SELECTIVE TREG STIMULATORS RUR20kD-IL-2 AND RELATED COMPOSITIONS

Abstract

The present disclosure provides selective Treg stimulator compositions, including RUR20kD-IL-2 and related compositions, as well as methods of using these compositions, for example, for the treatment of autoimmune diseases and / or other conditions responsive to therapies effective to provide a selective increase in the number and activation of regulatory T cells compared to effector T cells.

Description

This application is a divisional application of the application with application number 201980034052.1, and the filing date of the application with application number 201980034052.1 is May 20, 2019.

[0001] This application relates to long-acting interleukin-2 receptor (IL-2R) agonist Treg stimulator compositions that selectively increase the number and activation of regulatory T cells relative to effector T cells, and to methods of using these Treg stimulator compositions to treat autoimmune and inflammatory diseases and / or other conditions responsive to Treg-stimulatory therapy. Specifically, this application relates to the selective Treg stimulator composition RUR 20kD -IL-2 and related compositions, and methods for their preparation, their formulations, and the use of RUR 20kD -IL-2 and related compositions in methods for treating autoimmune diseases and inflammatory disorders.

[0002] The immune system is the body's primary defense against the invasion of infectious organisms. In a normally functioning immune system, an immune response does not occur against self-antigens; this is called self-tolerance. Autoimmune diseases occur when the body's own immune system attacks body tissues due to a loss of tolerance to self-antigens (Dejaco, C., et al., Immunology. 2006;117(3):289-300). In subjects with autoimmune diseases, antigen-specific cytotoxic T cells or autoantibodies damage body tissues, and the accompanying inflammation can cause dysfunction and, in some cases, death. Autoimmune diseases are a heterogeneous collection of diseases with a wide range of symptoms that affect approximately 6% of the population (Siatskas, C., et al., Curr Gene Ther. 2006;6(1):45-58). Although the clinical features of autoimmune diseases are very different, the immune-mediated mechanisms are related to the generation of an adaptive immune response against target antigens (Kuby, J., 1994: Autoimmunity. Immunology, 2nd ed., p445-467. WH Freeman and Company, New York).

[0003] Although various conventional treatments, such as corticosteroids, cyclophosphamide, azathioprine, and methotrexate, are slightly effective in some patients with autoimmune diseases, they are not consistently effective and are associated with side effects and toxicity (Jantunen, E., et al., Bone Marrow Transplant. 2000;25(4):351-6). Such conventional methods do not address the underlying pathology associated with autoreactive immunity.

[0004] In view of recent progress in the understanding of the pathophysiology of autoimmune diseases, new therapies potentially targeting cells or molecular targets have been developed and are currently being evaluated. Although the etiology of autoimmune diseases is unknown, it is believed to be caused by genetic factors, inappropriate immune regulation, and potential interactions between hormonal and environmental factors. Various mechanisms for inducing autoimmune diseases have been proposed, including sequestered antigens, molecular mimicry, aberrant expression of MHC class II molecules, cytokine imbalance, dysfunction of the idiotype network regulatory pathway, general regulatory T cell deficiency, and polyclonal B cell activation (Kuby, 1994, ibid.). Several methods for treating autoimmune diseases have been investigated, including B cell depletion, anti-cytokine therapy, and stem cell therapy. However, these methods have deficiencies in terms of efficacy, safety, and / or undesirable side effects. Conventional therapies for treating autoimmune diseases function by suppressing the overall immune system, thereby leading to a significant risk of infection and other serious side effects. Therefore, there remains a need for additional therapies to provide an improved combination of efficacy, safety, and / or tolerability for treating autoimmune diseases.

[0005] For many years, the role of IL-2 in autoimmune responses has been established as a pro-inflammatory cytokine. However, more recent studies have shown that IL-2 can play a protective role in chronic autoimmune inflammation under certain conditions. Specifically, the disrupted balance between regulatory T cells (Tregs) and effector T cells (Teffs) has been identified as a common feature of various autoimmune diseases, where this disrupted balance is thought to be influenced by homeostatic cytokines such as IL-2. Due to its pharmacokinetic profile, administration of unmodified IL-2 for autoimmune therapy requires frequent daily or every-other-day dosing, which is often accompanied by painful injection site reactions. In addition, due to discomfort and inconvenience, the need for frequent injections is often associated with poor patient compliance. Long-term repeated administration of IL-2 is also accompanied by an increased risk of unwanted pleiotropic and systemic activities of IL-2 and related risks and adverse effects. Moreover, due to the limited therapeutic window, using unmodified IL-2 to achieve immune homeostasis and maintain the desired Treg / Teff balance can prove challenging over an extended period of time, if not impossible. In addition, the narrow therapeutic margin of its autoimmune disease therapy requires administration of very low doses of IL-2, which adversely affects its potency. Although low-dose IL-2 can be used to stimulate Tregs to obtain some clinical benefits, adverse events are dose-limiting, and Treg increases are modest and short-lived. For example, administration of unmodified IL-2 for autoimmune disease therapy induces an unwanted increase in IL-5 and subsequent elevation of eosinophil levels, which can lead to inflammation. Accordingly, there remains a need for agents that can selectively modulate IL-2 signaling in a manner that promotes a disease-mitigating balance of regulatory and effector T cell activities in various autoimmune diseases.

[0006] Certain autoimmune diseases have underlying etiologies that include impaired IL-2 production and / or regulatory T cell defects, which have been implicated as immunological mechanisms prior to disease onset. There remains a need for alternative and more effective therapeutic compositions and treatment regimens to effectively alleviate autoimmune symptoms, improve quality of life, and preferably provide extended remission in various autoimmune diseases. The present disclosure addresses the limited availability and associated drawbacks of current options for treating chronic autoimmune diseases.

