Neo-2 / 15 variants and their use for preferentially stimulating T regulatory cells

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application Nos. 63 / 357,844, filed on July 1, 2022, and 63 / 395,437, filed on August 5, 2022, each of which is incorporated herein by reference in its entirety for any purpose.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on June 21, 2023, named "2023-06-21_01267-0005-00PCT_ST26", and is 95,337 bytes in size. Background Art

[0005] Regulatory T cells (Tregs) are essential for maintaining immune tolerance, and their dysfunction can lead to inflammation and autoimmune diseases. IL-2 is a pleiotropic cytokine that activates immunosuppressive Tregs and inflammatory cells, including NK cells, cytotoxic T cells, and helper T cells. Although low-dose IL-2 therapy is effective in expanding Tregs, this intervention is limited by the simultaneous stimulation of conventional T cells and natural killer cells. New therapies are needed to expand Treg cells without the concomitant stimulation of T cells and natural killer cells. Summary of the invention

[0006] Provided herein are polypeptides comprising a Neo-2 / 15 mutant, polypeptides comprising a Neo-2 / 15 mutant, and agents for targeting T regulatory cells, and methods of using the same to treat diseases.

[0007] In some embodiments, a polypeptide comprising a Neo-2 / 15 mutant is provided, wherein the Neo-2 / 15 mutant comprises an amino acid sequence that is 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 mutant comprises a lysine, arginine, threonine, serine, tyrosine, glutamic acid, alanine, or histidine substitution for glutamine at position 95 (Q95K or Q95R or Q95T or Q95S or Q95Y or Q95E or Q95A or Q95H), and at least one additional substitution selected from:

[0008] a) substitution of aspartic acid at position 15 by serine or alanine (D15S or D15A);

[0009] b) substitution of alanine or serine or glycine for asparagine at position 40 (N40A or N40S or N40G); and

[0010] c) substitution of the isoleucine at position 44 by alanine or serine or asparagine or threonine or tyrosine (I44A or I44S or I44N or I44T or I44Y);

[0011] 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.

[0012] In some embodiments, a polypeptide comprising a Neo-2 / 15 mutant is provided, wherein the Neo-2 / 15 mutant comprises an amino acid sequence that is 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 mutant comprises a substitution of glutamic acid, alanine, or histidine for glutamine at position 95 (Q95E or Q95A or Q95H), and at least one additional substitution selected from:

[0013] a) substitution of aspartic acid at position 15 by serine or alanine (D15S or D15A);

[0014] b) substitution of alanine or serine for asparagine at position 40 (N40A or N40S); and

[0015] c) substitution of isoleucine at position 44 with alanine or serine (I44A or I44S);

[0016] 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.

[0017] In some embodiments, a polypeptide comprising a Neo-2 / 15 mutant is provided, wherein the Neo-2 / 15 mutant comprises an amino acid sequence that is 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 mutant comprises a serine or alanine substitution for aspartic acid at position 15 (D15S or D15A), and at least one additional substitution selected from:

[0018] a) substitution of glutamine at position 95 by 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);

[0019] b) substitution of arginine for histidine at position 8 (H8R);

[0020] c) substitution of phenylalanine for histidine at position 11 (H11F);

[0021] d) substitution of lysine for tyrosine at position 14 (Y14K);

[0022] e) substitution of alanine or serine for asparagine at position 40 (N40A or N40S); and

[0023] f) substitution of the isoleucine at position 44 by alanine, serine or tyrosine (I44A or I44S or I44Y),

[0024] wherein 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.

[0025] In some embodiments, a polypeptide comprising a Neo-2 / 15 mutant is provided, wherein the Neo-2 / 15 mutant comprises an amino acid sequence that is 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 mutant comprises a serine or alanine substitution for aspartic acid at position 15 (D15S or D15A), and at least one additional substitution selected from:

[0026] a) substitution of glutamic acid, alanine or histidine for glutamine at position 95 (Q95E, Q95A or Q95H);

[0027] b) substitution of arginine for histidine at position 8 (H8R);

[0028] c) substitution of phenylalanine for histidine at position 11 (H11F);

[0029] d) substitution of lysine for tyrosine at position 14 (Y14K);

[0030] e) substitution of alanine or serine for asparagine at position 40 (N40A or N40S); and

[0031] f) substitution of the isoleucine at position 44 by alanine or serine (I44A or I44S),

[0032] wherein 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.

[0033] In some embodiments, a polypeptide comprising a Neo-2 / 15 mutant is provided, wherein the Neo-2 / 15 mutant comprises an amino acid sequence that is 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 mutant comprises:

[0034] a) substitution of phenylalanine for histidine at position 11 (H11F);

[0035] b) substitution of alanine or serine for asparagine at position 40 (N40A or N40S); and

[0036] c) substitution of the isoleucine at position 44 by alanine, serine or tyrosine (I44A or I44S or I44Y),

[0037] wherein 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.

[0038] In some embodiments, a polypeptide comprising a Neo-2 / 15 mutant is provided, wherein the Neo-2 / 15 mutant comprises an amino acid sequence that is 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 mutant comprises:

[0039] a) substitution of phenylalanine for histidine at position 11 (H11F);

[0040] b) substitution of alanine or serine for asparagine at position 40 (N40A or N40S); and

[0041] c) substitution of the isoleucine at position 44 by alanine or serine (I44A or I44S),

[0042] wherein 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.

[0043] In some embodiments, a polypeptide comprising a Neo-2 / 15 mutant is provided, wherein the Neo-2 / 15 mutant comprises domains D1, D2, D3, and D4; wherein:

[0044] (a) D1 contains the amino acid sequence: KIQLHAEHALYX 15 ALMILNI (SEQ ID NO:61) and D3 comprises the amino acid sequence LEDYAFNFELILEEIARLFESG (SEQ ID NO:64); or

[0045] (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); and

[0046] D2 comprises an amino acid sequence of at least 8 amino acids in length;

[0047] D4 contains the amino acid sequence EDEQEEMANAIITILX 95 SWIFS (SEQ ID NO: 66); wherein:

[0048] (i) In the Neo-2 / 15 mutant, D1, D2, D3 and D4 can be arranged in any order;

[0049] (ii) an amino acid linker may be present between any of the domains (a "domain linker");

[0050] (iii)X 15 is serine or alanine; X 95 is glutamate, alanine or histidine; X 40 is serine or alanine; and 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 or alanine or glycine; and X 44 is serine or alanine or asparagine or threonine or tyrosine;

[0051] (iv) wherein the Neo-2 / 15 mutant contains a total of no more than ten, no more than nine, no more than eight, no more than seven, no more than six, no more than five, no more than four, no more than three, no more than two, no more than one, or zero substitutions at amino acid positions not designated as X. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1A -B provides a graph showing reduced STAT5 signaling in Neo-2 / 15 mutants Neo-2 / 15_D15S_Q95E and Neo-2 / 15_D15A_Q95A. Figure 1AShown are 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 (dashed line, open shapes). Figure 1B Shown are the percentages of CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) showing STAT5 phosphorylation following titration stimulation with Neo-2 / 15_D15A_Q95A (solid line, filled shapes) and Neo-2 / 15 (dashed line, open shapes).

[0053] Figure 2A -C provides a representation of the fusion protein form. Figure 2A In the case of Neo-2 / 15 mutant and scFv, both are located at opposite ends of Fc. Figure 2B In the present invention, the Neo-2 / 15 mutant is linked to the N-terminus of the heavy chain of the antibody. Figure 2C In the present invention, the Neo-2 / 15 mutant is linked to the C-terminus of the heavy chain of the antibody.

[0054] Figure 3A -F provides a graph of STAT5 signaling for Neo-2 / 15 and Neo-2 / 15 mutants fused to anti-CD25 scFv. The percentage of all T cells (squares), CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) that exhibit STAT5 phosphorylation after titration stimulation with scFv fusion proteins (solid line, solid shapes) and human IL-2 (dashed line, open shapes) is provided. Figure 3A Neo-2 / 15 fused to anti-CD25 ScFV was provided; Figure 3B Neo-2 / 15_D15S_Q95E fused to anti-CD25 ScFV was provided; Figure 3C Neo-2 / 15_D15A_Q95A fused to anti-CD25 ScFV was provided; Figure 3D Neo-2 / 15_D15A_Q95H fused to anti-CD25 ScFV was provided; Figure 3E Neo-2 / 15_L13R_D15A_L17E_Q95H fused to anti-CD25 ScFV was provided; Figure 3F Neo-2 / 15_L13R_L17E_N40A_Q95H fused to anti-CD25 ScFV is provided.

[0055] Figure 4A-B provides a graph of STAT5 signaling for Neo-2 / 15 fused to anti-CD25 ScFV and Neo-2 / 15_D15S_Q95E fused to anti-CD25 ScFV in a second patient sample. The percentage of all T cells (circles), CD8+ cells (upright triangles), CD4+ cells (delta triangles), and Treg cells (squares) that exhibit STAT5 phosphorylation after stimulation with scFv fusion proteins (solid line, solid shapes) and human IL-2 control at 1 nM (dashed line) titration is provided. Figure 4A Neo-2 / 15 fused to anti-CD25 ScFV is provided. Figure 4B Neo-2 / 15_D15S_Q95E fused to anti-CD25 ScFv is provided.

[0056] Figure 5A -B provides a graph of STAT5 signaling of Neo-2 / 15 mutant D15S_Q95E without fusion (5A) and fusion with anti-CD25 ScFv (5B). The percentage of CD8+ cells (triangles), CD4+ cells (squares) and Treg cells (circles) showing STAT5 phosphorylation after titration stimulation with test article is provided.

[0057] Fig. 6A -C provides a graph of STAT5 signaling of Neo-2 / 15 mutant D15S_Q95E fused to anti-CD25 ScFv (6A); fused to the full-length antibody at the C-terminus (6B); and fused to the full-length antibody at the N-terminus (6C). The percentage of all T cells (diamonds), CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) that exhibit STAT5 phosphorylation after titration stimulation with the test article is provided.

[0058] Fig. 7A -D provides a diagram of STAT5 signaling of Neo-2 / 15 mutants fused to the C-terminus of the heavy chain of an anti-CD25 antibody ( Figure 2C The percentages of CD8+ cells (triangles), CD4+ cells (squares), and Treg cells (circles) that exhibit STAT5 phosphorylation following titration stimulation with scFv fusion proteins (solid line, filled shapes) and human IL-2 (dashed line, open shapes) are presented. Fig. 7A A fusion protein with Neo-2 / 15_H11F_N40S_I44S is provided; 7B provides a fusion protein with Neo-2 / 15_Y14K_D15S; 7C provides a fusion protein with Neo-2 / 15_N40S_Q95E; and 7D provides a fusion protein with Neo-2 / 15_D15S_Q95E.

[0059] Fig. 8A -D provides a diagram of STAT5 signaling of the Neo-2 / 15 mutant fused to the C-terminus of the heavy chain of the anti-CD25 antibody ( Figure 2C ). The percentages of CD8+ cells (triangles), CD4+ cells (squares) and Treg cells (circles) showing STAT5 phosphorylation after titration stimulation with scFv fusion protein (solid line, solid shapes) and human IL-2 (dashed line, open shapes) are provided. The experiment of Figure 7 was repeated with different patient samples. Fig. 7A A fusion protein with Neo-2 / 15_H11F_N40S_I44S is provided; 7B provides a fusion protein with Neo-2 / 15_Y14K_D15S; 7C provides a fusion protein with Neo-2 / 15_N40S_Q95E; and 7D provides a fusion protein with Neo-2 / 15_D15S_Q95E.

[0060] Fig. 9A -B provides a graph of STAT5 signaling for Neo-2 / 15_N40S_I44S_Q95E fused to the N-terminus of the heavy chain of an anti-CD25 antibody for two different patient samples (9A, 9B) (see Figure 2C The percentages of CD8+ (triangles), CD4+ (squares), and Treg cells (circles) that exhibit STAT5 phosphorylation following titration stimulation with scFv fusion proteins (solid line, filled shapes) and human IL-2 (dashed line, open shapes) are presented.

[0061] Fig. 10A -E provides a graph showing Treg expansion in an in vivo study in mice. Mice were transplanted with human CD34+ hematopoietic stem cells and had >25% human CD45+ cells in the peripheral blood. Mice were injected intraperitoneally with 5mg human IgG on days -1 and 13. Mice were injected intraperitoneally with 15-45ug of test article on days 0, 7, 14, and 21. On days -1, 6, 13, and 20, 100uL of whole blood was collected in K2EDTA by retro-orbital bleeding. Fig. 10A and 10B Shown is the expansion of Tregs over time in response to test article stimulation at the indicated dose levels, measured as the proportion of CD4+ cells. Fig. 10C Shown is the expansion of NK cells over time upon stimulation with the test article at the indicated dose levels. Fig. 10D and 10EShown are Treg Foxp3 MFI over time under stimulation of the test articles at the indicated dose levels. The test articles were vehicle control (open circles), distal fusion Treg agonist (TRA; open squares), and proximal fusion TRA. The distal fusion TRA was Neo-2 / 15_D15S_Q95E fused to the C-terminus of the heavy chain of the anti-CD25 antibody. The proximal fusion TRA was Neo-2 / 15_N40S_I44S_Q95E fused to the N-terminus of the heavy chain of the anti-CD25 antibody.

[0062] Fig.11A -G provides a graph of STAT5 signaling of C-terminally fused Neo-2 / 15 fusion proteins D15S_Q95K (11A), D15S_Q95T (11B), D15S_Q95Y (11C), N40G_I44S_Q95E (11D), N40S_I44T_Q95E (11E), N40S_I44Y_Q95E (11F) and N40S_I44N_Q95E (11G). Provides the pSTAT5 mean fluorescence index (MFI) of CD8+ cells (circles), CD4+ cells (squares) and Treg cells (triangles). DETAILED DESCRIPTION

[0063] 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 CAS registration number 2407798-79-0. NL-201 was developed from Neo-2 / 15 by introducing a cysteine ​​residue at position 62 to enable 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.

[0064] The inventors unexpectedly discovered that certain Neo-2 / 15 mutants can preferentially stimulate and expand T regulatory cells (Tregs) relative to non-Tregs when targeting Tregs. Specifically, by mutating selected amino acids of Neo-2 / 15 to weaken its binding to the medium-affinity IL-2 receptor IL-2Rβγ cBy combining the IL-2 / 15 mutant with the Treg-targeting agent, the inventors created 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. By combining the Treg binding site with IL-2Rβ and IL-2γ c Binding site decoupling allows manipulation and optimization of binding site orientation and proximity.

[0065] Regulatory T cells (Treg)

[0066] Regulatory T cells (Treg) are naturally occurring CD4+CD25+FOXP3+T lymphocytes, accounting for about 5-10% of the circulating CD4+T cell population, which mainly inhibit autoreactive lymphocytes and control innate and adaptive immune responses. Treg achieves this inhibition at least in part by inhibiting the proliferation, expansion and effector activity of T effector cells (Teff). While Foxp3 is a recognized marker of Treg cells, it is difficult to use the Foxp3 marker to isolate cells for functional studies. Studies have shown that CD4(+)CD25(+)CD127(low / -) cells express the highest levels of Foxp3 and have the strongest correlation with CD4(+)CD25(+)Foxp3(+)T cells (see Yu et al., Inflammation , December 2021; 35(6):1773-80). The inventors used CD4(+)CD25(+)CD127(low / -) as the identification features of Treg. Teff 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 the present invention, Teff are defined as non-Treg CD4+ and CD8+ T cells.

[0067] It is known that increasing the number of Tregs, increasing Treg activity and / or reducing Treg cell death (e.g., apoptosis) helps to suppress the undesirable immune response associated with a range of immune diseases and inflammation. Ideally, treatment with Treg agonists will preferentially enhance Tregs while minimizing or not activating Teff or other cells that may worsen inflammation. The examples and teachings provided herein demonstrate surprising and unexpected results, i.e., polypeptides comprising targeted Neo-2 / 15 mutants can be used to selectively and effectively activate Tregs relative to Teffs, indicating that the polypeptides can be used to treat or improve diseases and conditions that can benefit from suppressing immune responses, such as autoimmune diseases and diseases and conditions associated with inflammation.

[0068] Treg agonists (TRA)

[0069] The present invention particularly provides TRA that preferentially stimulates Treg cells. As used herein, "preferentially stimulates T regulatory cells" refers to TRA promoting the proliferation, survival, activation and / or function of Treg relative to non-Treg. In some aspects, TRA preferentially stimulates Treg relative to Teff or NK cells. In some embodiments, TRA comprises at least one Neo-2 / 15 mutant provided herein, which is connected to a targeting agent that binds to an antigen on the surface of a Treg cell.

[0070] Methods for measuring the ability to preferentially stimulate Tregs can be measured by flow cytometry of peripheral blood leukocytes, wherein an increase in the percentage of Tregs among total CD4+ T cells, an increase in the percentage of Tregs among total CD8+ T cells, an increase in the percentage of Tregs relative to NK cells, and / or a greater increase in the expression level of CD25 on the surface of Tregs relative to an increase in CD25 expression on other T cells is observed.

[0071] In some embodiments, TRA that preferentially stimulates Treg cells increases the percentage of Tregs in 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, effective Treg expansion is measured by at least a 2-fold increase in the percentage of Tregs in the total population of CD4+ cells when treated with TRA compared to a vehicle control.

