of STAT5 signaling of Neo-2 / 15_N40S_I44S_Q95E fused to the N-terminus of the heavy chain of the anti-CD25 antibody (shown in FIG. 2C) are provided for two different patient samples (A, B). The percentages of CD8+ (triangles), CD4+ (squares), and Treg cells (circles) showing STAT5 phosphorylation after stimulation with titrations of scFv fusion protein (solid line, filled shapes) and human IL-2 (dotted line, open shapes) are provided. [Figure 10A]This figure provides a graph showing Treg expansion in an in vivo mouse study. Mice were transplanted with human CD34+ hematopoietic stem cells and had greater than 25% human CD45+ cells in their peripheral blood. On days 1 and 13, mice were injected intraperitoneally with 5 mg of human IgG. On days 0, 7, 14, and 21, mice were injected intraperitoneally with 15-45 μg of test substance. On days -1, 6, 13, and 20, 100 μL of whole blood was collected by retroorbital bleeding into K2EDTA. The figure shows Treg expansion over time, measured as the ratio of CD4+ cells stimulated by the test substance at the indicated dose levels. The distal fusion TRA is Neo-2 / 15_D15S_Q95E fused to the C-terminus of the heavy chain of an anti-CD25 antibody. The proximal fusion TRA is Neo-2 / 15_N40S_I44S_Q95E fused to the N-terminus of the heavy chain of an anti-CD25 antibody. [Figure 10B] This figure provides a graph showing Treg expansion in an in vivo mouse study. Mice were transplanted with human CD34+ hematopoietic stem cells and had greater than 25% human CD45+ cells in their peripheral blood. On days 1 and 13, mice were injected intraperitoneally with 5 mg of human IgG. On days 0, 7, 14, and 21, mice were injected intraperitoneally with 15-45 μg of test substance. On days -1, 6, 13, and 20, 100 μL of whole blood was collected by retroorbital bleeding into K2EDTA. This figure shows Treg expansion over time, measured as the ratio of CD4+ cells stimulated by test substance at the indicated dose level. Test substances were vehicle control (open circles), a distal fusion Treg agonist (TRA; open squares), and a proximal fusion TRA. The distal fusion TRA is Neo-2 / 15_D15S_Q95E fused to the C-terminus of the heavy chain of an anti-CD25 antibody. The proximal fusion TRA is Neo-2 / 15_N40S_I44S_Q95E fused to the N-terminus of the heavy chain of an anti-CD25 antibody. [Figure 10C]This figure provides a graph showing Treg expansion in an in vivo mouse study. Mice were transplanted with human CD34+ hematopoietic stem cells and had more than 25% human CD45+ cells in their peripheral blood. On days 1 and 13, mice were intraperitoneally injected with 5 mg of human IgG. On days 0, 7, 14, and 21, mice were intraperitoneally injected with 15-45 μg of test substance. On days -1, 6, 13, and 20, 100 μL of whole blood was collected by retroorbital bleeding into K2EDTA. NK cell expansion over time upon stimulation with test substance at the indicated dose levels is shown. Test substances were a vehicle control (open circle), a distal fusion Treg agonist (TRA; open square), and a proximal fusion TRA. The distal fusion TRA was Neo-2 / 15_D15S_Q95E fused to the C-terminus of the heavy chain of an anti-CD25 antibody. The proximal fusion TRA is Neo-2 / 15_N40S_I44S_Q95E fused to the N-terminus of the heavy chain of an anti-CD25 antibody. [Figure 10D]

[0039] Figure 1 provides a graph showing Treg expansion in an in vivo mouse study. Mice were transplanted with human CD34+ hematopoietic stem cells and had greater than 25% human CD45+ cells in the peripheral blood. On days 1 and 13, mice were injected intraperitoneally with 5 mg of human IgG. On days 0, 7, 14, and 21, mice were injected intraperitoneally with 15-45 μg of test article. On days -1, 6, 13, and 20, 100 μL of whole blood was collected by retroorbital bleeding into K2EDTA. Treg Foxp3 MFI over time upon stimulation with test articles at the indicated dose levels is shown. Test articles were vehicle control (open circles), distal fusion Treg agonist (TRA; open squares), and proximal fusion TRA. The distal fusion TRA is Neo-2 / 15_D15S_Q95E fused to the C-terminus of the heavy chain of an anti-CD25 antibody. The proximal fusion TRA is Neo-2 / 15_N40S_I44S_Q95E fused to the N-terminus of the heavy chain of an anti-CD25 antibody. [Figure 10E]

[0039] Figure 1 provides a graph showing Treg expansion in an in vivo mouse study. Mice were transplanted with human CD34+ hematopoietic stem cells and had greater than 25% human CD45+ cells in the peripheral blood. On days 1 and 13, mice were injected intraperitoneally with 5 mg of human IgG. On days 0, 7, 14, and 21, mice were injected intraperitoneally with 15-45 μg of test article. On days -1, 6, 13, and 20, 100 μL of whole blood was collected by retroorbital bleeding into K2EDTA. Treg Foxp3 MFI over time upon stimulation with test articles at the indicated dose levels is shown. Test articles were vehicle control (open circles), distal fusion Treg agonist (TRA; open squares), and proximal fusion TRA. The distal fusion TRA is Neo-2 / 15_D15S_Q95E fused to the C-terminus of the heavy chain of an anti-CD25 antibody. The proximal fusion TRA is Neo-2 / 15_N40S_I44S_Q95E fused to the N-terminus of the heavy chain of an anti-CD25 antibody. [Figure 11A] 1 provides a graph of STAT5 signaling for the C-terminally fused Neo-2 / 15 fusion protein D15S_Q95K. pSTAT5 mean fluorescence index (MFI) is provided for CD8+ cells (circles), CD4+ cells (squares), and Treg cells (triangles). [Figure 11B] 1 provides a graph of STAT5 signaling for the C-terminally fused Neo-2 / 15 fusion protein D15S_Q95T. pSTAT5 mean fluorescence index (MFI) is provided for CD8+ cells (circles), CD4+ cells (squares), and Treg cells (triangles). [Figure 11C] 1 provides a graph of STAT5 signaling for the C-terminally fused Neo-2 / 15 fusion protein D15S_Q95Y. pSTAT5 mean fluorescence index (MFI) is provided for CD8+ cells (circles), CD4+ cells (squares), and Treg cells (triangles). [Figure 11D]1 provides a graph of STAT5 signaling for the C-terminally fused Neo-2 / 15 fusion protein N40G_I44S_Q95E. pSTAT5 mean fluorescence index (MFI) is provided for CD8+ cells (circles), CD4+ cells (squares), and Treg cells (triangles). [Figure 11E] 1 provides a graph of STAT5 signaling for the C-terminally fused Neo-2 / 15 fusion protein N40S_I44T_Q95E. pSTAT5 mean fluorescence index (MFI) is provided for CD8+ cells (circles), CD4+ cells (squares), and Treg cells (triangles). [Figure 11F] 1 provides a graph of STAT5 signaling for the C-terminally fused Neo-2 / 15 fusion protein N40S_I44Y_Q95E. pSTAT5 mean fluorescence index (MFI) is provided for CD8+ cells (circles), CD4+ cells (squares), and Treg cells (triangles). [Figure 11G] 1 provides a graph of STAT5 signaling for the C-terminally fused Neo-2 / 15 fusion protein N40S_I44N_Q95E. pSTAT5 mean fluorescence index (MFI) is provided for CD8+ cells (circles), CD4+ cells (squares), and Treg cells (triangles). DETAILED DESCRIPTION OF THE INVENTION

[0013] Neo-2 / 15 is a computationally designed, ultrastable, IL-2Rα-independent agonist of the IL-2 and IL-15 receptors, which share the IL-2R beta (IL-2Rβ or CD122) and IL-2R gamma (IL-2Rγ or CD132) signaling subunits. Neo-2 / 15 has been assigned the CAS registry number 2407798-79-0. NL-201 was developed from Neo-2 / 15 by introducing a cysteine residue at position 62 for site-specific conjugation of an unbranched 40 kDa polyethylene glycol (PEG) molecule and is being developed as a potent activator of CD8+ T cells, CD4+ T cells, and natural killer (NK) cells for cancer immunotherapy.

[0014] The present inventors have surprisingly discovered that certain Neo-2 / 15 mutants can preferentially stimulate and expand T regulatory cells (Tregs) compared to non-Tregs when targeting them. In particular, selective amino acids in Neo-2 / 15 are mutated to activate the intermediate-affinity IL-2 receptor (IL-2R). [ka] By attenuating binding to IL-2Rβ and conjugating the resulting Neo-2 / 15 mutant to a Treg-targeting agent, we have generated potent, selective T regulatory cell agonists, also referred to herein as TRAs. Unlike other IL-2-based Treg agonists, these TRAs consist of at least two distinct components: a Treg targeting domain and an attenuated Neo-2 / 15 mutant. The Treg binding site is linked to IL-2Rβ and [ka] By separating it from the binding site, the orientation and proximity to the binding site can be manipulated and optimized.

[0015] Regulatory T cells (Treg) Regulatory T cells (Tregs) are naturally occurring CD4+CD25+FOXP3+ T lymphocytes that comprise approximately 5-10% of the circulating CD4+ T cell population and predominantly suppress autoreactive lymphocytes, controlling innate and adaptive immune responses. Tregs achieve this suppression, at least in part, by inhibiting the proliferation, expansion, and effector activity of T effector cells (Teffs). While Foxp3 is an accepted marker for Treg cells, using the Foxp3 marker to isolate cells for functional studies has been challenging. CD4(+)CD25(+)CD127(low / -) cells have been shown to express the highest levels of Foxp3 and to have the strongest correlation with CD4(+)CD25(+)Foxp3(+) T cells (see Yu et al., Inflammation, 2021 Dec;35(6):1773-80). The present inventors use CD4(+)CD25(+)CD127(low / -) as the distinguishing characteristic of Tregs. Teffs are conventional T cells that have effector functions (e.g., cytokine secretion, cytotoxic activity, etc.) to enhance immune responses by expressing one or more T cell receptors. For the purposes of this invention, Teffs are defined as CD4+ and CD8+ T cells that are not Tregs.

[0016] Increasing the number of Tregs, increasing Treg activity, and / or reducing Treg cell death (e.g., apoptosis) is known to be useful in suppressing unwanted immune responses associated with a range of immune disorders and inflammation. Treatment with Treg agonists ideally preferentially enhances Tregs while minimizing or not activating Teffs or other cells that may exacerbate inflammation. The examples and teachings provided herein demonstrate the surprising and unexpected result that polypeptides comprising targeted Neo-2 / 15 mutants can be used to selectively and potently activate Tregs over Teffs, demonstrating that the polypeptides can be used to treat or ameliorate diseases and conditions that would benefit from suppressed immune responses, such as autoimmune diseases and diseases and conditions associated with inflammation.

[0017] Treg agonist (TRA) Provided herein, among other things, are TRAs that preferentially stimulate Treg cells. As used herein, "preferentially stimulates T regulatory cells" means that the TRA promotes the proliferation, survival, activation, and / or function of Tregs over non-Tregs. In some aspects, the TRA preferentially stimulates Tregs compared to Teff or NK cells. In some embodiments, the TRA comprises at least one Neo-2 / 15 variant provided herein linked to a targeting agent that binds to an antigen on the surface of Treg cells.

[0018] A method for measuring the ability to preferentially stimulate Tregs can be measured by flow cytometry of peripheral blood leukocytes, where an increased percentage of Tregs among all CD4+ T cells, an increased percentage of Tregs among all CD8+ T cells, an increased percentage of Tregs compared to NK cells, and / or a greater increase in the level of CD25 expression on the surface of Tregs compared to increased CD25 expression on other T cells is observed.

[0019] In some embodiments, TRAs that preferentially stimulate Treg cells increase the percentage of Tregs among total CD4+ T cells in a subject or peripheral blood sample by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, or at least 300%. In some aspects, potent Treg expansion is measured by at least a two-fold higher percentage of Tregs in the total CD4+ cell population upon treatment with a TRA compared to a vehicle control.

[0020] In some embodiments, methods of identifying TRAs of the invention include measuring the ability of the TRA to promote or stimulate STAT5 phosphorylation in Tregs relative to Teffs. Exemplary TRAs will have a significantly reduced ability to promote or stimulate STAT5 phosphorylation in Teffs relative to Neo-2 / 15 and / or IL-2, while maintaining the ability to promote or stimulate STAT5 phosphorylation in Tregs.

[0021] In some embodiments, exemplary TRAs of the present invention do not substantially activate Teffs or induce Teff proliferation. In some aspects, Teff activation or proliferation is measured using a STAT5 assay described herein. In some embodiments, TRAs preferably have maximal signaling in Teff cells that is less than 50%, less than 40%, less than 30%, or less than 20% of the maximal signaling resulting from IL-2 stimulation (e.g., at concentrations up to 10 nM). However, maximal signaling in Tregs is preferably at least 40%, at least 50%, at least 60%, or at least 70% of the maximal signaling resulting from IL-2 stimulation, with higher maximal Treg signaling indicating a more potent TRA. In some preferred embodiments, the EC50 of the Treg% pSTAT5+ curve should be less than 10 nM, less than 1 nM, less than 500 pM, less than 250 pM, or less than 100 pM, with lower Treg EC50s also indicating more potent TRAs. In some embodiments, the EC50 of the Treg% pSTAT5+ curve of the TRAs provided herein is below 1 nM and the maximal signaling in Tregs is greater than 50% of the maximal signaling resulting from IL-2 stimulation.

[0022] Polypeptides The present invention provides, inter alia, Neo-2 / 15 variants. Exemplary Neo-2 / 15 variants have, relative to Neo-2 / 15: [ka] The Neo-2 / 15 variants have attenuated binding to CD25. Such Neo-2 / 15 variants are optionally linked (e.g., by fusion or by chemical / enzymatic conjugation) to a targeting agent that binds to an antigen on the surface of Treg cells. Exemplary antigens for use in the present invention are CD25 or CD39. In an exemplary embodiment of the present invention, a polypeptide comprising a Neo-2 / 15 variant linked to a targeting agent that binds to an antigen on the surface of Treg cells is a Treg agonist (TRA). As used herein, when two polypeptides are "fused," it means that they are produced (e.g., translated) as a single, continuous polypeptide.

[0023] In some embodiments, the Neo-2 / 15 variant comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% identity to Neo-2 / 15 and one or more amino acid substitutions that reduce binding to IL-2Rβ, and / or [ka] In some embodiments, the Neo-2 / 15 variant has one or more amino acid substitutions that reduce binding to Neo-2 / 15. In some embodiments, the Neo-2 / 15 variant has an amino acid sequence that has at least 80%, at least 85%, at least 90%, or at least 95% identity to Neo-2 / 15, compared to Neo-2 / 15. [ka] and 1 to 5 amino acid substitutions that reduce binding to

[0024] In some embodiments, polypeptides of the invention include Neo-2 / 15 variants, which comprise an amino acid sequence at least 79%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% identical to SEQ ID NO:1 (truncated Neo-2 / 15 lacking the three N-terminal amino acids) or SEQ ID NO:2 (Neo-2 / 15). Such Neo-2 / 15 variants contain 2 to 20, 2 to 15, 2 to 10, 2 to 5, 2 to 4, or 2 to 3 amino acid substitutions compared to Neo-2 / 15. The amino acid sequences of exemplary Neo-2 / 15 variants are set forth in SEQ ID NOs:3-25 and 75-82. Particularly preferred are the amino acid sequences set forth in SEQ ID NO: 3 or 4, which contain Q95E and D15S substitutions, or SEQ ID NO: 6 or 7, which contain Q95E, N40S, and I44s substitutions, or SEQ ID NO: 80 or 81, which contain N40S, I44Y, and Q95E substitutions. While it may be advantageous to reduce the number of additional mutations, the present invention includes Neo-2 / 15 variants with truncations or additional insertions, deletions, or substitutions in addition to those described herein, provided that the Neo-2 / 15 variants maintain the activity of preferentially stimulating Tregs when targeted to Treg cells (e.g., by fusion to an anti-CD25 antibody in the manner described herein). To the extent that there are additions or deletions in the Neo-2 / 15 sequence, such additions or deletions preferably are not within the D1, D3, or D4 domains of the Neo-2 / 15 variant (i.e., not within amino acids 4-22, 34-55, and 80-100, numbered according to SEQ ID NO: 2). In some embodiments, as long as there are additional substitutions in Neo-2 / 15 (substitutions other than those at positions 8, 11, 14, 15, 40, 44, or 95 numbered according to SEQ ID NO:2), there are no more than 1-10, no more than 1-5, or no more than 1-3 such mutations within the amino acid sequence of the D1, D3, or D4 domain.