[0007] Overview The present disclosure is based on the discovery of the selective Treg stimulator RUR 20kD -IL-2 and related compositions. The RUR 20kD -IL-2 selective Treg stimulator compositions are mixtures of IL-2-PEG conjugates with defined heterogeneity. They are intended for low-dose subcutaneous administration to selectively restore Treg homeostasis with minimal impact on other immune cells.20kD - The IL-2 selective Treg stimulator composition is a conjugate mixture comprising recombinant human interleukin-2 (rhIL-2, and in particular the aldesleukin amino acid sequence without additional amino acid mutations or substitutions) stably covalently conjugated to a 20 kDa polyethylene glycol (PEG) moiety, wherein the mixture has defined fractions, each IL-2 moiety having a specific degree of PEGylation. The compositions of the present disclosure comprise a selected mixture of IL-2 PEG conjugates having defined fractions of predominantly di-PEGylated and tri-PEGylated IL-2, and defined lesser fractions of mono-PEGylated IL-2 and / or tetra- or higher PEGylated IL-2. Specifically, the compositions of the present disclosure provide the selective Treg stimulator RUR 20kD - IL-2 and related compositions, methods for their preparation, their formulations and uses RUR 20kD - Methods for using IL-2 and related compositions for the treatment of autoimmune diseases and inflammatory disorders. RUR 20kD - The IL-2 composition induces a durable response in immune-inflammatory diseases by activating and expanding antigen-specific T regulatory cells. Administered subcutaneously at low doses RUR 20kD - Treating autoimmune diseases with the IL-2 composition can provide a way to selectively restore Treg homeostasis with minimal impact on conventional T cell function, thereby providing an alternative and / or improved method for alleviating these conditions. The present invention relates to compositions and methods of administration and uses thereof as follows: 1. A composition comprising a PEGylated IL-2 conjugate having the following structure: Wherein: IL-2 is interleukin-2; n is independently an integer from about 3 to about 4000 each time it appears. 2. The composition of item 1 above, wherein IL-2 is aldesleukin. 3. The composition of item 2 above, wherein the composition comprises no more than about 20 mole % of the PEGylated IL-2 conjugate covered by the following formula when considered together Wherein n' is selected from 1, 4, 5, or an integer greater than 5. 4. The composition of item 3 above, wherein the composition comprises no more than about 15 mole % of the PEGylated IL-2 conjugate covered by the following formula when considered together Wherein n' is selected from 1, 4, 5, or an integer greater than 5. 5. The composition as described in item 3 above, wherein the composition comprises no more than about 10 mol% of the PEGylated IL-2 conjugate covered by the following formula when considered together wherein n' is selected from 1, 4, 5, or an integer greater than 5. 6. The composition according to any one of items 3 - 5 above, which comprises no more than about 10 mol% of the PEGylated IL-2 conjugate with n' equal to 1. 7. The composition according to any one of items 3 - 5 above, which comprises no more than about 7 mol% of the PEGylated IL-2 conjugate with n' equal to 1. 8. The composition according to any one of items 3 - 5 above, which comprises no more than about 5 mol% of the PEGylated IL-2 conjugate with n' equal to 1. 9. The composition according to any one of items 3 - 8 above, which comprises no more than about 10 mol% of the PEGylated IL-2 conjugate with n' equal to 4. 10. The composition according to any one of items 3 - 8 above, which comprises no more than about 7 mol% of the PEGylated IL-2 conjugate with n' equal to 4. 11. The composition according to any one of items 3 - 8 above, which comprises no more than about 5 mol% of the PEGylated IL-2 conjugate with n' equal to 4. 12. The composition of item 1 above comprising a mixture of PEGylated IL-2 conjugates, wherein the composition comprises approximately equimolar amounts of 13. The composition of item 2 above comprising a mixture of PEGylated IL-2 conjugates, wherein the composition comprises a PEGylated IL-2 conjugate having the following formula: wherein the molar ratio of (II) / (III) is selected from 1.4:1; 1.3:1; 1.2:1; 1.1:1; 1:1; 1:1.1; 1:1.2; 1:1.3; and 1:1.4. 14. The composition according to item 13 above, wherein the average number of branched polyethylene glycol moieties per aldesleukin is selected from 2; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.6; 2.7; 2.8; 2.9; and 3. 15. The composition according to item 13 above, wherein the average number of branched polyethylene glycol moieties per aldesleukin is about 2.5. 16. The composition according to any one of items 1 - 15 above, wherein the value of n ranges from 5 to 2000. 17. The composition according to any one of items 1 - 15 above, wherein the value of n ranges from 10 to 1000. 18. The composition according to any one of items 1 - 15 above, wherein the value of n ranges from 10 to 750. 19. The composition according to any one of items 1 - 15 above, wherein the value of n ranges from 10 to 500. 20. The composition according to any one of items 1 - 15 above, wherein the value of n ranges from 20 to 250. 21. The composition according to any one of items 1 - 15 above, wherein the average value of n is about 226. 22. The composition according to any one of items 1 - 15 above, wherein the nominal average molecular weight of each branched polyethylene glycol moiety ranges from about 250 Daltons to about 90,000 Daltons. 23. The composition according to any one of items 1 - 15 above, wherein the nominal average molecular weight of each branched polyethylene glycol moiety ranges from about 1000 Daltons to about 60,000 Daltons. 24. The composition according to any one of items 1 - 15 above, wherein the nominal average molecular weight of each branched polyethylene glycol moiety ranges from about 5,000 Daltons to about 60,000 Daltons. 25. The composition according to any one of items 1 - 15 above, wherein the nominal average molecular weight of each branched polyethylene glycol moiety ranges from about 10,000 Daltons to about 55,000 Daltons. 26. The composition according to item 1 or 2 above, on a molar basis, the composition comprises about 5 mol% or less of mono - PEGylated IL - 2 conjugate, and about 28 mol% to about 60 mol% of di - PEGylated IL - 2 conjugate, and about 24 mol% to about 65 mol% of tri - PEGylated IL - 2 conjugate, and about 12 mol% or less of higher PEGylated IL - 2 conjugate, and wherein the nominal average molecular weight of each branched polyethylene glycol moiety is about 20,000 Daltons. 27. The composition according to item 26 above, which further comprises 80% or more of the combined di - and tri - PEGylated IL - 2 conjugates. 28. The composition according to item 1 or 2 above, on a molar basis, the composition comprises about 2.5 to about 4.5 mol% of mono - PEGylated IL - 2 conjugate, and about 35 to about 50 mol% of di - PEGylated IL - 2 conjugate, and about 38 to about 46 mol% of tri - PEGylated IL - 2 conjugate, and about 3 to about 10 mol% of higher PEGylated IL - 2 conjugate, and wherein the nominal average molecular weight of each branched polyethylene glycol moiety is about 20,000 Daltons. 29. The composition of item 28 above, further comprising from about 80 to 95 mol% of the combined total of di-PEGylated and tri-PEGylated IL-2 conjugates. 30. The composition of item 1 or 2 above, on a molar basis, the composition comprising from about 2.8 to about 3.8 mol% of mono-PEGylated IL-2 conjugate, from about 44 to about 48 mol% of di-PEGylated IL-2 conjugate, from about 41 to about 44 mol% of tri-PEGylated IL-2 conjugate, and from about 7 to about 9 mol% of higher PEGylated IL-2 conjugate, and wherein the nominal average molecular weight of each branched polyethylene glycol moiety is about 20,000 daltons. 31. The composition of item 30 above, further comprising from about 87 to 90 mol% of the combined total of di-PEGylated and tri-PEGylated IL-2 conjugates. 32. The composition of item 1 or 2 above, on a molar basis, the composition comprising from about 2.8 to about 3.8 mol% of mono-PEGylated IL-2 conjugate, from about 44 to about 48 mol% of di-PEGylated IL-2 conjugate, from about 41 to about 44 mol% of tri-PEGylated IL-2 conjugate, and from about 7 to about 9 mol% of higher PEGylated IL-2 conjugate, and wherein the composition comprises a mixture of mono-PEGylated IL-2 conjugates having PEG moieties attached at one of lysines K7 or K8 or K31 or K75, and wherein the nominal average molecular weight of each branched polyethylene glycol moiety is about 20,000 daltons. 33. The composition of item 32 above, further comprising from about 87 to 90 mol% of the combined total of di-PEGylated and tri-PEGylated IL-2 conjugates. 34. The composition of item 1 or 2 above, on a molar basis, the composition comprising from about 2.8 to about 3.8 mol% of mono-PEGylated IL-2 conjugate, from about 44 to about 48 mol% of di-PEGylated IL-2 conjugate, from about 41 to about 44 mol% of tri-PEGylated IL-2 conjugate, and from about 7 to about 9 mol% of higher PEGylated IL-2 conjugate, and wherein the composition comprises a mono-PEGylated IL-2 conjugate having a PEG moiety attached at lysine K7, wherein the nominal average molecular weight of each branched polyethylene glycol moiety is about 20,000 daltons. 35. The composition of item 34 above, further comprising from about 87 to 90 mol% of the combined total of di-PEGylated and tri-PEGylated IL-2 conjugates. 36. The composition of any one of items 1 - 35 above, further comprising a pharmaceutically acceptable excipient. 37. A composition according to any one of items 1-35 above, which is in a form suitable for parenteral administration. 38. A composition according to any one of items 1-35 above, which is in a form suitable for subcutaneous administration. 39. A composition according to item 36 above, which comprises an aqueous diluent. 40. A composition according to item 39 above, which has a pH of about 5. 41. A composition according to item 39 or item 40 above, which further comprises sodium acetate, sodium chloride and sucrose. 42. A composition according to item 37 above, which comprises 1.5 mg / ml protein equivalent, 10 mM sodium acetate, 110 mM sodium chloride, 2% sucrose (w / v), pH 5.0. 43. A method of increasing the ratio of regulatory T cells to effector T cells in a subject by administering to the subject a therapeutically effective dose of a composition according to any one of items 1-42 above. 44. A method according to item 43 above, wherein the regulatory T cells are selected from Foxp3+ and CD25+ cells. 45. A method according to item 44 above, wherein the effector T cells are selected from CD4+ and CD8+ cells. 46. A method according to any one of items 43-45 above, wherein when evaluated in an in vivo mouse model, the fold increase in regulatory T cells reaches a value of at least about 2 when compared to baseline. 47. A method according to any one of items 43-45 above, wherein when evaluated in an in vivo mouse model, the fold increase in regulatory T cells reaches a value of at least about 4 when compared to baseline. 48. A method according to any one of items 43-47 above, wherein after administration, the increase in the number of regulatory T cells is maintained above the baseline level for at least 3 days. 49. A method according to item 43 above, wherein after administration, the increase in the number of regulatory T cells is maintained above the baseline level for at least 5 days. 50. A method of treating a subject having an autoimmune disease, which comprises administering to the subject a therapeutically effective amount of a composition according to any one of items 1-42 above. 51. A method according to item 50 above, wherein the administration is by subcutaneous injection. 52. A method according to any one of items 50-51 above, wherein the administration is carried out once every 2 weeks or once every 4 weeks. 53. A method according to any one of items 50-52 above, wherein the administration comprises a dose between 3-24 μg / kg once every two weeks. 54. A composition according to any one of items 1 - 42 above, for use in therapy. 55. A composition according to any one of items 1 - 42 above, for use in the treatment of an autoimmune disease. 56. Use of a composition according to any one of items 1 - 42 above for the manufacture of a medicament for the treatment of an autoimmune disease.

[0008] Brief Description of the Drawings Figure 1A and 1B are representative reverse phase HPLC chromatograms that illustrate the general composition of the RUR 20kD -IL-2 composition, the preparation of which is described in Examples 1 and 1A. Moving from left to right along the x-axis (elution time, minutes), the purified conjugate composition mainly comprises di-PEGylated and tri-PEGylated rIL-2.

[0009] Figure 2 is the amino acid sequence of aldesleukin (125-L-serine-2-133 interleukin 2, recombinant non-glycosylated interleukin-2 expressed in E. coli).

[0010] Figure 3A and 3B are graphs showing the pharmacokinetic analysis of murine Tregs in blood ( 20kD ) and spleen ( Figure 3A ) after administration of a single dose of the RUR Figure 3B -IL-2 composition in mice as described in Example 2.

[0011] Figure 4A 、 4B and 4C are graphs showing the levels of NK cells, CD4 T cells and CD8 T cells respectively in blood after administration of a single dose of the RUR 20kD -IL-2 composition in mice as described in Example 2.

[0012] Figure 5A and 5B are graphs of Treg function and activity as measured by mean fluorescence intensity (MFI) of CD25 and Foxp3 after administration of a single dose of the RUR 20kD -IL-2 composition in mice as described in Example 2.

[0013] Figures 6A - D are graphs of splenic Tregs isolated from vehicle-treated mice at 1 and 4 days in an in vitro Treg suppression assay as described in Example 3.

[0014] Figure 7It is a graph showing the relative inhibitory capacity of isolated Tregs cultured at a ratio of 1:2 with Tcon (conventional T cells) over time as described in Example 3.

[0015] Figure 8A and 8B showing the degree of ear swelling in mice treated with the RUR 20kD -IL-2 composition; this study was conducted to evaluate the ability of Tregs induced by RUR 20kD -IL-2 administration to inhibit T-cell antigen-driven inflammation in a mouse model of delayed-type hypersensitivity (DTH), as described in Example 4.

[0016] Figure 9A -C is a graph of the Treg levels (CD4, CD25, FOXP3 respectively) in the blood of cynomolgus monkeys after administration of a single dose of the RUR 20kD -IL-2 composition as described in Example 5.

[0017] Figure 10A and B is a graph showing the results of the pharmacodynamic analysis of murine Tregs after administration of the RUR 20kD -IL-2 composition or unmodified IL-2 (aldesleukin) in mice as described in Example 7.

[0018] Figure 11 is as described in detail in Example 8 when evaluated in a mouse model of systemic lupus erythematosus (SLE), the urinary protein level (g / L) of mice administered the RUR 20kD -IL-2 composition (0.3 mg / kg) over time.

[0019] Figure 12 is a graph showing the results of the pharmacodynamic analysis of CD4+FoxP3+CD25 20kD in peripheral blood samples over time (days) after administration of various doses of the RUR 亮 -IL-2 composition.

[0020] Figure 13 is a graph showing the results of the pharmacodynamic analysis of total CD4+FoxP3+CD25+Tregs (cells / μL) in peripheral blood samples over time (days) after single-dose administration of various doses of the RUR 20kD -IL-2 composition to human subjects as described in Example 10.

[0021] Figures 14A-D are graphs of single-dose administration of various doses of the RUR 20kD-Graphs of Tcon cell populations, CD4+ (Figure 14A) and CD8+ Tcon cells (Figure 14B) (expressed as a percentage of CD3 cells) in peripheral blood samples over time (days) following an -IL-2 composition. Figures 14C and 14D illustrate the amounts of various doses of RUR administered as a single dose to human subjects as described in Example 10 20kD -Graphs of the number of CD8+ T cells (cells / μL) and the number of Ki67+ CD8+ T cells (expressed as a percentage of CD8) in peripheral blood samples over time (days) following an -IL-2 composition, respectively..