[0072] In some embodiments, the method of identifying TRA of the present invention comprises measuring the ability of TRA to promote or stimulate STAT5 phosphorylation in Treg compared to Teff. Compared to Neo-2 / 15 and / or IL-2, the ability of exemplary TRA to promote or stimulate STAT5 phosphorylation in Teff is significantly reduced, but the ability to promote or stimulate STAT5 phosphorylation in Treg remains unchanged.

[0073] In some embodiments, exemplary TRA of the present invention does not substantially activate or induce Teff proliferation. In some aspects, the activation or proliferation of Teff is measured using STAT5 assay as described herein. In some embodiments, the maximum signal transduction of TRA in Teff cells is preferably less than 50%, less than 40%, less than 30% or less than 20% of the maximum signal transduction produced by IL-2 stimulation (e.g., at a concentration of up to 10nM). However, the maximum signal transduction in Treg should preferably be at least 40%, at least 50%, at least 60% or at least 70% of the maximum signal transduction produced by IL-2 stimulation, wherein the maximum Treg signal transduction is higher, indicating that TRA is stronger. In some preferred embodiments, the EC50 of Treg%pSTAT5+ curve should be lower than 10nM, lower than 1nM, lower than 500pM, lower than 250pM or even lower than 100pM, wherein the lower the Treg EC50, the more effective TRA is. In some embodiments, the EC50 of the Treg % pSTAT5+ curve for the TRA provided herein is less than 1 nM, wherein the maximal signaling in Tregs is greater than 50% of the maximal signaling caused by IL-2 stimulation.

[0074] Peptides

[0075] The present invention particularly provides Neo-2 / 15 mutants. Compared with Neo-2 / 15, exemplary Neo-2 / 15 mutants bind to IL-2Rβγ c The binding of Neo-2 / 15 mutants is weakened. Such Neo-2 / 15 mutants can be optionally connected (e.g., by fusion or by chemical / enzymatic binding) to a targeting agent that binds an antigen on the surface of a Treg cell. The exemplary antigen used in the present invention is CD25 or CD39. In an exemplary embodiment of the present invention, the polypeptide comprising a Neo-2 / 15 mutant that is connected to a targeting agent that binds an antigen on the surface of a Treg cell 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.

[0076] In some embodiments, the Neo-2 / 15 mutant comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to Neo-2 / 15 and has one or more amino acid substitutions that reduce binding to IL-2Rβ and / or one or more amino acid substitutions that reduce binding to IL-2Rγ. c In some embodiments, the Neo-2 / 15 mutant comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to Neo-2 / 15, and has reduced binding to IL-2Rβγ compared to Neo-2 / 15. c A combination of one to five amino acid substitutions.

[0077] In some embodiments, the polypeptides of the invention comprise Neo-2 / 15 mutants, wherein the Neo-2 / 15 mutants comprise an amino acid sequence that is 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 (a truncated Neo-2 / 15 without its 3 N-terminal amino acids) or SEQ ID NO: 2 (Neo-2 / 15). Such Neo-2 / 15 mutants comprise 2-20, 2-15, 2-10, 2-5, 2-4, or 2-3 amino acid substitutions compared to Neo-2 / 15. The amino acid sequences of exemplary Neo-2 / 15 mutants are set forth in SEQ ID NOs: 3-25 and 75-82. Particularly preferred are amino acid sequences listed in SEQ ID NO: 3 or 4 comprising Q95E, D15S substitutions, or SEQ ID NO: 6 or 7 comprising Q95E, N40S and I44s substitutions, or SEQ ID NO: 80 or 81 comprising N40S, I44Y and Q95E substitutions. Although it may be advantageous to reduce the number of further mutations, the present invention includes Neo-2 / 15 mutants with truncations or additional insertions, deletions or substitutions other than those described herein, provided that the Neo-2 / 15 mutant retains the activity of preferentially stimulating Tregs when targeting Treg cells (e.g., by fusion with an anti-CD25 antibody such as described herein). If there are additions or deletions in the Neo-2 / 15 sequence, such additions or deletions are preferably not within the D1, D3 or D4 domains of the Neo-2 / 15 mutant (i.e., not within amino acids 4-22, 34-55 and 80-100 numbered according to SEQ ID NO: 2). In some embodiments, if additional substitutions are present in Neo-2 / 15 (other than those at positions 8, 11, 14, 15, 40, 44, or 95, as numbered according to SEQ ID NO:2), no more than 1-10, no more than 1-5, or no more than 1-3 such mutations are present in the amino acid sequence of the D1, D3, or D4 domains.

[0078] The present invention includes polypeptides comprising a Neo-2 / 15 mutant, wherein the Neo-2 / 15 mutant comprises an amino acid sequence that is 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 mutant comprises a lysine, arginine, threonine, serine, tyrosine, glutamate, alanine or histidine substitution for glutamine at position 95 (Q95K or Q95R or Q95T or Q95S or Q95Y or Q95E or Q95A or Q95H), and at least one additional substitution selected from:

[0079] a) substitution of aspartic acid at position 15 by serine or alanine (D15S or D15A);

[0080] b) substitution of alanine or serine or glycine for asparagine at position 40 (N40A or N40S or N40G); and

[0081] c) substitution of the isoleucine at position 44 by alanine or serine or asparagine or threonine or tyrosine (I44A or I44S or I44N or I44T or I44Y);

[0082] wherein 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.

[0083] The present invention includes polypeptides comprising a Neo-2 / 15 mutant, wherein the Neo-2 / 15 mutant comprises an amino acid sequence that is 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 mutant comprises a substitution of glutamic acid, alanine, or histidine for glutamine at position 95 (Q95E or Q95A or Q95H), and at least one additional substitution selected from:

[0084] a) substitution of aspartic acid at position 15 by serine or alanine (D15S or D15A);

[0085] b) substitution of alanine or serine for asparagine at position 40 (N40A or N40S); and

[0086] c) Substitution of isoleucine at position 44 by alanine or serine (I44A or I44S)

[0087] wherein 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.

[0088] In some embodiments, the Neo2 / 15 mutant comprises a set of substitutions selected from the group consisting of: 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, N40S and I44A; Q95E, N40A and I44S; Q95A, N40S and I44S; Q95A, N40A and I44A; Q95A, N40S and I44A; Q95A, N40A and I44S; Q95H, N40S and I44S; Q95H, N40A and I44A; Q95H, N40S and I44A; Q95H, N40A and I44S; D15S and Q95K; D15S and Q95T; D15S and Q95Y; N40G, I44S and Q95E; N40S, I44T and Q95E; N40S, I44Y and Q95E; and N40S, I44N and Q95E.

[0089] In some such aspects, in addition to those substitutions mentioned above, additional 1-18, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2 substitutions are performed. In some aspects, no other substitutions are performed. Exemplary additional substituents include, for example, phenylalanine replaces the histidine at position 11 (H11F), lysine replaces the tyrosine at position 14 (Y14K), and arginine replaces the histidine at position 8 (H8R). In some embodiments, positions 8, 11 and 14 are not replaced. In some aspects, Neo-2 / 15 mutants include alanine replacing the aspartic acid at position 15. In some such aspects, Neo-2 / 15 mutants include arginine replacing the leucine at position 13, and / or glutamic acid replacing the leucine at position 17. In some embodiments, if the Neo-2 / 15 mutant comprises a serine or alanine substitution for aspartic acid at position 15, it does not comprise (i) a serine or alanine substitution for asparagine at position 40, or it does not comprise (ii) a serine or alanine substitution for isoleucine at position 44.

[0090] In some embodiments, if the Neo-2 / 15 mutant comprises a serine or alanine substitution for aspartic acid at position 15, it does not comprise (i) a serine or alanine or glycine substitution for asparagine at position 40, and / or it does not comprise (ii) a serine or alanine or asparagine or threonine or tyrosine substitution for isoleucine at position 44. In some embodiments, if the Neo-2 / 15 mutant comprises a serine or alanine substitution for aspartic acid at position 15, it does not comprise (i) a substitution at position 40 or (ii) a substitution at position 44. In some embodiments, if the Neo-2 / 15 mutant comprises a serine or alanine substitution for aspartic acid at position 15, it does not comprise (i) a serine or alanine substitution for asparagine at position 40, and it does not comprise (ii) a serine or alanine substitution for isoleucine at position 44. In some embodiments, if the Neo-2 / 15 mutant comprises a serine or alanine substitution for aspartic acid at position 15, it does not comprise (i) a substitution at position 40 and (ii) a substitution at position 44. In some embodiments, wherein the Neo-2 / 15 mutant comprises an alanine or serine substitution for asparagine at position 40 and / or an alanine or serine substitution for isoleucine at position 44, it comprises an acidic amino acid at position 15.

[0091] In some embodiments, the Neo-2 / 15 mutant comprises alanine or serine or asparagine or threonine or tyrosine substituted for isoleucine at position 44 (I44A or I44S or I44N or I44T or I44Y). In some embodiments, the Neo-2 / 15 mutant comprises alanine or serine or glycine substituted for asparagine at position 40 (N40A or N40S or N40G). In some embodiments, the Neo-2 / 15 mutant comprises a group of substitutions selected from the group consisting of: 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;

[0092] Q95H and D15A; and Q95A and D15A. In some aspects, the Neo-2 / 15 mutant comprises additional substituents at positions 8, 11, 14, 15, 40, 44, and 95 in addition to the substituents described herein. In some aspects, the Neo-2 / 15 mutant does not comprise additional substituents at positions 8, 11, 14, 15, 40, 44, and 95 in addition to the substituents described herein.

[0093] The present invention includes polypeptides comprising a Neo-2 / 15 mutant, wherein the polypeptide comprises an amino acid sequence that is 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 mutant comprises a serine or alanine substitution for aspartic acid at position 15 (D15S or D15A), and at least one additional substitution selected from:

[0094] a) substitution of glutamine at position 95 by 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);

[0095] b) substitution of arginine for histidine at position 8 (H8R);

[0096] c) substitution of phenylalanine for histidine at position 11 (H11F);

[0097] d) substitution of lysine for tyrosine at position 14 (Y14K);

[0098] e) substitution of alanine or serine for asparagine at position 40 (N40A or N40S); and

[0099] f) substitution of the isoleucine at position 44 by alanine, serine or tyrosine (I44A or I44S or I44Y),

[0100] Wherein 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 substitutions mentioned above, 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 the Neo-2 / 15 mutant comprises a substitution at position 95, it does not comprise a substitution at position 40 and / or 44. In some such exemplary embodiments, if Neo-2 / 15 comprises a substitution at position 40 or 44, it does not comprise a substitution at position 95, 11 or 8. In some such exemplary embodiments, if the Neo-2 / 15 mutant comprises a serine or alanine substitution for the aspartic acid at position 15, it does not comprise a substitution at position 14. In some exemplary embodiments, if the polypeptide comprises a serine or alanine substitution for aspartic acid at position 15, it 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, it does not comprise a substitution at position 8. In some such exemplary embodiments, if the polypeptide comprises a glutamic acid substitution for glutamine at position 15, it does not comprise an arginine substitution for histidine at position 8.

[0101] The present invention includes polypeptides comprising a Neo-2 / 15 mutant, wherein the polypeptide comprises an amino acid sequence that is 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 mutant comprises a serine or alanine substitution for aspartic acid at position 15 (D15S or D15A), and at least one additional substitution selected from:

[0102] g) substitution of glutamic acid, alanine or histidine for glutamine at position 95 (Q95E, Q95A or Q95H);

[0103] h) substitution of arginine for histidine at position 8 (H8R);

[0104] i) substitution of phenylalanine for histidine at position 11 (H11F);

[0105] j) substitution of lysine for tyrosine at position 14 (Y14K);

[0106] k) substitution of alanine or serine for asparagine at position 40 (N40A or N40S); and

[0107] 1) substitution of isoleucine at position 44 by alanine or serine (I44A or I44S),

[0108] wherein 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. The polypeptide may comprise 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 those substitutions mentioned above, 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 the Neo-2 / 15 mutant comprises a substitution at position 95, it does not comprise a substitution at position 40 and / or 44. In some such exemplary embodiments, if Neo-2 / 15 comprises a substitution at position 40 or 44, it does not comprise a substitution at position 95, 11 or 8. In some such exemplary embodiments, if the Neo-2 / 15 mutant comprises a serine or alanine substitution for aspartic acid at position 15, it does not comprise a substitution at position 14. In some such exemplary embodiments, if the polypeptide comprises a serine or alanine substitution for aspartic acid at position 15, it 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, it does not comprise a substitution at position 8. In some such exemplary embodiments, if the polypeptide comprises a glutamic acid substitution for glutamine at position 15, it does not comprise an arginine substitution for histidine at position 8.

[0109] The present invention includes polypeptides comprising a Neo-2 / 15 mutant, wherein the polypeptide comprises an amino acid sequence that is 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 comprises:

[0110] a) substitution of phenylalanine for histidine at position 11 (H11F);

[0111] b) substitution of alanine or serine for asparagine at position 40 (N40A or N40S); and

[0112] c) substitution of the isoleucine at position 44 by alanine, serine or tyrosine (I44A or I44S or I44Y),

[0113] wherein 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. The Neo-2 / 15 mutant may comprise a set of substitutions selected from N40S, I44S, and H11F. In some aspects, the Neo-2 / 15 mutant comprises additional substituents at positions 11, 40, and 44 in addition to the substituents described herein. In some such embodiments, the Neo-2 / 15 mutant does not comprise substitutions at 1, 2, 3, or all 4 positions of positions 8, 14, 15, and 95, and any combination thereof. In some aspects, the polypeptide comprises a phenylalanine substitution for histidine at position 11 (H11F); an alanine or serine substitution for asparagine at position 40 (N40A or N40S); and an alanine or serine substitution for isoleucine at position 44 (I44A or I44S).

[0114] In some exemplary embodiments, the substitution does 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, 17, 18, 33, 36, 37, 39, 43, 47, 84, 85, 88, 91, 92, 96, 98, or 99; and any combination thereof. In some exemplary embodiments, the substitution does not occur at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or all 20 of 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, the substitution does not occur at 1, 2, 3, 4, or all 4 of positions 91, 92, 96, and 99. In some exemplary embodiments, the substitution does not occur at 1, 2, 3, 4, or all 5 of positions 17, 91, 92, 96, and 99. In some exemplary embodiments, if the Neo-2 / 15 mutant has a substitution at a position other than position 8, 11, 14, 15, 40, 44, or 95, the substitution does not significantly interfere with the binding of the Neo-2 / 15 mutant to IL-2Rγ. c combination.

[0115] In some exemplary embodiments, the Neo-2 / 15 mutant comprises three amino acids N-terminally and linked to the amino acid at position 4, wherein the amino acids are proline-lysine-lysine.

[0116] Neo-2 / 15 contains 4 helical domains in the order D1-D3-D2-D4. D1 is from approximately amino acids 1-22 of Neo-2 / 15; D3 is from approximately amino acids 33-55 of Neo-2 / 15; D2 is from approximately amino acids 58-76 of Neo-2 / 15; and D4 is from approximately amino acids 80-100 of Neo-2 / 15. Although the Neo-2 / 15 mutants of the embodiments have the same domain order as Neo-2 / 15, the present invention also includes Neo-2 / 15 mutants that have been reordered with helical domains. Those skilled in the art will appreciate that the domains can be reordered and still produce a similarly attenuated polypeptide that can be connected to a Treg targeting domain. In some embodiments, the domains are reordered by circular permutations, which creates new N-termini and C-termini. The domains were reordered by circular permutation, resulting in domain orders including D4-D1-D2-D3, D3-D4-D1-D2, and D2-D3-D4-D1. The linkers between domains can be altered to accommodate the reordering. Exemplary Neo-2 / 15 mutants contain amino acid linkers between one or more domains. Such linkers are generally not involved in binding to IL-2Rβγ c The combination of the four domains is not a combination, but plays a role in connecting the four domains. The length of the joint and the identity of the amino acid of the joint allow great variability. In various embodiments, the joint can be any length. In some aspects, the joint is 1 to 100 amino acid lengths, such as 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 2-10 or 1-5 amino acid lengths. Skilled practitioners can use the teachings of the art (see, for example, Silva et al., Nature, January 2019; 565(7738): 186-191) in conjunction with the teachings of this specification to transfer the domain and / or construct a joint for connecting the domain while maintaining the desired properties of the polypeptide. In the included embodiments, the order of the domains is D1-D3-D2-D4, wherein there is a first joint between domains D1 and D3, a second joint between domains D3 and D2, and a third joint 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 of the first linker is VKTNSPPAEE (SEQ ID NO: 67). An exemplary sequence of the second linker is DQ, and an exemplary sequence of the third linker is TAS (SEQ ID NO: 68).

[0117] In some embodiments, the polypeptide of the invention comprises a Neo-2 / 15 mutant, wherein the Neo-2 / 15 mutant comprises domains D1, D2, D3, and D4; wherein:

[0118] (a) D1 contains the amino acid sequence: KIQLHAEHALYX 15 ALMILNI (SEQ ID NO:61) and D3 comprises the amino acid sequence LEDYAFNFELILEEIARLFESG (SEQ ID NO:64); or

[0119] (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); and

[0120] D2 comprises an amino acid sequence of at least 8 amino acids in length;

[0121] D4 contains the amino acid sequence EDEQEEMANAIITILX 95 SWIFS (SEQ ID NO: 66); wherein:

[0122] (i) D1, D2, D3 and D4 can be in any order in the polypeptide;

[0123] (ii) an amino acid linker may be present between any of the domains (a "domain linker");

[0124] (iii)X 15 is serine or alanine; X 95 is glutamate, alanine or histidine; X 40 is serine or alanine; and 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 or alanine or glycine; and X 44 is serine or alanine or asparagine or threonine or tyrosine; wherein the polypeptide contains no more than ten, no more than nine, no more than eight, no more than seven, no more than six, no more than five, no more than four, no more than three, no more than two, no more than one, or zero substitutions at amino acid positions not designated as X. In some embodiments, X 40 is serine or alanine; and X 44 X is serine or alanine or tyrosine. 95 is glutamate, alanine or histidine; X 40 is serine or alanine; and X 44 It is serine or alanine.