[0025] The invention includes polypeptides comprising Neo-2 / 15 variants, wherein the Neo-2 / 15 variants comprise an amino acid sequence at least 80%, at least 85%, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% identical to SEQ ID NO:1 or SEQ ID NO:2, wherein the Neo-2 / 15 variants have a lysine, arginine, threonine, serine, tyrosine, glutamic acid, alanine, or histidine in place of glutamine at position 95 (Q95K or Q95R or Q95T or Q95S or Q95Y or Q95E or Q95A or Q95H); a) serine or alanine instead of aspartic acid at position 15 (D15S or D15A); b) alanine or serine or glycine instead of asparagine at position 40 (N40A or N40S or N40G), and c) at least one additional substitution selected from alanine or serine or asparagine or threonine or tyrosine for isoleucine at position 44 (I44A or I44S or I44N or I44T or I44Y), Polypeptides are provided in which the first amino acid of SEQ ID NO:1 is designated as position 4 and the first amino acid of SEQ ID NO:2 is designated as position 1.

[0026] The invention includes polypeptides comprising Neo-2 / 15 variants, wherein the Neo-2 / 15 variants comprise an amino acid sequence at least 80%, at least 85%, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% identical to SEQ ID NO:1 or SEQ ID NO:2, wherein the Neo-2 / 15 variants comprise a glutamic acid, alanine, or histidine in place of glutamine at position 95 (Q95E or Q95A or Q95H); a) serine or alanine instead of aspartic acid at position 15 (D15S or D15A); b) alanine or serine instead of asparagine at position 40 (N40A or N40S), and c) at least one additional substitution selected from alanine or serine for isoleucine at position 44 (I44A or I44S), Polypeptides are provided in which the first amino acid of SEQ ID NO:1 is designated as position 4 and the first amino acid of SEQ ID NO:2 is designated as position 1.

[0027] In some embodiments, the Neo2 / 15 variants are Q95E and D15S; Q95E and D15A; Q95A and D15S; Q95A and D15A; Q95H and D15S; Q95H and D15A; Q95E, N40S and I44S; Q95E, N40A and I44A; Q95E, N40A and I44S; Q95A, N40S and I44S; Q95A, N40A and I44A; Q95A, N40S and I44A and N40S, I44N, and Q95E.

[0028] In some such aspects, in addition to the above substitutions, an additional 1 to 18, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 substitutions are made. In some aspects, no other substitutions are made. Exemplary additional substitutions include, for example, phenylalanine for histidine at position 11 (H11F), lysine for tyrosine at position 14 (Y14K), and arginine for histidine at position 8 (H8R). In some embodiments, positions 8, 11, and 14 are unsubstituted. In some aspects, the Neo-2 / 15 variant contains an alanine for aspartic acid at position 15. In some such aspects, the Neo-2 / 15 variant contains an arginine for leucine at position 13 and / or a glutamic acid for leucine at position 17. In some embodiments, when a Neo-2 / 15 variant comprises serine or alanine in place of aspartic acid at position 15, the Neo-2 / 15 variant (i) does not comprise a serine or alanine substitution for asparagine at position 40, or the Neo-2 / 15 variant (ii) does not comprise a serine or alanine substitution for isoleucine at position 44. In some embodiments, when a Neo-2 / 15 variant comprises serine or alanine in place of aspartic acid at position 15, the Neo-2 / 15 variant (i) does not comprise a serine, alanine, or glycine substitution for asparagine at position 40, and / or the Neo-2 / 15 variant (ii) does not comprise a serine, alanine, asparagine, threonine, or tyrosine substitution for isoleucine at position 44. In some embodiments, when a Neo-2 / 15 variant comprises a serine or alanine in place of aspartic acid at position 15, the Neo-2 / 15 variant does not comprise (i) a substitution at position 40 or (ii) a substitution at position 44. In some embodiments, when a Neo-2 / 15 variant comprises a serine or alanine in place of aspartic acid at position 15, the Neo-2 / 15 variant does not comprise (i) a serine or alanine substitution for asparagine at position 40, and the Neo-2 / 15 variant does not comprise (ii) a serine or alanine substitution for isoleucine at position 44.In some embodiments, when a Neo-2 / 15 variant comprises serine or alanine in place of aspartic acid at position 15, the Neo-2 / 15 variant does not comprise (i) a substitution at position 40 and (ii) a substitution at position 44. In some embodiments, when a Neo-2 / 15 variant comprises alanine or serine in place of asparagine at position 40 and / or alanine or serine in place of isoleucine at position 44, the Neo-2 / 15 variant comprises an acidic amino acid at position 15.

[0029] In some embodiments, the Neo-2 / 15 variant comprises an alanine or serine or asparagine or threonine or tyrosine in place of the isoleucine at position 44 (I44A or I44S or I44N or I44T or I44Y). In some embodiments, the Neo-2 / 15 variant comprises an alanine or serine or glycine in place of the asparagine at position 40 (N40A or N40S or N40G). In some embodiments, the Neo-2 / 15 variant comprises a set of substitutions selected from: Q95E and D15S; Q95E, D15S, and H11F; Q95E, N40S, and I44S; Q95E, N40S, I44S, and H11F; Q95E, N40S, I44S, Q95H, D15A, L17E, and L13R; and Y14K; Q95E, D15S, Y14K; Q95H and D15A; and Q95A and D15A. In some aspects, the Neo-2 / 15 variant comprises additional substitutions in addition to those described herein at positions 8, 11, 14, 15, 40, 44, and 95. In some embodiments, the Neo-2 / 15 variant does not contain additional substituents beyond those described herein at positions 8, 11, 14, 15, 40, 44, and 95.

[0030] The invention includes polypeptides comprising a Neo-2 / 15 variant, wherein the polypeptide has an amino acid sequence at least 80%, at least 85%, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% identical to SEQ ID NO:1 or SEQ ID NO:2 (wherein the Neo-2 / 15 variant contains serine or alanine instead of aspartic acid at position 15 (D15S or D15A)); (a) lysine, arginine, threonine, serine, tyrosine, glutamic acid, alanine, or histidine in place of glutamine at position 95 (Q95K or Q95R or Q95T or Q95S or Q95Y or Q95E or Q95A or Q95H); b) arginine instead of histidine at position 8 (H8R); c) phenylalanine instead of histidine at position 11 (H11F); d) lysine instead of tyrosine at position 14 (Y14K); e) alanine or serine instead of asparagine at position 40 (N40A or N40S), and f) at least one additional substitution selected from alanine or serine or tyrosine for isoleucine at position 44 (I44A or I44S or I44Y), The first amino acid of SEQ ID NO:1 is designated as position 4, and the first amino acid of SEQ ID NO:2 is designated as position 1. In some such aspects, in addition to those listed above, an additional 1 to 18, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 substitutions are made. In some aspects, no other substitutions are made. In some such exemplary embodiments, if a Neo-2 / 15 variant contains a substitution at position 95, the Neo-2 / 15 variant does not contain a substitution at positions 40 and / or 44. In some such exemplary embodiments, if a Neo-2 / 15 variant contains a substitution at positions 40 or 44, the Neo-2 / 15 variant does not contain a substitution at positions 95, 11, or 8. In some such exemplary embodiments, if a Neo-2 / 15 variant contains a serine or alanine instead of the aspartic acid at position 15, the Neo-2 / 15 variant does not contain a substitution at position 14. In some exemplary embodiments, if the polypeptide comprises a serine or alanine in place of aspartic acid at position 15, the polypeptide does not comprise a lysine substitution for tyrosine at position 14. In some such exemplary embodiments, if the polypeptide comprises a substitution at position 95, the polypeptide does not comprise a substitution at position 8. In some such exemplary embodiments, if the polypeptide comprises a glutamic acid in place of glutamine at position 15, the polypeptide does not comprise an arginine substitution for histidine at position 8.

[0031] The invention includes polypeptides comprising a Neo-2 / 15 variant, wherein the polypeptide has an amino acid sequence at least 80%, at least 85%, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% identical to SEQ ID NO:1 or SEQ ID NO:2 (wherein the Neo-2 / 15 variant contains serine or alanine instead of aspartic acid at position 15 (D15S or D15A)); g) glutamic acid, alanine, or histidine in place of glutamine at position 95 (Q95E, Q95A, or Q95H); h) arginine instead of histidine at position 8 (H8R); i) phenylalanine instead of histidine at position 11 (H11F); j) lysine instead of tyrosine at position 14 (Y14K); k) alanine or serine instead of asparagine at position 40 (N40A or N40S), and l) at least one additional substitution selected from alanine or serine for isoleucine at position 44 (I44A or I44S), The first amino acid of SEQ ID NO:1 is designated as position 4, and the first amino acid of SEQ ID NO:2 is designated as position 1. A polypeptide can include a set of substitutions selected from, for example, D15S and Y14K; N40S, I44S, and D15S; N40S, I44S, Y14K, and D15S; N40S and D15S; H8R and D15S; and H11F and D15S. In some such aspects, in addition to the substitutions described above, an additional 1-18, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 substitutions are made. In some aspects, no other substitutions are made. In some such exemplary embodiments, if a Neo-2 / 15 variant includes a substitution at position 95, the Neo-2 / 15 variant does not include substitutions at positions 40 and / or 44. In some such exemplary embodiments, when Neo-2 / 15 comprises a substitution at position 40 or 44, the Neo-2 / 15 does not comprise a substitution at position 95, 11, or 8. In some such exemplary embodiments, when a Neo-2 / 15 variant comprises a serine or alanine in place of aspartic acid at position 15, the Neo-2 / 15 variant does not comprise a substitution at position 14. In some exemplary embodiments, when a polypeptide comprises a serine or alanine in place of aspartic acid at position 15, the polypeptide does not comprise a lysine substitution for tyrosine at position 14. In some such exemplary embodiments, when a polypeptide comprises a substitution at position 95, the polypeptide does not comprise a substitution at position 8. In some such exemplary embodiments, when a polypeptide comprises a glutamic acid in place of glutamine at position 15, the polypeptide does not comprise an arginine substitution for histidine at position 8.

[0032] The invention includes polypeptides comprising a Neo-2 / 15 variant, wherein the polypeptide comprises an amino acid sequence at least 80%, at least 85%, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% identical to SEQ ID NO:1 or SEQ ID NO:2, wherein the polypeptide is a) phenylalanine instead of histidine at position 11 (H11F); b) alanine or serine instead of asparagine at position 40 (N40A or N40S), and c) containing alanine or serine or tyrosine instead of isoleucine at position 44 (I44A or I44S or I44Y), The first amino acid of SEQ ID NO:1 is designated as position 4, and the first amino acid of SEQ ID NO:2 is designated as position 1. Neo-2 / 15 variants can include a set of substitutions selected from N40S, I44S, and H11F. In some aspects, Neo-2 / 15 variants include additional substitutions in addition to those described herein at positions 11, 40, and 44. In some such embodiments, Neo-2 / 15 variants do not include substitutions at one, two, three, or all four of positions 8, 14, 15, and 95, and any combination thereof. In some aspects, the polypeptide includes a phenylalanine (H11F) in place of the histidine at position 11, an alanine or serine (N40A or N40S) in place of the asparagine at position 40, and an alanine or serine (I44A or I44S) in place of the isoleucine at position 44.

[0033] In some exemplary embodiments, substitutions do not occur at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or all 23 of positions 6, 7, 8, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 of positions 6, 7, 8, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some exemplary embodiments, substitutions do not occur at one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirty, four, fifteen, six, seven, eight, nine, ten, eleven, twelve, thirty, four, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty of the following positions: 6, 7, 10, 13, 17, 18, 33, 36, 37, 39, 43, 47, 84, 85, 88, 91, 92, 96, 98, or 99. In some exemplary embodiments, substitutions do not occur at one, two, three, or four of the following positions: 91, 92, 96, and 99. In some exemplary embodiments, substitutions do not occur at one, two, three, four, or five of the following positions: 17, 91, 92, 96, and 99. In some exemplary embodiments, as long as the Neo-2 / 15 variant has a substitution at a position other than 8, 11, 14, 15, 40, 44, or 95, [ka] Substitutions that do not substantially interfere with binding of the Neo-2 / 15 mutant to the nucleotide sequence of the target gene are preferred.

[0034] In some exemplary embodiments, the Neo-2 / 15 variant comprises three amino acids N-terminal to and attached to the amino acid at position 4, wherein the amino acids are proline-lysine-lysine.

[0035] Neo-2 / 15 contains four helical domains in the order D1-D3-D2-D4. D1 is located at approximately amino acids 1-22 of Neo-2 / 15, D3 is located at approximately amino acids 33-55 of Neo-2 / 15, D2 is located at approximately amino acids 58-76 of Neo-2 / 15, and D4 is located at approximately amino acids 80-100 of Neo-2 / 15. While the exemplary Neo-2 / 15 variants have the same domain order as Neo-2 / 15, the present invention also encompasses Neo-2 / 15 variants that have undergone helical domain rearrangement. One skilled in the art will appreciate that domain rearrangements can be used to produce similarly attenuated polypeptides that can still be linked to a Treg targeting domain. In some embodiments, domains are rearranged by circular permutation, creating new N- and C-termini. Reordering the domains by circular permutation results in domain orders including D4-D1-D2-D3, D3-D4-D1-D2, and D2-D3-D4-D1. The linkers between the domains can be altered to accommodate the reordering. Exemplary Neo-2 / 15 mutants include an amino acid linker between one or more of the domains. Such linkers generally include: [ka] The linker is not involved in binding to the nucleotide sequence of the polypeptide, but rather functions to connect the four domains. There is a large degree of variation permitted in the length of the linker and the identity of the linker amino acids. In various embodiments, the linker can be of any length. In some aspects, the linker is 1 to 100 amino acids in length, e.g., 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 2 to 10, or 1 to 5 amino acids in length. Using teachings in the art (e.g., Silva et al., Nature, 2019 Jan;565(7738):186-191) in combination with the teachings herein, one of skill in the art can construct linkers for shifting and / or connecting domains while maintaining desirable properties of the polypeptide. Included are embodiments in which the domains are ordered as D1-D3-D2-D4, with a first linker between domains D1 and D3, a second linker between domains D3 and D2, and a third linker between D2 and D4. In some aspects, the first linker is 10 amino acids in length, the second linker is 2 amino acids in length, and the third linker is 3 amino acids in length. An exemplary sequence for the first linker is VKTNSPPAEE (SEQ ID NO: 67). An exemplary sequence for the second linker is DQ, and an exemplary sequence for the third linker is TAS (SEQ ID NO: 68).

[0036] In some embodiments, a polypeptide of the invention comprises a Neo-2 / 15 variant, wherein the Neo-2 / 15 variant comprises domains D1, D2, D3, and D4; (a) D1 has the amino acid sequence: KIQLHAEHALYX 15 ALMILNI (SEQ ID NO: 61), and D3 comprises the amino acid sequence LEDYAFNFELILEEIARLFESG (SEQ ID NO: 64), or (b) D1 comprises the amino acid sequence: KIQLHAEHALYDALMILNI (SEQ ID NO: 62) and D3 comprises the amino acid sequence LEDYAFX 40 FELX 44 LEEIARLFESG (SEQ ID NO: 65), D2 comprises an amino acid sequence at least 8 amino acids in length, D4 has the amino acid sequence EDEQEEMANAIITILX 95 SWIFS (SEQ ID NO: 66), wherein (i) D1, D2, D3, and D4 can be in any order in the polypeptide; (ii) an amino acid linker may be present between any of the domains (a "domain linker"); (iii)X 15 is serine or alanine; X 95 is glutamic acid, alanine, or histidine; X 40 is serine or alanine; X 44 is serine or alanine; or X 15 is serine or alanine; X 95 is glutamic acid, lysine, arginine, threonine, serine, tyrosine, alanine, or histidine; X 40 is serine, alanine, or glycine; X 44 is serine or alanine or asparagine or threonine or tyrosine; and the polypeptide contains a total of 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or fewer, or 0 substitutions at amino acid positions not designated as X. In some embodiments, X 40 is serine or alanine; X 44 is serine or alanine or tyrosine. 95 is glutamic acid, alanine, or histidine, and X 40 is serine or alanine, and X 44 is serine or alanine.