[0022] Figure 15A 、 15B -Graph of CD25bright+ / FoxP3+ Tregs counted using flow cytometry. Whole blood was collected from human subjects at multiple time points before treatment and after treatment with various doses of RUR administered as a single dose as described in Example 10 20kD -IL-2. Whole blood was collected from human subjects at multiple time points before treatment and after treatment with various doses of RUR administered as a single dose as described in Example 10 Figure 15A -Illustrating the median peak effect of each dose on the number of CD25bright+ / FoxP3+ Tregs (cells / μl), while Figure 15B -Providing the absolute number of CD25bright+ / FoxP3+ Tregs over time (days) after treatment

[0023] Figure 16A 、 16B -Graphs of CD4+ and CD8+ T cells counted using flow cytometry, respectively. Whole blood was collected from human subjects at multiple time points before treatment and after treatment with various doses of RUR administered as a single dose as described in Example 10. Results are presented as the proportion (%) of each cell population and fold change calculated based on pre-treatment values 20kD -IL-2. Whole blood was collected from human subjects at multiple time points before treatment and after treatment with various doses of RUR administered as a single dose as described in Example 10. Results are presented as the proportion (%) of each cell population and fold change calculated based on pre-treatment values

[0024] Figure 17A 、 17B -Graph of the Treg to Tcon dose-response ratio ( Figure 17A ) and CD25bright+ / FoxP3+ Tregs and CD8+ T cells ( Figure 17B ) counted using flow cytometry. Whole blood was collected from human subjects at multiple time points before treatment and after treatment with various doses of RUR administered as a single dose as described in Example 10. Results are presented as the ratio of the proportion (%) of each cell population and fold change calculated based on pre-treatment values. Tcon cells are CD8+ T cells 20kD -IL-2. Whole blood was collected from human subjects at multiple time points before treatment and after treatment with various doses of RUR administered as a single dose as described in Example 10. Results are presented as the ratio of the proportion (%) of each cell population and fold change calculated based on pre-treatment values. Tcon cells are CD8+ T cells

[0025] Details -The present disclosure provides a selective Treg stimulant composition comprising RUR 20kD-IL-2 embodiments and related compositions. Generally, the chemically modified IL-2 conjugate compositions provided herein are characterized by having a specific and predominant number of branched polyethylene glycol moieties stably covalently attached to IL-2 via their amino groups. The compositions provided herein comprise a selected mixture of IL-2 PEG conjugates having defined fractions of predominantly di-PEGylated and tri-PEGylated IL-2, and defined lesser fractions of mono-PEGylated IL-2 and / or tetra- or higher PEGylated IL-2.

[0026] In one aspect, the present disclosure provides a composition comprising a PEGylated IL-2 conjugate having the following structure: Wherein: IL-2 is interleukin-2; n is independently at each occurrence an integer from about 3 to about 4000.

[0027] In a specific embodiment of the composition, IL-2 is aldesleukin. In a specific embodiment of the composition, the nominal average molecular weight of each branched polyethylene glycol moiety is about 20,000 daltons. In a further specific embodiment of the composition, the PEGylated IL-2 conjugate of the composition has a PEG moiety attached at lysine 31.

[0028] In one aspect, the present disclosure provides a composition comprising a conjugate of the following formula: Wherein IL-2 is interleukin-2, n is an integer from about 3 to about 4000, and n' is 2 and 3.

[0029] The polymeric moiety of formula (I) is also referred to as 1,3-bis(methoxypoly(ethylene glycol) MW 10,000 carbamoyl)-2-propoxy)-4-butyryl (up to and including the carbonyl group covalently attached to the amino nitrogen of the IL-2 moiety). The mixture composition according to formula (I) is generally referred to herein as RUR-IL2, which encompasses a range of PEG sizes. In addition to about 3 to about 4000, illustrative ranges of n include, for example, about 5 - 2000, or about 10 - 1000, or about 10 - 750, or about 10 - 500, or about 10 - 400, or about 10 - 300, or about 10 - 250, or about 20 - 250. In some embodiments, n averages about 226.

[0030] In another aspect, the present disclosure provides a composition of the following formula: Wherein IL-2 is interleukin-2, n is an integer from about 3 to about 4000, and n' is 1, 2, and 3.

[0031] In some embodiments, the selective Treg stimulant composition of Formula I comprises an IL-2R stably covalently linked to a branched polyethylene glycol moiety, wherein the number of branched PEG moieties per IL-2 moiety (degree of PEGylation) is the distribution of the major 2- and 3-polymers (di- and tri-PEGylated) in the mixture, and wherein the minor fractions include 1-polymers (mono-PEGylated) and 4-polymers (tetra-PEGylated). Thus, in some embodiments, the minor fractions in the composition according to Formula I will include conjugates wherein n' is 1, 4, 5 or higher, but not greater than 11.

[0032] For example, in one embodiment, the selective Treg stimulant composition is encompassed by the following structure: Wherein IL-2 is one of the amino acid residues of IL-2, and the "NH" shown in structure (Ib) is the amino group of the IL-2 residue; wherein "n" is an integer from about 3 to about 4000; and n' is 2 and 3.

[0033] In some embodiments, provided herein are selective Treg stimulant compositions and related compositions referred to as RUR 20kD -IL-2. These compositions comprise IL-2 conjugates having individual covalent PEG attachments, which have a nominal molecular weight of about 20 kD in total, as described herein. Preferably, the IL-2 moiety is aldesleukin. These compositions further comprise a selected mixture of IL-2 PEG conjugates having a defined fraction of predominantly di-PEGylated and tri-PEGylated IL-2, and a defined lesser fraction of mono-PEGylated IL-2 and / or tetra- or higher PEGylated IL-2. Specific formulations of the RUR 20kD -IL-2 compositions are described below and throughout the application. As used herein, the compositions of RUR 20kD -IL-2 of Formulation A, the compositions of RUR 20kD -IL-2 of Formulation B, the compositions of RUR 20kD -IL-2 of Formulation C, the compositions of RUR 20kD -IL-2 of Formulation D, and the compositions of RUR 20kD -IL-2 of Formulation E represent certain embodiments of the selective Treg stimulant RUR 20kD -IL-2 and related compositions, and in these embodiments, the IL-2 moiety is aldesleukin (as described herein). Optionally, these compositions comprise their pharmaceutically acceptable salts.

[0034] In one embodiment, provided herein is a composition of RUR 20kD -IL-2, wherein on a molar basis, the composition comprises about 5 mol% or less of mono-PEGylated IL-2 conjugate, and about 28 mol% to about 60 mol% of di-PEGylated IL-2 conjugate, and about 24 mol% to about 65 mol% of tri-PEGylated IL-2 conjugate, and about 12 mol% or less of higher PEGylated IL-2 conjugate, and wherein the nominal average molecular weight of each branched polyethylene glycol moiety is about 20,000 daltons. Preferably, the composition of RUR 20kD -IL-2 comprises 80 mol% or more of the combined di- and tri-PEGylated IL-2 conjugates.

[0035] In one embodiment, provided herein is a composition of RUR 20kD -IL-2, wherein on a molar basis, the composition comprises about 2.5 to about 4.5 mol% of mono-PEGylated IL-2 conjugate, and about 35 to about 50 mol% of di-PEGylated IL-2 conjugate, and about 38 to about 46 mol% of tri-PEGylated IL-2 conjugate, and about 3 to about 10 mol% of higher PEGylated IL-2 conjugate, and wherein the nominal average molecular weight of each branched polyethylene glycol moiety is about 20,000 daltons. Preferably, the composition of RUR 20kD -IL-2 comprises about 80 to 95 mol% of the combined total of di-PEGylated and tri-PEGylated IL-2 conjugates.

[0036] In one embodiment, provided herein is a composition of RUR 20kD -IL-2, wherein on a molar basis, the composition comprises about 2.8 to about 3.8 mol% of mono-PEGylated IL-2 conjugate, and about 44 to about 48 mol% of di-PEGylated IL-2 conjugate, and about 41 to about 44 mol% of tri-PEGylated IL-2 conjugate, and about 7 to about 9 mol% of higher PEGylated IL-2 conjugate, and wherein the nominal average molecular weight of each branched polyethylene glycol moiety is about 20,000 daltons. Preferably, the composition of RUR 20kD -IL-2 comprises about 87 to 90 mol% of the combined total of di-PEGylated and tri-PEGylated IL-2 conjugates.

[0037] In one embodiment, provided herein is a composition of RUR 20kD- Compositions of IL-2, wherein on a molar basis, the composition comprises from about 2.8 to about 3.8 mol% of mono-PEGylated IL-2 conjugates, from about 44 to about 48 mol% of di-PEGylated IL-2 conjugates, from about 41 to about 44 mol% of tri-PEGylated IL-2 conjugates, and from about 7 to about 9 mol% of higher PEGylated IL-2 conjugates, and wherein the composition comprises a mixture of mono-PEGylated IL-2 conjugates having a PEG moiety attached at one of lysine K7 or K8 or K31 or K75, and wherein the nominal average molecular weight of each branched polyethylene glycol moiety is about 20,000 daltons. Preferably, the RUR of Formulation D 20kD - The composition of IL-2 comprises from about 87 to 90 mol% of the combined total of di-PEGylated and tri-PEGylated IL-2 conjugates.

[0038] In one embodiment, provided herein is the RUR of Formulation E 20kD - Compositions of IL-2, wherein on a molar basis, the composition comprises from about 2.8 to about 3.8 mol% of mono-PEGylated IL-2 conjugates, from about 44 to about 48 mol% of di-PEGylated IL-2 conjugates, from about 41 to about 44 mol% of tri-PEGylated IL-2 conjugates, and from about 7 to about 9 mol% of higher PEGylated IL-2 conjugates, and wherein the composition comprises a mono-PEGylated IL-2 conjugate having a PEG moiety attached at lysine K7, wherein the nominal average molecular weight of each branched polyethylene glycol moiety is about 20,000 daltons. Preferably, the RUR of Formulation E 20kD - The composition of IL-2 comprises from about 87 to 90 mol% of the combined total of di-PEGylated and tri-PEGylated IL-2 conjugates.

[0039] As used herein, "RUR 20kD - IL-2 and related compositions" may refer to the RUR according to Formulation A 20kD - IL-2 and / or the RUR of Formulation B 20kD - IL-2 and / or the RUR of Formulation C 20kD - IL-2 and / or the RUR of Formulation D 20kD - IL-2 and / or the RUR of Formulation E 20kD - One or more compositions of any of the IL-2s and / or pharmaceutically acceptable salts of these compositions. The formulations of Example 1 and / or Example 1A are non-limiting examples of the "RUR 20kD - IL-2 and related compositions" of the present disclosure.

[0040] Further embodiments of the selective Treg stimulant compositions provided herein: The compositions provided herein may comprise conjugates in which n is equal to 2, for example, such di-PEGylated conjugates in which two branched polyethylene glycol polymers (each having the 1,3-bis(methoxypoly(ethylene glycol) 10kD carbamoyl)-2-propoxy)-4-butanoyl structure shown above) are attached at the same relative position for substantially all of the di-PEGylated IL-2 conjugates in the composition. Alternatively, the di-PEGylated conjugate may comprise a mixture of di-PEGylated conjugates, for example, such a mixture of di-PEGylated conjugates in which the attachment of the branched polyethylene glycol moieties occurs at two sites on IL-2, where the specific attachment sites are not the same for all of the di-PEGylated IL-2 conjugates comprised in the composition. Thus, such di-PEGylated compositions are homogeneous with respect to the degree of PEGylation, particularly the number of branched PEG moieties attached (e.g., the 2-mer), but heterogeneous with respect to the position at which the PEG is attached to IL-2, and in this case represent positional isomers of PEG attachment.