[0125] In some embodiments, D1 comprises the amino acid sequence: KIQLHAEHALYX 15 A LMILNI (SEQ ID NO: 61) and D3 comprises the amino acid sequence LEDYAFNFELILE EIARLFESG (SEQ ID NO: 64) and the histidine at position 8 of SEQ ID NO: 61 is substituted. In some such embodiments, the substituent is phenylalanine.

[0126] In some embodiments, D1 comprises the amino acid sequence: KIQLHAEHALYX 15 A LMILNI (SEQ ID NO:61) and D3 comprises the amino acid sequence LEDYAFNFELILEE IARLFESG (SEQ ID NO:64) and 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.

[0127] In some embodiments, D1 comprises the amino acid sequence: KIQLHAEHALYDAL MILNI (SEQ ID NO: 62) and D3 comprises the amino acid sequence LEDYAFX 40 FELX 44 L EEIARLFESG (SEQ ID NO: 65). In some such embodiments, X 40 and X 44 In some embodiments, at least one or two of the SEQ ID NO:62 positions are serine. In some embodiments, the histidine at position 8 of SEQ ID NO:62 is optionally substituted. An optional substituent may be, for example, phenylalanine. In some embodiments, the tyrosine at position 11 of SEQ ID NO:62 is optionally substituted. An optional substituent 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 not substituted. In some embodiments, the aspartic acid at position 12 of SEQ ID NO:62 is not substituted.

[0128] 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 mutant does not comprise a substitution of glutamine at position 3, leucine at position 4, glutamic acid at position 7, leucine at position 10, and methionine at position 15 in SEQ ID NO:61, and a substitution of aspartic acid at position 3, tyrosine at position 4, phenylalanine at position 6, leucine at position 10, and glutamic acid at position 14 in SEQ ID NO:64. In some embodiments, the Neo-2 / 15 mutant also does not comprise a substitution of leucine at position 14 in SEQ ID NO:61.

[0129] 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, and the polypeptide does not comprise a substitution of glutamine at position 3, leucine at position 4, glutamic acid at position 7, leucine at position 10, and methionine at position 15 in SEQ ID NO:62, and a substitution of aspartic acid at position 3, tyrosine at position 4, phenylalanine at position 6, leucine at position 10, and glutamic acid at position 14 in SEQ ID NO:65. In some embodiments, the Neo-2 / 15 mutant also does not comprise a substitution of leucine at position 14 in SEQ ID NO:62.

[0130] The present invention includes Neo-2 / 15 mutants, wherein 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 mutants, wherein 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 mutants, wherein 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.

[0131] 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, including any X residues, unless otherwise indicated. For example, position 14 of SEQ ID NO: 65 has the sequence LEDYAFX 40 FELX44 LE E IAR LFESG, 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 those amino acid sequences where the N-terminal amino acid is designated as position 4, the sequence numbering starts at 4. For example, position 95 of SEQ ID NO: 3 has the sequence KIQLHAEHALYSALMILNIVKTNSPPAEEKLEDYAFNFELILEEIA RLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITIL E SWIFS, where the first amino acid is designated as position 4, is glutamic acid (underlined). For some sequences, the sequential numbering begins at position 4 simply for consistency of position numbering between sequences.

[0132] In any of these embodiments, D2 can comprise an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or 100% identical to KDEAEKAKRMKE WMKRIKT (SEQ ID NO: 63).

[0133] In any of these embodiments, the domain linkers can independently be 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, or 2-10 amino acids in length.

[0134] 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.

[0135] In some embodiments, the Neo-2 / 15 mutant 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 and is linked to the amino acid at position 1 of SEQ ID NO: 61 or 62. In some such aspects, the three amino acids are proline-lysine-lysine.

[0136] In some embodiments, the Neo-2 / 15 mutant 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.

[0137] In some embodiments, the invention provides a polypeptide comprising a Neo-2 / 15 mutant, wherein the binding affinity of the Neo-2 / 15 mutant to IL-2Rβγ 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 less than that of Neo-2 / 15. In some embodiments, the binding affinity of the Neo-2 / 15 mutant to IL-2Rβγ is at least 10-fold, or at least 100-fold less than that of Neo-2 / 15, but not more than 500-fold less than that of Neo-2 / 15. In some embodiments, the binding affinity of the Neo-2 / 15 mutant to IL-2Rβγ is at least 100-fold, or at least 500-fold less than that of Neo-2 / 15, but not more than 1000-fold less than that of Neo-2 / 15. In some embodiments, the binding affinity of the Neo-2 / 15 mutant to IL-2Rβγ is at least 100-fold, or at least 500-fold, or at least 1000-fold less than that of Neo-2 / 15, but is no more than 10,000-fold less than that of Neo-2 / 15.

[0138] In some embodiments, the present invention provides a polypeptide comprising a Neo-2 / 15 mutant, wherein the binding affinity of the Neo-2 / 15 mutant to IL-2Rβγ is attenuated by 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 as compared to IL-2. In some embodiments, the binding affinity of the Neo-2 / 15 mutant to IL-2Rβγ is attenuated by at least 10 times, or at least 100 times, but not more than 500 times as compared to IL-2. In some embodiments, the binding affinity of the Neo-2 / 15 mutant to IL-2Rβγ is attenuated by at least 100 times, or at least 500 times, but not more than 1000 times as compared to IL-2. In some embodiments, the binding affinity of the Neo-2 / 15 mutant to IL-2Rβγ is at least 100-fold, or at least 500-fold, or at least 1000-fold less than that of IL-2, but is no more than 10,000-fold less than that of IL-2.

[0139] In some embodiments, the present invention provides a polypeptide comprising a Neo-2 / 15 mutant, wherein the binding affinity of the Neo-2 / 15 mutant to IL-2Rβγ is within 2-fold, 3-fold, 4-fold, or 5-fold of the affinity of the Neo-2 / 15 mutant consisting of the sequence set forth in SEQ ID NO: 4 for IL-2Rβγ. In some embodiments, 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), the affinity of the Neo-2 / 15 mutant to IL-2Rβγ is within 2-fold, 3-fold, 4-fold, or 5-fold of the affinity of a reference polypeptide to IL-2Rβγ, wherein the reference polypeptide comprises a Neo-2 / 15 mutant fused to the C-terminus of an antibody, wherein the reference polypeptide comprises the amino acid sequences of SEQ ID NOs: 69 and 70.

[0140] In some embodiments, the present invention provides a polypeptide comprising a Neo-2 / 15 mutant, wherein the EC50 for the Neo-2 / 15 mutant to stimulate STAT5 phosphorylation in Treg cells is substantially equal to or less than that of the Neo-2 / 15 mutant consisting of the sequence set forth in SEQ ID NO: 4. In some embodiments, 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), the EC50 for the Neo-2 / 15 mutant to stimulate STAT5 phosphorylation in Treg cells is substantially equal to or less than that of a reference polypeptide comprising a Neo-2 / 15 mutant fused to the C-terminus of an antibody, wherein the reference polypeptide comprises the amino acid sequences of SEQ ID NOs: 69 and 70.

[0141] In some embodiments, the present invention provides a polypeptide comprising a Neo-2 / 15 mutant, wherein the maximum signaling value of the Neo-2 / 15 mutant in stimulating STAT5 phosphorylation in Treg cells is substantially equal to or greater than that of the Neo-2 / 15 mutant consisting of the sequence set forth in SEQ ID NO: 4. In some embodiments, 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), the maximum signaling value of the Neo-2 / 15 mutant in stimulating STAT5 phosphorylation in Treg cells is substantially equal to or greater than that of a reference polypeptide comprising a Neo-2 / 15 mutant fused to the C-terminus of the antibody, wherein the reference polypeptide comprises the amino acid sequences of SEQ ID NOs: 69 and 70.

[0142] In some embodiments, the present invention provides a polypeptide comprising a Neo-2 / 15 mutant, wherein the Neo-2 / 15 mutant stimulates STAT5 phosphorylation in Treg cells with a maximal signaling value that is at least 50%, at least 75%, at least 100% of the maximal signaling caused by Neo-2 / 15 and / or IL-2 stimulation.

[0143] In some embodiments, the present invention provides a polypeptide comprising a Neo-2 / 15 mutant, wherein 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), the Neo-2 / 15 mutant 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 greater.

[0144] In some embodiments, the present invention provides a polypeptide comprising a Neo-2 / 15 mutant, wherein the Neo-2 / 15 mutant stimulates NK cells at an EC50 substantially equal to or higher than the Neo-2 / 15 mutant consisting of the sequence set forth in SEQ ID NO: 4. In some embodiments, 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), the Neo-2 / 15 mutant stimulates NK cells at an EC50 substantially equal to or higher than a reference polypeptide comprising a Neo-2 / 15 mutant fused to the C-terminus of an antibody, wherein the reference polypeptide comprises the amino acid sequences of SEQ ID NOs: 69 and 70.

[0145] As used herein, the term "identity" with respect to a polypeptide sequence refers to the subunit sequence identity between two molecules. When the subunit position in two molecules is occupied by the same monomer subunit (i.e., the same amino acid residue or nucleotide), the molecule is identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. In general, the sequences are aligned to obtain the highest order match (including vacancies if necessary). In various embodiments, disclosed techniques and widely available computer programs, such as GCG program packages, can be used to calculate identity (Devereux et al., Nucleic Acids Res. 12: 387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215: 403, 1990). For example, sequence analysis software (such as the sequence analysis software package of the genetics computer group of the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705)) can be used to measure sequence identity using default parameters. Unless otherwise stated, percent identity is determined over the length of the reference sequence.

[0146] Substitution, deletion, insertion or any combination thereof can be used to obtain a final derivative or variant. Generally, these changes are made on a few amino acids to minimize the changes in the molecule, particularly the specificity of the antigen-binding protein. However, larger changes may be tolerated in certain circumstances.

[0147] In some aspects, when it is necessary to replace amino acids, but it is not desired that amino acid substitution significantly change the properties of proteins, conservative amino acid substitutions are performed. As used herein, "conservative amino acid substitutions" means that a given amino acid can be replaced by an amino acid with similar physicochemical properties, such as replacing another with an acidic residue (such as E or D), replacing another with a basic amino acid (such as K, R or H), replacing another with two amino acids with backbone distorted side chains (G or P), replacing another with a hydrophobic amino acid (L, I, V, A or M), replacing another with an aromatic hydrophobic amino acid (Y, F or W), or replacing another with a polar amino acid with an uncharged side chain (T, S, N, Q, C). Polypeptides comprising conservative amino acid substitutions can be tested in any of the assays described herein to confirm that the desired activity is retained. It can also be grouped according to the similarity of the amino acid side chain properties. Alternatively, naturally occurring residues can be divided into several groups according to common side chain properties. Non-conservative substitutions will require replacing members of another category with members of one of these categories. Specific conservative substitutions include those substitutions listed in, for example, Table 1.

[0148] Table 1

[0149] feature Conservative substitution Ala Ser Arg Lys Asn Gln,His Asp Glu Cys Ser,Ala Gln Asn Glu Asp Gly Pro His Asn,Gln Ile Leu,Val Leu Ile, Val Lys Arg,Gln,Glu Met Leu,Ile Phe Met,Leu,Tyr,Trp Ser Thr Thr Ser Trp Tyr,Phe Tyr Trp,Phe Val Ile,Leu

[0150] As used herein, the natural 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).

[0151] Treg targeting domain

[0152] The Neo-2 / 15 mutant of the present invention can be coupled with a targeting agent for Treg (i.e., a Treg targeting agent) to give it the best ability to preferentially amplify and activate T regulatory cells. Therefore, the present invention provides a polypeptide comprising a Neo-2 / 15 mutant and a targeting agent as described in any embodiment herein. In a particularly preferred embodiment, the targeting agent binds to an antigen on the surface of a Treg cell. The binding is preferably specific binding. Exemplary antigens are CD25 or CD39.

[0153] Treg targeting agents can be any agent capable of directing Neo-2 / 15 mutants to T regulatory cells. In other words, targeting agents can be any agent capable of directing cellular localization of Neo-2 / 15 mutants. The target of a targeting agent is typically a surface marker that is significantly more expressed on Tregs than on any other cell type and is easily targeted.

[0154] In an exemplary embodiment, 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 molecules. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. The antibody can be murine, human, humanized, chimeric or derived from other species (such as rabbit, goat, sheep, horse or camel), but is preferably a human antibody or a humanized antibody. The term "antibody," as used herein, also includes antibody fragments capable of binding to antigens, such as Fab, Fab' and F(ab')2 fragments, etc.

[0155] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible naturally occurring mutations that may exist in small amounts, the individual antibodies constituting the population are identical. Monoclonal antibodies have a high degree of specificity (for a single antigenic site). In addition, in contrast to the polyclonal antibody preparations comprising different antibodies for different determinants (epitopes), each monoclonal antibody is for a single determinant on an antigen. In addition to its specificity, the advantage of monoclonal antibodies is that they can be synthesized but not contaminated by other antibodies. The modifier "monoclonal" represents that the antibody is characterized by obtaining from a substantially homogeneous antibody population, and should not be construed as requiring the production of the antibody by any ad hoc method. For example, the monoclonal antibody used according to the present disclosure can be prepared by the hybridoma method described by Kohler et al. (1975) Nature 256:495, or can be prepared by a recombinant DNA method (see, US 4816567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described by 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).

[0156] As used herein, a full-length antibody comprises an antigen-binding domain directly fused 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.

[0157] "Antigen-binding fragments" comprise a portion of a full-length antibody capable of binding to an antigen, typically its antigen-binding or variable region, but may also comprise a 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 Fab expression libraries, anti-idiotypic (anti-ld) antibodies, CDRs (complementarity determining regions), and epitope-binding fragments of any of the above that specifically bind to Treg cells immunologically.

[0158] The constant domains or antibodies may be involved in various effector functions, such as mediating 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), longer in vivo half-life, and protein A binding. In IgG, IgA, and IgD antibody isotypes, the Fc region consists 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.

[0159] As used herein, the term "Fc domain" includes native and mutant forms of Fc regions, including truncated forms. The Fc domain can provide extended serum half-life. Herein, specific residues within the Fc domain are identified by position according to the EU numbering scheme.

[0160] IgG subclasses differ in their ability to mediate effector functions. For example, IgG1 is superior to IgG2 and IgG4 in mediating ADCC and CDC. Therefore, in embodiments where effector functions are undesirable, IgG2 or IgG4 Fc regions will be preferred. However, it is known that molecules containing IgG2 Fc are more difficult to manufacture and have less attractive biophysical properties, such as shorter half-lives, compared to molecules containing IgG1 Fc. Therefore, molecules containing IgG1 Fc (which are mutated to reduce effector functions) can be used to replace the IgG2 Fc domain.

[0161] By introducing one or more mutations in Fc, the effector function of the antibody can be increased or decreased. In a preferred embodiment of the invention, 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). It is known that human IgG1 has a glycosylation site at N297 (EU numbering system), and glycosylation contributes to the effector function of IgG1 antibodies. Exemplary IgG1 sequences have a mutated N297, such as glutamine (N297Q) or alanine (N297A) or glycine (N297G). The Fc comprising a human IgG1 Fc having the N297G mutation may also comprise further insertions, deletions and substitutions.

[0162] In some embodiments, the binding affinity of the Fc domain substantially lacking effector function to Fcγ receptors is reduced by at least about 100 times or at least about 1000 times. In some embodiments, the Fc domain includes known LALA mutations, L234A and L235A mutations (EU numbering). In some embodiments, the Fc region includes G237A, P329G or P329A mutations (EU numbering). In some embodiments, using the EU numbering system, the Fc domain includes L234A mutations, L235A mutations and / or G237A mutations. The examples of amino acids reported to reduce effector function include LALA-PG mutations (L234A, L235A, P329G), LALA-PA mutations (L234A, L235A, P329A) or LALA-GA mutations (L234A, L235A, G237A). Exemplary Fc domains are listed in SEQ ID NO:71. In certain embodiments, the human IgG1 Fc comprises a LALA-PG or 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.

[0163] In some aspects, the polypeptide of the present invention comprises a full-length antibody containing a wild-type or mutant Fc domain. In other aspects, the polypeptide of the present invention comprises an antibody fragment, such as a fusion of the variable region of the heavy chain and light chain of an antibody (e.g., scFv). The polypeptide of the present invention comprising an antibody fragment may also optionally comprise a heterologous polypeptide capable of increasing the circulating half-life of a Treg agonist in vivo. Such heterologous polypeptides may be, for example, a wild-type or mutant Fc domain.

[0164] 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 in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, ed., Harlow and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)).

[0165] In addition, naked DNA immunization techniques known in the art to generate an immune response to the encoded immunogen are also used (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15:617-648).