[0037] In some embodiments, D1 has the amino acid sequence: KIQLHAEHALYX 15ALMILNI (SEQ ID NO: 61), and D3 comprises the amino acid sequence LEDYAFNFELILEEIARLFESG (SEQ ID NO: 64), wherein the histidine at position 8 of SEQ ID NO: 61 is substituted. In some such embodiments, the substitution is phenylalanine.

[0038] In some embodiments, D1 has the amino acid sequence: KIQLHAEHALYX 15 ALMILNI (SEQ ID NO: 61), and D3 comprises the amino acid sequence LEDYAFNFELILEEIARLFESG (SEQ ID NO: 64), in which the histidine at position 5, the histidine at position 8, and the tyrosine at position 11 of SEQ ID NO: 61, and the asparagine at position 7 and the isoleucine at position 11 of SEQ ID NO: 64 are not substituted.

[0039] In some embodiments, D1 comprises the amino acid sequence: KIQLHAEHALYDALMILNI (SEQ ID NO: 62) and D3 comprises the amino acid sequence LEDYAFX 40 FELX 44 In some such embodiments, X 40 and X 44 At least one or both of are serine. In some embodiments, the histidine at position 8 of SEQ ID NO:62 is optionally substituted. The optional substitution may be, for example, phenylalanine. In some embodiments, the histidine at position 11 of SEQ ID NO:62 is optionally substituted. The optional substitution may be, for example, lysine. In some embodiments, the histidine at position 5, the histidine at position 8, and the tyrosine at position 11 of SEQ ID NO:62 are unsubstituted. In some embodiments, the aspartic acid at position 12 of SEQ ID NO:62 is unsubstituted.

[0040] In some embodiments, D1 comprises the amino acid sequence set forth in SEQ ID NO:61 and D3 comprises the amino acid sequence set forth in SEQ ID NO:64, and the Neo-2 / 15 variant does not contain a substitution at glutamine at position 3, leucine at position 4, glutamic acid at position 7, leucine at position 10, and methionine at position 15 of SEQ ID NO:61, and at aspartic acid at position 3, tyrosine at position 4, phenylalanine at position 6, leucine at position 10, and glutamic acid at position 14 of SEQ ID NO:64. In some embodiments, the Neo-2 / 15 variant also does not contain a substitution at leucine at position 14 of SEQ ID NO:61.

[0041] In some embodiments, D1 comprises the amino acid sequence set forth in SEQ ID NO:62, and D3 comprises the amino acid sequence set forth in SEQ ID NO:65, wherein the polypeptide does not comprise a substitution at glutamine at position 3, leucine at position 4, glutamic acid at position 7, leucine at position 10, and methionine at position 15 of SEQ ID NO:62, and at aspartic acid at position 3, tyrosine at position 4, phenylalanine at position 6, leucine at position 10, and glutamic acid at position 14 of SEQ ID NO:65. In some embodiments, the Neo-2 / 15 variant also does not comprise a substitution at leucine at position 14 of SEQ ID NO:62.

[0042] The present invention includes Neo-2 / 15 variants in which glutamic acid at position 5, glutamic acid at position 6, asparagine at position 9, isoleucine at position 12, threonine at position 13, serine at position 17, isoleucine at position 19, and phenylalanine at position 20 of SEQ ID NO: 66 are not substituted. The present invention includes Neo-2 / 15 variants in which leucine at position 14 of SEQ ID NO: 61 or 62 and phenylalanine at position 20 of SEQ ID NO: 66 are not substituted. The present invention includes Neo-2 / 15 variants in which leucine at position 14 of SEQ ID NO: 61 or 62 and isoleucine at position 12, threonine at position 13, serine at position 17, isoleucine at position 19, and phenylalanine at position 20 of SEQ ID NO: 66 are not substituted.

[0043] As used herein, a "position" in a SEQ ID NO: refers to a consecutive position in the amino acid sequence identified by the SEQ ID NO:, unless otherwise indicated, and includes any X residue. For example, position 14 of SEQ ID NO: 65 has the sequence [ka] is glutamic acid (underlined). Notwithstanding the above, for certain amino acid sequences (such as SEQ ID NOS: 1, 3, 6, 14, 16, 20, and 81), the N-terminal amino acid may be designated as position 4. For amino acid sequences in which the N-terminal amino acid is designated as position 4, the consecutive numbering begins at position 4. For example, [ka] Position 95 of SEQ ID NO: 3, where the first amino acid is designated as position 4, is glutamic acid (underlined). Consecutive numbering begins at position 4 for a particular sequence solely for consistency of position numbering between sequences.

[0044] In any of these embodiments, D2 may comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, or 100% identity to KDEAEKAKRMKEWMKRIKT (SEQ ID NO: 63).

[0045] In any of these embodiments, the domain linker can independently be 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 2 to 10 amino acids.

[0046] In some embodiments, the order of the four domains is D1-D3-D2-D4, D4-D1-D3-D2, D2-D4-D1-D3, or D3-D2-D4-D1.

[0047] In some embodiments, the Neo-2 / 15 variant comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, or at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 3, 6, 14, 16, or 20. In some such aspects, D1 comprises three amino acids N-terminal to and attached to amino acid position 1 of either SEQ ID NO: 61 or 62. In some such aspects, the three amino acids are proline-lysine-lysine.

[0048] In some embodiments, the Neo-2 / 15 variant comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 2-25 or 75-82.

[0049] In some embodiments, the invention provides polypeptides comprising a Neo-2 / 15 variant, wherein the Neo-2 / 15 variant binds to IL-2Rβγ with an affinity that is at least 10-fold, or at least 100-fold, or at least 500-fold, or at least 1000-fold, or at least 10,000-fold diminished compared to Neo-2 / 15. In some embodiments, the Neo-2 / 15 variant binds to IL-2Rβγ with an affinity that is at least 10-fold, or at least 100-fold diminished compared to Neo-2 / 15, but not more than 500-fold diminished compared to Neo-2 / 15. In some embodiments, the Neo-2 / 15 variant binds to IL-2Rβγ with an affinity that is at least 100-fold or at least 500-fold diminished compared to Neo-2 / 15, but not more than 1000-fold diminished compared to Neo-2 / 15. In some embodiments, the Neo-2 / 15 variant binds to IL-2Rβγ with an affinity that is at least 100-fold, or at least 500-fold, or at least 1000-fold diminished compared to Neo-2 / 15, but not more than 10,000-fold diminished compared to Neo-2 / 15.

[0050] In some embodiments, the invention provides polypeptides comprising a Neo-2 / 15 variant, wherein the Neo-2 / 15 variant binds to IL-2Rβγ with an affinity that is at least 10-fold, or at least 100-fold, or at least 500-fold, or at least 1000-fold, or at least 10,000-fold diminished compared to IL-2. In some embodiments, the Neo-2 / 15 variant binds to IL-2Rβγ with an affinity that is at least 10-fold, or at least 100-fold diminished compared to IL-2, but no more than 500-fold diminished compared to IL-2. In some embodiments, the Neo-2 / 15 variant binds to IL-2Rβγ with an affinity that is at least 100-fold or at least 500-fold diminished compared to IL-2, but no more than 1000-fold diminished compared to IL-2. In some embodiments, the Neo-2 / 15 variant binds to IL-2Rβγ with an affinity that is at least 100-fold, or at least 500-fold, or at least 1000-fold attenuated compared to IL-2, but not more than 10,000-fold attenuated compared to IL-2.

[0051] In some embodiments, the invention provides polypeptides comprising a Neo-2 / 15 variant that binds to IL-2Rβγ with an affinity that is within 2-fold, 3-fold, 4-fold, or 5-fold of the affinity of a Neo-2 / 15 variant consisting of the sequence set forth for IL-2Rβγ in SEQ ID NO: 4. In some embodiments, the Neo-2 / 15 variant binds to IL-2Rβγ with an affinity that is within 2-fold, 3-fold, 4-fold, or 5-fold of the affinity of a reference polypeptide for IL-2Rβγ when fused to the N- and / or C-terminus of an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 73 and a light chain comprising the amino acid sequence of SEQ ID NO: 70, wherein the reference polypeptide comprises a Neo-2 / 15 variant fused to the C-terminus of the antibody, and wherein the reference polypeptide comprises the amino acid sequences of SEQ ID NOs: 69 and 70.

[0052] In some embodiments, the invention provides polypeptides comprising a Neo-2 / 15 variant, which stimulate STAT5 phosphorylation in Treg cells with an EC50 that is substantially the same as or less than a Neo-2 / 15 variant consisting of the sequence set forth in SEQ ID NO: 4. In some embodiments, the Neo-2 / 15 variant, when fused to the N- and / or C-terminus of an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 73 and a light chain comprising the amino acid sequence of SEQ ID NO: 70, stimulates STAT5 phosphorylation in Treg cells with an EC50 that is substantially the same as or less than a reference polypeptide comprising a Neo-2 / 15 variant fused to the C-terminus of the antibody, wherein the reference polypeptide comprises the amino acid sequences of SEQ ID NOs: 69 and 70.

[0053] In some embodiments, the invention provides polypeptides comprising a Neo-2 / 15 variant, which stimulate STAT5 phosphorylation in Treg cells at a maximum signaling level substantially equal to or greater than that of a Neo-2 / 15 variant consisting of the sequence set forth in SEQ ID NO: 4. In some embodiments, the Neo-2 / 15 variant, when fused to the N- and / or C-terminus of an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 73 and a light chain comprising the amino acid sequence of SEQ ID NO: 70, stimulates STAT5 phosphorylation in Treg cells at a maximum signaling level substantially equal to or greater than that of a reference polypeptide comprising a Neo-2 / 15 variant fused to the C-terminus of the antibody, wherein the reference polypeptide comprises the amino acid sequences of SEQ ID NOs: 69 and 70.

[0054] In some embodiments, the present invention provides a polypeptide comprising a Neo-2 / 15 variant, wherein the Neo-2 / 15 variant stimulates STAT5 phosphorylation in Treg cells at a maximal signaling level that is at least 50%, at least 75%, or at least 100% of the maximal signaling resulting from Neo-2 / 15 and / or IL-2 stimulation.

[0055] In some embodiments, the present invention provides a polypeptide comprising a Neo-2 / 15 variant, which, when fused to the N-terminus and / or C-terminus of an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 73 and a light chain comprising the amino acid sequence of SEQ ID NO: 70, stimulates STAT5 phosphorylation in Treg cells with an EC50 of 1 nM or less, 0.5 nM or less, or 0.1 nM or less, and / or stimulates STAT5 phosphorylation in Teff cells with an EC50 of 1 nM or more.

[0056] In some embodiments, the invention provides polypeptides comprising a Neo-2 / 15 variant, which stimulates NK cells with substantially the same or a higher EC50 as a Neo-2 / 15 variant consisting of the sequence set forth in SEQ ID NO: 4. In some embodiments, the Neo-2 / 15 variant, when fused to the N- and / or C-terminus of an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 73 and a light chain comprising the amino acid sequence of SEQ ID NO: 70, stimulates NK cells with substantially the same or a higher EC50 as a reference polypeptide comprising a Neo-2 / 15 variant fused to the C-terminus of the antibody, wherein the reference polypeptide comprises the amino acid sequences of SEQ ID NOs: 69 and 70.

[0057] The term "identity," as used herein with respect to polypeptide sequences, refers to subunit sequence identity between two molecules. If a subunit position in both molecules is occupied by the same monomer subunit (i.e., the same amino acid residue or nucleotide), then the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. Generally, sequences are aligned (including gaps, if necessary) to obtain the top match. Identity, in various embodiments, can be calculated using published techniques and widely available computer programs such as the GCG program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990). Sequence identity can be measured, for example, using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705) using default parameters. Unless otherwise specified, percent identity is determined over the entire length of the reference sequence.

[0058] Substitution, deletion, insertion, or any combination thereof may be used to arrive at the final derivative or variant. Generally, these changes are made to a few amino acids to minimize the change in the molecule, especially the specificity of the antigen-binding protein. However, in certain circumstances, larger changes may be tolerated.

[0059] In some embodiments, conservative amino acid substitutions are made when it is desirable to replace an amino acid, but the amino acid substitution does not significantly alter the protein profile. As used herein, "conservative amino acid substitution" means that a given amino acid can be replaced with an amino acid having similar physiochemical properties, such as replacing an acidic residue with another (such as E or D), replacing a basic amino acid with another (such as K, R, or H), replacing two amino acids with backbone-contorted side chains with another (such as G or P), replacing a hydrophobic amino acid with another (such as L, I, V, A, or M), replacing an aromatic hydrophobic amino acid with another (such as Y, F, or W), or replacing a polar amino acid with an uncharged side chain with another (such as T, S, N, Q, or C). Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to determine whether they maintain the desired activity. Amino acids can also be classified according to the similarity of their side chain properties. Alternatively, naturally occurring residues can be classified based on common side chain properties. Non-conservative substitutions involve exchanging a member of one of these classes for a member of another class. Specific conservative substitutions include, for example, those shown in Table 1. [Table 1]

[0060] As used herein, naturally occurring amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0061] Treg targeting domain The Neo-2 / 15 variants of the present invention can be coupled to a targeting agent directed at Tregs, i.e., a Treg targeting agent, to provide them with optimal ability to preferentially expand and activate T regulatory cells. Accordingly, the present invention provides a polypeptide comprising a Neo-2 / 15 variant and a targeting agent as described in any of the embodiments herein. In a particularly preferred embodiment, the targeting agent binds to an antigen on the surface of Treg cells. Binding is preferably specific binding. Exemplary antigens are CD25 or CD39.

[0062] The Treg targeting agent can be any agent that can direct the Neo-2 / 15 variant to T regulatory cells. In other words, the targeting agent can be any agent that can direct the cellular localization of the Neo-2 / 15 variant. The target of the targeting agent is typically a surface marker that is expressed on Tregs to a significantly higher extent than any other cell type and is accessible for targeting.

[0063] In exemplary embodiments, the targeting agent is an antibody or an antibody-binding fragment of an antibody. The term "antibody" specifically encompasses monoclonal antibodies, polyclonal antibodies, and bispecific antibodies. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass, or allotype (e.g., human G1m1, G1m2, G1m3, non-G1m1 [i.e., any allotype other than G1m1], G1m17, G2m23, G3m21, G3m28, G3m11, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1, A2m2, Km1, Km2, and Km3) of immunoglobulin molecule. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Antibodies may be murine, human, humanized, chimeric, or derived from other species such as rabbit, goat, sheep, horse, or camel, but are preferably human or humanized. The term "antibody," as used herein, also includes antibody fragments capable of binding to antigen, such as Fab, Fab', and F(ab')2 fragments.

[0064] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present disclosure may be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628, Marks et al (1991) J. Mol. Biol., 222:581-597, or from transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008) Curr. Opinion 20(4):450-459).

[0065] As used herein, a full-length antibody comprises an antigen-binding domain fused directly to a constant region. For conventional antibodies, a full-length antibody comprises two light chain polypeptides, each comprising a light chain variable region (VL) fused to a light chain constant region, and two heavy chain polypeptides, each comprising a heavy chain variable region (VH) fused to a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Certain antibodies, such as camelid antibodies, comprise a single chain, typically a heavy chain comprising a variable region (or antigen-binding portion) and a constant region.