[0041] The composition may further comprise a single conjugate in which n is equal to 3, for example, such a tri-PEGylated conjugate in which three branched polyethylene glycol moieties are attached at the same relative position for substantially all of the IL-2 conjugates in the composition. Alternatively, the tri-PEGylated conjugate may comprise a mixture of tri-PEGylated conjugates, for example, such a mixture of tri-PEGylated conjugates in which the attachment sites of the branched polyethylene glycol moieties are at different sites on IL-2 for the conjugates comprised in the composition. Thus, such tri-PEGylated compositions are homogeneous with respect to the degree of PEGylation, particularly the number of branched PEG moieties attached, but heterogeneous with respect to the position at which the PEG is attached to IL-2, and in this case represent positional isomers of PEG attachment.

[0042] The composition may further comprise a single conjugate in which n is equal to 1, for example, such a mono-PEGylated conjugate in which one branched polyethylene glycol moiety is attached at the same relative position for substantially all of the IL-2 conjugates in the composition. Alternatively, the mono-PEGylated conjugate may comprise a mixture of mono-PEGylated conjugates, for example, such a mixture of mono-PEGylated conjugates in which the attachment sites of the branched polyethylene glycol moieties are at different sites on IL-2 for the conjugates comprised in the composition. Thus, such mono-PEGylated compositions are homogeneous with respect to the degree of PEGylation, particularly the number of branched PEG moieties attached, but heterogeneous with respect to the position at which the PEG is attached to IL-2, and in this case represent positional isomers of PEG attachment.

[0043] Certain positions of PEG attachment on the IL-2 molecule are more prevalent in the compositions described herein. For example, lysines K7 or K8 or K31 or K75 are typically sites of PEGylation. RUR 20kD Compositions of -IL-2 and related compositions can include conjugates in which lysines K7 or K8 or K31 or K75 are sites of PEGylation. RUR 20kD Compositions of -IL-2 and related compositions can include mono-PEGylated conjugates in which lysines K7 or K8 or K31 or K75 are sites of PEGylation. RUR 20kD Compositions of -IL-2 and related compositions can include mono-PEGylated conjugates in which lysine K7 is the site of PEGylation. RUR 20kD Compositions of -IL-2 and related compositions can include mono-PEGylated conjugates in which lysine K31 is the site of PEGylation.

[0044] In some embodiments, the composition contains no more than about 20 mol%, and preferably no more than about 15 mol% of the conjugates covered by formula (I) when considered together, where n' is an integer selected from 1, 4, 5, or an integer greater than 5, and where the mole percentage is based on the total PEG-IL-2 conjugate. In some embodiments, the composition contains no more than about 10 mol% of the conjugates covered by formula (I) when considered together, where n' is an integer selected from 1, 4, 5, or an integer greater than 5, and where the mole percentage is based on the total PEG-IL-2 conjugate. In some additional embodiments, the composition contains no more than about 10 mol% of monomers, and preferably no more than about 7 mol% of monomers, or no more than about 5 mol% of monomers (i.e., according to structure (I), where n equals 1). In some further embodiments, the composition contains no more than about 10 mol% of tetramers, and preferably no more than about 7 mol% of tetramers, or no more than about 5 mol% of tetramers (i.e., according to structure (I), where n equals 4). In certain additional embodiments, the composition contains no more than about 10 mol% of monomers and no more than about 10 mol% of tetramers. Alternatively, the composition contains no more than about 7 mol% of monomers and no more than about 7 mol% of tetramers, or can contain no more than about 5 mol% of monomers and no more than about 5 mol% of tetramers.

[0045] In some embodiments, with respect to the PEGylated IL-2 in the composition, the composition will generally meet one or more of the following characteristics: at least about 80% of the conjugates in the composition will comprise a mixture of di-PEGylated and tri-PEGylated conjugates, some having 2 branched polymers with the structure shown in formula (I) above attached to the IL-2 moiety, and some having 3 branched polymers with the structure shown in formula (I) above attached to the IL-2 moiety; at least about 85% of the conjugates in the composition will comprise a mixture of di-PEGylated and tri-PEGylated conjugates, some having 2 branched polymers with the structure shown in formula (I) above attached to the IL-2 moiety, and some having 3 branched polymers with the structure shown in formula (I) above attached to the IL-2 moiety; at least about 90% of the conjugates in the composition will comprise a mixture of di-PEGylated and tri-PEGylated conjugates, some having 2 branched polymers with the structure shown in formula (I) above attached to the IL-2 moiety, and some having 3 branched polymers with the structure shown in formula (I) above attached to the IL-2 moiety; and at least about 95% of the conjugates in the composition will comprise a mixture of di-PEGylated and tri-PEGylated conjugates, some having 2 branched polymers with the structure shown in formula (I) above attached to the IL-2 moiety, and some having 3 branched polymers with the structure shown in formula (I) above attached to the IL-2 moiety; no more than about 20% of the conjugates in the composition will have 1 or 4 or more branched polymers with the structure shown in formula (I) above attached to the IL-2 moiety; no more than about 15% of the conjugates in the composition will have 1 or 4 or more branched polymers with the structure shown in formula (I) above attached to the IL-2 moiety; no more than about 10% of the conjugates in the composition will have 1, 4 or more branched polymers with the structure shown in formula (I) above attached to the IL-2 moiety; and no more than about 7% of the conjugates in the composition will have 1 or 4 or more branched polymers with the structure shown in formula (I) above attached to the IL-2 moiety.

[0046] In some embodiments, the composition contains no more than about 20 mol%, and preferably no more than about 15 mol% of the compounds covered by formula (I) when considered together, where n’ is an integer selected from 1, 4, 5, or an integer greater than 5, wherein the mole percentages are based on the total PEG-IL-2 conjugate. In some embodiments, the composition contains no more than about 10 mol% of the conjugate covered by formula (I) when considered together, where n’ is an integer selected from 1, 4, 5, or an integer greater than 5, wherein the mole percentages are based on the total PEG-IL-2 conjugate. In some additional embodiments, the composition contains no more than about 10 mol% of monomers, and preferably no more than about 7 mol% of monomers, or no more than about 5 mol% of monomers (i.e., according to structure (I), where n equals 1). In some further embodiments, the composition contains no more than about 10 mol% of tetramers, and preferably no more than about 7 mol% of tetramers, or no more than about 5 mol% of tetramers (i.e., according to structure (I), where n equals 4). In certain additional embodiments, the composition comprises no more than about 10 mol% of monomers and no more than about 10 mol% of tetramers. Alternatively, the composition comprises no more than about 7 mol% of monomers and no more than about 7 mol% of tetramers, or may comprise no more than about 5 mol% of monomers and no more than about 5 mol% of tetramers.

[0047] In some further embodiments, the composition comprises approximately equimolar amounts of

[0048] For example, illustrative compositions can comprise any one or more of the following approximate ratios of di-PEGylated species to tri-PEGylated species: 1.4:1; 1.3:1; 1.2:1; 1.1:1; 1:1; 1:1.1; 1:1.2; 1:1.3; or 1:1.4. The average number of PEG moieties per IL-2 in such compositions is selected from, for example, 2; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.6; 2.7; 2.8; 2.9; and 3. In certain embodiments, the average number of PEG moieties per IL-2 is about 2.5.

[0049] For example, in some embodiments, the composition contains no more than about 20 mole percent (mol%) of the IL-2 conjugate covered by the following formula where n’ is selected from 1, 4, 5, or an integer greater than 5.

[0050] In some additional embodiments, the composition comprises no more than about 15 mole percent (mol%) of an IL-2 conjugate covered by the following formula when considered together: and wherein n' is selected from 1, 4, 5, or an integer greater than 5.

[0051] In some further embodiments, the composition comprises no more than about 10 mole percent (mol%) of an IL-2 conjugate covered by the following formula when considered together: and wherein n' is selected from 1, 4, 5, or an integer greater than 5.

[0052] In some of the foregoing additional embodiments, the composition comprises no more than about 10 mol% of an IL-2 conjugate where n' is equal to 1. In some other embodiments, the composition comprises no more than about 7 mol% of an IL-2 conjugate where n' is equal to 1.

[0053] In some further embodiments, the composition comprises no more than about 5 mol% of an IL-2 conjugate where n' is equal to 1. In some alternative embodiments, the composition comprises less than about 5 mol% of an IL-2 conjugate where n' is equal to 1.

[0054] In some further embodiments related to any one or more of the foregoing, the composition comprises no more than about 10 mol% of an IL-2 conjugate where n' is equal to 4. Alternatively, in some other embodiments, the composition comprises no more than about 7 mol% of an IL-2 conjugate where n' is equal to 4. In some further embodiments, the composition comprises no more than about 5 mol% of an IL-2 conjugate where n' is equal to 4.

[0055] Also provided herein are compositions that comprise approximately equimolar amounts of

[0056] In some additional embodiments, provided herein are compositions comprising an IL-2 conjugate of the following formula: wherein the molar ratio of diPEG / triPEG conjugate is selected from 1.4:1; 1.3:1; 1.2:1; 1.1:1; 1:1; 1:1.1; 1:1.2; 1:1.3; and 1:1.4.

[0057] In still further embodiments, the average number of branched polyethylene glycol moieties (having the structure shown above) per IL-2 residue of the composition is selected from 2; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.6; 2.7; 2.8; 2.9; and 3. In a specific embodiment, the average number of branched polyethylene glycol moieties (having the structure shown above) per IL-2 moiety is about 2.5. In some embodiments related to one or more of the foregoing, the value of n ranges from 5 - 2000. In some other embodiments, the value of n ranges from 10 - 1000. In still some additional embodiments, the value of n ranges from 10 - 750. In some embodiments, the value of n ranges from 10 - 500, or 20 - 250.

[0058] In the embodiments provided herein, the value of n can vary independently at each occurrence. In one or more embodiments described herein, the value of n in each polyethylene glycol arm of the branched polymer is substantially the same. In some further embodiments, the value of n in each polymer arm constituting the branched polymer ranges from about 170 to 285. In still further embodiments, the value of n in each polymer arm constituting the branched polymer ranges from about 204 to about 250. In one or more specific embodiments, the value of n in each polymer arm constituting the branched polymer is about 226.

[0059] In one or more embodiments related to any one or more of the aspects or embodiments provided herein, the nominal average molecular weight of each branched polyethylene glycol moiety is in the range of about 250 Daltons to about 90,000 Daltons. In some other embodiments, the nominal average molecular weight of each branched polyethylene glycol moiety is in the range of about 1000 Daltons to about 60,000 Daltons. In still further embodiments, the nominal average molecular weight of each branched polyethylene glycol moiety is in the range of about 5,000 Daltons to about 60,000 Daltons. In some other embodiments, the nominal average molecular weight of each branched polyethylene glycol moiety is in the range of about 10,000 Daltons to about 55,000 Daltons.