[0166] The amino acid sequence of the protein to which the antibody response is to be generated (e.g., CD25 of an anti-CD25 antibody) can be analyzed to select specific regions of the protein for generating antibodies. For example, hydrophobicity and hydrophilicity analysis of the amino acid sequence of the protein are used to identify hydrophilic regions in the protein structure. Various other methods known in the art (such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis) can be used to easily identify protein regions and other regions and domains that exhibit immunogenic structures. The hydrophilicity profile can be generated using the method of Hopp, TP and Woods, KR, 1981, Proc. Natl. Acad. Sci. USA 78: 3824-3828. The hydrophilicity profile can be generated using the method of Kyte, J. and Doolittle, RF, 1982, J. Mol. Biol. 157: 105-132. Percent (%) accessible residue features can be generated using the method of Janin J., 1979, Nature 277: 491-492. Average flexibility features can be generated using the method of Bhaskaran R., Ponnuswamy PK, 1988, Int. J. Pept Protein Res. 32: 242-255. β-turn features can be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1: 289-294. Therefore, 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 (e.g., BSA, KLH or other carrier proteins) with carriers are also well known in the art. In some cases, direct conjugation is performed using, for example, carbodiimide reagents; in other cases, linking reagents such as those provided by Pierce Chemical Co., Rockford, Ill. are effective. Administration of the immunogen is typically carried out by injection over an appropriate period of time and using a suitable adjuvant, as understood in the art.During the immunization protocol, antibody titers may be measured to determine the adequacy of antibody formation.

[0167] Monoclonal antibodies can be produced by various methods well known in the art. For example, it is well known to prepare immortalized cell lines that secrete the desired monoclonal antibodies using the standard hybridoma technology of Kohler and Milstein or modified techniques that immortalize antibody-producing B cells. Immortalized cell lines that secrete the desired antibodies are screened by immunoassays. Once the appropriate immortalized cell culture is determined, the cells can be expanded and antibodies produced from in vitro cultures or ascites.

[0168] Antibodies and fragments thereof can also be produced by recombinant means. Regions specifically binding to the desired region of the target can also be produced in the context of chimeric or complementary determining region (CDR) transplanted antibodies of multiple species sources. Humanized or human antibodies can also be produced and are preferentially used in therapeutic environments. Methods for humanizing murine and other non-human antibodies by replacing one or more non-human antibody CDRs with corresponding human antibody sequences are well known (see, for example, 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.

[0169] The antibodies used in the present invention can be fully human antibodies. Various methods in the art provide ways to produce fully human monoclonal antibodies. For example, a preferred embodiment provides a technique using transgenic mice that are inactivated for antibody production and engineered with human heavy and light chain loci, known as Xenomouse (Amgen Fremont, Inc.). Exemplary descriptions of preparing transgenic mice that produce human antibodies can be found in, for example, Mendez et al. Nature Genetics, 15: 146-156 (1998); Kellerman, SA & Green, LL, Curr. Opin. Biotechnol 13, 593-597 (2002).

[0170] In addition, human antibodies of the invention can be generated using HuMAb mice (Medarex, Inc.), which contain human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, as well as targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg et al. (1994) Nature 368(6474): 856-859).

[0171] In another embodiment, mice carrying human immunoglobulin sequences on transgenic and transchromosomes (such as mice carrying human heavy chain transgenic and human light chain transchromosomes) can be used to produce fully human antibodies of the present invention. Such mice are referred to herein as "KM mice", and such mice are described in Tomizuka et al. (2000) Proc.Natl.Acad.Sci.USA97:722-727 and Tomizuka et al. PCT Publication WO 02 / 43478.

[0172] The human monoclonal antibodies of the present invention can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Phage display methods for isolating human antibodies have been established in the art. See, for example: U.S. Pat. Nos. 5,223,409, 5,403,484, and 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081 to Griffiths et al.

[0173] Human monoclonal antibodies of the invention can also be prepared using SCID mice into which human immune cells have been reconstituted so that a human antibody response can be generated upon immunization. Such mice are described, for example, in U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.

[0174] In exemplary aspects, targeting agents are antibodies or their antigen binding fragments for surface markers, and the degree of expression of the surface markers on Treg is significantly higher than the expression on any other cell type, and is easy to target. Exemplary Treg targeting agents are antibodies or their antigen binding fragments (i.e., anti-CD25 antibodies) in conjunction with CD25. Exemplary human CD25 polypeptide sequences are shown in Genbank accession number NP_000408, version number NP_000408.1, and Uniprot / Swiss-Prot accession number P01589.

[0175] As used herein, "binds to CD25" is used to mean that the antibody binds to CD25 with an affinity that is greater than the affinity with which the antibody binds to an unrelated antigen (e.g., bovine serum albumin). In some embodiments, the antibody binds to CD25 with an association constant (K) of 10,000 when measured under physiological conditions. a ) is at least 100, 200, 500, 1000, 2000, 5000, 104 , 10 5 or 10 6 The antibodies of the present disclosure may bind CD25 with high affinity. For example, in some embodiments, the antibodies may bind CD25 with an affinity equal to or less than about 10 -6 M, equal to or less than about 10 -7 M, equal to or less than about 10 -8 M or equal to or less than about 10 -9 M's KD Binding to CD25. Binding can be determined by ELISA, flow cytometry or surface plasmon resonance (SPR) techniques, for example in a BIAcore 3000 instrument using recombinant human IL-2Rα as ligand and the antibody as analyte.

[0176] Anti-CD25 antibodies or antigen-binding fragments thereof suitable for use in the present invention can be blocking antibodies or non-blocking antibodies. Blocking antibodies are those antibodies that bind to CD25 and inhibit or block the binding of IL-2 to CD25. For example, the binding can be inhibited by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. Non-blocking antibodies do not inhibit or block the binding of IL-2 to CD25.

[0177] Anti-CD25 antibodies or antigen-binding fragments thereof are known in the art and can be used in the methods disclosed herein. Methods for preparing anti-CD25 antibodies or antigen-binding fragments thereof are also known in the art and can be used to prepare anti-CD25 antibodies or antigen-binding fragments thereof for use in the present invention.

[0178] Anti-CD25 antibodies for use in the TRA provided herein include, but are not limited to, anti-CD25 antibodies described in U.S. Pat. No. 7,438,907, such as, for example, antibodies AB1, AB7, Abl1 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 US 6,383,487; and anti-CD25 antibodies described in U.S. Pat. No. 20200010554, including 7D4, MA251 or 7G7B6.

[0179] Other anti-CD25 antibodies for use in the present invention include the chimeric antibody basiliximab and its humanized version, and the anti-CD25 antibodies BT563 (see Baan et al., Transplant. Proc. 33:224-2246, 2001) and 7G8. Exemplary human antibodies used in the methods of the present invention are HUMAX- HUMAX- It is a human monoclonal antibody of IgG1, kappa isotype, specific for human CD25, and has VH and VL domains of the sequences specified in SEQ ID NO.47 and 48 disclosed herein. Antibody 4C9 (available from Ventana Medical Systems, Inc.) and antibody RFT5 (described in US6383487) can also be used 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 VT-072) and M-A251 (available from BioLegend, catalog number IVA053).

[0180] Other anti-CD25 antibodies useful in the present invention include the PC61 antibody described in US Patent Application No. 20080025947, the murine monoclonal antibody S4B6, and the monoclonal antibody MAB602 disclosed in Boyman et al., Science, (2006), 311: 1924-1927.

[0181] In some embodiments, the antibody is a humanized, deimmunized, or resurfaced version of an antibody disclosed herein.

[0182] 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 sold under the trade name ZENAPAX. An exemplary anti-CD25 antibody is an antibody comprising a daclizumab variable domain with an Fc domain with reduced / no effector function.

[0183] The heavy chain variable region of Daclizumab (SEQ ID NO: 26) comprises three heavy chain complementary determining regions (CDRs), referred to herein as (in amino- to carboxyl-terminal order) CDR-H1, CDR-H2, and CDR-H3, designated as SEQ ID NO: 27 (CDR-H1); SEQ ID NO: 28 (CDR-H2); and SEQ ID NO: 29 (CDR-H3). The heavy chain framework (FR) sequence of Daclizumab is designated as 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).

[0184] In some aspects, the polypeptide of the invention comprises a heavy chain variable domain and a light chain variable domain of a daclizumab antibody or a mutant form of the heavy chain and light chain variable domain of a daclizumab antibody (also referred to herein as a daclizumab-related antibody) or an antigen-binding fragment thereof. Such polypeptides may also comprise a wild-type FC domain, a variant FC domain, such as, for example, an FC domain with reduced effector function, a truncated FC domain (whether wild-type or variant), or no FC domain.

[0185] Dalizumab-related antibodies are known in the art, see, for example, International Application No. WO2014144935 and U.S. Patent No. 8,314,213 (incorporated herein by reference in their entirety). In some such embodiments, the polypeptide of the present invention comprises a dalizumab-related antibody or an antigen-binding fragment thereof, which has reduced T cell immunogenicity compared to dalizumab. In some embodiments, the polypeptide of the present invention comprises dalizumab or a dalizumab-related antibody, or an antigen-binding fragment thereof, characterized by having one, two, three or four of the following properties (i)-(iv):

[0186] (i) including the six CDRs listed in NOs: 27, 28, 29, 30, 31 and 32;

[0187] (ii) comprising the six CDRs listed in SEQ ID NOs: 27, 28, 29, 30, 31 and 32, but having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 13, 14, 15, 16 or 17 amino acid substitutions in the CDRs, provided that any single CDR has no more than 4 or no more than 3 amino acid substitutions compared to the corresponding CDR sequence of an antibody having the CDRs of SEQ ID NOs: 27, 28, 29, 30, 31 and 32, or any single CDR other than CDR-H2 has no more than 3 or no more than 2 amino acid substitutions compared to the corresponding CDR sequence of an antibody having the CDRs of SEQ ID NOs: 27, 28, 29, 30, 31 and 32;

[0188] (iii) individual framework regions comprising SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39 and 40 or framework regions having 1-10, preferably 1-10 or 1-5 amino acid substitutions in these individual framework regions

[0189] (iv) comprises 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 cases, comprises the CDRs of (i) or (ii).

[0190] In various embodiments, the daclizumab-related antibody or antigen-binding fragment thereof comprises one or more specific substitutions, including 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, amino acid substitution S29K in CDR-L1 compared to CDR-L1 of SEQ ID NO:30, amino acid substitution 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, amino acid substitution N53E in CDR-L2 compared to CDR-L2 of SEQ ID NO:31; amino acid substitutions N52S, S53R and T54K in CDR-H2 compared to CDR-H2 of SEQ ID NO:28; Compared with the CDR-H2 of SEQ ID NO:28, the amino acid substitution T54S in CDR-H2; compared with the CDR-H2 of SEQ ID NO:30, the amino acid substitution S29K in CDR-L1, and compared with the CDR-L2 of SEQ ID NO:31, the amino acid substitution N53D in CDR-L2; compared with the CDR-H2 of SEQ ID NO:28, the amino acid substitutions S53R and T54K in CDR-H2; compared with the CDR-L1 of SEQ ID NO:30, the amino acid substitution S29K in CDR-L1, and compared with the CDR-L2 of SEQ ID NO:31, the amino acid substitution N53D in CDR-L2; and combinations thereof.

[0191] In various embodiments, the daclizumab-related antibody or antigen-binding fragment thereof comprises the following amino acid substitutions: (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 comprises one or more of the following: (iii) S53R, S53K or S53N in CDR-H2 compared to CDR-H2 of SEQ ID NO: 28, (iv) 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) E63K in CDR-H2 compared to CDR-H2 of SEQ ID NO: 28, (viii) 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.

[0192] In various embodiments, the daclizumab-related antibody or antigen-binding fragment thereof comprises the following amino acid substitutions: (i) N52S, N52K, N52R or N52V in CDR-H2 compared to CDR-H2 of SEQ ID NO:28; (ii) S53K, S53R or S53N; and (iii) T54R, T54S or T54K; (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 the following amino acid substitutions: (iv) Y56R in CDR-H2 compared to CDR-H2 of SEQ ID NO:28.

[0193] 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.

[0194] In various embodiments, the daclizumab-related antibody or antigen-binding fragment thereof comprises the following amino acid substitutions: (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 comprises one or more of the following: (iii) S53R, S53K or S53N in CDR-H2 compared to CDR-H2 of SEQ ID NO: 28, (iv) 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) E63K in CDR-H2 compared to CDR-H2 of SEQ ID NO: 28, In some such embodiments, the framework regions have up to 4 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 a specific embodiment, the framework regions comprise the amino acid substitution I48M in FR-H2 compared to FR-H2 of SEQ ID NO: 5. In a specific embodiment, the framework regions do not comprise the substitution at 148 in FR-H2 compared to FR-H2 of SEQ ID NO: 5.

[0195] In various embodiments, the daclizumab-related antibody or antigen-binding fragment thereof comprises the following amino acid substitutions: (i) N52S, N52K, N52R or N52V in CDR-H2 compared to CDR-H2 of SEQ ID NO:28; (ii) S53K, S53R or S53N; and (iii) T54R, T54S or T54K; (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 the following amino acid substitutions: (vi) Y56R in CDR-H2 compared to CDR-H2 of SEQ ID NO:28. In some such embodiments, the framework regions have up to 4 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 specific embodiments, the framework regions comprise amino acid substitution I48M in FR-H2 compared to FR-H2 of SEQ ID NO: 34. In specific embodiments, the framework regions do not comprise a 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, wherein the heavy chain variable domain has substitutions N52S, S53R, T54K, and the light chain variable domain has substitutions N53E; or the heavy chain variable domain has substitutions N52K, T54R, and the light chain variable domain has substitutions N53E. The numbering of the heavy and light chain variable regions is by Kabat numbering (see Tables 1 and 2 of WO2014 / 144935).

[0196] In various embodiments, the daclizumab-related antibody or antigen-binding fragment thereof has a heavy chain CDR1 having an amino acid sequence selected from SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 27; CDR2 having an amino acid sequence of SEQ ID NO: 44; and CDR3 having an amino acid sequence of SEQ ID NO: 29; and a light chain comprising CDR1 having an amino acid sequence of SEQ ID NO: 45; CDR2 having an amino acid sequence of SEQ ID NO: 46; and CDR3 having an amino acid sequence of SEQ ID NO: 32. (See, e.g., U.S. Pat. No. 8,314,213, the entire contents of which are incorporated herein and used for all purposes.)

[0197] In some embodiments, a polypeptide of the invention comprises an antibody or antigen-binding fragment thereof comprising a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 27, 42, or 43; a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 28 or 44; a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 29; a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 30 or 45; a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 31 or 46; and a CDR-L3 comprising an 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 an amino acid sequence of SEQ ID NO: 26 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 41.

[0198] In addition to or in lieu of simply binding to CD25, an anti-CD25 antibody or antigen-binding fragment thereof may be selected to retain 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 will be an antibody or antigen-binding fragment thereof that will not induce elimination of CD25-expressing T cells or inhibit proliferation of CD25-expressing T cells when administered alone. In some aspects, the anti-CD25 antibody or antigen-binding fragment thereof will bind to CD25 with the same or greater affinity as daclizumab.

[0199] Exemplary TRA Configuration

[0200] In some aspects, the TRA of the present invention comprises a Neo-2 / 15 mutant connected to an antibody or an antigen-binding fragment thereof (e.g., via translational fusion or chemical / enzymatic binding). TRA may optionally comprise an amino acid linker between one or more components constituting TRA (e.g., between a Neo-2 / 15 mutant and an antibody or antibody binding fragment). A non-limiting example of a linker connecting different components of TRA is a glycine / serine linker. For example, a glycine / serine linker may comprise the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 49) or comprise the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 50). This is merely a non-limiting example, and the linker may have a different number of GGGGS (SEQ ID NO: 51) repeats. In some embodiments, the linker comprises 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 linker used to connect the different components of TRA can be a flexible linker or a rigid linker. Non-limiting examples of rigid linkers are 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 (SE Q ID NO:58); AEAAAKEAAAKA (SEQ ID NO:59); PAPAP (SEQ ID NO:60); and (ALA-PRO) 10-34 (SEQ ID NO:83).

[0201] In some aspects, the polypeptide of the present invention comprises an antibody or an antigen-binding fragment of an antibody that targets a surface marker that is expressed at a significantly higher level on Treg than on any other cell type and is easy to target, wherein the Neo-2 / 15 mutant is connected to one of the C-terminus or N-terminus of the antibody, optionally through an amino acid linker. In some aspects, the polypeptide of the present invention comprises a Neo-2 / 15 mutant and a scFv, wherein the Neo-2 / 15 mutant and the scFv are both located at opposite ends of the Fc. In some such aspects, the polypeptide comprises a homodimer containing two Neo-2 / 15 mutants, each of which is connected to the N-terminus of the Fc domain via its C-terminus, and the Fc domain is connected to the variable heavy chain of the scFv via its C-terminus. In some aspects, the polypeptide of the present invention comprises a Neo-2 / 15 mutant and an antibody, wherein the Neo-2 / 15 mutant is connected to the N-terminus of the heavy chain of the antibody. In some such aspects, the polypeptide comprises a heterotetramer comprising two heavy chains (VH + constant region) and two light chains (VL + constant region), each heavy chain variable region being linked to the C-terminus of the Neo-2 / 15 mutant via its N-terminus. In some aspects, the polypeptide of the invention comprises a Neo-2 / 15 mutant and an antibody, wherein the Neo-2 / 15 mutant is linked to the C-terminus of the heavy chain of the antibody. In some such aspects, the polypeptide comprises a heterotetramer comprising two heavy chains (VH + constant region) and two light chains (VL + constant region), each heavy chain constant region being linked to the N-terminus of the Neo-2 / 15 mutant via its C-terminus. For example, the connection can be made via a linker. The linker can be, for example, (GGGGS) n Linker, wherein n is 1-10.