[0066] An "antigen-binding fragment" includes a portion of a full-length antibody that is capable of binding to an antigen, generally the antigen binding or variable region thereof, but may also include the constant region or a portion thereof. Examples of antibody fragments include Fab, Fab', F(ab')2 fragments; scFv fragments; antigen-binding portions of single-domain antibodies; Fv fragments; minibodies; diabodies; triabodies; tetrabodies; linear antibodies; fragments produced by a Fab expression library; anti-idiotypic (anti-Id) antibodies; CDRs (complementarity-determining regions); and epitope-binding fragments of any of the above that immunospecifically bind to Treg cells.

[0067] The constant domains or antibodies may be responsible for mediating various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), ADCP (antibody-dependent cellular phagocytosis), CDC (complement-dependent cytotoxicity), and complement fixation, binding to Fc receptors (e.g., CD16, CD32, FcRn), greater in vivo half-life, and protein A binding. In IgG, IgA, and IgD antibody isotypes, the Fc region is composed of two identical protein fragments derived from the second and third constant domains, while IgM and IgE Fc regions contain three heavy chain constant domains.

[0068] As used herein, the term "Fc domain(s)" includes native and variant Fc regions, including truncated forms. Fc domains can provide extended serum half-life. As used herein, specific residues within an Fc domain are identified by their position according to the EU numbering scheme.

[0069] IgG subclasses differ in their ability to mediate effector function. For example, IgG1 is superior to IgG2 and IgG4 in mediating ADCC and CDC. Therefore, in embodiments where effector function is undesirable, IgG2 or IgG4 Fc regions are preferred. However, IgG2 Fc-containing molecules are known to be more difficult to manufacture and have less attractive biophysical properties, such as a shorter half-life, compared to IgG1 Fc-containing molecules. Therefore, IgG1 Fc-containing molecules with mutations that reduce effector function may be used instead of the IgG2 Fc domain.

[0070] The effector function of an antibody can be increased or decreased by introducing one or more mutations into the Fc domain. In a preferred embodiment of the present invention, the TRA comprises an Fc domain engineered to reduce effector function. Exemplary Fc molecules with reduced effector function include those with one or more substitutions in the Fc domain corresponding to E233P, L234V, L234A, L235A, L235E, ΔG236, G237A, E318A, K320A, K322A, A327G, A330S, or P331S according to the EU index. In some exemplary embodiments, the Fc domain has the following substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P238S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); S267E / L328F (IgG1). Human IgG1 has a glycosylation site at N297 (EU numbering system), and glycosylation is known to contribute to the effector function of IgG1 antibodies. Exemplary IgG1 sequences have a mutated N297, such as glutamine (N297Q) or alanine (N297A) or glycine (N297G). Fcs, including human IgG1 Fc with the N297G mutation, can also contain additional insertions, deletions, and substitutions.

[0071] In some embodiments, the Fc domain substantially lacking effector function has at least about 100-fold or at least about 1000-fold reduced binding affinity to an Fcγ receptor. In some embodiments, the Fc domain comprises what are known as LALA mutations, L234A and L235A mutations (EU numbering). In some embodiments, the Fc region comprises G237A, P329G, or P329A mutations (EU numbering). In some embodiments, using the EU numbering system, the Fc domain comprises L234A, L235A, and / or G237A mutations. Examples of amino acids that have been reported to reduce effector function include the LALA-PG mutation (L234A, L235A, P329G), the LALA-PA mutation (L234A, L235A, P329A), or the LALA-GA mutation (L234A, L235A, G237A). An exemplary Fc domain is set forth in SEQ ID NO: 71. In certain embodiments, the human IgG1 Fc comprises a LALA-PG or a LALA-GA substitution and is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 71.

[0072] In some embodiments, polypeptides of the invention comprise full-length antibodies comprising a wild-type or variant Fc domain. In other embodiments, polypeptides of the invention comprise antibody fragments, such as fusions of the variable regions of the heavy and light chains of an antibody (e.g., scFv). Polypeptides of the invention comprising antibody fragments can also optionally comprise a heterologous polypeptide capable of increasing the circulating half-life of the Treg agonist in vivo. Such a heterologous polypeptide can be, for example, a wild-type or variant Fc domain.

[0073] Methods for preparing antibodies are known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host with an immunogenic protein, peptide, or fragment, either isolated or immunoconjugated (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)).

[0074] Additionally, naked DNA immunization techniques known in the art can be used to generate an immune response to the encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15:617-648).

[0075] The amino acid sequence of a protein to which an antibody response is desired (e.g., CD25 for anti-CD25 antibodies) can be analyzed to select specific regions of the protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of the protein's amino acid sequence can be used to identify hydrophilic regions within the protein structure. Regions of proteins that exhibit immunogenic structures, as well as other regions and domains, can be readily identified using a variety of other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz, or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the method of Hopp, TP, and Woods, KR, 1981, Proc. Natl. Acad. Sci. USA 78:3824-3828. Hydropathicity profiles can be generated using the method of Kyte, J., and Doolittle, RF, 1982, J. Mol. Biol. 157:105-132. Percent (%) accessible residue profiles can be generated using the method of Janin J., 1979, Nature 277:491-492. Average flexibility profiles can be generated using the method of Bhaskaran R., Ponnuswamy PK, 1988, Int. J. Pept. Protein Res. 32:242-255. β-turn profiles can be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Methods for preparing proteins or polypeptides for use as immunogens are well known in the art. Methods for preparing immunogenic conjugates of proteins with carriers such as BSA, KLH, or other carrier proteins are also well known in the art.In some situations, direct conjugation, for example, using carbodiimide reagents, is used; in others, linking reagents such as those supplied by Pierce Chemical Co. (Rockford, Ill.) are effective. Administration of the immunogen is often carried out by injection over a suitable period of time and with the use of a suitable adjuvant, as is understood in the art. Antibody titers can be taken during the immunization schedule to determine adequacy of antibody formation.

[0076] Monoclonal antibodies can be produced by various means well known in the art. For example, immortalized cell lines secreting the desired monoclonal antibodies can be prepared using the standard hybridoma technique of Kohler and Milstein or modifications to immortalize antibody-producing B cells, as is commonly known. Immortalized cell lines secreting the desired antibodies are screened by immunoassay. Once a suitable immortalized cell culture is identified, the cells can be expanded and antibody can be produced either from in vitro culture or ascites fluid.

[0077] Antibodies and fragments thereof can also be produced by recombinant means. Regions that specifically bind to desired regions of a target can also be produced in the context of chimeric or complementarity-determining-region (CDR)-grafted antibodies of multiple species origin. Humanized or human antibodies can also be produced and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies by replacing one or more of the non-human antibody CDRs with the corresponding human antibody sequence are well known (see, e.g., Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-327; Verhoeyen et al., 1988, Science 239:1534-1536). See also Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89:4285 and Sims et al., 1993, J. Immunol. 151:2296.

[0078] Antibodies for use in the present invention can be fully human. Various methods in the art provide means for producing fully human monoclonal antibodies. For example, a preferred embodiment provides a technique using inactivated transgenic mice for antibody production engineered with human heavy and light chain loci, called Xenomouse (Amgen Fremont, Inc.). Exemplary descriptions of the preparation of transgenic mice producing human antibodies can be found, for example, in Mendez, et al., Nature Genetics, 15:146-156 (1998) and Kellerman, SA & Green, LL, Curr. Opin. Biotechnol 13, 593-597 (2002).

[0079] Additionally, human antibodies of the invention can be generated using HuMAb mice (Medarex, Inc.), which contain human immunoglobulin gene minilocuses encoding unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences, along with targeted mutations that inactivate the endogenous mu and kappa chain loci (see, e.g., Lonberg, et al. (1994) Nature 368(6474):856-859).

[0080] In another embodiment, fully human antibodies of the invention can be raised using mice carrying human immunoglobulin sequences on transgenes and transchromosomes, e.g., mice carrying a human heavy chain transgene and a human light chain transchromosome. Such mice are referred to herein as "KM mice," and are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727 and PCT Publication No. WO 02 / 43478 to Tomizuka et al.

[0081] Human monoclonal antibodies of the invention can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are established in the art. See, for example, U.S. Patent Nos. 5,223,409, 5,403,484, and 5,571,698 to Ladner et al.; 5,427,908 and 5,580,717 to Dower et al.; 5,969,108 and 6,172,197 to McCafferty et al.; and 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081 to Griffiths et al.

[0082] Human monoclonal antibodies of the invention can also be prepared using SCID mice reconstituted with human immune cells so that they can generate a human antibody response upon immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.

[0083] In an exemplary embodiment, the targeting agent is an antibody or antigen-binding fragment thereof that is directed to and accessible for targeting a surface marker expressed on Tregs to a significantly higher extent than any other cell type. An exemplary Treg targeting agent is an antibody or antigen-binding fragment thereof that binds to CD25 (i.e., an anti-CD25 antibody). An exemplary human CD25 polypeptide sequence is set forth in Genbank accession number NP_000408, version number NP_000408.1, and Uniprot / Swiss-Prot accession number P01589.

[0084] As used herein, "binds to CD25" is used to mean that the antibody binds to CD25 with higher affinity than it binds to an unrelated antigen, such as bovine serum albumin. In some embodiments, the antibody binds to CD25 with an affinity at least 100, 200, 500, 1000, 2000, 5000, 1000, 15000, 2000, 25000, 3000, 3500, 4000, 5000, 6000, 7000, 8000, 9000, 1000, 15000, 25000, 35000, 4000, 5000, 6000, 7000, 8000, 9000, 1000, 15 ... 4 , 10 5 , or 10 6 times higher association constant (K a ) binds to CD25. The antibodies of the present disclosure can bind to CD25 with high affinity. For example, in some embodiments, the antibodies bind to CD25 with a binding affinity of about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, or about 10 -9 K below M D The antibody can bind to CD25 at 1000 kJ / s. Binding can be determined by ELISA, or flow cytometry, or surface plasmon resonance (SPR) technology, for example, on a BIAcore 3000, using recombinant human IL-2Rα as the ligand and the antibody as the analyte.

[0085] Anti-CD25 antibodies or antigen-binding fragments thereof suitable for use in the present invention can be blocking or non-blocking antibodies. A blocking antibody is an antibody that binds to CD25 and inhibits or blocks the binding of IL-2 to CD25. For example, binding can be inhibited by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. A non-blocking antibody does not inhibit or block the binding of IL-2 to CD25.

[0086] Anti-CD25 antibodies or antigen-binding fragments thereof are known in the art and can be used in the methods disclosed herein. Methods of making anti-CD25 antibodies or antigen-binding fragments thereof are also known in the art and can be used to make anti-CD25 antibodies or antigen-binding fragments thereof for use in the present invention.

[0087] Anti-CD25 antibodies for use in the TRAs provided herein include, but are not limited to, anti-CD25 antibodies described in U.S. Pat. No. 7,438,907, such as antibodies AB1, AB7, Ab11, or AB12; anti-CD25 antibodies described in International Application No. WO2020102591, such as D17, AH04526, AH04750, AH05285, AH05256, AH04527, AH05251, AH05285, AH05259, AH04750; anti-CD25 antibodies described in U.S. Pat. No. 6,383,487; and anti-CD25 antibodies described in U.S. Pat. No. 20200010554, including 7D4, MA251, or 7G7B6.

[0088] Other anti-CD25 antibodies for use in the present invention include the chimeric antibody basiliximab and its humanized forms, as well as the anti-CD25 antibodies BT563 (see Baan et al., Transplant. Proc. 33:224-2246, 2001) and 7G8). An exemplary human antibody for use in the methods of the present invention is HUMAX-TAC®, being developed by Genmab. HUMAX-TAC® is a human monoclonal antibody of the IgG1 kappa isotype specific for human CD25 and having VH and VL domains with the sequences set forth in SEQ ID NOs: 47 and 48, as disclosed herein. Antibody 4C9 (available from Ventana Medical Systems, Inc.) and antibody RFT5 (described in US Pat. No. 6,383,487) are also useful in the present invention. Other suitable antibodies include B489(143-13) (available from Life Technologies, catalog number MA1-91221), SP176 (available from Novus, catalog number NBP2-21755), 1B5D12 (available from Novus, catalog number NBP2-37349), 2R12 (available from Novus, catalog number NBP2-21755), BC96 (available from BioLegend, catalog number V-072), and M-A251 (available from BioLegend, catalog number IV A053).

[0089] Additional anti-CD25 antibodies for use in the present invention include the PC61 antibody described in U.S. Patent Application No. 20080025947, the murine monoclonal antibody S4B6, and the monoclonal antibody MAB602 disclosed in Boyman et al., Science, (2006), 311:1924-1927.

[0090] In some embodiments, the antibody is a humanized, deimmunized, or resurfaced version of an antibody disclosed herein.

[0091] The anti-CD25 antibody daclizumab is an example of a Treg targeting agent that can be used in the present invention. The anti-CD25 antibody daclizumab is a humanized anti-CD25 antibody previously marketed under the trade name ZENAPAX. An exemplary anti-CD25 antibody is an antibody comprising a daclizumab variable domain with reduced / no effector function Fc domain.

[0092] The daclizumab heavy chain variable region (SEQ ID NO:26) contains three heavy chain complementarity-determining regions (CDRs), designated herein (from amino to carboxy terminus) as CDR-H1, CDR-H2, and CDR-H3, and are designated SEQ ID NO:27 (CDR-H1); SEQ ID NO:28 (CDR-H2); and SEQ ID NO:29 (CDR-H3). The heavy chain framework (FR) sequences of daclizumab are set forth in SEQ ID NO:33 (FR-H1), SEQ ID NO:34 (FR-H2), SEQ ID NO:35 (FR-H3), and SEQ ID NO:36 (FR-H4).

[0093] In some embodiments, the polypeptides of the invention comprise the heavy and light chain variable domains of a daclizumab antibody, or variants of the heavy and light chain variable domains of a daclizumab antibody (also referred to herein as daclizumab-related antibodies), or antigen-binding fragments thereof. Such polypeptides may also comprise a wild-type F C domain, a variant F C domain, e.g., an F C domain with reduced effector function, a truncated F C domain (whether wild-type or variant), or no F C domain.

[0094] Daclizumab-related antibodies are known in the art; see, e.g., International Application No. WO2014144935 and U.S. Patent No. 8,314,213, which are incorporated by reference in their entireties. In some such embodiments, polypeptides of the invention comprise daclizumab-related antibodies, or antigen-binding fragments thereof, that have reduced T-cell immunogenicity compared to daclizumab. In some embodiments, polypeptides of the invention comprise daclizumab or daclizumab-related antibodies, or antigen-binding fragments thereof, characterized by one, two, three, or four of the following properties (i)-(iv): (i) comprising the six CDRs shown in numbers 27, 28, 29, 30, 31, and 32; (ii) comprising the six CDRs set forth in SEQ ID NOs: 27, 28, 29, 30, 31 and 32, except that any individual CDR has no more than four or no more than three amino acid substitutions compared to the corresponding CDR sequences of an antibody having the CDRs of SEQ ID NOs: 27, 28, 29, 30, 31 and 32, or has a maximum of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen or seventeen amino acid substitutions in the CDRs, provided that any individual CDR has no more than four or no more than three amino acid substitutions compared to the corresponding CDR sequences of an antibody having the CDRs of SEQ ID NOs: 27, 28, 29, 30, 31 and 32, or that any individual CDR other than CDR-H2 has no more than three or no more than two amino acid substitutions compared to the corresponding CDR sequences of an antibody having the CDRs of SEQ ID NOs: 27, 28, 29, 30, 31 and 32, (iii) comprising an individual framework region of SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, and 40, or a framework region having 1 to 10, preferably 1 to 10 or 1 to 5 amino acid substitutions in such an individual framework region; (iv) VH and VL sequences having at least 75% sequence identity (and in certain embodiments, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the VH and VL sequences of daclizumab (SEQ ID NO: 26 and SEQ ID NO: 41), and in some examples, comprising the CDRs of (i) or (ii).