[0060] In still some additional embodiments, the nominal average molecular weight of each branched polyethylene glycol moiety is in the range of about 15,000 Daltons to about 25,000 Daltons. In still one or more further embodiments, the nominal average molecular weight of each branched polyethylene glycol moiety is in the range of about 18,000 Daltons to about 22,000 Daltons. In still some further embodiments, the nominal average molecular weight of each branched polyethylene glycol moiety is about 20,000 Daltons.

[0061] Additional exemplary compositions include the compositions according to the above formula, wherein the overall polymeric portion of the molecule has a nominal average molecular weight in the range of about 250 Daltons to about 90,000 Daltons. Additional suitable ranges for the polymeric portion of the molecule include nominal average molecular weights selected from the range of about 1,000 Daltons to about 60,000 Daltons, the range of about 5,000 Daltons to about 60,000 Daltons, the range of about 10,000 Daltons to about 55,000 Daltons, the range of about 15,000 Daltons to about 50,000 Daltons, and the range of about 20,000 Daltons to about 50,000 Daltons.

[0062] Additional illustrative weight-average molecular weights of the polyethylene glycol polymeric portion include about 200 Daltons, about 300 Daltons, about 400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1,000 Daltons, about 1,500 Daltons, about 2,000 Daltons, about 2,200 Daltons, about 2,500 Daltons, about 3,000 Daltons, about 4,000 Daltons, about 4,400 Daltons, about 4,500 Daltons, about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 55,000 Daltons, about 60,000 Daltons, about 65,000 Daltons, about 70,000 Daltons, and about 75,000 Daltons. In some preferred embodiments, the weight-average molecular weight of the branched polyethylene glycol polymer is about 20,000 Daltons. In some specific embodiments in which each branched PEG portion has a nominal molecular weight of about 20,000 Daltons, when characterized for the overall composition, the resulting molecular weight range of the composition is about 55 to 75 kDa.

[0063] Further embodiments of the selective Treg stimulant compositions provided herein include pharmaceutically acceptable salts thereof. As described above, the IL-2 conjugate compositions can be in the form of pharmaceutically acceptable salts. Generally, such salts are formed by reaction with a pharmaceutically acceptable acid or acid equivalent. In this regard, the term "pharmaceutically acceptable salt" generally refers to relatively non-toxic inorganic and organic acid addition salts. These salts can be prepared in situ during the preparation of the dosage form or the administration vehicle, or by reacting a long-acting interleukin-2 composition as described herein with a suitable organic or inorganic acid and isolating the salt so formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napsylate, oxalate, mesylate, glucoheptonate, lactobionate, and lauryl sulfonate, etc. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19). Thus, the described salts can be derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc.; or prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfamic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isothionic acid, etc. As used herein, the term "composition" or "compositions", includes the RUR 20kD -IL-2 embodiments and related compositions, including any and / or all pharmaceutically acceptable salts of the PEGylated IL-2 conjugate. This description applies whether or not the term "or a pharmaceutically acceptable salt thereof" is added to the description of the composition.

[0064] Method of Use Embodiment Compared to unmodified IL-2, the selective Treg stimulant compositions (including the RUR 20kD-IL-2 embodiments and related compositions) address potential pathologies associated with autoreactive immunity, as well as target-specific mechanisms that yield beneficial T cell function, and provide significant improvements compared to the administration of unmodified IL-2. To address the deficiencies in existing autoimmune disease therapies, the compositions of the present invention provide sustained exposure following administration and have a unique pharmacological profile. The compositions of the present invention selectively expand and activate endogenous Tregs in vivo, where the expansion of conventional T cells and / or natural killer cells is restricted, and thereby provide an excellent method for treating autoimmune diseases.

[0065] More specifically, it has been found that selective Treg stimulant compositions having a specific and predominant number of branched polyethylene glycol moieties stably covalently linked to IL-2 via its amino group (including the RUR provided herein) 20kD -IL-2 embodiments and related compositions) are particularly effective when administered at low doses. The compositions of the present invention effectively bind and activate the IL-2 receptor to preferentially increase the cell population and immunosuppressive function of regulatory T cells (Tregs), while having minimal stimulatory effects on T effector cells (Teffs). Sustained exposure to the compositions of the present invention (commonly referred to herein as RUR 20kD -IL-2 and related compositions, or otherwise referred to as RUR-IL-2 compositions) in rodent, non-human primate studies, and human clinical studies effectively provides the magnitude, duration, and specificity of the Treg to Teff response that cannot be achieved with equidose unmodified IL-2.

[0066] Administration of a single low escalating subcutaneous dose of the selective Treg stimulant composition RUR 20kD -IL-2 (as described in the supporting examples) does not result in dose-limiting toxicity, serious adverse events, or clinically significant abnormalities. Preliminary pharmacokinetic analysis showed that in most subjects, the composition reached its maximum concentration at approximately 4 - 6 days after dosing, where the concentration changed little for up to approximately 2 weeks after dosing, after which the concentration decreased, with a half-life of approximately 8 - 9 days. Preliminary pharmacodynamic evaluation revealed that administration of the selective long-acting IL-2 receptor agonist Treg stimulant composition resulted in a dose-dependent increase in circulating CD4+FoxP3+CD25 亮 Tregs, i.e., the absolute number of circulating CD4+FoxP3+CD25 亮 Tregs continued to increase, where the levels did not return to baseline until approximately 20 to 25 days after administration. Compared to before dosing, CD4+FoxP3+CD25 亮The number of Tregs increases on average several-fold (where the magnitude depends on the dose). The overall CD4+FoxP3+CD25+ Treg population also increases, although the magnitude of the change is less than that observed for CD4+FoxP3+CD25bright Tregs. For the lowest dose, there was no change in the number of Tregs in the treated subjects compared to placebo subjects. The major effect on Tregs was seen since no change in the percentage or number of T cell populations (CD4+, CD8+) was observed with the RUR 20kD -IL-2 compositions. Thus, the compositions and methods of the present invention are surprisingly effective in increasing the inhibitory capacity of Tregs in in vivo / in vitro bioassays (even when compared to alternative chemically modified IL-2 compounds) and also in human studies (as will be described), along with other features in the following sections.

[0067] The selective Treg stimulant compositions provided herein (including the RUR 20kD -IL-2 embodiments and related compositions) can be used (among other things) to treat autoimmune diseases and disorders. Exemplary autoimmune diseases that can be treated by administering RUR-IL-2 or RUR 20kD -IL-2 compositions include systemic conditions such as systemic lupus erythematosus (SLE), ulcerative colitis, Crohn's disease, rheumatoid arthritis, atopic dermatitis, systemic sclerosis, ankylosing spondylitis, graft-versus-host disease, and polymyositis; or organ-specific autoimmune diseases including type 1 diabetes, Addison's disease, Hashimoto's thyroiditis, Graves' disease, Sjogren's syndrome, vitiligo, pernicious anemia, glomerulonephritis, myasthenia gravis, Goodpasture's syndrome, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, peanut allergy, and pulmonary fibrosis.

[0068] In some embodiments, the condition being treated is systemic lupus erythematosus (SLE). Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease that primarily affects middle-aged women. Features of SLE include, for example, skin rashes, joint pain, recurrent pleurisy, and kidney disease. The progressive homeostatic imbalance of Tregs relative to Tcons is common to many autoimmune diseases, including SLE. Overall, the therapeutic hypothesis linking Treg homeostasis to the pathology of SLE, the activity of low-dose IL-2 in SLE patients, and the superior Treg-inducing properties of the RUR 20kD -IL-2 and related compositions relative to IL-2, support the use of RUR-IL-2 or RUR 20kD- The use of IL-2 and related compositions in the treatment of SLE and other autoimmune diseases and conditions is well supported. In one or more further embodiments, provided herein is a method of treating a condition by administering an RUR-IL-2 or RUR 20kD - IL-2-related composition, wherein the condition is selected from, for example, allergies, GVHD, Crohn's disease, ulcerative colitis, rheumatoid arthritis, type 1 diabetes, multiple sclerosis, and psoriasis.

[0069] In still some further embodiments, when administered to a subject in a therapeutically effective dose, the RUR-IL-2 or RUR 20kD - IL-2-related composition effectively preferentially expands and activates regulatory T cells compared to conventional T cells and natural killer cells.

[0070] In another aspect, provided herein is a method of increasing the ratio of regulatory T cells to effector T cells in a subject by administering to the subject a therapeutically effective dose of an RUR-IL-2 or RUR 20kD - IL-2-related composition as described herein.

[0071] In some embodiments related to the foregoing methods, the regulatory T cells are selected from Foxp3+ and CD25+ cells. In one or more embodiments related to the previous embodiments or methods, the effector T cells are selected from CD4+ and CD8+ cells.

[0072] In some further embodiments related to the above methods or related embodiments, when evaluated in an in vivo mouse model, the fold increase in regulatory T cells reaches a value of at least about 2, or at least about 4, or even at least about 6 when compared to baseline.

[0073] In some embodiments of the method, after administration, the increase in the number of regulatory T cells is maintained above the baseline level for at least 3 days. In some additional embodiments, after administration, the increase in the number of regulatory T cells is maintained above the baseline level for at least 5 days. Preferably, the increase in the number of regulatory T cells is maintained above the baseline level for at least 7 days.

[0074] In yet another further aspect, provided herein is a method of treating a subject having an autoimmune disease, which comprises administering to the subject a therapeutically effective amount of a selective Treg stimulant composition, including an RUR-IL-2 or RUR 20kD - IL-2-related composition embodiment as described above or elsewhere herein.

[0075] In yet a further aspect, the present disclosure provides a method of treating a subject having an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of a composition selected from: RUR 20kD -IL-2 formulation A, RUR 20kD -IL-2 formulation B, RUR 20kD -IL-2 formulation C, RUR 20kD -IL-2 formulation D, and RUR 20kD -IL-2 formulation E.

[0076] In yet a further aspect, the present disclosure provides a method of treating a subject having an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of a composition selected from: RUR 20kD -IL-2 formulation A, RUR 20kD -IL-2 formulation B, and RUR 20kD -IL-2 formulation C.

[0077] In yet a further aspect, the present disclosure provides a method of treating a subject having an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of RUR 20kD -a composition of IL-2 formulation A.

[0078] In yet a further aspect, the present disclosure provides a method of treating a subject having an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of RUR 20kD -a composition of IL-2 formulation B.

[0079] In yet a further aspect, the present disclosure provides a method of treating a subject having an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of RUR 20kD -a composition of IL-2 formulation C.

[0080] In yet a further aspect, the present disclosure provides the use of a composition selected from: RUR 20kD -IL-2 formulation A, RUR 20kD -IL-2 formulation B, RUR 20kD -IL-2 formulation C, RUR 20kD -IL-2 formulation D, and RUR 20kD -IL-2 formulation E, in therapy.