[0202] In some embodiments, the polypeptide of the present invention comprises a Neo-2 / 15 mutant; a Neo-2 / 15 mutant linked to an anti-CD25 antibody as described herein; a Neo-2 / 15 mutant linked to an antigen-binding fragment of an anti-CD25 antibody as described herein; or a Neo-2 / 15 mutant linked to an antigen-binding fragment of an anti-CD25 antibody as described herein and an Fc domain as described herein. In some embodiments, the polypeptide of the present invention consists of or consists essentially of a Neo-2 / 15 mutant; a Neo-2 / 15 mutant linked to an anti-CD25 antibody as described herein; a Neo-2 / 15 mutant linked to an antigen-binding fragment of an anti-CD25 antibody as described herein; or a Neo-2 / 15 mutant linked to an antigen-binding fragment of an anti-CD25 antibody as described herein and an Fc domain as described herein. In some embodiments, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 69 and the amino acid sequence of SEQ ID NO: 70. In some embodiments, the polypeptide of the present invention comprises an amino acid sequence of SEQ ID NO: 74 and an amino acid sequence of SEQ ID NO: 70. In some embodiments, the polypeptide of the present invention comprises a Neo-2 / 15 mutant linked to the C-terminus of a targeting agent or an Fc domain, wherein the polypeptide comprises an amino acid sequence of SEQ ID NO: 69 and an amino acid sequence of SEQ ID NO: 70. In some embodiments, the polypeptide of the present invention comprises a Neo-2 / 15 mutant linked to the N-terminus of a targeting agent or an Fc domain, wherein the polypeptide comprises an amino acid sequence of SEQ ID NO: 74 and an amino acid sequence of SEQ ID NO: 70.

[0203] In some embodiments, the polypeptides of the invention do not substantially activate Teff cells, or the EC50 for activating Teff cells by the polypeptides is greater than 5 times, greater than 10 times, greater than 50 times, greater than 100 times, greater than 500 times, or greater than 1000 times higher than the EC50 for activating Treg cells.

[0204] In some embodiments, the polypeptide of the present invention does not substantially induce the proliferation of Teff cells, or the EC50 of the polypeptide inducing Teff cell proliferation is greater than 5 times, greater than 10 times, greater than 50 times, greater than 100 times, greater than 500 times, or greater than 1000 times higher than the EC50 of inducing Treg cell proliferation.

[0205] In some embodiments, the maximum signaling of the polypeptide of the invention in non-Treg cells is less than 30% or less than 20% of the maximum signaling of IL-2 to non-Treg cells (e.g., at a polypeptide concentration of 10 nM or less). In some embodiments, non-Treg cells are Teff cells. In some embodiments, non-Treg cells are NK cells.

[0206] In some embodiments, the maximal signaling of a polypeptide of the invention in Tregs is at least 50%, at least 60%, or at least 70% of the maximal signaling of IL-2 in Tregs (eg, at a polypeptide concentration of 10 nM or less).

[0207] In various embodiments, signaling in Tregs and non-Tregs is measured as % pSTAT5+ compared to IL-2. The EC50 can be determined from a % pSTAT5+ curve obtained using a titration of the test article.

[0208] In some embodiments, the polypeptides of the 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 (ie, 1 nM).

[0209] In some embodiments, the polypeptide of the present invention does not substantially activate or stimulate NK cells, or the EC50 of the polypeptide activating NK cells is greater than 5 times, greater than 10 times, greater than 50 times, greater than 100 times, greater than 500 times, or greater than 1000 times higher than the EC50 of activating Treg cells. In some embodiments, the polypeptide does not substantially induce the proliferation of NK cells, or the EC50 of the polypeptide inducing NK cell proliferation is greater than 5 times, greater than 10 times, greater than 50 times, greater than 100 times, greater than 500 times, or greater than 1000 times higher than the EC50 of inducing Treg cell proliferation. In some embodiments, the polypeptide induces STAT5 phosphorylation in Treg cells with an EC50 lower than 1nM, lower than 500pM, or lower than 100pM, and / or the polypeptide induces STAT5 phosphorylation in NK cells with an EC50 higher than 1000pM.

[0210] Nucleic Acids

[0211] Provided herein are nucleic acids comprising a nucleotide sequence encoding a polypeptide of the invention, whether it is, for example, a Neo-2 / 15 mutant, or a Neo-2 / 15 mutant fused to an antibody, a Neo-2 / 15 mutant fused to an antibody antigen-binding fragment, a Neo-2 / 15 mutant fused to an antibody antigen-binding fragment and an Fc domain, or another polypeptide of the invention. The antibody or its antibody fragment may be, for example, an anti-CD25 antibody or an antibody fragment thereof, or an anti-CD39 antibody or an antibody binding fragment thereof, or another antibody capable of targeting Treg cells.

[0212] In some aspects, nucleic acids of the invention will encode a polypeptide comprising any one or more of sequences 1-82.

[0213] The nucleic acids and polypeptides of the invention can be produced using any suitable method known in the art.

[0214] In exemplary aspects, the nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide of the present disclosure. As used herein, "nucleic acid" includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," generally meaning a polymer of DNA or RNA, or a modified form thereof, which may be single-stranded or double-stranded. The nucleic acid may comprise any nucleotide sequence encoding any antigen-binding protein or polypeptide of the present disclosure.

[0215] In some aspects, the nucleic acids of the present disclosure are recombinant. As used herein, the term "recombinant" refers to (i) molecules constructed outside of living cells by connecting natural or synthetic nucleic acid fragments to nucleic acid molecules that can replicate in living cells, or (ii) molecules produced by replication of the molecules described in (i) above. For purposes of this article, replication can be in vitro replication or in vivo replication.

[0216] In some aspects, nucleic acid is constructed based on chemical synthesis and / or enzyme ligation reaction using procedures known in the art. In some aspects, nucleic acid of the present disclosure is incorporated into a vector. In this respect, the present disclosure provides any one comprising any nucleic acid disclosed by the present invention. In exemplary aspects, the vector is a recombinant expression vector. For purposes of this paper, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct, when the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide, and the vector is sufficient to allow mRNA, protein, polypeptide or peptide to be expressed in the cell under conditions of contact with the cell, allowing the host cell to express mRNA, protein, polypeptide or peptide.

[0217] The vector 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 reproduction and amplification or for expression or both, such as plasmids and viruses. The vector of the present disclosure can be prepared using standard recombinant DNA technology. Circular or linear expression vector constructs can be prepared to contain a replication system that works in a prokaryotic or eukaryotic host cell. The replication system can be derived from, for example, CoIE1, 2μ plasmids, λ, SV40, bovine papilloma virus, etc. In some aspects, depending on the circumstances and considering whether the vector is based on DNA or RNA, the vector contains regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the host type (e.g., bacteria, fungi, plants or animals) to be introduced by the vector. The vector can include one or more marker genes that allow selection of transformed or transfected hosts. The vector can include a natural or standardized promoter, which is operably linked to a nucleotide sequence encoding a polypeptide (including its functional part and functional variant), or is linked to a nucleotide sequence complementary or hybridized to a nucleotide sequence encoding a TRA conjugate or fusion protein. The selection of promoters, such as strong promoters, weak promoters, inducible promoters, tissue-specific promoters and development-specific promoters, is within the ordinary skill of the skilled person. Similarly, the combination of nucleotide sequences and promoters is also within the skill of the skilled person.

[0218] Provided herein is a host cell comprising nucleic acid or vector disclosed herein.As used herein, the term "host cell" refers to any type of cell that can contain a vector disclosed herein and can produce an expression product (e.g., mRNA, protein) encoded by nucleic acid. In some aspects, the host cell is an adherent cell or a suspension cell, i.e., a cell grown in a suspended state. In exemplary aspects, the host cell is a cultured cell or a primary cell, i.e., a cell directly separated from an organism (e.g., a human). The host cell can be any cell type, can be derived from any type of tissue, and can be in any developmental stage. The host cell can be a prokaryotic cell, such as a bacterial cell (e.g., Escherichia coli) or a eukaryotic cell, such as a yeast cell or a mammalian cell. Although unfused attenuated IL-2 / IL-15 mimetics can be expressed in prokaryotic or eukaryotic cells, TRA is preferably expressed in mammalian cells.

[0219] Provided herein are methods for preparing polypeptides of the present invention. In an exemplary embodiment, the method includes culturing a host cell of the present disclosure to express a polypeptide and harvesting the expressed polypeptide. The host cell may be any host cell described herein. In exemplary aspects, the host cell is selected from: CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells. In exemplary aspects, 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 exemplary aspects, the growth medium increases cell density, culture viability, and yield in a timely manner. In exemplary aspects, the growth medium contains amino acids, vitamins, inorganic salts, glucose, and serum as sources of growth factors, hormones, and attachment factors. In exemplary aspects, the growth medium is a completely chemically defined medium consisting of amino acids, vitamins, trace elements, inorganic salts, lipids, and insulin or insulin-like growth factor. In addition to nutrients, the growth medium also helps maintain pH and osmotic pressure. A variety of growth media are commercially available and described in the art. See, for example, Arora, "Cell Culture Media: A Review" MATER METHODS 3: 175 (2013)

[0220] Pharmaceutical composition

[0221] Provided herein are pharmaceutical compositions comprising polypeptides of the present disclosure. In exemplary aspects, the compositions comprise agents that enhance the chemical-physical characteristics of the polypeptide, such as by stabilizing, for example, TRA at certain temperatures (e.g., room temperature), increasing shelf life, reducing degradation (e.g., oxidative protease-mediated degradation), increasing the half-life of, for example, TRA.

[0222] In exemplary aspects of the present disclosure, the composition further comprises a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the polypeptide is formulated into a pharmaceutical composition comprising an activating agent and a pharmaceutically acceptable carrier, diluent or excipient. In this regard, the present disclosure further provides a pharmaceutical composition comprising an activating agent, which is intended to be administered to a subject, such as a mammal (such as a human).

[0223] In exemplary aspects, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions (such as oil / water emulsions or water / oil emulsions), and various types of wetting agents. The term also encompasses any medicament approved by the U.S. federal government regulatory agency or listed in the United States Pharmacopeia (USP) for use in animals (including humans). The pharmaceutical composition may contain any pharmaceutically acceptable ingredient, including, for example, acidifiers, additives, adsorbents, aerosol propellants, air displacers, alkalizers, anti-caking agents, anticoagulants, antimicrobial preservatives, antioxidants, preservatives, bases, binders, buffers, chelating agents, coating agents, colorants, desiccants, detergents, diluents, disinfectants, disintegrants, dispersants, solubility enhancers, dyes, softeners, emulsifiers, emulsion stabilizers, fillers, film formers, flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oily vehicles, organic bases, tablet bases, pigments, plasticizers, polishing agents, preservatives, chelating agents, skin penetrants, solubilizers, solvents, stabilizers, suppository bases, surfactants (surface Active agents, surfactants, suspending agents, sweeteners, therapeutic agents, thickeners, tonicity agents, toxicants, viscosity enhancers, water absorbents, water miscible cosolvents, water softeners or wetting agents. See, for example, Handbook of Pharmaceutical Excipients, Third Edition, AH Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated herein by reference in its entirety. Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated by reference in its entirety.

[0224] It is obvious to those skilled in the art that the optimal dosage of one or more active ingredients in a pharmaceutical composition will depend on a variety of factors. Relevant factors include, but are not limited to, the type of animal (e.g., human), the specific form of the polypeptide of the present invention, the mode of administration, and the composition used.

[0225] In order to treat a disease, a therapeutically effective amount of the polypeptide of the present invention is provided. This refers to an effective amount of the polypeptide for treating a disease or having a desired effect, that is, the amount of biological or medical response of cells, tissues, systems or animals (such as humans) sought by researchers, veterinarians, doctors or other treatment providers. The data obtained from cell culture assays and animal studies can be used to formulate a dosage range for people. Clinicians can adjust the dosage regimen to provide the best desired response. Those skilled in the art will understand that certain factors may affect the dosage and time required for the effective treatment of a subject, including but not limited to the severity of the disease or disorder, previous treatment, the general health and / or age of the subject, and other existing diseases. In addition, the treatment of a subject with a therapeutically effective amount of the polypeptide of the present invention may include a single treatment, or may include a series of treatments. The subject may be a mammal, including a human.

[0226] How to use

[0227] Exemplary polypeptides of the present invention can be used to expand Treg in a subject or sample. Provided herein is a method for increasing the ratio of Treg to non-Treg. In some embodiments, provided herein is a method for increasing the ratio of Treg to Teff cells and / or the ratio of Treg to NK cells. In some aspects, the method includes contacting a T cell population with an effective amount of the polypeptide of the present invention. In some aspects, the T cell population is located in the peripheral blood of the subject. The typical Treg frequency in human blood is 5-10% of total CD4+CD3+T cells, however, in diseases treatable by this method, the frequency may be lower or higher. In a preferred embodiment, the percentage of Treg increases 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, the polypeptide of the present invention is administered to a subject and the ratio of regulatory Treg to Teff in the subject's peripheral blood increases.

[0228] Since the exemplary Treg agonists of the present invention will preferentially expand Tregs relative to non-Tregs, the exemplary TRA can be used to increase the ratio of regulatory T cells (Tregs) to natural killer (NK) cells in the peripheral blood of a subject. For example, the ratio can be measured by determining the ratio of Tregs to CD16+ and / or CD56+ lymphocytes (CD19- and CD3-).

[0229] The method of the present invention includes a method for activating Treg cells and / or a method for inducing proliferation of Treg cells, the method comprising the step of contacting Treg cells with the polypeptide of the present invention.

[0230] It is envisioned that the polypeptides of the invention may have a therapeutic effect on a patient's disease or condition without significantly increasing the ratio of Treg 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.

[0231] Exemplary Treg agonists of the invention can be used, for example, to treat diseases, disorders, or conditions that would benefit from Treg expansion without a corresponding expansion of non-regulatory T cells or NK cells in the patient's peripheral blood.

[0232] Exemplary Treg agonists of the invention can be used, for example, to treat a disease, disorder, or condition associated with Teff cell activity.

[0233] Exemplary Treg agonists of the invention can be used, for example, to treat a disease, disorder, or condition associated with B cell activity.

[0234] Diseases, disorders or conditions particularly suitable for treatment with TRA include, but are not limited to, inflammation, autoimmune diseases, atopic diseases, paraneoplastic autoimmune diseases, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular 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, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome (seronegative, tendinopathy, arthropathy syndrome psoriasis, dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myositis, polymyositis, dermatomyositis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjögren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, psoriasis retrograde psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, systemic lupus erythematosus systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, sinusitis, sinusitis with polyps, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barré disease, type I diabetes, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, transplant rejection, kidney damage, vasculitis caused by hepatitis C, spontaneous abortion, etc.

[0235] In preferred embodiments, the autoimmune or inflammatory disorder is lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type I diabetes, multiple sclerosis, spontaneous abortion, atopic disease, and inflammatory bowel disease.

[0236] In another embodiment, a patient who has, is at risk of, or has an autoimmune or inflammatory disorder is treated with the TRA of the invention and the patient's response to treatment is monitored. The patient response monitored can be any detectable or measurable response of the patient to treatment, or any combination of such responses.

[0237] In some exemplary embodiments, the polypeptides of the present invention will be administered in combination with one or more other agents. The other agents may be other therapeutic agents for treating a condition or disease or may be agents administered to reduce side effects associated with polypeptide therapy.

[0238] In some embodiments, the subject or patient treated by the present method is a mammal. In some aspects, the subject or patient is a human.

[0239] Pill Box

[0240] The present disclosure further provides a medicine box comprising a polypeptide of the present invention. In exemplary aspects, the medicine box includes a polypeptide in a container. In exemplary aspects, the polypeptide is provided in the form of a unit dose in the medicine box. For the purposes of this article, "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 (e.g., to treat any of the conditions or illnesses described herein) to a subject. In exemplary aspects, the medicine box includes several unit doses, such as a unit dose of a weekly or monthly supply, and optionally, each unit dose is individually packaged or otherwise separated from other unit doses. In some embodiments, the components of the medicine box / unit dose are packaged together with instructions for use to a patient. In some embodiments, the medicine box includes one or more devices for use to a patient, such as a needle and a syringe, etc. In some aspects, the polypeptide is prepackaged into a ready-to-use form. In exemplary aspects, the ready-to-use form is for single use. In exemplary aspects, the medicine box includes a variety of single-use, ready-to-use forms of polypeptides. In some aspects, the kit further comprises other therapeutic or diagnostic agents or a pharmaceutically acceptable carrier (eg, solvent, buffer, diluent, etc.), including any of those described herein.

[0241] Example

[0242] In all examples referring to anti-CD25 antibodies, the anti-CD25 antibodies comprise daclizumab heavy and light chain variable region binding domains. Unless otherwise specified, the Fc domains and full-length antibodies are IgG1, whose effector function is reduced by LALA-GA mutations (L234A, L235A and G237A, according to the EU system numbering), and optionally include K447S modification (according to the EU system numbering) to prevent potential cleavage of the C-terminal fusion domain.