[0095] In various embodiments, the daclizumab-related antibody, or antigen-binding fragment thereof, has the amino acid substitution I48M in FR-H2 compared to FR-H2 of SEQ ID NO: 34; amino acid substitutions N52K and T54R in CDR-H2 compared to CDR-H2 of SEQ ID NO: 28, and S29K in CDR-L1 compared to CDR-L1 of SEQ ID NO: 30, and N53D in CDR-L2 compared to CDR-L2 of SEQ ID NO: 31; amino acid substitutions N52K and T54R in CDR-H2 compared to CDR-H2 of SEQ ID NO: 28, and N53E in CDR-L2 compared to CDR-L2 of SEQ ID NO: 31; The amino acid substitutions include one or more specific substitutions, including amino acid substitutions N52S, S53R, and T54K in CDR-H2 compared to CDR-H2 of SEQ ID NO: 28; amino acid substitution T54S in CDR-H2 compared to CDR-H2 of SEQ ID NO: 28; amino acid substitution S29K in CDR-L1 compared to CDR-L1 of SEQ ID NO: 30, and N53D in CDR-L2 compared to CDR-L2 of SEQ ID NO: 31; amino acid substitutions S53R and T54K in CDR-H2 compared to CDR-H2 of SEQ ID NO: 28; amino acid substitution S29K in CDR-L1 compared to CDR-L1 of SEQ ID NO: 30; and N53D in CDR-L2 compared to CDR-L2 of SEQ ID NO: 31; and combinations thereof.

[0096] In various embodiments, the daclizumab-related antibody, or antigen-binding fragment thereof, has the following amino acid substitutions in CDR-H2 relative to CDR-H2 of SEQ ID NO:28: (i) N52S, N52K, N52R, or N52V, and (ii) T54R, T54S, or T54K, and optionally (iii) S53R, S53K, or S53N in CDR-H2 relative to CDR-H2 of SEQ ID NO:28; (iv) S53R, S53K, or S53N in CDR-H2 relative to CDR-H2 of SEQ ID NO:28; (v) Y56R in CDR-H2 compared to CDR-H2 of SEQ ID NO: 28; (vi) E73K in CDR-H2 compared to CDR-H2 of SEQ ID NO: 28; (vii) S29K in CDR-L1 compared to CDR-L1 of SEQ ID NO: 30; and (viii) N53D or N53E in CDR-L2 compared to CDR-L2 of SEQ ID NO: 31.

[0097] In various embodiments, the daclizumab-related antibody, or antigen-binding fragment thereof, comprises amino acid substitutions: (i) N52S, N52K, N52R, or N52V; (ii) S53K, S53R, or S53N; and (iii) T54R, T54S, or T54K in CDR-H2 compared to CDR-H2 of SEQ ID NO: 28; (iv) S29K in CDR-L1 compared to CDR-L1 of SEQ ID NO: 30; and (v) N53D or N53E in CDR-L2 compared to CDR-L2 of SEQ ID NO: 31; and optionally, further comprises amino acid substitution: (iv) Y56R in CDR-H2 compared to CDR-H2 of SEQ ID NO: 28.

[0098] In various embodiments, the daclizumab-related antibody, or antigen-binding fragment thereof, comprises the following amino acid substitutions: (i) N52S, (ii) S53R or S53K, (iii) T54S or T54K, and (iv) Y56R in CDR-H2 compared to CDR-H2 of SEQ ID NO: 28; (v) S29K in CDR-L1 compared to CDR-L1 of SEQ ID NO: 30; and (vi) N53D in CDR-L2 compared to CDR-L2 of SEQ ID NO: 31.

[0099] In various embodiments, the daclizumab-related antibody, or antigen-binding fragment thereof, comprises amino acid substitutions of (i) N52S, N52K, N52R, or N52V and (ii) T54R, T54S, or T54K in CDR-H2 compared to CDR-H2 of SEQ ID NO:28, and optionally, (iii) S53R, S53K, or S53N in CDR-H2 compared to CDR-H2 of SEQ ID NO:28; (iv) S53R, S53K, or S53N in CDR-H2 compared to CDR-H2 of SEQ ID NO:28; (v) Y56R in CDR-H2 compared to CDR-H2 of SEQ ID NO: 28, (v) E58Q in CDR-H2 compared to CDR-H2 of SEQ ID NO: 28, (vi) E73K in CDR-H2 compared to CDR-H2 of SEQ ID NO: 28, (vii) S29K in CDR-L1 compared to CDR-L1 of SEQ ID NO: 30, and (viii) N53D or N53E in CDR-L2 compared to CDR-L2 of SEQ ID NO: 31. In some such embodiments, the framework regions have up to four amino acid substitutions compared to the frameworks of SEQ ID NO: 3 (FR-H1), SEQ ID NO: 5 (FR-H2), SEQ ID NO: 7 (FR-H3), SEQ ID NO: 9 (FR-H4), SEQ ID NO: 10 (FR-L1), SEQ ID NO: 12 (FR-L2), SEQ ID NO: 14 (FR-L3), and SEQ ID NO: 16 (FR-L4). In certain embodiments, the framework region comprises the amino acid substitution 148M in FR-H2 compared to FR-H2 of SEQ ID NO: 5. In certain embodiments, the framework region does not comprise a substitution at 148 in FR-H2 compared to FR-H2 of SEQ ID NO: 5.

[0100] In various embodiments, the daclizumab-related antibody, or antigen-binding fragment thereof, comprises amino acid substitutions: (i) N52S, N52K, N52R, or N52V; (ii) S53K, S53R, or S53N; and (iii) T54R, T54S, or T54K in CDR-H2 compared to CDR-H2 of SEQ ID NO:28; (iv) S29K in CDR-L1 compared to CDR-L1 of SEQ ID NO:30; and (v) N53D or N53E in CDR-L2 compared to CDR-L2 of SEQ ID NO:31; and optionally, (vi) a Y56R amino acid substitution in CDR-H2 compared to CDR-H2 of SEQ ID NO:28. In some such embodiments, the framework regions have up to four amino acid substitutions compared to the frameworks of SEQ ID NO:33 (FR-H1), SEQ ID NO:34 (FR-H2), SEQ ID NO:35 (FR-H3), SEQ ID NO:36 (FR-H4), SEQ ID NO:37 (FR-L1), SEQ ID NO:38 (FR-L2), SEQ ID NO:39 (FR-L3), and SEQ ID NO:40 (FR-L4). In certain embodiments, the framework regions comprise the amino acid substitution 148M in FR-H2 compared to FR-H2 of SEQ ID NO:34. In certain embodiments, the framework regions do not comprise the substitution at 148 in FR-H2 compared to FR-H2 of SEQ ID NO:34. In various embodiments, the anti-CD25 antibody or anti-CD25 binding fragment comprises the heavy and light chain variable domains of daclizumab with N52S, S53R, and T54K substitutions in the heavy chain variable domain and N53E substitution in the light chain variable domain; or with N52K and T54R substitutions in the heavy chain variable domain and N53E substitution in the light chain variable domain. The numbering of the heavy and light chain variable regions is done according to the Kabat numbering system (see Tables 1 and 2 of WO2014 / 144935).

[0101] In various embodiments, a daclizumab-related antibody, or antigen-binding fragment thereof, has a heavy chain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 27, a CDR2 having the amino acid sequence of SEQ ID NO: 44, and a CDR3 having the amino acid sequence of SEQ ID NO: 29, and a light chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 45, a CDR2 having the amino acid sequence of SEQ ID NO: 46, and a CDR3 having the amino acid sequence of SEQ ID NO: 32. (See, e.g., U.S. Patent No. 8,314,213, incorporated herein in its entirety for all purposes.)

[0102] In some embodiments, polypeptides of the invention comprise an antibody or antigen-binding fragment thereof comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27, 42, or 43, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28 or 44, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30 or 45, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31 or 46, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32. In some such embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 41.

[0103] In addition to, or as an alternative to, simply binding CD25, an anti-CD25 antibody, or antigen-binding fragment thereof, can be selected to possess other functional properties, such as high-affinity binding to CD25 and / or inhibition or blocking of CD25 binding to IL-2. In some embodiments, the selected anti-CD25 antibody, or antigen-binding fragment thereof, is one that, when administered alone, does not induce depletion of CD25-expressing T cells or inhibit the proliferation of CD25-expressing T cells. In some aspects, the anti-CD25 antibody, or antigen-binding fragment thereof, binds to CD25 with the same or greater affinity as daclizumab.

[0104] Exemplary TRA Configuration In some aspects, the TRAs of the present invention comprise a Neo-2 / 15 variant linked (e.g., via translational fusion or chemical / enzymatic conjugation) to an antibody or antigen-binding fragment thereof. The TRAs can optionally include an amino acid linker between one or more of the components that make up the TRA (e.g., between the Neo-2 / 15 variant and the antibody or antibody-binding fragment). A non-limiting example of a linker connecting different components of a TRA is a glycine / serine linker. For example, the glycine / serine linker can include the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 49) or the sequence GGGSGGGGSGGGGGS (SEQ ID NO: 50). This is merely a non-limiting example; the linker can have a varying number of GGGGS (SEQ ID NO: 51) repeats. In some embodiments, the linker includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 GGGGS (SEQ ID NO: 51) repeats. In some embodiments, the linker comprises multiple glycine repeats, such as GGGGGG (SEQ ID NO: 52) or GGGGGGGG (SEQ ID NO: 53). The linkers used to connect the different components of the TRA can be flexible or rigid linkers. 。 Non-limiting examples of rigid linkers include EAAK (SEQ ID NO: 54), (EAAK)2 (SEQ ID NO: 55) 、 (EAAK)3 (SEQ ID NO: 56) 、 (EAAAK)3 (SEQ ID NO: 57) 、 A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 58), AEAAAKEAAAKA (SEQ ID NO: 59), PAPAP (SEQ ID NO: 60), and (ALA-PRO) 10-34 (SEQ ID NO: 83).

[0105] In some embodiments, polypeptides of the invention target surface markers expressed on Tregs to a significantly greater extent than any other cell type and comprise a targeting-accessible antibody or antigen-binding fragment of an antibody, wherein the Neo-2 / 15 variant is linked to either the C-terminus or the N-terminus of the antibody, optionally via an amino acid linker. In some embodiments, polypeptides of the invention comprise a Neo-2 / 15 variant and an scFv, wherein both the Neo-2 / 15 variant and the scFv are disposed at opposite ends of an Fc domain. In some such embodiments, the polypeptide comprises a homodimer comprising two Neo-2 / 15 variants, each linked via its C-terminus to the N-terminus of an Fc domain, which is linked via its C-terminus to the variable heavy chain of the scFv. In some embodiments, polypeptides of the invention comprise a Neo-2 / 15 variant and an antibody, wherein the Neo-2 / 15 variant is linked to the N-terminus of the heavy chain of the antibody. In some such embodiments, the polypeptide comprises a heterotetramer comprising two heavy chains (VH+ constant regions) and two light chains (VL+ constant regions), wherein each heavy chain variable region is linked via its N-terminus to the C-terminus of a Neo-2 / 15 variant. In some embodiments, the polypeptide of the invention comprises a Neo-2 / 15 variant and an antibody, wherein the Neo-2 / 15 variant is linked to the C-terminus of a heavy chain of the antibody. In some such embodiments, the polypeptide comprises a heterotetramer comprising two heavy chains (VH+ constant regions) and two light chains (VL+ constant regions), wherein each heavy chain constant region is linked via its C-terminus to the N-terminus of a Neo-2 / 15 variant. Linkage can be achieved, for example, via a linker. The linker can be, for example, (GGGGS) n It may be a linker, and n is 1 to 10.

[0106] In some embodiments, a polypeptide of the invention comprises a Neo-2 / 15 variant, a Neo-2 / 15 variant linked to an anti-CD25 antibody described herein, a Neo-2 / 15 variant linked to an antigen-binding fragment of an anti-CD25 antibody described herein, a Neo-2 / 15 variant linked to an antigen-binding fragment of an anti-CD25 antibody described herein, and an Fc domain described herein. In some embodiments, a polypeptide of the invention consists of, or consists essentially of, a Neo-2 / 15 variant, a Neo-2 / 15 variant linked to an anti-CD25 antibody described herein, a Neo-2 / 15 variant linked to an antigen-binding fragment of an anti-CD25 antibody described herein, a Neo-2 / 15 variant linked to an antigen-binding fragment of an anti-CD25 antibody described herein, and an Fc domain described herein. In some embodiments, a polypeptide of the invention comprises the amino acid sequence of SEQ ID NO:69 and the amino acid sequence of SEQ ID NO:70. In some embodiments, a polypeptide of the invention comprises the amino acid sequence of SEQ ID NO:74 and the amino acid sequence of SEQ ID NO:70. In some embodiments, a polypeptide of the invention comprises a Neo-2 / 15 variant linked to the C-terminus of a targeting agent or an Fc domain, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 69 and the amino acid sequence of SEQ ID NO: 70. In some embodiments, a polypeptide of the invention comprises a Neo-2 / 15 variant linked to the N-terminus of a targeting agent or an Fc domain, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 74 and the amino acid sequence of SEQ ID NO: 70.

[0107] In some embodiments, the polypeptides of the present invention do not substantially activate Teff cells, or the polypeptides activate Teff cells with an EC50 that is more than 5-fold, more than 10-fold, more than 50-fold, more than 100-fold, more than 500-fold, or more than 1000-fold higher than the EC50 for activating Treg cells.

[0108] In some embodiments, the polypeptides of the invention do not substantially induce proliferation of Teff cells, or the polypeptides induce proliferation of Teff cells with an EC50 that is more than 5-fold, more than 10-fold, more than 50-fold, more than 100-fold, more than 500-fold, or more than 1000-fold higher than the EC50 for inducing proliferation of Treg cells.

[0109] In some embodiments, the maximal signaling by a polypeptide of the present invention in non-Treg cells is less than 30% or less than 20% of the maximal signaling by IL-2 in non-Treg cells (e.g., at a concentration of 10 nM or less of the polypeptide). In some embodiments, the non-Treg cells are Teff cells. In some embodiments, the non-Treg cells are NK cells.

[0110] In some embodiments, the maximal signaling in Tregs by a polypeptide of the invention is at least 50%, at least 60%, or at least 70% of the maximal signaling in Tregs by IL-2 (e.g., at a concentration of 10 nM or less of the polypeptide).

[0111] In various embodiments, signaling in Tregs and non-Tregs is measured as %pSTAT5+ relative to IL-2. The EC50 may be determined from the %pSTAT5+ curve obtained using a titration of the test substance.

[0112] In some embodiments, the polypeptides of the present invention induce STAT5 phosphorylation in Treg cells with an EC50 of less than 1 nM, less than 500 pM, or less than 100 pM, and / or the polypeptides induce STAT5 phosphorylation in Teff cells with an EC50 of greater than 1000 pM (i.e., 1 nM).

[0113] In some embodiments, the polypeptides of the invention do not substantially activate or stimulate NK cells, or the polypeptides activate NK cells with an EC50 that is more than 5-fold, more than 10-fold, more than 50-fold, more than 100-fold, more than 500-fold, or more than 1000-fold higher than the EC50 for activating Treg cells. In some embodiments, the polypeptides do not substantially induce NK cell proliferation, or the polypeptides induce NK cell proliferation with an EC50 that is more than 5-fold, more than 10-fold, more than 50-fold, more than 100-fold, more than 500-fold, or more than 1000-fold higher than the EC50 for inducing Treg cell proliferation. In some embodiments, the polypeptides induce STAT5 phosphorylation in Treg cells with an EC50 of less than 1 nM, less than 500 pM, or less than 100 pM, and / or the polypeptides induce STAT5 phosphorylation in NK cells with an EC50 of more than 1000 pM.

[0114] nucleic acid Provided herein are nucleic acids comprising a nucleotide sequence encoding a polypeptide of the invention, e.g., a Neo-2 / 15 variant or a Neo-2 / 15 variant fused to an antibody, a Neo-2 / 15 variant fused to an antigen-binding fragment of an antibody, a Neo-2 / 15 variant fused to an antigen-binding fragment of an antibody and an Fc domain, or another polypeptide of the invention. The antibody or antibody fragment can be, for example, an anti-CD25 antibody or antibody fragment thereof, or an anti-CD39 antibody or antibody-binding fragment thereof, or another antibody capable of targeting Treg cells.