[0081] In yet a further aspect, the present disclosure provides RUR 20kD -the use of a composition of IL-2 formulation A in therapy.

[0082] In yet a further aspect, the present disclosure provides RUR 20kD -the use of a composition of IL-2 formulation B in therapy.

[0083] In yet a further aspect, the present disclosure provides the use of a composition of RUR 20kD -IL-2 formulation C in therapy.

[0084] In yet a further aspect, the present disclosure provides the use of a composition of RUR 20kD -IL-2 formulation D in therapy.

[0085] In yet a further aspect, the present disclosure provides the use of a composition of RUR 20kD -IL-2 formulation E in therapy.

[0086] In yet a further aspect, the present disclosure provides the use of a selective Treg stimulant composition selected from the following for the preparation of a medicament for treating an autoimmune disease: RUR 20kD -IL-2 formulation A, RUR 20kD -IL-2 formulation B, RUR 20kD -IL-2 formulation C, RUR 20kD -IL-2 formulation D, and RUR 20kD -IL-2 formulation E.

[0087] In a more specific embodiment, the treatment of systemic lupus erythematosus (SLE) includes subcutaneous administration of a formulation comprising a therapeutically effective amount of RUR-IL-2 or a RUR 20kD -IL-2-related composition. See, for example, the results described in Example 8, which illustrate the role of Tregs induced by RUR 20kD -IL-2 compositions in controlling the physiological immune response and disease progression in a representative animal model of SLE. As described therein, RUR 20kD -IL-2 compositions effectively inhibit biomarkers of kidney injury (one of the characteristics of patients with SLE) to levels close to those observed in normal mice.

[0088] In embodiments involving the treatment methods described herein, such embodiments are also further embodiments for use in such treatment or alternatively for the preparation of a medicament for use in such treatment. The present disclosure further provides a composition according to any one of the embodiments of the compositions (including their formulations) described herein for use in therapy. The present disclosure further provides a composition according to any one of the embodiments of the compositions (including their formulations) described herein for the treatment of autoimmune diseases.

[0089] In one aspect, the present disclosure provides a composition comprising a PEGylated IL-2 conjugate having the following structure: Wherein: IL-2 is interleukin-2; n is independently, each time it appears, an integer from about 3 to about 4000; It is used in therapy. In a specific embodiment of the composition for use in therapy, IL-2 is aldesleukin. In a specific embodiment of the composition for use in therapy, the nominal average molecular weight of each branched polyethylene glycol moiety is about 20,000 Daltons. In a further specific embodiment of the composition for use in therapy, the PEGylated IL-2 conjugate of the composition has a PEG moiety attached at lysine 31. In a specific embodiment of the composition for use in therapy, the therapy is for autoimmune diseases.

[0090] The term In describing and claiming certain features of the present disclosure, the following terms will be used according to the definitions described below, unless otherwise indicated.

[0091] As used and described herein, the term "selective" refers to an in vivo immune response that, in some respects, is characteristic of an induced immune cell or immune signaling response, but not in other respects. Specifically, "selective" with respect to Treg induction and / or activation refers to an immune response that exhibits an increase in the number of Treg cells (by flow cytometry, CD25 high and overall), and / or an increase in the activation state of Treg, as indicated by one or more activation markers, such as ICOS or Ki67 or Stat5, and / or activation refers to a downstream induced immunosuppressive response and / or an induced immunotolerance response, while lacking certain other immune responses. In this context, "selective Treg induction" refers to an immune response of Treg as described, while lacking significant and / or clinically important effector T cells and related immune activation responses. Significant and / or clinically important effector T cells and related immune activation responses include, for example, CD4-positive T effector cell and / or CD8-positive T effector cell proliferation, and / or activation markers, such as ICOS or Ki67, or other well-known effector immune responses. Other effector immune response signals may include certain pro-inflammatory cytokines, such as those referred to as "cytokine syndrome" and / or such as IL-5, INF γ 、IL-6、IFN α 、IL-17、IL-22、IL-19 elevation. Selective Treg stimulation can also be reflected in the average Treg:Tcon ratio. Preferably, the average Treg:Tcon ratio achieved in response to the RUR-IL-2 or RUR20kD-IL-2 related compositions described herein is at least 5-fold, and preferably 7-fold, and more preferably 10-fold or more.

[0092] As used herein, the term "degree of PEGylation" refers to the number of stable PEG substituents covalently linked to one or more amino groups of an individual aldesleukin polypeptide.

[0093] As used herein, the term "about" means reasonably near the stated value, such as within plus or minus 10% of the stated value. Preferably, as used herein, "about" or "approximately" means within plus or minus 5% of a given amount.

[0094] As used herein, the term "n' is 2 and 3" refers to a mixture of IL-2 conjugates, wherein the mixture comprises di-PEGylated and tri-PEGylated conjugates as described herein.

[0095] The term "regulatory T cell" or "Treg" refers to a T cell, such as a CD4+FoxP3+CD25 亮 phenotype. (See, e.g., Jeffrey A. Bluestone and Qizhi Tang, Treg cells—the next frontier of cell therapy, Science, 12 October 2018·Vol. 362 Issue 6411, p154-155.) The term "T con" or "conventional T cell" refers to a T lymphocyte that expresses an αβ T cell receptor (TCR) as well as the co-receptors CD4 or CD8 and performs well-established adaptive immune effector functions, such as T helper cell function and cytotoxic T cell effector function. For example, Tcon can refer to CD4 + CD25 - naive conventional T cell. "Effector T cell (Teff)" refers to CD4+ and CD8+ cell effector phenotypes, such as helper T cells, cytotoxic T cells, and others, as known to the person skilled in the art. "NK cell", also known as "natural killer cell", "K cell" or "killer cell", is a type of lymphocyte (white blood cell) and a component of the innate immune system. NK cells play a major role in the host rejection of tumor and virus-infected cells.

[0096] "IL-2 intermediate" refers to an IL-2 polypeptide, particularly aldesleukin. "RUR 20kD -IL-2" refers to an IL-2 PEG conjugate, wherein the IL-2 moiety is aldesleukin as described herein, and the PEG moiety is as described herein. RUR 20kD -IL-2 compositions can also be referred to generically by the chemical name (1,3-bis(methoxypoly(ethylene glycol) 10kD(carbamoyl)-2-propoxy)-4-butanamide) interleukin-2) is used to refer to, recognizing that this does not fully describe the composition. As used herein, aldesleukin refers to 125-L-serine-2-133 interleukin-2, a recombinant non-glycosylated interleukin-2 expressed in Escherichia coli. The sequence of the amino acid sequence of aldesleukin is shown in Figure 2 . Aldesleukin expressed in other host systems known to those skilled in the art is also within the meaning of the term as used herein.

[0097] As used herein, the term "IL-2" refers to the moiety having human IL-2 activity. The term "IL-2 moiety" refers to the IL-2 moiety before attachment to the branched polyethylene glycol moiety and the IL-2 moiety after covalent attachment. It should be understood that when the native IL-2 moiety is attached to a polyethylene glycol polymer, such as the branched polyethylene glycol polymer provided herein, the IL-2 moiety is slightly altered due to the presence of one or more covalent bonds associated with the attachment to the polyethylene glycol moiety. This slightly altered form of the IL-2 moiety attached to another molecule is referred to herein as the "residue" of the IL-2 moiety. In the context of the residue of IL-2, the term'residue' means the portion of the IL-2 molecule that remains after covalent attachment to a polymer, such as polyethylene glycol, at one or more covalent attachment sites, as shown in the formulas herein. Typically, the site of attachment will be one of the 11 amine groups of lysine in IL-2.

[0098] It should be understood that when unmodified IL-2 is attached to a polymer, such as polyethylene glycol, the IL-2 is slightly altered due to the presence of one or more covalent bonds associated with the attachment to the polymer. This slightly altered form of IL-2 attached to another molecule, such as a branched PEG moiety, may in some cases be referred to as the "residue" of IL-2, or may simply be referred to as "IL-2", etc., where it is understood that the IL-2 contained in such a polymer conjugate is slightly altered due to the presence of one or more covalent bonds (each covalent bond linking the branched PEG moiety to IL-2). The term "higher PEGylated IL-2 conjugate" refers to a tetra-PEG conjugate or a penta-PEG conjugate or a conjugate with up to 11 PEG moieties. Preferably, the "higher PEGylated IL-2 conjugate" refers to a tetra-PEG conjugate or a penta-PEG conjugate.

[0099] For example, a protein having an amino acid sequence corresponding to any one of SEQ ID NO:1 to 4 described in International Patent Publication No. WO 2012 / 065086 is an exemplary IL-2 protein, as is any protein or polypeptide that is substantially homologous thereto. The term substantially homologous means that a particular subject sequence, such as a mutant sequence, differs from a reference sequence by one or more substitutions, deletions or additions, the net effect of which does not result in an adverse functional difference between the reference sequence and the subject sequence. For the purposes of this disclosure, sequences having greater than 95% homology, equivalent biological activity (although not necessarily equivalent strength of biological activity) and equivalent expression characteristics are considered to be substantially homologous. For the purpose of determining homology, truncation of the mature sequence should be ignored. As used herein, the term "IL-2" includes such proteins that are deliberately modified, for example, by site-directed mutagenesis or accidentally by mutation. These terms also include analogs having 1 to 6 additional glycosylation sites, analogs having at least one additional amino acid at the carboxyl terminus of the protein (wherein the additional amino acid includes at least one glycosylation site), and analogs having an amino acid sequence including at least one glycosylation site. The term includes both native and recombinantly produced moieties. Additionally, IL-2 can be derived from human sources, animal sources, and plant sources. An exemplary IL-2 is the human recombinant IL-2 called aldesleukin (see Figure 2 ). References to the long-acting IL-2R agonists as described herein are intended to cover their pharmaceutically acceptable salt forms.

[0100] In one aspect, the RUR-IL-2 or RUR 20kD -IL-2 related compositions described herein are long-acting agents. The long-acting nature of the RUR-IL-2 or RUR 20kD -IL-2 related compositions provided herein means that such compositions have an extended circulating half-life in plasma relative to the circulating half-life of the same unmodified IL-2R agonist (e.g., aldesleukin or other suitable IL-2 sequence). For example, the comparator agonist is not modified by covalent attachment to one or more water-soluble polymer moieties, such as polyethylene glycol moieties, and is compared when administered to the same subject at a protein-equivalent dose of the IL-2R agonist and evaluated by the same pharmacokinetic analysis.