[0243] In 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 density gradient medium and SepMate tubes. Pan-CD3+ T cells were isolated from PBMCs using the StemCell EasySep Human T Cell Isolation Kit and seeded into each well of a 96-well round-bottom plate with X-VIVO 15 medium. Serial dilutions of the test article were diluted in X-VIVO 15 medium and added to each well, and the cells were stimulated for 30 minutes at 37°C. Cells were fixed by adding paraformaldehyde to 1.5% and incubating at room temperature for 10 minutes. Cells were permeabilized in ice-cold 100% methanol at 4°C for 30 minutes. Cells were then washed twice with FACS buffer (phosphate-buffered saline with 2% fetal bovine serum and 1 mM EDTA). Cells were stained for 1 hour at 4°C using fluorophore-conjugated CD4, CD8, CD25, CD127, and pSTAT5 antibodies. The cells were washed with FACS buffer and fixed again with 1.5% paraformaldehyde. To evaluate NK cell signaling, the same procedure was performed with PBMCs that had not been isolated from CD3+T cells, but CD56 antibodies were added during the staining step. Cells were analyzed by flow cytometry on Cytek Aurora and analyzed using the OMIQ flow cytometry analysis platform. The percentage of pSTAT5+ cells in different cell populations was determined by gating the unstimulated cell control. The dose-response curves of %pSTAT5+ cells were fitted to a logistic model, and the half-maximal effective concentration (EC50 value) was calculated using GraphPad Prism data analysis software. The dose-response curves were performed in the range of 0.01pM to 100nM. An IL-2 control curve was included to determine the responsiveness of the donor cells, measured by %pSTAT5+ gating. Each candidate was titrated at least three times to confirm consistent performance. In order to compare two different test products or test products containing Neo-2 / 15 and / or IL-2, cells from the same blood draw should be used for testing. There is significant signaling variability in patient blood draws. Tregs are defined as those T cells that are CD4+, CD25+, and CD127(low / -). Annotation of all T cells includes all CD3+ cells, including CD8+, CD4+, and Tregs.

[0244] When selecting a specific fusion protein for use as a TRA, the following parameters should be considered. First, the candidate preferably has a low maximum signal transduction in non-Treg cells (such as Teff cells and / or NK cells) at a concentration of, for example, up to 10 nM (i.e., less than 30%, less than 20%, or less than 10%, or even lower, of the maximum signal caused by IL-2 stimulation). In some embodiments, lower signal transduction in non-Treg is associated with higher Treg selectivity. Second, the maximum signal transduction in Treg should preferably be at least 40%, at least 50%, or at least 60% of the maximum signal transduction produced by IL-2 stimulation, wherein the higher the maximum Treg signal transduction, the stronger the TRA. Third, the EC50 of the Treg% pSTAT5+ curve is preferably lower than 1 nM, preferably even lower, for example, lower than 500 pM, or lower than about 100 pM, and the lower the Treg EC50, the more effective the TRA is. In addition to these three parameters, it is also helpful to consider the Treg pSTAT5 MFI (mean fluorescence intensity) when selecting TRA. Higher MFI corresponds to stronger signal transduction and downstream events, such as cell proliferation and activation. In some embodiments, when evaluating the Emax of non-Tregs, a test article may still be considered to have Treg-selective characteristics if a high Emax is achieved only at a high EC50 (e.g., greater than 20% of the maximal signaling produced by IL-2 stimulation); the precise evaluation will rely on the assessment of the entire pSTAT5 titration curve between cell subsets.

[0245] Example 1 - Modulating Neo-2 / 15 potency to obtain attenuated Neo-2 / 15 mutants that retain binding to IL-2RBG and can be used as TRA when fused to a Treg targeting agent

[0246] To identify the residues involved in the binding interaction between huIL2Rβ and / or IL2Rβγ and Neo-2 / 15, the PDB structure 6DG5 was examined in PyMol to select Neo-2 / 15 residues within 5.5 angstroms of either receptor. Twenty-seven positions were identified, and samples of Neo-2 / 15 mutant proteins with single alanine mutations at these 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 had binding affinity for both huIL2Rβ and huIL2Rβγ as analyzed by Octet Biolayer Interferometry. Binding data were collected in an Octet RED96 instrument and processed using the integrated Octet software. At concentrations equal to or greater than the analyte concentration, binding to huIL2Rβ was collected using human IL-2Rβ / CD122 protein, Fc tag (validated by MALS and SPR) (ILB-H5253), and binding to huIL2Rβγ was collected using human IL-2Rγ / CD132 protein, Fc tag (ILG-H5256) and human IL-2Rβ / CD122 protein, His tag (validated by SPR) (CD2-H5221). Anti-human Fc capture (AHC) sensors were equilibrated in 1X Cytiva HBS-EP + buffer 10 binding buffer for 90s. The receptors were loaded by mixing the sensor with wells containing 0.5ug / ml of the above receptors until the threshold response reached 1.0nm. The sensor was then benchmarked in binding buffer for 90s. After the baseline measurement, the binding kinetics (kon) were monitored by immersing the biosensor in the wells containing the analyte for 400s. Analyte concentrations were decreased with 3-fold dilutions to 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 biosensors in separate wells of binding buffer for 800 s. Dissociation constants (Kd) were calculated using a 1:1 steady-state binding model using Octet software.

[0247] After calculating the binding affinity of 27 positions to huIL2Rβ and huIL2Rβγ through single alanine mutation screening, four positions were identified for adjustment. Each variant was characterized for folding and Tm to confirm that the reduction in affinity was not due to misfolding, and SEC traces were followed to confirm that there was no aggregation in the test samples. The 27 positions identified within 5.5 angstroms of one of the two receptors were 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, F99. Of these 27 positions, 4 positions were identified for adjustment in the first round of attenuation. Table 2 shows the Kd of Neo-2 / 15 mutants with significant single alanine mutations.

[0248] Table 2

[0249] protein IL-2RB Kd(nM) IL-2RBG Kd(nM) Neo-2 / 15 1.6 6 Neo-2 / 15_D15A 140 230 Neo-2 / 15_N40A 60 44 Neo-2 / 15_I44A 84 58 Neo-2 / 15_Q95A 3.0 190

[0250] To identify combinations of alanine mutations on Neo-2 / 15 that further attenuate its binding to huIL2Rβ and huIL2Rβγ, the four identified positions were combined with different probabilities. Low attenuation measures were 1-10X reduction relative to Neo-2 / 15 Kd; medium attenuation measures were 10-100X reduction relative to Neo-2 / 15 Kd; high attenuation measures were 100X+ reduction relative to Neo-2 / 15 Kd (usually between 100X and 1000X reduction relative to Neo-2 / 15 Kd).

[0251] Mutations at position 95 reduced binding to IL-2G, whereas mutations at positions 15, 40, and 44 reduced binding to IL-2B.

[0252] Attenuation of the γ domain is difficult to measure alone because Neo-2 / 15 binds weakly to IL2RG, in the low uM range, thus requiring affinity to be measured in the presence of IL2Rβ. Additionally, in embodiments where mutants have moderate to high attenuation, binding is typically measured by octet under high affinity conditions.

[0253] For high affinity conditions, the attenuated Neo-2 / 15 mutant was fused to the full-length antibody. The antibody was a control antibody (i.e., it was not an anti-IL-2RB or anti-IL-2RG binding antibody). For the attenuated Neo-2 / 15 mutant, an anti-CD25 antibody with reduced effector function was used, while 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 on 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% skim milk powder). After baseline measurements were made in binding buffer only, the binding kinetics were monitored by immersing the biosensor into wells containing a defined concentration of Neo-2 / 15 mutants (333-3000 nM) to measure association, and then immersing the sensor back into the baseline wells to measure dissociation. The dissociation constant was calculated using the steady-state binding model of the Octet software. The results showed that for the anti-CD25 fusion protein containing the Neo-2 / 15D15S_Q95E mutant, the fusion protein bound to IL-2RBG, but with a weaker affinity than that of Neo-2 / 15 or Neo-2 / 15 binding to trastuzumab.

[0254] Example 2 - Neo-2 / 15 mutant exhibits reduced pSTAT5 activity

[0255] Neo-2 / 15 mutants were recombinantly expressed and purified from E. coli using the methods described in U.S. Patent No. 10,703,791. Neo-2 / 15 mutants with Q95E and D15S mutations (Neo-2 / 15_D15S_Q95E) or Q95AD15A (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, CD8+ cell types, 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 retained a Treg EC50 of ~9 nM and ~40% of Treg maximal pSTAT5 signaling. Figure 1A and 1B ; Table 3. Higher attenuation levels of Neo-2 / 15_D15S_Q95E translated into better TRA activity (see Examples below).

[0256] Table 3

[0257]

[0258] Example 3 - Neo-2 / 15 mutants can be fused with Treg targeting agents and Fc domains in different configurations.

[0259] Neo-2 / 15 mutants were cloned into pcDNA3.4 as various daclizumab binding domain fusion proteins and expressed in Expi293 cells. These designs differed in the format of the binding domain (scFv or full-length mAb with reduced / no effector function) and the site of fusion of the Neo-2 / 15 mutant and scFv. Figure 2A -C provides an example configuration.

[0260] exist Figure 2A In the TRA, the Neo-2 / 15 mutant and the scFv are located at opposite ends of the Fc. TRA is a fusion protein comprising a homodimer, which comprises two Neo-2 / 15 mutants, each of which is linked to the N-terminus of the Fc domain via its C-terminus, and the Fc domain is linked to the variable heavy chain of the ScFv via its C-terminus.

[0261] exist Figure 2B In the TRA, the Neo-2 / 15 mutant is fused to the N-terminus of the heavy chain variable region. TRA is a fusion protein comprising a heterotetramer comprising two heavy chains (VH + constant region) and two light chains (VL + constant region), each heavy chain variable region being linked to the C-terminus of the Neo-2 / 15 mutant via its N-terminus.

[0262] exist Figure 2C In TRA, the Neo-2 / 15 mutant is fused to the C-terminus of the heavy chain. TRA is a fusion protein comprising a heterotetramer comprising two heavy chains (VH + constant region) and two light chains (VL + constant region), each heavy chain constant region being linked by its C-terminus to the N-terminus of the Neo-2 / 15 mutant.

[0263] A linker (GGGGSGGGGSGGGGS) was used.

[0264] Example 4 - Neo-2 / 15 mutant-ScFv fusion protein exhibits TRA activity in pSTAT5 signaling assay

[0265] Neo-2 / 15 mutants were fused to anti-CD25 ScFv antibody fragments (without the Fc domain) and tested in the pSTAT5 assay to identify fusion proteins that were able to achieve a reduction in Teff signaling while maintaining Treg signaling.

[0266] Among the ScFv fusion protein groups shown in Table 4, the Neo-2 / 15 mutant_D15S_Q95E fusion protein exhibited the best reduction in Teff signaling while maintaining Treg signaling. Figure 3A -F. For Figure 4A -B, Addition of IL-2 control at a constant concentration of 1 nM resulted 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 indicated by the dotted lines).

[0267] Table 4

[0268]

[0269]

[0270]

[0271] Example 5 - Fusion proteins of the invention can be optimized to achieve higher affinity for CD25 than IL-2

[0272] The binding to human CD25 was determined by Octet. Unlike IL-2 and IL-2 mutant Treg agonists, the Neo-2 / 15 mutant of the present invention does not have a binding site for CD25. The affinity for CD25 can be adjusted by the selection of anti-CD25 binding agents. The TRA comprising the Neo-2 / 15 mutant fused with an anti-CD25 antibody exhibits a higher affinity for CD25 than IL-2. The binding force of the ScFv Neo-2 / 15_D15S_Q95E fusion protein (without Fc domain) to human CD25 is about 25 times higher than that of hIL-2 and human CD25, and the Kd is 0.5nM relative to 13nM (data not shown).

[0273] Example 6 - Fusion of Neo-2 / 15 mutants with anti-CD25 Treg targeting agents enhances Treg selectivity

[0274] Unfused Neo-2 / 15Neo-2 / 15 mutant_D15S-Q95E (an anti-CD25 Neo-2 / 15 mutant fusion protein containing Neo-2 / 15_D15S-Q95E and IL-2) was evaluated for pSTAT signaling in Treg and Teff cells. Fusion of the Neo-2 / 15_D15S-Q95E mutant to an anti-CD25 ScFv resulted in enhanced potency against Treg cells while reducing potency against CD8+ and CD4+ T cells. The fusion protein exhibited TRA activity. This was an unexpected result as it was not known that fusion of a T-reg targeting agent to the attenuated Neo-2 / 15 mutant would result in a differential reduction in Teff signaling compared to Treg signaling. See Figure 5A -B and Table 5.

[0275] Table 5

[0276]

[0277] *Please note that these experiments were performed with different donors.

[0278] Example 7 - Fusion proteins of the invention comprising Neo-2 / 15_D15S_Q95E exhibit Treg selectivity in different antibody configurations as measured in a pSTAT5 assay.

[0279] Neo-2 / 15_D15S_Q95E mutant was fused to anti-CD25 antibody in different configurations as shown in Figure 2. The fusion protein showing the best TRA activity is shown in Fig. 6A -C and Table 6. For the Neo-2 / 15_D15S_Q95E fusion protein, the mutant bound to the C-terminus of the full-length antibody was the preferred configuration.

[0280] Table 6

[0281]

[0282] Example 8 - Additional Adjustments to the Neo-2 / 15 Mutant Components of TRA

[0283] Another set of mutations was performed to determine if mutations at other positions associated with Il-2RB or IL-2RBG binding 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 other positions identified were positions 8, 11, and 14. Table 7 shows the Kd for mutants with one significant mutation in the single alanine mutation scan. The mutation combinations tested were D15S+N40S; D15S+N40S+I44S; H8R+D15S; H8R+D15S+N40S+I44S; H8R+N40S+I44S;

[0284] H11F+D15S; H11F+D15S+N40S+I44S; H11F+N40S+I44S; Y14K+D15S+N40S+I44S; Y14K+D15S; H11F+N4 0S+I44S; D15S+N40S+Q95E; D15S+N40S+I44S+Q95E; H8R+D15S+Q95E; H8R+D15S+N40S+I44S+Q95E; H 8R+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.

[0285] Table 7

[0286] protein IL-2RB Kd(nM) IL-2RBG Kd(nM) Neo-2 / 15 1.6 6 Neo-2 / 15_H8A 1.4 11 Neo-2 / 15_H11A 3.3 15 Neo-2 / 15_Y14A 0.7 10-20*

[0287] Each mutant was made into a fusion protein, expressed as a C-terminal or N-terminal fusion to a full-length anti-CD25 antibody, to determine if the position of the fusion had an effect on activity. When fused to the C-terminus, the Neo-2 / 15 mutant was located distal to the IL-2A receptor, whereas when fused at the N-terminus, the Neo-2 / 15 mutant was located proximal to the receptor. Certain fusions were effective as TRAs when fused to one terminus, but not as effective at the other. In general, the fusion proteins exhibited higher Teff activity when fused at the N-terminus than at the C-terminus.

[0288] When fused to the C-terminus, if the D15S and Q95E mutations are present, any additional mutations made will reduce TregSTAT5 phosphorylation activity to undesirable levels. Without being bound by theory, it is believed that the level of attenuation of the D15S_Q95E mutant is optimal for a C-terminal fusion, and any additional mutations made to this mutant are preferably made at positions unrelated to IL-2RB or IL-2RBG binding, provided that the goal is a C-terminal fusion to a full-length antibody.

[0289] The previous C-terminal fusions included 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 properties of the C-terminally fused TRA. See below Fig. 7A -D and 8A-D and Tables 8 and 9. Tables 8 and 9 are values ​​from two different donor samples.

[0290] Note that for the fusion proteins with N40S and Q95E mutations at the C-terminus, additional mutations of I44S, I44S+H11F, I44S+8HR, I44S+Y14K would reduce Treg STAT5 phosphorylation activity to undesirable levels if fused at the C-terminus.

[0291] When fused to the N-terminus, the first ten 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 below Fig. 9A -B and Tables 8 and 9. Tables 8 and 9 are values ​​from two different donor samples.

[0292] Table 8

[0293]

[0294] **This donor showed a lower response compared to the previous donor.

[0295] Table 9

[0296]

[0297] C-terminal fusions to the Neo-2 / 15_D15S_Q95E mutant showed a consistent strong preferential Treg pSTAT5 signaling signature across donors with low levels of Teff signaling, whereas N-terminal fusions showed a less consistent signature across donors, with the exception of protein fusions to the Neo-2 / 15_Q95E_N40S_I44S mutant. Fig. 9A -B.

[0298] Example 9 - Treg expansion in mice

[0299] Fusion proteins comprising Neo-2 / 15_D15S_Q95E fused to a full-length anti-CD25 antibody at either the C-terminus or the N-terminus were tested for their ability to expand Treg cells and NK cells in vivo.

[0300] Humanized NSG mice were purchased from Jackson Labs. These mice were transplanted with human CD34+ hematopoietic stem cells and contained >25% human CD45+ cells in the peripheral blood. Mice were injected intraperitoneally with 5mg human IgG on days -1 and 13. Mice were injected intraperitoneally with 15-45ug of test article on days 0, 7, 14, and 21. On days -1, 6, 13, and 20, 100uL of whole blood was collected in K2EDTA by retro-orbital bleeding. Red blood cells were lysed using BioLegend RBC Lysis Buffer, and the remaining PBMCs were washed with PBS and then stained with viability dye 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. Cells were subjected to flow cytometry analysis on a Cytek Aurora and analyzed using the OMIQ flow cytometry analysis platform.