[0115] In some embodiments, a nucleic acid of the invention encodes a polypeptide comprising any one or more of sequences 1-82.

[0116] The nucleic acids and polypeptides of the invention can be produced using any suitable method known in the art.

[0117] In exemplary aspects, a nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide of the present disclosure. As used herein, "nucleic acid" includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally refers to a polymer of DNA or RNA, or modified forms thereof, which may be single-stranded or double-stranded. A nucleic acid can comprise any nucleotide sequence that encodes any of the antigen-binding proteins or polypeptides of the present disclosure.

[0118] In some embodiments, the nucleic acids of the present disclosure are recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by joining natural or synthetic nucleic acid segments to a nucleic acid molecule that can replicate within the living cell, or (ii) a molecule resulting from replication of the above (i). For purposes of this specification, replication can be in vitro replication or in vivo replication.

[0119] In some embodiments, nucleic acids are constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. In some embodiments, the nucleic acids of the present disclosure are incorporated into vectors. In this regard, the present disclosure provides vectors comprising any of the nucleic acids disclosed herein. In an exemplary embodiment, the vector is a recombinant expression vector. For purposes of this specification, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that enables expression of an mRNA, protein, polypeptide, or peptide by a host cell, wherein the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed in the cell.

[0120] The vectors of the present disclosure can be any suitable vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation or propagation, for expression, or both, such as plasmids and viruses. The vectors of the present disclosure can be prepared using standard recombinant DNA techniques. Circular or linear expression vector constructs can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from CoIE1, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like. In some embodiments, the vector contains regulatory sequences, such as transcription and translation initiation and termination codons, that are specific to the type of host (e.g., bacteria, fungi, plants, or animals) into which the vector will be introduced, as needed, and taking into account whether the vector is DNA- or RNA-based. The vector can also contain one or more marker genes to allow for selection of transformed or transfected hosts. The vector can include a native or canonical promoter operably linked to a nucleotide sequence that is complementary to or hybridizes with a nucleotide sequence encoding a polypeptide (including functional portions and variants thereof) or a nucleotide sequence encoding a TRA conjugate or fusion protein. For example, the selection of strong, weak, inducible, tissue-specific, and developmentally specific promoters is within the ordinary skill of one of ordinary skill in the art. Similarly, combining a nucleotide sequence with a promoter is also within the ordinary skill of one of ordinary skill in the art.

[0121] Host cells containing the nucleic acids or vectors of the present disclosure are provided herein. As used herein, the term "host cell" refers to any type of cell that can contain the vectors of the present disclosure and produce expression products encoded by the nucleic acids (e.g., mRNA, protein). In some embodiments, the host cells are adherent or suspension cells, i.e., cells that grow in suspension. In exemplary embodiments, the host cells are cultured or primary cells, i.e., directly isolated from an organism, e.g., a human. The host cells can be of any cell type, derived from any type of tissue, and at any developmental stage. The host cells can be prokaryotic cells, such as bacterial cells (e.g., E. coli), or eukaryotic cells, such as yeast cells or mammalian cells. The non-fusion attenuated IL-2 / 1L-15 mimics can be expressed in prokaryotic or eukaryotic cells, while TRAs are preferably expressed in mammalian cells.

[0122] A method for producing a polypeptide of the invention is provided herein. In an exemplary embodiment, the method includes culturing a host cell of the present disclosure to express the polypeptide and harvesting the expressed polypeptide. The host cell can be any of the host cells described herein. In an exemplary embodiment, the host cell is selected from the group consisting of CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells. In an exemplary embodiment, the step of culturing the host cell includes culturing the host cell in a growth medium to support the growth and expansion of the host cell. In an exemplary embodiment, the growth medium increases cell density, culture viability, and productivity in a timely manner. In an exemplary embodiment, the growth medium includes amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones, and binding factors. In an exemplary embodiment, the growth medium is a fully chemically defined medium consisting of amino acids, vitamins, trace elements, inorganic salts, lipids, and insulin or insulin-like growth factors. In addition to nutrients, the growth medium also serves to maintain pH and osmolality. Several growth media are commercially available and described in the art. See, for example, Arora, “Cell Culture Media: A Review” MATER METHODS 3:175 (2013).

[0123] Pharmaceutical Composition Pharmaceutical compositions comprising the polypeptides of the present disclosure are provided herein. In exemplary embodiments, the compositions include agents that enhance the chemophysical characteristics of the polypeptide, for example, by stabilizing the TRA at a certain temperature (e.g., room temperature), extending its shelf life, reducing degradation (e.g., oxidative protease-mediated degradation), or by extending the half-life of the TRA.

[0124] In exemplary aspects of the present disclosure, the composition additionally comprises a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the polypeptide is formulated into a pharmaceutical composition comprising an active agent together with a pharmaceutically acceptable carrier, diluent, or excipient. In this regard, the present disclosure further provides a pharmaceutical composition comprising an active agent, wherein the pharmaceutical composition is intended for administration to a subject, e.g., a mammal (e.g., a human).

[0125] In exemplary embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate-buffered saline, water, emulsions such as oil / water or water / oil emulsions, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the U.S. federal government for use in animals, including humans, or listed in the United States Pharmacopeia (USP). Pharmaceutical compositions may contain, for example, acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, preservatives, bases, binders, buffers, chelating agents, coating agents, colorants, desiccants, detergents, diluents, disinfectants, disintegrants, dispersants, dissolution enhancers, dyes, emollients, emulsifiers, emulsion stabilizers, fillers, film-forming agents, flavor enhancers, flavoring agents, flavoring agents, and the like. Any pharmaceutically acceptable ingredient may be included, including: ointment enhancer, gelling agent, granulating agent, moisturizing agent, lubricant, mucoadhesive agent, ointment base, ointment, oily vehicle, organic base, pastille base, pigment, plasticizer, abrasive, preservative, sequestering agent, skin penetration agent, solubilizer, solvent, stabilizer, suppository base, surface active agent, surfactant, suspending agent, sweetener, therapeutic agent, thickener, tonicity agent, toxicity agent, thickener, water-absorbing agent, water-miscible cosolvent, water softener or humectant.For example, see Handbook of Pharmaceutical Excipients, Third Edition, AH Kibbe (Pharmaceutical Press, London, UK, 2000) (incorporated by reference in its entirety), Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) (incorporated by reference in its entirety).

[0126] It will be apparent to those skilled in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors, including, but not limited to, the type of animal (e.g., human), the particular form of the polypeptide of the invention, the mode of administration, and the composition used.

[0127] To treat a disease, the polypeptides of the present invention are provided in a therapeutically effective amount. This refers to the amount of polypeptide that is effective in treating the disease or has the desired effect, i.e., the amount that elicits the biological or medical response in a cell, tissue, system, or animal, such as a human, that is being sought by a researcher, veterinarian, physician, or other treatment provider. Data obtained from cell culture assays and animal studies can be used in calculating a range of dosages for use in humans. Dosage regimens can be adjusted by a clinician to provide the optimum desired response. One of skill in the art will appreciate that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatments, the subject's general health and / or age, and the presence of other diseases, can affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of the polypeptides of the present invention can include a single treatment or a series of treatments. The subject can be a mammal, including a human.

[0128] How to use Exemplary polypeptides of the present invention can be used to expand Tregs in a subject or sample. Methods for increasing the ratio of Tregs to non-Tregs are provided herein. In some embodiments, methods for increasing the ratio of Tregs to Teff cells and / or the ratio of Tregs to NK cells are provided herein. In some aspects, the methods comprise contacting a population of T cells with an effective amount of a polypeptide of the present invention. In some aspects, the population of T cells is in the peripheral blood of a subject. The typical frequency of Tregs in human blood is 5-10% of all CD4+CD3+ T cells, but may be lower or higher in diseases treatable by the present methods. In preferred embodiments, the percentage of Tregs is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. In certain embodiments, a polypeptide of the present invention is administered to a subject, increasing the ratio of regulatory Tregs to Teff cells in the peripheral blood of the subject.

[0129] Because the exemplary Treg agonists of the invention preferentially expand Tregs over non-Tregs, the exemplary TRAs are useful for increasing the ratio of regulatory T cells (Tregs) to natural killer (NK) cells in the peripheral blood of a subject, which can be measured, for example, by determining the ratio of Tregs to CD16+ and / or CD56+ lymphocytes that are CD19- and CD3-.

[0130] Methods of the present invention include methods of activating Treg cells and / or methods of inducing proliferation of Treg cells, comprising the step of contacting Treg cells with a polypeptide of the present invention.

[0131] It is contemplated that the polypeptides of the present invention may have a therapeutic effect on a disease or disorder in a patient without significantly expanding the ratio of Tregs to non-regulatory T cells or NK cells in the patient's peripheral blood. The therapeutic effect may be due to the local activity of the polypeptide at the site of inflammation or autoimmunity.

[0132] Exemplary Treg agonists of the invention can be used, for example, to treat diseases, disorders, or conditions that would benefit from the expansion of Tregs without a corresponding expansion of non-regulatory T cells or NK cells within the patient's peripheral blood.

[0133] Exemplary Treg agonists of the present invention can be used, for example, to treat diseases, disorders, or conditions associated with Teff cell activity.

[0134] Exemplary Treg agonists of the present invention can be used, for example, to treat diseases, disorders, or conditions associated with B cell activity.

[0135] Diseases, disorders, or conditions that are particularly suitable for treatment with TRA include inflammation, autoimmune diseases, atopic diseases, paraneoplastic autoimmune diseases, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, small-articular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic-onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative, Cartilage syndrome, articular syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small-articular juvenile rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic-onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome (seronegative, cartilage syndrome, articular syndrome), dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myositis, polymyositis , dermatomyositis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatoid arthritis, sarcoidosis, sclerosis, primary gallbladder sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, gut acid psoriasis, inverse psoriasis, pustular psoriasis, erythematous psoriasis, dermatitis, atopic dermatitis, arteriosclerosis, lupus, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease These include, but are not limited to, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, rhinosinusitis, rhinosinusitis with polyps, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barré disease, type 1 diabetes, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, transplant rejection, kidney damage, hepatitis C-induced vasculitis, and spontaneous pregnancy loss.

[0136] In preferred embodiments, the autoimmune or inflammatory disorder is lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type I diabetes, multiple sclerosis, spontaneous loss of pregnancy, atopic disease, and inflammatory bowel disease.

[0137] In another embodiment, a patient having, at risk of developing, or at risk of having an autoimmune or inflammatory disorder is treated with a TRA of the invention and the patient's response to the treatment is monitored. The patient response monitored can be any detectable or measurable response of the patient to the treatment, or any combination of such responses.

[0138] In some exemplary embodiments, the polypeptides of the invention are administered in combination with one or more additional agents, which may be additional therapeutic agents used to treat a disorder or disease, or may be agents administered to reduce side effects associated with treatment with the polypeptide.

[0139] In some embodiments, the subject or patient treated by the method is a mammal. In some aspects, the subject or patient is human.

[0140] kit The present disclosure additionally provides kits comprising a polypeptide of the present invention. In exemplary aspects, the kit comprises the polypeptide in a container. In exemplary aspects, the polypeptide is provided in the kit as a unit dose. For purposes of this specification, a "unit dose" refers to a discrete amount dispersed in a suitable carrier. In exemplary aspects, a unit dose is an amount sufficient to provide a desired effect in a subject, e.g., treatment of any one of the conditions or disorders described herein. In exemplary aspects, the kit comprises several unit doses, e.g., a weekly or monthly supply of unit doses, optionally with each unit dose individually packaged or separated from the other unit doses. In some embodiments, the kit / unit dose components are packaged with instructions for administration to a patient. In some embodiments, the kit includes one or more devices for administration to a patient, such as a needle and syringe. In some aspects, the polypeptide is pre-packaged in a ready-to-use form. In exemplary aspects, the ready-to-use form is for single use. In exemplary aspects, the kit includes multiple single-use, ready-to-use forms of the polypeptide. In some embodiments, the kit further comprises other therapeutic or diagnostic agents, including any of those described herein, or a pharmaceutically acceptable carrier (e.g., a solvent, buffer, diluent, etc.). [Example]

[0141] In all examples referencing anti-CD25 antibodies, the anti-CD25 antibodies comprised daclizumab heavy and light chain variable region binding domains. The Fc domain and full-length antibody were IgG1s with reduced effector function conferred by LALA-GA mutations (L234A, L235A, and G237A, numbered according to the EU system), and optionally contained a K447S modification (numbered according to the EU system) to prevent potential cleavage of the C-terminal fusion domain, unless otherwise specified.

[0142] For all examples with pSTAT5 data, the following pSTAT5 titration protocol was used: Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized human whole blood using Lymphoprep gradient medium and SepMate tubes. Pan CD3+ T cells were isolated from PBMCs using the StemCell EasySep Human T Cell Isolation Kit and placed in each well of a 96-well round-bottom plate with X-VIVO 15 medium. Serial dilutions of test substances were diluted in X-VIVO 15 medium and added to each well, and cells were stimulated for 30 minutes at 37°C. Cells were fixed by adding paraformaldehyde to 1.5% and incubated at room temperature for 10 minutes. Cells were permeabilized in ice-cold 100% methanol for 30 minutes at 4°C. Cells were then washed twice with FACS buffer (phosphate-buffered saline with 2% fetal bovine serum and 1 mM EDTA). Cells were stained with fluorophore-conjugated CD4, CD8, CD25, CD127, and pSTAT5 antibodies for 1 hour at 4°C. Cells were washed with FACS buffer and fixed again with 1.5% paraformaldehyde. To assess NK cell signaling, the same procedure was performed using PBMCs that had not undergone CD3+ T cell isolation, with the addition of CD56 antibody during the staining step. Flow cytometry analysis of cells was performed on a Cytek Aurora and analyzed using the OMIQ cytometry analysis platform. The percentage of pSTAT5+ cells in different cell populations was determined by gating on unstimulated cell controls. Dose-response curves for %pSTAT5+ cells were fitted to a logistic model, and half-maximal effective concentrations (EC50 values) were calculated using GraphPad Prism data analysis software. Dose-response curves were performed in the range of 0.01 pM to 100 nM. An IL-2 control curve was included to determine donor cell responsiveness, measured by gating on %pSTAT5+. Titrations were repeated at least three times per candidate to ensure consistent performance. To compare two different test substances or test substances with Neo-2 / 15 and / or IL-2, testing should be performed using cells from the same blood draw. There is significant signaling variability in patient blood draws.Tregs are defined as T cells that are CD4+, CD25+, and CD127(low / -). The all T cell designation includes all CD3+ cells, including CD8+, CD4+, and Tregs.

[0143] When selecting a specific fusion protein for use as a TRA, the following parameters should be considered. First, candidates preferably have low maximal signaling in non-Treg cells, such as Teff cells and / or NK cells, at concentrations up to 10 nM (i.e., less than 30%, 20%, or 10%, or even lower, than the maximal signaling resulting from IL-2 stimulation). In some embodiments, lower signaling in non-Treg cells correlates with higher Treg selectivity. Second, maximal signaling in Tregs is preferably at least 40%, at least 50%, or at least 60% of the maximal signaling resulting from IL-2 stimulation, with higher maximal Treg signaling indicating a more potent TRA. Third, the EC50 of the Treg %pSTAT5+ curve is preferably less than 1 nM, preferably less than 500 pM, or even lower, such as less than about 100 pM, with lower Treg EC50s also indicating more potent TRAs. In addition to these three parameters, it is useful to consider Treg pSTAT5 MFI (mean fluorescence intensity) when selecting a TRA. A higher MFI corresponds to stronger signaling and downstream events, such as cell proliferation and activation. In some embodiments, when assessing Emax in non-Tregs, if a high Emax (e.g., greater than 20% of maximal signaling resulting from IL-2 stimulation) is achieved only at a high EC50, the test substance may still be considered to have a Treg-selective profile; accurate assessment depends on evaluation of the entire pSTAT5 titration curve among cell subsets.