[0101] As used herein, "PEG" or "polyethylene glycol" is intended to cover any water-soluble poly(ethylene oxide). Unless otherwise indicated, "PEG polymer" or polyethylene glycol is a polyethylene glycol in which substantially all (preferably all) monomeric subunits are ethylene oxide subunits, however, the polymer can contain different end-capping moieties or functional groups, such as for conjugation. The PEG polymers used in this disclosure will comprise one of the following two structures: "-(CH 2 CH2 O) n - "or" -(CH 2 CH 2 O) n- 1 CH 2 CH 2 -," which depends on whether the terminal oxygen has been replaced, for example, during synthetic transformation. As described above, for PEG polymers, the variable (n) ranges from about 3 to 4000, and the end groups and structure of the overall PEG can vary. Preferably, PEG has the specific meaning as described in detail herein.

[0102] "Branched" with respect to the geometry or overall structure of a polymer refers to a polymer having two or more polymer "arms" or "chains" extending from a branch point or central structural feature. As an example, the illustrative PEG reagent, mPEG2-butyric acid, N-hydroxysuccinimide ester (1,3-bis(methoxypoly(ethylene glycol)carbamoyl)-2-propoxy)-4-succinimidyloxybutyrate) is a branched polyethylene glycol polymer composed of two linear PEG chains, each linear PEG chain being covalently attached via a carbamate linkage (~NHC(O)O~) to the 1- and 3-carbons of a central propyl group from which succinimidyloxybutyrate extends.

[0103] The molecular weight in the context of water-soluble polymers, such as PEG, can be expressed as the number-average (nominal average) molecular weight or the weight-average molecular weight. Unless otherwise specified, all references to molecular weight herein are to the nominal average molecular weight. Both molecular weight determinations (number-average and weight-average) can be measured using gel permeation chromatography, gel filtration chromatography, or other liquid chromatography techniques. Other methods for measuring molecular weight values can also be used, such as using end-group analysis or measuring colligative properties (e.g., freezing point depression, boiling point elevation, or osmotic pressure) to determine the number-average molecular weight or using light scattering techniques, ultracentrifugation, or viscometry to determine the weight-average molecular weight. Gel filtration chromatography is often used to determine the average molecular weight of branched polymers. PEG polymers are typically polydisperse (i.e., the number-average molecular weight and the weight-average molecular weight of the polymer are not equal), having a low polydispersity value preferably less than about 1.2, more preferably less than about 1.15, still more preferably less than about 1.10, yet still more preferably less than about 1.05, and most preferably less than about 1.03.

[0104] A "stable" linkage or bond refers to a chemical bond that is substantially stable in water, i.e., does not undergo hydrolysis to any appreciable extent over an extended period of time under physiological conditions. Examples of hydrolytically stable linkages typically include, but are not limited to, the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, amines, etc. Generally, a stable linkage is one that exhibits a hydrolysis rate of less than about 1-2% per day under physiological conditions. The hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks.

[0105] As used in this specification, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0106] "Substantially" or "essentially" means close to the whole or all, e.g., 95% or more of a given quantity, unless stated to the contrary.

[0107] Preparations and Examples It should be understood that the preparations and examples are set forth by way of illustration and not limitation, and various modifications can be made by one of ordinary skill in the art. Methods for preparing selective Treg stimulant compositions (including RUR 20kD -IL-2 embodiments and related compositions) are described herein. The reagents and starting materials are readily available or can be easily synthesized by one of ordinary skill in the art. Suitable conditions for the steps of these methods are well known, and suitable substitutions of buffers and reagents are within the skill of the art. In addition, one of ordinary skill in the art will understand that, in some cases, the steps and order for generating the compositions can be modified and are well understood by a skilled biochemist. Similarly, it should be understood that preparations can be isolated and / or purified by various well-known techniques as needed or desired.

[0108] Preparation of IL-2 Intermediates: The IL-2 moiety can be derived from non-recombinant methods and / or recombinant methods, and the present disclosure is not limited in this regard. The IL-2 moiety can be derived from human sources, animal sources, and plant sources. For example, it may be possible to isolate IL-2 from biological systems and otherwise obtain IL-2 from culture media. See, for example, the procedures described in U.S. Patent No. 4,401,756 and Pauly et al. (1984) J. Immunol. Methods 75(1):73-84.

[0109] Methods for generating and expressing recombinant polypeptides in vitro and in prokaryotic and eukaryotic host cells are well known to those of ordinary skill in the art. See, e.g., U.S. Patent No. 5,614,185. The IL-2 moiety can be expressed in bacterial [e.g., Escherichia coli, see, e.g., Fischer et al. (1995) Biotechnol. Appl. Biochem. 21(3):295-311], mammalian [see, e.g., Kronman et al. (1992) Gene 121:295-304], yeast [e.g., Pichia pastoris, see, e.g., Morel et al. (1997) Biochem. J. 328(1):121-129], and plant [see, e.g., Mor et al. (2001) Biotechnol. Bioeng. 75(3):259-266] expression systems. Although recombinant-based protein preparation methods can vary, recombinant methods generally involve constructing a nucleic acid encoding the desired polypeptide or fragment, cloning the nucleic acid into an expression vector, transforming a host cell (e.g., a plant, bacterium, yeast, transgenic animal cell, or mammalian cell such as a Chinese hamster ovary cell or baby hamster kidney cell), and expressing the nucleic acid to produce the desired polypeptide or fragment. A variety of protein purification methods can be employed to purify the compositions of the present disclosure, and such methods are known in the art and are described, for example, in Scopes, Protein Purification: Principles and Practice, 3rd ed., Springer, NY (1994). To facilitate the identification and purification of the recombinant polypeptide, a nucleic acid sequence encoding an epitope tag or other affinity binding sequence can be inserted or added in-frame with the coding sequence, thereby producing a fusion protein consisting of the desired polypeptide and a polypeptide suitable for binding.

[0110] Depending on the system used to express the protein having IL-2 activity, the IL-2 moiety can be unglycosylated or glycosylated, and either can be used. That is, the IL-2 moiety can be unglycosylated, or the IL-2 moiety can be glycosylated, and in one or more preferred embodiments, the IL-2 moiety is unglycosylated. The IL-2 moiety can also be advantageously modified to include and / or substitute one or more amino acid residues, such as, for example, lysine, cysteine, and / or arginine, to provide facile attachment of the polymer to atoms within the side chains of the amino acids. Examples of substitutions of the IL-2 moiety are described in U.S. Patent No. 5,206,344. Additionally, the IL-2 moiety can be modified to include non-naturally occurring amino acid residues. Techniques for adding amino acid residues and non-naturally occurring amino acid residues are well known to those of ordinary skill in the art.

[0111] In addition, the IL-2 moiety can advantageously be modified to include the attachment of a functional group (other than by addition of an amino acid residue containing the functional group). For example, the IL-2 moiety can be modified to include a thiol group. In addition, the IL-2 moiety can be modified to include an N-terminal alpha carbon. In addition, the IL-2 moiety can be modified to include one or more carbohydrate moieties. In addition, the IL-2 moiety can be modified to include an aldehyde group. In addition, the IL-2 moiety can be modified to include a ketone group. In some embodiments of the present disclosure, preferably the IL-2 moiety is not modified to include one or more of a thiol group, an N-terminal alpha carbon, a carbohydrate, an aldehyde group, and a ketone group.

[0112] Exemplary IL-2 moieties are described in the literature, and for example, in U.S. Patent Nos. 5,116,943, 5,153,310, 5,635,597, 7,101,965, and 7,567,215, and U.S. Patent Application Publication Nos. 2010 / 0036097 and 2004 / 0175337. Preferred IL-2 moieties have Figure 2 the amino acid sequences provided therein and represent the amino acid sequences of aldesleukin as used herein.

[0113] In some cases, the IL-2 moiety will be in a "monomeric" form, where a single expression of the corresponding peptide is organized into discrete units. In other cases, the IL-2 moiety will be in the form of a "dimer" (e.g., a dimer of recombinant IL-2), where two monomeric forms of the protein associate with each other (e.g., are bonded by disulfide bonds). For example, in the context of a dimer of recombinant human IL-2, the dimer can be in the form of two monomers associated with each other by disulfide bonds formed by the Cys125 residues of each monomer.

[0114] For any given peptide or protein moiety or composition, it may be determined whether the moiety has IL-2 activity. Various methods for determining IL-2 activity in vitro are described in the art and herein. One exemplary method is the CTTL-2 cell proliferation assay described herein. Exemplary methods are also described in Moreau et al. (1995) Mol. Immunol. 32:1047-1056). Briefly, in a non-specific binding assay, the proposed IL-2 moiety or composition is allowed to pre-incubate for 1 hour at 4°C in the presence of a cell line carrying the IL-2 receptor. Thereafter, 125 125I-labeled IL-2 is incubated in the system for 3 hours at 4°C. The data are expressed as the % inhibitory capacity of the proposed IL-2 moiety activity compared to wild-type IL-2. Other methods known in the art can also be used to evaluate IL-2 function, including electrometric methods, spectrophotometric methods, chromatographic methods, and radiometric methods.

[0115] Preparation of Selective Treg Stimulator Compositions (Including RUR 20kD -IL-2 Embodiments and Related Compositions): RUR 20kD -Exemplary selective Treg stimulator compositions of IL-2 are generally prepared by reacting purified IL-2 with a molar excess of a PEG reagent (in molar equivalents excess relative to IL-2), mPEG2(20kD)-butyric acid, N-hydroxysuccinimide ester (or any other suitable activated ester) (1,3-bis(methoxypoly(ethylene glycol) MW 10,000 carbamoyl)-2-propoxy)-4-succinimidyl butyrate) in a bicine solution at a high pH of about 9. Generally under mild conditions, such as about 20 °C to about 65 °C, or about 20 °C to about 40 °C, or at ambient temperature or room temperature, the reactants are mixed for about 30 minutes to about 5 hours, or about 30 minutes to 4 hours, or about 30 minutes to 2 hours, or about 30 minutes to 1 hour. The reaction is quenched by acidifying to a low pH via addition of a suitable acid, such as acetic acid.

[0116] The PEGylated rIL-2 reaction product is then purified by a suitable method, such as ion exchange chromatography. For example, when ion exchange chromatography is employed, RUR 20kD -IL-2 compositions bind to the resin and are then eluted with a suitable gradient, such as a sodium chloride gradient. The chromatographic product pools are then concentrated and diafiltered into a suitable formulation buffer (e.g., sodium acetate buffer with sucrose) using, for example, tangential flow filtration (TFF).

[0117] If desired, the product pools can be further separated into positional isomers by reverse phase chromatography using reverse phase high performance liquid chromatography (RP-HPLC) using a suitable column (e.g., a C18 column or a C3 column, commercially available from companies such as Amersham Biosciences or Vydac) or by ion exchange chromatography using an ion exchange column (e.g., Sepharose TM ion exchange column) from Amersham Biosciences. Either method can be used to separate polymer-activator isomers (i.e., positional isotypes) having the same molecular weight.

[0118] Selective Treg stimulator compositions (including RUR 20kD -IL-2 embodiments and related compositions) can be characterized by various analytical and biological assay techniques described herein and / or known to those skilled in the art, including analytical HPLC, SDS-Page, LCMS, and biological assays such as CTLL-2 proliferation and in vivo Treg induction.