[0301] Treg expansion was determined by gating on live cells for CD3 expression and then gating on CD4 expression. Treg gating was then performed on CD4+ cells by CD25+Foxp3+ staining. Effective Treg expansion was defined as the percentage of Tregs that were at least 2x higher in CD4+ cells compared to control mice and the day -1 time point. Treg CD25 and Foxp3 MFI were also compared between groups, with higher MFIs corresponding to a more stable and more effective Treg phenotype. NK cells were defined as CD56+ cells when gating on live cells. An increase in %CD56+ cells is considered an undesirable effect of a potential Treg agonist. High Treg expansion was observed with both fusion proteins containing the full-length antibody, however, the N-terminal fusion showed significant NK cell expansion at high doses. See Fig. 10A-E.

[0302] Anti-drug antibody (ADA) analysis was performed to determine whether TRA would generate anti-drug antibodies. For this experiment, a ScFv fusion protein of Neo-2 / 15_D15S_Q95E without the Fc domain was used. No anti-drug antibodies were detected. (Data not shown).

[0303] Example 10 - Alternative Treg Targeting Agents

[0304] Neo-2 / 15_D15S_Q95E was fused to an anti-CD39 antibody to test whether fusion proteins targeting T-reg markers other than CD25 would also result in a PSTAT5 signature characterized by low Teff signaling and preferential Treg signaling. Although the Treg signal was not as high as with the CD25 fusion, preferential Treg signaling was observed. (Data not shown).

[0305] Example 11 - IL-2 / IL-15 antagonists are not TRA

[0306] Protein P5 in International Application No. WO2021 / 188374 was fused with anti-CD25 ScFV and tested for pSTAT5 Treg signaling activity. No pSTAT5 signaling was detected in all T cell types tested, CD8+ cells, CD4+ cells, and Tregs. (Data not shown).

[0307] Example 12 - Generation of Additional TRA

[0308] Another set of mutations was performed to identify additional substitutions at positions 95, 40, and 44 that might result in active T reg agonists. Mutants that exhibited Treg-selective pSTAT5 characteristics 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.

[0309] Fusion proteins were prepared and each mutant was expressed as a C-terminal fusion to a full-length anti-CD25 antibody as described previously. pSTAT5 signaling was plotted using the pSTAT5 mean fluorescence index for each cell subset. Fig.11A -G.

[0310] Example 13 - TRA of the present invention exhibits reduced binding to IL-2RBG in a cell binding assay.

[0311] HEK293 cell lines expressing human CD122 and CD132 were used for saturation binding studies. Cells were collected with Versene (Gibco, catalog number 15040-066), washed twice in PBS (Gibco, catalog number 10010-23), and blocked on ice for 20 minutes with 2% BSA (Sigma-Aldrich, catalog number A2153-100G) in PBS. After blocking, cells were dispensed into 96-well U-bottom plates (Corning, catalog number 3799) at a density of 2 × 105 cells per well. Antibody fusions were added to cells with a concentration range of 0.5mM to about 150pM or 0.05mM to about 15pM in 1% BSA / PBS. Cells were incubated on ice for 30 minutes, then precipitated and washed twice with cold PBS. The cells were precipitated and resuspended in 200ng / mL APC conjugated mouse anti-human IgG Fc secondary antibody (Jackson ImmunoResearch, catalog number 709-605-149) solution, which was pre-diluted in 2% BSA / PBS. After incubation on ice for 30 minutes, the cells were precipitated and washed twice in cold PBS. After being resuspended in 1×106 cells / mL in 2% paraformaldehyde solution (Acros, catalog number 41073-0010), fluorescence was detected using CytoFLEX flow cytometer (Beckman-Coulter). GraphPad Prism (GraphPad, San Diego, CA) was used to calculate the apparent dissociation constant (KD).

[0312] Exemplary Sequence Listing

[0313]

[0314] Table 10

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

Claims

1. A polypeptide comprising a Neo-2 / 15 mutant, wherein the Neo-2 / 15 mutant comprises an amino acid sequence that is at least 80%, at least 85% or at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 2, and wherein the Neo-2 / 15 mutant comprises a lysine, arginine, threonine, serine, tyrosine, glutamic acid, alanine or histidine substitution for glutamine at position 95 (Q95K or Q95R or Q95T or Q95S or Q95Y or Q95E or Q95A or Q95H), and at least one additional substitution selected from: (a) substitution of aspartic acid at position 15 by serine or alanine (D15S or D15A); (b) substitution of alanine or serine or glycine for asparagine at position 40 (N40A or N40S or N40G); and (c) substitution of the isoleucine at position 44 by alanine or serine or asparagine or threonine or tyrosine (I44A or I44S or I44N or I44T or I44Y); 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 polypeptide of claim 1, wherein the Neo-2 / 15 mutant comprises a lysine, threonine, tyrosine, glutamic acid, alanine or histidine substitution for glutamine at position 95 (Q95K, Q95T or Q95Y or Q95E or Q95A or Q95H).

3. The polypeptide of claim 1, wherein the at least one additional substituent is selected from: (a) substitution of aspartic acid at position 15 by serine or alanine (D15S or D15A); (b) substitution of alanine or serine or glycine for asparagine at position 40 (N40A or N40S); and (c) substitution of the isoleucine at position 44 by alanine or serine or asparagine or threonine or serine or tyrosine (I44A or I44S or I44Y).

4. The polypeptide of claim 1, wherein the Neo-2 / 15 mutant comprises a substitution of glutamic acid, alanine or histidine for glutamine at position 95 (Q95E or Q95A or Q95H), and at least one additional substitution selected from: a) substitution of aspartic acid at position 15 by serine or alanine (D15S or D15A); b) substitution of alanine or serine for asparagine at position 40 (N40A or N40S); and c) Substitution of the isoleucine at position 44 with alanine or serine (I44A or I44S).

5. The polypeptide of any one of claims 1-3, wherein the Neo-2 / 15 mutant comprises a tyrosine substitution for glutamine at position 95 (Q95Y).

6. The polypeptide of any one of claims 1-4, wherein the Neo-2 / 15 mutant comprises a substitution of glutamic acid for glutamine at position 95 (Q95E).

7. The polypeptide of any one of claims 1 to 6, wherein the Neo-2 / 15 mutant comprises a serine or alanine substitution for aspartic acid at position 15 (D15S or D15A).

8. The polypeptide of any one of claims 1-7, wherein the Neo-2 / 15 mutant comprises an alanine substitution for aspartic acid at position 15 (D15A).

9. The polypeptide of claim 8, wherein the Neo-2 / 15 mutant comprises a leucine substitution for the arginine at position 13.

10. The polypeptide of claim 8 or claim 9, wherein the Neo-2 / 15 mutant comprises a substitution of glutamic acid for leucine at position 17.

11. The polypeptide of any one of claims 1-7, wherein the Neo-2 / 15 mutant comprises a serine substitution for aspartic acid at position 15 (D15S).

12. The polypeptide of any one of claims 1-11, wherein the Neo-2 / 15 mutant comprises a phenylalanine substitution for histidine at position 11 (H11F).

13. The polypeptide of any one of claims 1-11, wherein the Neo-2 / 15 mutant comprises a lysine substitution for a tyrosine at position 14 (Y14K).

14. The polypeptide of any one of claims 1-12, wherein the Neo-2 / 15 mutant does not comprise a substitution at any of positions 8, 14, 40 and 44.

15. The polypeptide of claim 14, wherein the Neo-2 / 15 mutant does not comprise a substitution at any of positions 8, 11, 14, 40 and 44.

16. The polypeptide of any one of claims 1 to 15, wherein if the Neo-2 / 15 mutant comprises a serine or alanine substitution for aspartic acid at position 15, it does not comprise (i) a serine or alanine or glycine substitution for asparagine at position 40, and / or it does not comprise (ii) a serine or alanine or asparagine or threonine or tyrosine substitution for isoleucine at position 44.

17. The polypeptide of any one of claims 1-15, wherein if the Neo-2 / 15 mutant comprises a serine or alanine substitution for the aspartic acid at position 15, it does not comprise (i) a serine or alanine substitution for the asparagine at position 40, and / or it does not comprise (ii) a serine or alanine substitution for the isoleucine at position 44.

18. The polypeptide of any one of claims 1-15, wherein if the Neo-2 / 15 mutant comprises a serine or alanine substitution for aspartic acid at position 15, it does not comprise (i) a substitution at position 40 and / or (ii) a substitution at position 44.

19. The polypeptide of any one of claims 16-18, wherein both (i) and (ii) are true.

20. The polypeptide of any one of claims 1 to 6, wherein the Neo-2 / 15 mutant comprises an acidic amino acid at position 15.

21. The polypeptide of any one of claims 1 to 6, wherein the Neo-2 / 15 mutant does not comprise a substitution at position 15.

22. The polypeptide of claim 20 or 21, wherein the Neo-2 / 15 mutant comprises alanine or serine or glycine substituted for asparagine at position 40 (N40A or N40S or N40G).

23. The polypeptide of claim 22, wherein the Neo-2 / 15 mutant comprises an alanine or serine substitution for asparagine at position 40 (N40A or N40S).

24. The polypeptide of any one of claims 20-23, wherein the Neo-2 / 15 mutant comprises alanine or serine or asparagine or threonine or tyrosine substituted for isoleucine at position 44 (I44A or I44S or I44N or I44T or I44Y).

25. The polypeptide of claim 24, wherein the Neo-2 / 15 mutant comprises an alanine or serine substitution for isoleucine at position 44 (I44A or I44S).

26. The polypeptide of claim 24, wherein the Neo-2 / 15 mutant comprises a tyrosine substitution for isoleucine at position 44 (I44Y).

27. The polypeptide of any one of claims 20-26, wherein the Neo-2 / 15 mutant comprises a phenylalanine substitution for histidine at position 11 (H11F).

28. The polypeptide of any one of claims 20-27, wherein the Neo-2 / 15 mutant comprises a lysine substitution for a tyrosine at position 11 (Y14K).

29. The polypeptide of any one of claims 1-28, wherein the Neo-2 / 15 mutant does not comprise a substitution at position 8 (8HR).

30. The polypeptide of any one of claims 1-29, wherein the Neo-2 / 15 mutant does not comprise a substitution at one or both of positions 17 and 99.

31. A polypeptide as described in any of the preceding claims, wherein the Neo-2 / 15 mutant comprises a set of substitutions selected from the group consisting of: a) Q95E and D15S; b) Q95E, D15S and H11F; c) Q95E, N40S and I44S; d) Q95E, N40S, I44S and H11F; e) Q95E, N40S, I44S and Y14K; f) Q95E, D15S and Y14K; g) Q95H, D15A, L17E and L13R; h) Q95H and D15A; and i) Q95A and D15A.

32. The polypeptide of any one of claims 1 to 30, wherein the Neo-2 / 15 mutant comprises a set of substitutions selected from the group consisting of: (a) D15S and Q95K; (b) D15S and Q95T; (c) D15S and Q95Y; (d) N40G, I44S and Q95E; (e) N40S, I44T and Q95E; (f) N40S, I44Y and Q95E; and (g) N40S, I44N and Q95E.

33. The polypeptide of claim 31, wherein the Neo-2 / 15 mutant comprises substitutions Q95E and D15S.

34. The polypeptide of any one of claims 31-33, wherein the Neo-2 / 15 mutant does not comprise any additional substitutions.

35. A polypeptide comprising a Neo-2 / 15 mutant, wherein the Neo-2 / 15 mutant comprises an amino acid sequence that is 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 mutant comprises a serine or alanine substitution for aspartic acid at position 15 (D15S or D15A), and at least one additional substitution selected from: (a) a lysine, arginine, threonine, serine, tyrosine, glutamic acid, alanine or histidine substitution for the glutamine at position 95 (Q95K or Q95R or Q95T or Q95S or Q95Y or Q95E or Q95A or Q95H); (b) arginine substituted for histidine at position 8 (H8R); (c) phenylalanine substituted for histidine at position 11 (H11F); (d) substitution of lysine for tyrosine at position 14 (Y14K); (e) substitution of alanine or serine for asparagine at position 40 (N40A or N40S); and (f) substitution of the isoleucine at position 44 by alanine, serine or tyrosine (I44A or I44S or I44Y), wherein 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.

36. The polypeptide of claim 35, wherein the at least one additional substitution is selected from: (a) substitution of glutamic acid, alanine or histidine for glutamine at position 95 (Q95E, Q95A or Q95H); (b) arginine substituted for histidine at position 8 (H8R); (c) phenylalanine substituted for histidine at position 11 (H11F); (d) substitution of lysine for tyrosine at position 14 (Y14K); (e) substitution of alanine or serine for asparagine at position 40 (N40A or N40S); and (f) Substitution of isoleucine at position 44 by alanine or serine (I44A or I44S).

37. The polypeptide of claim 35 or 36, wherein if the Neo-2 / 15 mutant comprises a substitution at position 95, it does not comprise a substitution at position 40 and / or 44.

38. The polypeptide of any one of claims 35-37, wherein if the Neo-2 / 15 mutant comprises a substitution at position 40 or 44, it does not comprise a substitution at position 95, 11 or 8.

39. The polypeptide of any one of claims 35-38, wherein if the Neo-2 / 15 mutant comprises a substitution at position 40 or 44, it does not comprise a substitution at position 14.

40. The polypeptide of any one of claims 35-39, wherein if the Neo-2 / 15 mutant comprises a serine or alanine substitution for aspartic acid at position 15, it does not comprise a substitution at position 14.

41. The polypeptide of any one of claims 1-40, wherein if the Neo-2 / 15 mutant comprises a serine or alanine substitution for the aspartic acid at position 15, it does not comprise a lysine substitution for the tyrosine at position 14.

42. The polypeptide of any one of claims 1-41, wherein if the Neo-2 / 15 mutant comprises a substitution at position 95, it does not comprise a substitution at position 8.

43. The polypeptide of any one of claims 1-42, wherein if the Neo-2 / 15 mutant comprises a substitution of glutamic acid for glutamine at position 95, it does not comprise a substitution of arginine for histidine at position 8.

44. A polypeptide comprising a Neo-2 / 15 mutant, wherein the Neo-2 / 15 mutant comprises an amino acid sequence that is 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 mutant comprises: (a) Phenylalanine substituted for histidine at position 11 (H11F); (b) substitution of alanine or serine for asparagine at position 40 (N40A or N40S); and (c) substitution of the isoleucine at position 44 by alanine, serine or tyrosine (I44A or I44S or I44Y), wherein 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.

45. The polypeptide of claim 44, wherein the Neo-2 / 15 mutant comprises: (a) Phenylalanine substituted for histidine at position 11 (H11F); (b) substitution of alanine or serine for asparagine at position 40 (N40A or N40S); and (c) Substitution of isoleucine at position 44 with alanine or serine (I44A or I44S).

46. ​​A polypeptide as claimed in claim 44 or claim 45, wherein the Neo-2 / 15 mutant does not comprise a substitution at position 15.

47. The polypeptide of any one of claims 44-46, wherein the Neo-2 / 15 mutant does not comprise a substitution at position 95.

48. The polypeptide of any one of claims 44-47, wherein the Neo-2 / 15 mutant does not comprise a substitution at position 14 and / or 8.

49. The polypeptide of any one of claims 35-38, wherein the Neo-2 / 15 mutant comprises a set of substitutions selected from the group consisting of: D15S and Y14K; N40S, I44S, and H11F; N40S, I44S and D15S; N40S, I44S, Y14K and D15S; N40S and D15S; H8R and D15S; and H11F and D15S.

50. The polypeptide of claim 49, wherein the Neo-2 / 15 mutant comprises the substitutions N40S, I44S and D15S.

51. The polypeptide of claim 49 or 50, wherein the Neo-2 / 15 mutant does not comprise any additional substitutions.

52. A polypeptide as described in any of the preceding claims, wherein the Neo-2 / 15 mutant 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% or 98% identical to SEQ ID NO: 1 or SEQ ID NO:

2.

53. A polypeptide as described in any of the preceding claims, wherein the Neo-2 / 15 mutant 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.

54. A polypeptide as described in any of the preceding claims, wherein the Neo-2 / 15 mutant 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 the group consisting of SEQ ID NOs: 2-25 and SEQ ID Nos: 75-82.

55. The polypeptide of any one of claims 1-41, wherein the Neo-2 / 15 mutant comprises three amino acids N-terminal and linked to the amino acid at position 4, wherein the three amino acids are proline-lysine-lysine, wherein the positions are numbered according to SEQ ID NO:

1.

56. The polypeptide of any one of claims 1-55, wherein the Neo-2 / 15 mutant does not comprise a substitution at 1, 2, 3 or all 4 of positions 91, 92, 96 and 99.

57. The polypeptide of claim 56, wherein the Neo-2 / 15 mutant does not comprise a substitution at 1, 2, 3 or all 4 of positions 91, 92, 96 or 99.

58. The polypeptide of any one of claims 1-57, wherein the Neo-2 / 15 mutant does not comprise a substitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of positions 6, 7, 10, 18, 33, 36, 37, 39, 43, 47, 84, 85, 88 or 98.

59. The polypeptide of any one of claims 1-58, wherein the Neo-2 / 15 mutant does not comprise a substitution at position 91 or 92.

60. The polypeptide of any one of claims 1-59, wherein the Neo-2 / 15 mutant does not comprise a substitution at position 8, 11 or 14.

61. The polypeptide of any one of claims 1-60, wherein the Neo-2 / 15 mutant does not comprise a substitution at position 13 or 17.

62. The polypeptide of any one of claims 1-61, wherein the Neo-2 / 15 mutant has a substitution at a position other than position 8, 11, 14, 15, 40, 44 or 95, and the substitution does not substantially interfere with the binding of the Neo-2 / 15 mutant to IL-2Rβγ.