[0144] Example 1 - Adjusting Neo-2 / 15 titers to obtain attenuated Neo-2 / 15 variants that retain binding to IL-2RBG and can be used as TRAs when fused to Treg targeting agents To identify residues involved in the binding interactions between huIL2Rβ and / or IL2Rβγ and Neo-2 / 15, the PDB structure 6DG5 was inspected in PyMol to select Neo-2 / 15 residues within 5.5 Å of either receptor. Twenty-seven positions were identified, and Neo-2 / 15 mutant protein samples with single alanine mutations at those positions were cloned into the pET28 vector using Gibson assembly, expressed in Lemo21 cells, and purified by NTA-Ni and size-exclusion chromatography. Neo-2 / 15 mutants with single alanine mutations at each receptor interface position exhibited binding affinity for huIL2Rβ and huIL2Rβγ, which were analyzed by Octet Biolayer interferometry. Binding data were collected on an Octet RED96 instrument and processed using integrated Octet software. Binding to huIL2Rβ was collected using human IL-2R beta / CD122 protein, Fc tag (MALS & SPR validated) (ILB-H5253), and binding to huIL2Rβγ was collected using human IL-2R beta / CD122 protein, His tag (SPR validated) (CD2-H5221) and human IL-2R gamma / CD132 protein, Fc tag (ILG-H5256) at concentrations equal to or greater than the analyte concentration. Anti-human Fc capture (AHC) sensors were equilibrated for 90 seconds in 1x Cytiva HBS-EP + Buffer 10 binding buffer. The sensors were loaded with receptor by mixing with wells containing 0.5 μg / ml of the aforementioned receptor until a threshold response of 1.0 nM was reached. The sensors were then baselined in binding buffer for 90 seconds. After the baseline measurement, association kinetics (k) were monitored by immersing the biosensor in wells containing the analyte for 400 seconds. Analyte concentrations were decreased as three-fold dilutions from 90, 30, 10, 3.3, 1.1, and 0.4 nM for huIL2Rβ and 600, 200, 66.6, 22.2, 7.4, and 2.5 nM for huIL2Rβγ. Dissociation kinetics (koff) were monitored by immersing the biosensor in separate wells of binding buffer for 800 seconds. Dissociation constants (Kd) were calculated using a 1:1 steady-state binding model using Octet software.

[0145] Using single-alanine mutation screening, we calculated the binding affinity of 27 positions for huIL2Rβ and huIL2Rβγ, and then identified four positions for tuning. Each variant had a characterized Tm to ensure that the reduced affinity was not the result of misfolding, and SEC traces were followed to confirm the absence of aggregation in the tested samples. The 27 positions identified within 5.5 Å of either receptor were positions Q6, L7, H8, E10, H11, L13, Y14, D15, L17, M18, K33, D36, Y37, F39, N40, L43, I44, E47, E84, E85, N88, I91, T92, Q95, S96, I98, and F99. Of the 27 positions, four were identified for tuning in the first attenuation round. Table 2 shows the Kd for Neo-2 / 15 mutants with single alanine mutations of interest. [Table 2]

[0146] The four identified positions were combined in different possibilities to identify combinations of alanine mutations on Neo-2 / 15 to further attenuate its binding to huIL2Rβ and huIL2Rβγ: low attenuation was measured as a 1- to 10-fold reduced Kd relative to Neo-2 / 15, moderate attenuation was measured as a 10- to 100-fold reduced Kd relative to Neo-2 / 15, and high attenuation was measured as a 100-fold or greater reduced Kd relative to Neo-2 / 15 (typically a 100- to 1000-fold reduced Kd relative to Neo-2 / 15).

[0147] The mutation at position 95 was to attenuate binding to IL-2G, whereas the mutations at positions 15, 40, and 44 were to attenuate binding to IL-2B.

[0148] Because binding of Neo-2 / 15 to IL2RG alone is weak, in the low uM range, it is difficult to measure attenuation to the gamma domain by itself, and therefore affinity must be measured in the presence of IL2Rβ. Furthermore, in embodiments where the mutant has moderate to high attenuation, binding via the octet is typically measured under high affinity conditions.

[0149] For high-affinity conditions, attenuated Neo-2 / 15 mutants were fused to full-length antibodies. The antibodies were control antibodies (i.e., not anti-IL-2RB or anti-IL-2RG binding antibodies). For the attenuated Neo-2 / 15 mutants, an anti-CD25 antibody with reduced effector function was used, and for the Neo-2 / 15 control, trastuzumab was used. Biotinylated human IL-2Rb or heterodimeric biotinylated human IL-2Rb + IL-2Rg-Fc (Acro Biosystems, ILG-H82F3) was immobilized to an anti-streptavidin sensor at 2 μg / mL in binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, 0.5% nonfat dry milk). After baseline measurements in binding buffer alone, the binding kinetics was monitored by immersing the biosensor in wells containing defined concentrations of Neo-2 / 15 variants (333-3000 nM) to measure association, followed by immersing the sensor in the baseline well to measure dissociation. Dissociation constants were calculated using a steady-state binding model with Octet software. Results demonstrated that for anti-CD25 fusion proteins containing the Neo-2 / 15 D15S_Q95E variant, the fusion protein bound to IL-2RBG but with weaker affinity than Neo-2 / 15 or Neo-2 / 15 bound to trastuzumab.

[0150] Example 2 - Neo-2 / 15 mutants exhibit attenuated pSTAT5 activity. Neo-2 / 15 mutants were recombinantly expressed and purified from E. coli using the method described in U.S. Patent No. 10,703,791. Neo-2 / 15 mutants with Q95E and D15S mutations (Neo-2 / 15_D15S_Q95E) or Q95A D15A (Neo-2 / 15_D15A_Q95A) mutations (Neo-2 / 15_D15A_Q95A) were tested for their ability to stimulate STAT5 phosphorylation in T cells in vitro. Both mutants showed attenuated pSTAT5 activity in all cell types tested, including CD8+ and CD4+ cell types and T-regs, with Neo-2 / 15_D15S_Q95E showing greater attenuation than Neo-2 / 15_D15A_Q95A. Notably, Neo-2 / 15_D15S_Q95E still maintained a Treg EC50 of approximately 9 nM and approximately 40% of Treg maximal pSTAT5 signaling (see Figures 1A and 1B, Table 3). The higher level of attenuation of Neo-2 / 15_D15S_Q95E translated into better TRA activity (see Examples below). [Table 3]

[0151] Example 3 - Neo-2 / 15 variants can be fused in different configurations to Treg targeting agents and Fc domains. Neo-2 / 15 variants were cloned into pcDNA3.4 and expressed in Expi293 cells in various daclizumab binding domain fusion protein formats. Designs varied in binding domain format (effector-reduced / effector-free scFv or full-length mAb) and site of Neo-2 / 15 variant and scFv fusion. Exemplary configurations are provided in Figures 2A-C.

[0152] In Figure 2A, the Neo-2 / 15 variant and the scFv are both positioned at opposite ends of the Fc. The TRA is a fusion protein comprising a homodimer containing two Neo-2 / 15 variants, each linked via its C-terminus to the N-terminus of an Fc domain, which in turn is linked via its C-terminus to the variable heavy chain of an ScFv.

[0153] In Figure 2B, the Neo-2 / 15 variant is fused to the N-terminus of the heavy chain variable region. The TRA comprises a heterotetramer containing two heavy chains (VH + constant regions) and two light chains (VL + constant regions), with each heavy chain variable region linked via its N-terminus to the C-terminus of the Neo-2 / 15 variant.

[0154] In Figure 2C, the Neo-2 / 15 variant is fused to the C-terminus of the heavy chain. The TRA comprises a heterotetramer containing two heavy chains (VH + constant regions) and two light chains (VL + constant regions), with each heavy chain constant region linked via its C-terminus to the N-terminus of the Neo-2 / 15 variant.

[0155] The linker (GGGGSGGGGSGGGGS) was used.

[0156] Example 4 - Neo-2 / 15 mutant-ScFv fusion protein exhibited TRA activity in pSTAT5 signaling assay Neo-2 / 15 variants were fused to an anti-CD25 ScFv antibody fragment (without the Fc domain) and tested in a pSTAT5 assay to identify fusion proteins that could achieve reduced Teff signaling while maintaining Treg signaling.

[0157] Among the panel of ScFv fusion proteins shown in Table 4, the Neo-2 / 15 mutant_D15S_Q95E fusion protein showed the best reduction in Teff signaling while maintaining Treg signaling. See also Figures 3A-F. For Figures 4A-B, IL-2 control was added at a constant concentration of 1 nM, resulting in an average %pSTAT5+ signaling of 7.44% for all T cells, 9.45% for CD8+ T cells, 6.71% for CD4+ non-Tregs, and 14.2% for Tregs, as represented by the dotted lines. [Table 4]

[0158] Example 5 - Fusion proteins of the invention can be optimized for higher affinity to CD25 than to IL-2 Binding to human CD25 was determined by Octet. Unlike IL-2 and IL-2 mutant Treg agonists, the Neo-2 / 15 mutants of the present invention do not have a binding site for CD25. Affinity for CD25 can be modulated by the choice of anti-CD25 binder. TRAs containing Neo-2 / 15 mutants fused to anti-CD25 antibodies exhibit higher affinity for CD25 than IL-2. The ScFv Neo-2 / 15_D15S_Q95E fusion protein (without the Fc domain) exhibited approximately 25-fold higher binding to human CD25 than hIL-2, with a Kd of 0.5 nM compared to 13 nM (data not shown).

[0159] Example 6 - Fusing Neo-2 / 15 variants to anti-CD25 Treg targeting agents enhances Treg selectivity We evaluated the pSTAT signaling of Treg and Teff cells for anti-CD25 Neo-2 / 15 mutant fusion proteins, including unfused Neo-2 / 15 Neo-2 / 15 mutant_D15S-Q95E and Neo-2 / 15_D15S-Q95E, and IL-2. Fusion of the Neo-2 / 15_D15S-Q95E mutant to an anti-CD25 ScFv enhanced its potency in Treg cells and reduced its potency in CD8+ and CD4+ T cells. The fusion proteins exhibited TRA activity. This was an unexpected result, as fusing a T-reg targeting agent to an attenuated Neo-2 / 15 mutant was not known to cause a differential reduction in Teff signaling compared to Treg signaling. See Figure 5A-B and Table 5. [Table 5]

[0160] Example 7 - Fusion proteins of the invention, including Neo-2 / 15_D15S_Q95E, demonstrated Treg selectivity as measured in the pSTAT5 assay in different antibody configurations. The Neo-2 / 15_D15S_Q95E variant was fused to an anti-CD25 antibody in different configurations, as shown in Figure 2. The fusion proteins that demonstrated the best TRA activity are shown in Figures 6A-C and Table 6. For the Neo-2 / 15_D15S_Q95E fusion protein, the variant attached to the C-terminus of the full-length antibody is the preferred configuration. [Table 6]

[0161] Example 8 - Additional preparation of the Neo-2 / 15 mutant component of TRA Another panel of mutations was generated to identify whether mutations at additional positions involved in binding to IL-2RB or IL-2RBG could be added to the Neo_2 / 15_D15S_Q95E mutant while still maintaining its pSTAT activity profile, and whether other combinations could result in alternative TRAs. The additional positions identified were positions 8, 11, and 14. Table 7 shows the Kd of mutants with one notable mutation from the single alanine mutation scan. The mutation combinations tested were D15S+N40S, D15S+N40S+I44S, H8R+D15S, H8R+D15S+N40S+I44S, H8R+N40S+I44S, H11F+D15S, H11F+D15S+N40S+I44S, H11F+N40S+I44S, Y14K+D15S+N40S+I44S, Y14K+D15S, H11F+N40 S+I44S, D15S+N40S+Q95E, D15S+N40S+I44S+Q95E, H8R+D15S+Q95E, H8R+D15S+N40S+I44S+Q95E, H8R +N40S+I44S+Q95E, H11F+D15S+Q95E, H11F+D15S+N40S+I44S+Q95E, H11F+N40S+I44S+Q95E, N40S+I44S+Q95E, N40S+Q95E, Y14K+D15S+Q95E, Y14K+D15S+N40S+I44S+Q95E, and Y14K+N40S+I44S+Q95E. [Table 7]

[0162] To determine whether the fusion position affected activity, fusion proteins were generated using each mutant expressed as a C- or N-terminal fusion to the full-length anti-CD25 antibody. When fused to the C-terminus, the Neo-2 / 15 mutant is distal to the IL-2A receptor, whereas when fused at the N-terminus, the Neo-2 / 15 mutant is proximal to the receptor. Certain fusions were effective as TRAs when fused to one terminus but less effective at the other. In general, fusion proteins fused at the N-terminus exhibited higher Teff activity than fusions at the C-terminus.

[0163] When fused to the C-terminus, in the presence of the D15S and Q95E mutations, all additional mutations made had the effect of reducing Treg STAT5 phosphorylation activity to undesirable levels. Without being bound by theory, it is believed that the level of attenuation of the D15S_Q95E mutant is at an optimal level for C-terminal fusions, and that any additional mutations made in this mutant are preferably made at positions not involved in binding to IL-2RB or IL-2RBG, provided that the intended C-terminal fusion is to a full-length antibody.

[0164] The upper C-terminal fusions contained the following mutations: D15S_Q95E, H11F_N40S_I44S, Y14K_D15S, and N40S_Q95E. The fusion protein containing Neo-2 / 15_D15S_Q95E exhibited the best pSTAT5 signaling profile of the C-terminally fused TRAs. See Figures 7A-D and 8A-D below, and Tables 8 and 9. Tables 8 and 9 are from two different donor samples.

[0165] It should be noted that for the fusion protein with the N40S and Q95E mutations at the C-terminus, the additional mutations I44S, I44S+H11F, I44S+8HR, and I44S+Y14K had the effect of reducing Treg STAT5 phosphorylation activity to undesirable levels when fused at the C-terminus.

[0166] When fused to the N-terminus, the top 10 fusion proteins contained the following mutations: (i) D15S_N40S, (ii) D15S_N40S_I44S, (iii) H8R_D15S, (iv) H11F_D15S, (v) H11F_N40S_I44S, (vi) H11F_D15S_Q95E, (vii) H11F_N40S_I44S_Q95E, (viii) N40S_I44S_Q95E, (ix) Y14K_D15S_Q95E, and (x) Y14K_N40S_I44S_Q95E. See Figures 9A-B and Tables 8 and 9 below. Tables 8 and 9 are from two different donor samples. [Table 8] [Table 9]

[0167] C-terminal fusions with the Neo-2 / 15_D15S_Q95E mutant consistently demonstrated a strong preferential Treg pSTAT5 signaling profile among donors with low levels of Teff signaling, whereas N-terminal fusions demonstrated a more inconsistent profile among donors, with the exception of protein fusions with the Neo-2 / 15_Q95E_N40S_I44S mutant (see Figure 9A-B).

[0168] Example 9 - Treg expansion in mice Fusion proteins containing Neo-2 / 15_D15S_Q95E fused at either the C-terminus or N-terminus to a full-length anti-CD25 antibody were tested for their ability to expand Treg and NK cells in vivo.

[0169] Humanized NSG mice were purchased from Jackson Labs. These mice were transplanted with human CD34+ hematopoietic stem cells and had more than 25% human CD45+ cells in their peripheral blood. On days 1 and 13, mice were intraperitoneally injected with 5 mg of human IgG. On days 0, 7, 14, and 21, mice were intraperitoneally injected with 15-45 μg of test substance. On days -1, 6, 13, and 20, 100 μL of whole blood was collected by retroorbital bleeding into K2EDTA. Red blood cells were lysed using BioLegend RBC lysis buffer, and the remaining PBMCs were washed with PBS and then stained with viability stain and human Fc-blocking antibodies. Cells were then fixed using eBioscience Foxp3 / transcription factor staining buffer set. Cells were washed with FACS buffer and then stained with CD3, CD4, CD8, CD25, CD56, and Foxp3 antibodies. Flow cytometric analysis of cells was performed on a Cytek Aurora and analyzed using the OMIQ cytometry analysis platform.