63. A polypeptide comprising a polypeptide of a Neo-2 / 15 mutant, wherein the Neo-2 / 15 mutant comprises domains D1, D2, D3 and D4; wherein: (c) D1 contains the amino acid sequence: KIQLHAEHALYX 15 ALMILNI (SEQ ID NO:61) and D3 comprises the amino acid sequence LEDYAFNFELILEEIARLFESG (SEQ ID NO:64); or (d) 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); and D2 comprises an amino acid sequence of at least 8 amino acids in length; D4 contains the amino acid sequence EDEQEEMANAIITILX 95 SWIFS (SEQ ID NO: 66); wherein: (v) In the Neo-2 / 15 mutant, D1, D2, D3 and D4 can be arranged in any order; (vi) an amino acid linker may be present between any of the domains (a "domain linker"); (vii)X 15 is serine or alanine; X 95 is glutamic acid, lysine, arginine, threonine, serine, tyrosine, alanine or histidine; X 40 is serine or alanine or glycine; and X 44 is serine or alanine or asparagine or threonine or tyrosine; (viii) wherein the Neo-2 / 15 mutant contains a total of no more than ten, no more than nine, no more than eight, no more than seven, no more than six, no more than five, no more than four, no more than three, no more than two, no more than one, or zero substitutions at amino acid positions not designated as X.

64. The polypeptide of claim 63, wherein X 40 is serine or alanine; and X 44 It is serine or alanine or tyrosine.

65. The polypeptide of claim 63, wherein X 95 is glutamate, alanine or histidine; X 40 is serine or alanine; and X 44 It is serine or alanine.

66. The polypeptide of any one of claims 63-65, wherein D1 comprises the amino acid sequence: KIQLHAEHALYX 15 ALMILNI (SEQ ID NO:61) and D3 comprises the amino acid sequence LEDYAFNFELILEEIARLFESG (SEQ ID NO:64).

67. The polypeptide of any one of claims 63-66, wherein X 15 It's serine.

68. The polypeptide of any one of claims 63-67, wherein the asparagine at position 7 and the isoleucine at position 11 in SEQ ID NO:64 are not substituted.

69. The polypeptide of any one of claims 63-65, wherein 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).

70. The polypeptide of claim 69, wherein X 40 It's serine.

71. The polypeptide of claim 69 or claim 70, wherein X 44 It's serine.

72. The polypeptide of any one of claims 69-71, wherein the aspartic acid at position 12 of SEQ ID NO:62 is not substituted.

73. The polypeptide of any one of claims 69-72, wherein the tyrosine at position 11 of SEQ ID NO:62 is substituted.

74. The polypeptide of claim 73, wherein the tyrosine at position 11 of SEQ ID NO:62 is substituted with lysine.

75. The polypeptide of any one of claims 63-74, wherein the histidine at position 8 of SEQ ID NO:61 or SEQ ID NO:62 is substituted.

76. The polypeptide of claim 75, wherein the substituent is phenylalanine (H11F).

77. The polypeptide of any one of claims 63-76, wherein the histidine at position 5, the histidine at position 8, and the tyrosine at position 11 of SEQ ID NO:61 or SEQ ID NO:62 are not substituted.

78. The polypeptide of any one of claims 63-77, wherein X 95 It's glutamate.

79. The polypeptide of any one of claims 63-78, wherein (ii) 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, wherein the glutamine at position 3, the leucine at position 4, the glutamic acid at position 7, the leucine at position 10, and the methionine at position 15 of SEQ ID NO:61; and the aspartic acid at position 3, the tyrosine at position 4, the phenylalanine at position 6, the leucine at position 10, and the glutamic acid at position 14 of SEQ ID NO:64 are not substituted; or (ii) 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 glutamine at position 3, the leucine at position 4, the glutamic acid at position 7, the leucine at position 10, and the methionine at position 15 of SEQ ID NO:62; and the aspartic acid at position 3, the tyrosine at position 4, the phenylalanine at position 6, the leucine at position 10, and the glutamic acid at position 14 of SEQ ID NO:65 are not substituted.

80. The polypeptide of any one of claims 63-79, wherein the glutamic acid at position 5, the glutamic acid at position 6, the asparagine at position 9, the isoleucine at position 12, the threonine at position 13, the serine at position 17, the isoleucine at position 19, and the phenylalanine at position 20 of SEQ ID NO:66 are not substituted.

81. The polypeptide of any one of claims 63-80, wherein the leucine at position 14 of SEQ ID NO:61 or SEQ ID NO:62 and the phenylalanine at position 20 of SEQ ID NO:66 are not substituted.

82. The polypeptide of any one of claims 63-81, wherein the leucine at position 14 in SEQ ID NO:61 or SEQ ID NO:62 and the isoleucine at position 12, the threonine at position 13, the serine at position 17, the isoleucine at position 19 and the phenylalanine at position 20 of SEQ ID NO:66 are not substituted.

83. The polypeptide of any one of claims 63-82, wherein D3 comprises a lysine at the N-terminus and is linked to the amino acid at position 1.

84. The polypeptide of any one of claims 63-83, wherein D2 comprises an amino acid sequence at least 80% identical to KDEAEKAKRMKEWMKRIKT (SEQ ID NO: 63).

85. The polypeptide of claim 84, wherein D2 comprises the amino acid sequence KDEAEKAKRMKEWMKRIKT (SEQ ID NO: 63).

86. The polypeptide of any one of claims 63-85, wherein each amino acid linker is independently 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10 or 2-10 amino acids in length.

87. The polypeptide of any one of claims 63-86, wherein the order of the four domains is D1-D3-D2-D4.

88. The polypeptide of any one of claims 63-86, wherein the order of the four domains is D1-D3-D2-D4, D4-D1-D3-D2, D2-D4-D1-D3 or D3-D2-D4-D1.

89. A polypeptide as described in any of claims 63-88, wherein the Neo-2 / 15 mutant 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.

90. The polypeptide of any one of claims 63-89, wherein D1 comprises a three amino acid N-terminus and is linked to the amino acid at position 1 of SEQ ID NO:61 or SEQ ID NO:

62.

91. The polypeptide of claim 90, wherein the three amino acids are proline-lysine-lysine.

92. The polypeptide of any one of claims 1-91, wherein the Neo-2 / 15 mutant 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: 1-25 and SEQ ID Nos: 75-82.

93. The polypeptide of any one of claims 1-92, wherein the Neo-2 / 15 mutant 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 less than that of Neo-2 / 15 and / or IL-2.

94. The polypeptide of any one of claims 1-92, wherein the polypeptide 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 less than that of Neo-2 / 15 and / or IL-2.

95. A polypeptide as described in claim 93 or claim 94, wherein the affinity is no less than 500-fold reduced compared to Neo-2 / 15 and / or IL-2.

96. The polypeptide of claim 93 or claim 94, wherein the affinity is no more than 1000-fold or no more than 10,000-fold reduced compared to Neo-2 / 15 and / or IL-2.

97. The polypeptide of any one of claims 1-96, wherein the affinity of the Neo-2 / 15 mutant binding to IL-2Rβγ is within 2-fold, 3-fold, 4-fold or 5-fold of the affinity of the Neo-2 / 15 mutant consisting of the sequence listed in SEQ ID NO:4 for IL-2Rβγ.

98. The polypeptide of any one of claims 1-97, wherein the Neo-2 / 15 mutant 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-terminus and / or the C-terminus of an antibody comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:73, the light chain comprising the amino acid sequence of SEQ ID NO:70, wherein the reference polypeptide comprises the Neo-2 / 15 mutant fused to the C-terminus of an antibody, wherein the reference polypeptide comprises the amino acid sequences of SEQ ID NOs:69 and 70.

99. The polypeptide of any one of claims 1-98, wherein 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 set forth in SEQ ID NO:

4.

100. A polypeptide as described in any of claims 1-99, wherein when fused to the N-terminus and / or C-terminus of an antibody comprising a heavy chain and a light chain, the EC50 of the Neo-2 / 15 mutant to stimulate STAT5 phosphorylation in Treg cells is substantially equal to or less than a reference polypeptide comprising the Neo-2 / 15 mutant fused to the C-terminus of the antibody, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:73, and the light chain comprises the amino acid sequence of SEQ ID NO:70, wherein the reference polypeptide comprises the amino acid sequences of SEQ ID NOs:69 and 70.

101. The polypeptide of any one of claims 1-100, wherein the Neo-2 / 15 mutant stimulates STAT5 phosphorylation in Treg cells with a maximum signaling value that is substantially the same as or greater than that of the Neo-2 / 15 mutant consisting of the sequence set forth in SEQ ID NO:

4.

102. A polypeptide as described in any of claims 1-101, wherein when fused to the N-terminus and / or C-terminus of an antibody comprising a heavy chain and a light chain, the maximum signaling value of the Neo-2 / 15 mutant to stimulate STAT5 phosphorylation in Treg cells is substantially equal to or greater than a reference polypeptide comprising the Neo-2 / 15 mutant fused to the C-terminus of the antibody, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 73, and the light chain comprises the amino acid sequence of SEQ ID NO: 70, wherein the reference polypeptide comprises the amino acid sequences of SEQ ID NOs: 69 and 70.

103. A polypeptide as described in any of claims 1-102, wherein the Neo-2 / 15 mutant stimulates STAT5 phosphorylation in Treg cells with a maximum signaling value, which is at least 50%, at least 75%, at least 100% of the maximum signaling caused by Neo-2 / 15 and / or IL-2 stimulation.

104. A polypeptide as described in any of claims 1-103, wherein when fused to the N-terminus and / or the 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, the Neo-2 / 15 mutant 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 greater.

105. The polypeptide of any one of claims 1-104, wherein the polypeptide consists of the Neo-2 / 15 mutant.

106. The polypeptide of any one of claims 1-104, wherein the polypeptide comprises an Fc domain.

107. The polypeptide of any one of claims 1-104 or 106, wherein the polypeptide comprises a targeting agent.

108. The polypeptide of claim 107, wherein the targeting agent binds to an antigen on the surface of a Treg cell.

109. The polypeptide of claim 108, wherein the antigen is CD25 or CD39.

110. The polypeptide of any one of claims 107-109, wherein the targeting agent is an antibody or an antigen-binding fragment thereof.

111. The polypeptide of claim 110, wherein the targeting agent is an antigen-binding fragment of an antibody.

112. The polypeptide of claim 111, wherein the antigen binding fragment is an antigen binding portion of a Fab, (Fab')2, scFv, or a single domain antibody.

113. The polypeptide of claim 112, wherein the antigen binding fragment is a scFv.

114. The polypeptide of claim 110, wherein the targeting agent is a full-length antibody.

115. A polypeptide as described in any one of claims 110-114, wherein the antibody or antigen-binding fragment thereof comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:27, 42 or 43; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:28 or 44; a CDR-H3 comprising the amino acid sequence of SEQ ID NO:29; a CDR-L1 comprising the amino acid sequence of SEQ ID NO:30 or 45; a CDR-L2 comprising the amino acid sequence of SEQ ID NO:31 or 46; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

32.

116. The polypeptide of any one of claims 110-115, wherein 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.

117. The polypeptide of any one of claims 106-116, wherein the polypeptide comprises an Fc domain substantially lacking effector function.

118. The polypeptide of claim 117, wherein the Fc domain is an IgG1 Fc domain comprising substitutions L234A and L235A.

119. The polypeptide of claim 118, wherein the Fc domain comprises the substitution G237A.

120. The polypeptide of any one of claims 117-119, wherein the Fc domain comprises the substitution P329G or P329A.

121. The polypeptide of any one of claims 106-120, wherein the polypeptide comprises a heavy chain IgG1 constant region comprising the amino acid sequence of SEQ ID NO:

71.

122. The polypeptide of any one of claims 106-120, wherein the Neo-2 / 15 mutant is linked to the N-terminus of the targeting agent or the Fc domain.

123. The polypeptide of any one of claims 106-120, wherein the Neo-2 / 15 mutant is linked to the C-terminus of the targeting agent or the Fc domain.

124. The polypeptide of claim 122 or claim 123, wherein the Neo-2 / 15 mutant is fused to a targeting agent or an Fc domain.

125. The polypeptide of any one of claims 122-124, wherein the Neo-2 / 15 mutant is linked to the C-terminus of the targeting agent or Fc domain via an amino acid linker.

126. The polypeptide of claim 125, wherein the amino acid linker comprises the sequence (GGGGS) n , where n is 1-10.

127. The polypeptide of claim 126, wherein n is 1, 2, 3 or 4.

128. The polypeptide of claim 123, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:69 and the amino acid sequence of SEQ ID NO:

70.

129. The polypeptide of claim 122, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:74 and the amino acid sequence of SEQ ID NO:

70.

130. The polypeptide of any one of claims 1-129, wherein the polypeptide does not substantially activate Teff cells, or wherein the EC50 for activation of Teff cells by the polypeptide is greater than 5 times, greater than 10 times, greater than 50 times, greater than 100 times, greater than 500 times, or greater than 1000 times higher than the EC50 for activation of Treg cells.

131. A polypeptide as described in any of claims 1-130, wherein the polypeptide does not substantially induce the proliferation of Teff cells, or wherein the EC50 of the polypeptide inducing Teff cell proliferation is greater than 5 times, greater than 10 times, greater than 50 times, greater than 100 times, greater than 500 times, or greater than 1000 times higher than the EC50 of inducing Treg cell proliferation.

132. A polypeptide as described in any of claims 1-131, wherein the maximum signal transduction of the polypeptide in non-Treg cells is less than 30% or less than 20% of the maximum signal transduction of IL-2 in the non-Treg cells when the concentration of the polypeptide in the non-Treg cells is 10nM or lower.

133. The polypeptide of claim 132, wherein the non-Treg cells are Teff cells.

134. The polypeptide of claim 132, wherein the non-Treg cells are NK cells.

135. The polypeptide of any one of claims 1-134, wherein the maximal signaling of the polypeptide in Tregs is at least 50% of the maximal signaling of IL-2 in Tregs when the concentration of the polypeptide is 10 nM or less.

136. A polypeptide as described in any of claims 1-135, wherein the polypeptide induces 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 polypeptide induces STAT5 phosphorylation in Teff cells with an EC50 of greater than 1000 pM.

137. The polypeptide of any one of claims 1-136, wherein the polypeptide does not substantially activate NK cells, or wherein the EC50 for activating NK cells of the polypeptide is greater than 5 times, greater than 10 times, greater than 50 times, greater than 100 times, greater than 500 times, or greater than 1000 times higher than the EC50 for activating Treg cells.

138. A polypeptide as described in any of claims 1-137, wherein the polypeptide does not substantially induce proliferation of NK cells, or wherein the EC50 of the polypeptide inducing NK cell proliferation is greater than 5 times, greater than 10 times, greater than 50 times, greater than 100 times, greater than 500 times, or greater than 1000 times higher than the EC50 of inducing Treg cell proliferation.

139. The polypeptide of any one of claims 1-138, wherein the polypeptide induces 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 polypeptide induces STAT5 phosphorylation in NK cells with an EC50 of greater than 1000 pM.

140. A pharmaceutical composition comprising the polypeptide of any one of claims 1-139 and a pharmaceutically acceptable carrier or diluent.

141. An isolated polynucleotide comprising a polynucleotide sequence encoding the polypeptide of any one of claims 1-139.

142. A vector comprising the polynucleotide of claim 141.

143. An isolated host cell comprising the polynucleotide of claim 141 or the vector of claim 142.

144. An isolated host cell expressing the polypeptide of any one of claims 1-139.

145. A method of producing a polypeptide, the method comprising incubating the host cell of claim 143 or claim 144 under conditions suitable for expression of the polypeptide.

146. The method of claim 145, further comprising isolating the polypeptide.

147. A method of inducing Treg cell proliferation, the method comprising contacting the Treg cell with the polypeptide of any one of claims 1-139.

148. A method of activating a Treg cell, the method comprising contacting the Treg cell with a polypeptide of any one of claims 1-139.

149. The method of claim 147 or claim 148, wherein the Treg cells are in vitro or in vivo.

150. A method for treating a disease associated with Teff cell activity and / or B cell activity, the method comprising administering a polypeptide as described in any one of claims 1-139 or a pharmaceutical composition as described in claim 140 to a subject in need thereof.

151. The method of claim 150, wherein the subject suffers from an autoimmune disease.

152. A method for treating an autoimmune disease, the method comprising administering to a subject in need thereof the polypeptide of any one of claims 1-139 or the pharmaceutical composition of claim 140.

153. The method of claim 151 or claim 152, wherein the autoimmune disease is selected from: 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 Behcet's disease; type II diabetes; thyroid autoimmune diseases, including but not limited to Hashimoto's thyroiditis or Graves' disease; central nervous system autoimmune diseases, including but not limited to multiple sclerosis, myasthenia gravis, or encephalomyelitis; (5) pemphigus, including but not limited to pemphigus vulgaris, pemphigus vegetative, pemphigus foliaceus, Senear-Usher syndrome, or pemphigus brazilianus; psoriasis; inflammatory bowel disease, including but not limited to ulcerative colitis or Crohn's disease; and celiac disease.

154. A method of treating a subject who has received a transplant of biological material, such as an organ, tissue or cell transplant, the method comprising administering to the subject a polypeptide as described in any one of claims 1-139 or a pharmaceutical composition as described in claim 140.

155. A method of treating graft-versus-host disease in a subject, the method comprising administering to a subject in need thereof the polypeptide of any one of claims 1-139 or the pharmaceutical composition of claim 140.

156. The method of any one of claims 140-155, wherein the subject is a human.

Images