[0170] Treg expansion was determined by gating on live cells for CD3 expression, followed by gating on CD4 expression. CD4+ cells were then gated for Tregs by CD25+Foxp3+ staining. Strong Treg expansion was defined as at least a two-fold higher percentage of Tregs among CD4+ cells compared to control mice and the day 1 time point. Treg CD25 and Foxp3 MFI were also compared between groups, with higher MFI corresponding to a more stable and potent Treg phenotype. NK cells were defined as CD56+ cells when gated on live cells. An increase in %CD56+ cells was considered an undesirable effect of potential Treg agonists. While high Treg expansion was observed with both fusion proteins containing full-length antibodies, the N-terminal fusion showed significant NK cell expansion at higher doses. See Figures 10A-E.

[0171] To determine whether TRA elicits anti-drug antibodies, we performed anti-drug antibody (ADA) assays. In this experiment, we used an ScFv fusion protein with Neo-2 / 15_D15S_Q95E without the Fc domain. No anti-drug antibodies were detected (data not shown).

[0172] Example 10 - Alternative Treg targeting agents Neo-2 / 15_D15S_Q95E was fused to anti-CD39 antibody to test whether fusion proteins targeting T-reg markers other than CD25 would result in a PSTAT5 profile characterized by low Teff signaling with preferential Treg signaling. Preferential Treg signaling was observed, although the Treg signaling was not as high as with the CD25 fusion (data not shown).

[0173] Example 11 - IL-2 / IL-15 antagonists are not TRAs Protein P5 from International Application No. WO2021 / 188374 was fused to anti-CD25 ScFV and tested for pSTAT5 Treg signaling activity. There was no pSTAT5 signaling for all T cell types tested, including CD8+ cells, CD4+ cells, and Tregs (data not shown).

[0174] Example 12 - Generation of additional TRAs Another panel of mutations was generated to identify additional substitutions at positions 95, 40, and 44 that may result in active Treg agonists. Mutants exhibiting a Treg-selective pSTAT5 profile were identified, including Neo-2 / 15_D15S_Q95K, Neo-2 / 15_D15S_Q95T, Neo-2 / 15_D15S_Q95Y, Neo-2 / 15_N40G_I44S_Q95E, Neo-2 / 15_N40S_I44T_Q95E, Neo-2 / 15_N40S_I44Y_Q95E, and Neo-2 / 15_N40S_I44N_Q95E.

[0175] Fusion proteins were generated with each mutant expressed as a C-terminal fusion to a full-length anti-CD25 antibody, as previously described. pSTAT5 signaling was plotted using the pSTAT5 mean fluorescence index for each cell subset. See Figures 11A-G.

[0176] Example 13 - TRAs of the invention demonstrate attenuated binding to IL-2RBG in cell binding assays. Saturation binding studies were performed using the HEK293 cell line expressing human CD122 and CD132. Cells were harvested with Versene (Gibco, Catalog No. 15040-066), washed twice in PBS (Gibco, Catalog No. 10010-23), and blocked for 20 minutes in 2% BSA (Sigma-Aldrich, Catalog No. A2153-100G) in PBS on ice. After blocking, cells were aliquoted into 96-well U-bottom plates (Corning, Catalog No. 3799) at 2 x 10 cells per well. Antibody fusions were added to cells at concentrations ranging from 0.5 mM to approximately 150 pM or 0.05 mM to approximately 15 pM in 1% BSA / PBS. Cells were incubated on ice for 30 minutes, then pelleted and washed twice with cold PBS. Cells were pelleted and resuspended in a 200 ng / mL solution of APC-conjugated mouse anti-human IgG Fc secondary antibody (Jackson ImmunoResearch, catalog number 709-605-149) prediluted in 2% BSA / PBS. After a 30-minute incubation on ice, cells were pelleted and washed twice in cold PBS. Fluorescence was detected using a CytoFLEX flow cytometer (Beckman-Coulter) when resuspended at 1 x 106 cells / mL in 2% paraformaldehyde solution (Acros, catalog number 41073-0010). Apparent dissociation constants (KD) were calculated using GraphPad Prism (GraphPad, San Diego, CA). [Table 10] [Table 11-1]

Table 11-2

Table 11-3

Table 11-4

Table 11-5

Table 11-6

Claims

1. A polypeptide for inducing the proliferation of regulatory T cells (Treg), It contains the Neo-2 / 15 mutant and a Treg targeting agent, The Neo-2 / 15 mutant contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 2, The Neo-2 / 15 variant comprises lysine, arginine, threonine, serine, tyrosine, glutamic acid, alanine, or histidine (Q95K or Q95R or Q95T or Q95S or Q95Y or Q95E or Q95A or Q95H) instead of glutamine at position 95, and serine or alanine (D15S or D15A) instead of aspartic acid at position 15, and optionally, the Neo-2 / 15 variant comprises tyrosine or glutamic acid (Q95Y or Q95E) instead of glutamine at position 95, and serine or alanine (D15S or D15A) instead of aspartic acid at position 15, The polypeptide wherein the first amino acid of SEQ ID NO: 2 is designated as position 1, and the first amino acid of SEQ ID NO: 1 is designated as position 4.

2. The Neo-2 / 15 mutant is further, a. Alanine, serine, or glycine (N40A or N40S) instead of asparagine at position 40, and / or b. Alanine, serine, asparagine, threonine, serine, or tyrosine (I44A, I44S, or I44Y) instead of isoleucine at position 44. The polypeptide according to claim 1, comprising:

3. The Neo-2 / 15 mutant is further, a. Instead of leucine, which is ranked 13th, use arginine (L13R). b. Instead of leucine, which is ranked 17th, glutamic acid (L17E) c. Phenylalanine (H11F) instead of histidine at position 11, and / or d. Replace tyrosine (Y14K) with lysine (Y14K), which is ranked 14th. The polypeptide according to claim 1, comprising:

4. The Neo-2 / 15 mutant described above is a) Q95E and D15S, b) Q95E, D15S, and H11F, c) Q95E, D15S, and Y14K, d) Q95H, D15A, L17E, and L13R, e) Q95H and D15A, f) Q95A and D15A, g) D15S and Q95K, h) D15S and Q95T, or i) D15S and Q95Y The polypeptide according to claim 1, comprising a set of substitutions selected from.

5. The Neo-2 / 15 variant contains an amino acid sequence that is at least 90%, at least 95%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 3-5, 13-17, and 75-77, and / or The Neo-2 / 15 mutant contains three amino acids at the N-terminus and bound to the amino acid at position 4, wherein the three amino acids are proline-lysine-lysine, and the positional numbering follows Sequence ID No.

1. The polypeptide according to claim 1.

6. a. The Neo-2 / 15 mutant and / or polypeptide binds to IL-2Rβγ with an affinity at least 10 times, or at least 100 times, or at least 500 times, or at least 1000 times, or at least 10,000 times, compared to Neo-2 / 15 and / or IL-2, and optionally, the affinity is attenuated by 500 times or less compared to Neo-2 / 15 and / or IL-2, and / or the affinity is attenuated by 1000 times or less or 10,000 times or less compared to Neo-2 / 15 and / or IL-2. b. The Neo-2 / 15 mutant binds to IL-2Rβγ with an affinity of up to 2, 3, 4, or 5 times that of the Neo-2 / 15 mutant consisting of the sequence shown in Sequence ID No. 4 for IL-2Rβγ. c. When the Neo-2 / 15 variant is fused to the N-terminus and / or C-terminus of an antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 73 and a light chain containing the amino acid sequence of SEQ ID NO: 70, it binds to IL-2Rβγ with an affinity of 2, 3, 4, or 5 times the affinity of the reference polypeptide to IL-2Rβγ, wherein the reference polypeptide comprises the Neo-2 / 15 variant fused to the C-terminus of the antibody, and the reference polypeptide comprises the amino acid sequences of SEQ ID NOs: 69 and 70. d. The Neo-2 / 15 mutant stimulates STAT5 phosphorylation in Treg cells with an EC50 substantially the same as or less than that of the Neo-2 / 15 mutant consisting of the sequence shown in Sequence ID No.

4. e. When the Neo-2 / 15 variant is fused to the N-terminus and / or C-terminus of an antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 73 and a light chain containing the amino acid sequence of SEQ ID NO: 70, it stimulates STAT5 phosphorylation in Treg cells at an EC50 substantially the same as or less than that of a reference polypeptide containing the Neo-2 / 15 variant fused to the C-terminus of the antibody, wherein the reference polypeptide comprises the amino acid sequences of SEQ ID NOs: 69 and 70. f. The Neo-2 / 15 mutant stimulates STAT5 phosphorylation in Treg cells with a maximum signal transduction value substantially the same as or greater than that of the Neo-2 / 15 mutant consisting of the sequence shown in Sequence ID No.

4. g. When the Neo-2 / 15 variant is fused to the N-terminus and / or C-terminus of an antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 73 and a light chain containing the amino acid sequence of SEQ ID NO: 70, it stimulates STAT5 phosphorylation in Treg cells at a maximum signal transduction value substantially the same as or greater than that of a reference polypeptide comprising the Neo-2 / 15 variant fused to the C-terminus of the antibody, wherein the reference polypeptide comprises the amino acid sequences of SEQ ID NOs: 69 and 70, h. The Neo-2 / 15 mutant stimulates STAT5 phosphorylation in Treg cells at a maximum signaling value that is at least 50%, at least 75%, and at least 100% of the maximum signaling resulting from Neo-2 / 15 and / or IL-2 stimulation, and / or i. When the Neo-2 / 15 mutant is fused with the N-terminus and / or C-terminus of an antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 73 and a light chain containing the amino acid sequence of SEQ ID NO: 70, it stimulates STAT5 phosphorylation in Treg cells at an EC50 of 1 nM or less, 0.5 nM or less, or 0.1 nM or less, and / or stimulates STAT5 phosphorylation in T effector (Teff) cells at an EC50 of 1 nM or more. The polypeptide according to claim 1.

7. The polypeptide comprises an Fc domain, Optionally, the polypeptide includes an Fc domain that substantially lacks effector function. Optionally, the Fc domain is a. An IgG1 Fc domain containing substitutions L234A and L235A, b. Containing substitution G237A, or c. Substitutions including P329G or P329A, The polypeptide according to claim 1.

8. The aforementioned targeting agent, a. Binds to an antigen on the surface of Treg cells, optionally the antigen being CD25 or CD39, and / or b. An antibody or its antigen-binding fragment, optionally, i. The targeting agent is an antigen-binding fragment of an antibody, and optionally the antigen-binding fragment is Fab, (Fab')2, scFv, or antigen-binding moiety of a single-domain antibody, and optionally the antigen-binding fragment is scFv. ii. The targeting agent is a full-length antibody. iii. The antibody or its antigen-binding fragment includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 27, 42, or 43, CDR-H2 containing the amino acid sequence of SEQ ID NO: 28 or 44, CDR-H3 containing the amino acid sequence of SEQ ID NO: 29, CDR-L1 containing the amino acid sequence of SEQ ID NO: 30 or 45, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31 or 46, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32, or iv. The antibody or its antigen-binding fragment comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 26 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

41. The polypeptide according to claim 1.

9. The polypeptide according to claim 1, wherein the polypeptide comprises a heavy chain IgG1 constant region having the amino acid sequence of SEQ ID NO:

71.

10. The Neo-2 / 15 variant is linked to the targeting agent or the N-terminus of the Fc domain, Optionally, the polypeptide comprises the amino acid sequence of SEQ ID NO: 74 and the amino acid sequence of SEQ ID NO: 70, or the Neo-2 / 15 variant is linked to the C-terminus of the targeting agent or the Fc domain. Optionally, the polypeptide comprises the amino acid sequence of SEQ ID NO: 69 and the amino acid sequence of SEQ ID NO:

70. Optionally, a. The Neo-2 / 15 variant is fused to the targeting agent or the Fc domain, or b. The Neo-2 / 15 variant is linked to the targeting agent or the Fc domain via an amino acid linker, and optionally the amino acid linker contains the sequence (GGGGS) n, where n is 1 to 10, and optionally n is 1, 2, 3, or 4. The polypeptide according to claim 1.

11. a. The polypeptide does not substantially activate Teff cells, or the polypeptide activates Teff cells at an EC50 that is more than 5 times, more than 10 times, more than 50 times, more than 100 times, more than 500 times, or more than 1000 times higher than the EC50 required to activate Treg cells. b. The polypeptide does not substantially induce the proliferation of Teff cells, or the polypeptide induces the proliferation of Teff cells having an EC50 that is more than 5 times, more than 10 times, more than 50 times, more than 100 times, more than 500 times, or more than 1000 times higher than the EC50 required to induce the proliferation of Treg cells. c. The maximum signal transduction by the polypeptide in non-Treg cells is less than 30% or less than 20% of the maximum signal transduction by IL-2 in non-Treg cells at a polypeptide concentration of 10 nM or less, and optionally, the non-Treg cells are Teff cells or NK cells. d. The maximum signal transduction by the polypeptide in Treg is at least 50% of the maximum signal transduction by IL-2 in Treg at a concentration of the polypeptide of 10 nM or less. e. The polypeptide induces STAT5 phosphorylation in Treg cells at an EC50 below 1 nM, below 500 pM, or below 100 pM, and / or the polypeptide induces STAT5 phosphorylation in Teff cells at an EC50 above 1000 pM. f. The polypeptide does not substantially activate NK cells, or the polypeptide activates NK cells at an EC50 that is more than 5 times, 10 times, 50 times, 100 times, 500 times, or 1000 times higher than the EC50 required to activate Treg cells. g. The polypeptide does not substantially induce the proliferation of NK cells, or the polypeptide induces the proliferation of NK cells having an EC50 that is more than 5 times, more than 10 times, more than 50 times, more than 100 times, more than 500 times, or more than 1000 times higher than the EC50 required to induce the proliferation of Treg cells, and / or h. The polypeptide induces STAT5 phosphorylation in Treg cells at an EC50 below 1 nM, below 500 pM, or below 100 pM, and / or the polypeptide induces STAT5 phosphorylation in NK cells at an EC50 above 1000 pM. The polypeptide according to claim 1.

12. A pharmaceutical composition comprising the polypeptide described in claim 1 and a pharmaceutically acceptable carrier or diluent.

13. A polypeptide or pharmaceutical composition for inducing proliferation of Treg cells and / or activating Treg cells in vitro, wherein the polypeptide comprises the polypeptide described in any one of claims 1 to 11, or the pharmaceutical composition comprises the pharmaceutical composition described in claim 12.

14. A polypeptide or pharmaceutical composition for inducing proliferation of Treg cells and / or activating Treg cells in vivo, wherein the polypeptide comprises the polypeptide described in any one of claims 1 to 11, or the pharmaceutical composition comprises the pharmaceutical composition described in claim 12.

15. A polypeptide or pharmaceutical composition for treating diseases related to Teff cell activity and / or B cell activity, The polypeptide comprises the polypeptide described in any one of claims 1 to 11, or the pharmaceutical composition comprises the pharmaceutical composition described in claim 12. Optionally, the disease is an autoimmune disease, and optionally, the autoimmune disease is selected from the group consisting of: rheumatic diseases including but not limited to rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, scleroderma, mixed connective tissue disease, dermatomyositis, polymyositis, Reiter's syndrome, or Behçet's disease; type II diabetes mellitus; autoimmune diseases of the thyroid gland including but not limited to Hashimoto's thyroiditis or Graves' disease; autoimmune diseases of the central nervous system including but not limited to multiple sclerosis, myasthenia gravis, or encephalomyelitis; pemphigus including but not limited to pemphigus vulgaris, pemphigus proliferative, pemphigus foliaceus, sinea-Usher syndrome, or pemphigus breviated; psoriasis; inflammatory bowel diseases including but not limited to ulcerative colitis or Crohn's disease; and celiac disease. The polypeptide or pharmaceutical composition.

16. A polypeptide or pharmaceutical composition for treating a subject who has received a transplant of a biomaterial such as an organ, tissue, or cell transplant, The polypeptide comprises the polypeptide described in any one of claims 1 to 11, or the pharmaceutical composition comprises the pharmaceutical composition described in claim 12. Optionally, if the subject has graft-versus-host disease, The polypeptide or pharmaceutical composition.

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