[0119] Formulation: In yet another or multiple embodiments, provided herein are selective Treg stimulant compositions, including RUR 20kD -IL-2 embodiments and related compositions, which comprise an IL-2 conjugate composition as described herein, and a pharmaceutically acceptable excipient.

[0120] "Pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" means a component that can be included in the compositions described herein and that does not cause significant adverse toxicological effects to a subject. The compositions of the present disclosure are preferably formulated as pharmaceutical compositions for administration by any route that renders the composition bioavailable (such as parenteral administration, including intravenous, intramuscular, or subcutaneous). Such pharmaceutical compositions and methods for their preparation are well known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (D.B. Troy, ed., 21st ed., Lippincott, Williams & Wilkins, 2006)). Optionally, the compositions provided herein may further comprise pharmaceutically acceptable excipients, and exemplary excipients include, but are not limited to, those selected from the following: carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, amino acids, and combinations thereof. The amount of any individual excipient in the composition will vary depending on the activity of the excipient and the specific needs of the composition. Generally, the optimal amount of any individual excipient is determined experimentally, i.e., by preparing compositions containing different amounts of the excipient (ranging from low to high), examining stability and other parameters, and then determining the range that achieves optimal performance without significant adverse effects. Carbohydrates, such as sugars, derived sugars, such as alditols, aldonic acids, esterified sugars, and / or sugar polymers, may be present as excipients. Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides, such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides, such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, inositol, cyclodextrin, etc. The excipients may also include inorganic salts or buffers, such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and combinations thereof. The compositions may also include antimicrobial agents for preventing or arresting the growth of microorganisms. Non-limiting examples of antimicrobial agents suitable for one or more embodiments of the present disclosure include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenethyl alcohol, phenylmercuric nitrate, thimerosal, and combinations thereof. Antioxidants may likewise be present in the compositions. Antioxidants are used to prevent oxidation and thereby prevent the deterioration of the conjugates or other components of the formulation. Suitable antioxidants for one or more embodiments of the present disclosure include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof. Surfactants may be present as excipients.Exemplary surfactants include: polysorbates such as "Tween 20" and "Tween 80", and pluronics such as F68 and F88 (both of which are available from BASF, Mount Olive, New Jersey); sorbitan esters; lipids such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposome form), fatty acids and fatty esters; steroids such as cholesterol; and chelating agents such as EDTA; zinc and other such suitable cations. An acid or a base may be present in the composition as an excipient. Non-limiting examples of acids that may be used include those acids selected from: hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, but are not limited to, those bases selected from: sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and combinations thereof. One or more amino acids may be present in the compositions described herein as excipients. In this regard, exemplary amino acids include arginine, lysine, and glycine. Additional suitable pharmaceutically acceptable excipients include, for example, those described in Handbook of Pharmaceutical Excipients, 7th Edition, Rowe, R.C., ed., Pharmaceutical Press, 2012. The preferred formulation of the selective Treg stimulant composition (including the RUR 20kD -IL-2 embodiments and related compositions) is 1.5 mg / ml protein equivalent, 10 mM sodium acetate, 110 mM sodium chloride, 2% sucrose (w / v), pH 5.0. The RUR 20kD -IL-2 composition can be stored in a sterile, ready-to-use, disposable polycarbonate bottle of appropriate volume with a polypropylene cap with a silicone liner.

[0121] Administration: The dosage of the selective Treg stimulant composition (including the RUR 20kD -IL-2 embodiments and related compositions) will vary depending on a number of factors, but will optimally be a therapeutically effective dose when the composition is stored in a unit dose container (e.g., vial). Additionally, the pharmaceutical formulation may be contained in a syringe. The therapeutically effective dose can be experimentally determined by repeated administration of increasing amounts of the selective Treg stimulant composition (including the RUR 20kD -IL-2 embodiments and related compositions) to determine the amount that produces a clinically desired endpoint as described herein, such as remission of autoimmune symptoms and / or immunosuppression, and / or induction of tolerance.

[0122] The amount of the preferred dose is a low dose that effectively preferentially expands and activates regulatory T cells in a subject compared to conventional T cells and natural killer cells. Activation of regulatory T cells can be measured by many different methods. For example, given the overall role of STAT5 in IL-2-dependent T cell processes, detection of increased STAT5 in lymphocytes can be used as a key marker of Treg activation. Phenotypically, activation of Tregs can also be measured by flow cytometry by increased cell surface IL-2Rα (CD25) and / or increased intracellular expression of the protein forkhead box P3 (Foxp3), a major regulator of the Treg lineage, and / or increased expression of the protein Ki67 associated with cell proliferation. Collectively, these markers are associated with the function of Treg cells and are often dysregulated in such cells in autoimmune diseases. In this article, the preferred detection of Treg cell induction and activation is performed by flow cytometry. The functionality of Tregs can also be evaluated by in vitro inhibition assays that measure their ability to inhibit the proliferation of conventional T cells. The results of Treg mobilization and activation can also be directly measured in vivo using antigen-driven inflammation models.

[0123] The RUR provided herein 20kD -IL-2 embodiments and related compositions are generally administered via injection. Other modes of administration are also contemplated, such as pulmonary, nasal, buccal, rectal, sublingual, and transdermal. As used herein, the term "parenteral" includes subcutaneous, intravenous, intraarterial, intratumoral, intralymphatic, intraperitoneal, intracardiac, intrathecal, and intramuscular injection, as well as infusion injection. In a specific embodiment, the injection is subcutaneous. For example, administration to a patient can be achieved by injecting a composition comprising the RUR 20kD -IL-2 embodiments and related compositions and a diluent. Regarding possible diluents, the diluent can be selected from, for example, bacteriostatic water for injection, 5% aqueous glucose solution, phosphate buffered saline, Ringer's solution, lactated Ringer's solution, saline, sterile water, deionized water, and combinations thereof. One of ordinary skill in the art can determine by testing whether two given pharmacological components are compatible together in a given formulation. An exemplary composition for administration to a patient, for example a subcutaneous formulation, comprises, for example, a therapeutically effective dose of the RUR 20kD -IL-2 embodiments and related compositions, water, sodium acetate, sodium chloride, and sucrose. The liquid composition will have a pH in the range of about 4.5 - 7.5; or about 4.5 - 6.

[0124] In certain embodiments, the selective Treg stimulant composition (including the RUR provided herein 20kD-IL-2 embodiments and related compositions) are in solid form. Preferred solid forms are those as solid dry forms, such as those containing less than 5% by weight of water or preferably less than 2% by weight of water. The solid forms are generally suitable for reconstitution in aqueous diluents. Preferred solid formulations are stable for at least about 24 months when stored in a sealed container at a temperature of about 0 - 10°C.

[0125] As used herein, the terms "patient" or "subject" refer to a living organism that has or is susceptible to a condition, such as an autoimmune disease, that can be prevented or treated by administration of a composition as provided herein, and includes both humans and animals. Subjects include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), and preferably humans. In certain embodiments, the patient, preferably a human, is further characterized as having a disease, disorder, or condition, such as an autoimmune disease, that would benefit from administration of the compositions of the present disclosure.

[0126] As used herein, the terms "treatment" or "treating" refer to the management and care of a patient having a condition, for the purpose of combating or alleviating the symptoms and complications of those conditions, and administration of the compositions of the present disclosure is indicated for those conditions. Treatment includes administering the compositions of the present disclosure to a patient in need thereof to prevent the onset of symptoms or complications, alleviate the symptoms or complications, or eliminate the disease, condition, or disorder. For example, an autoimmune disorder. Preferably, treatment includes administering the compositions of the present disclosure to a patient in need thereof to result in immunosuppression and / or tolerance. The patient to be treated is an animal, and preferably a human. As used herein, administration includes the situation where the patient takes the composition and / or the situation where the patient is directed to take the composition.

[0127] The phrases "pharmaceutically effective amount" and "pharmacologically effective amount" and "therapeutically effective amount" and "physiologically effective amount" are used interchangeably herein and refer to the amount of RUR provided herein 20kD -IL-2 and related compositions required to achieve the desired level of the substance in the bloodstream or target tissue. The exact amount will depend on many factors, such as, for example, the specific condition being treated, the expected patient population, individual patient considerations, the components and physical characteristics of the therapeutic composition to be administered, etc.

[0128] A pharmaceutical composition comprising a compound of the present disclosure can be administered parenterally to a patient in need of such treatment. Parenteral administration can be effected by subcutaneous, intramuscular, or intravenous injection by means of a syringe, an optional pen-like syringe, or a mechanically driven injector. Alternatively, parenteral administration can be effected by means of an infusion pump. Embodiments of the present disclosure provide pharmaceutical compositions suitable for administration to a patient, said administration comprising administering to a patient in need thereof a therapeutically effective amount of a composition of the present disclosure and one or more pharmaceutically acceptable excipients. Such pharmaceutical compositions can be prepared by any of a variety of techniques using conventional excipients well known in the art for pharmaceutical products. (Remington’s Pharmaceutical Sciences, 21st Edition, University of the Sciences in Philadelphia, Philadelphia, PA, USA (2006)).

[0129] The dosage of the selective Treg stimulant composition (including the RUR 20kD -IL-2 and related compositions) provided herein and the dosing regimens associated with the methods and compositions will vary depending on the age, weight, and general condition of the subject, the type and status of the condition being treated, the judgment of the health care professional, and the particular selective Treg stimulant composition to be administered.

[0130] As used herein, the term "effective amount" means an amount or dose of a composition of the present disclosure that, upon administration to a patient or subject in a single dose or multiple doses, will elicit a biological or medical response or a desired therapeutic effect on a tissue, system, animal, mammal, or human being being sought by a researcher, veterinarian, physician, or other clinician. Preferably, an effective amount means an amount or dose of a composition of the present disclosure that, upon administration to a patient or subject in a single or multiple doses, will induce an increase in selective Treg cells of at least 10-fold over pre-dose levels. The dose can include a higher initial loading dose, followed by lower doses. In one or more instances, the selective Treg stimulant composition (including the RUR 20kD-A therapeutically effective amount of IL-2 and related compositions) is an amount covered by one or more of the following ranges expressed in terms of the amount of IL-2: from about 0.10 to about 700 μg / kg; from about 0.20 to about 650 μg / kg, from about 0.30 to about 600 μg / kg; from about 1.0 to about 550 μg / kg, from about 2.0 to about 500 μg / kg, from about 10 to about 450 μg / kg, from about 25 to about 400 μg / kg, from about 50 to about 350 μg / kg or from about 100 to about 300 μg / kg, including any and all combinations of the foregoing starting and ending values from each and every one of the foregoing ranges. In some embodiments, for example, for treating an autoimmune disease in a subject or a disease or disorder that may benefit from preferential expansion and activation of regulatory T cells compared to conventional T cells and natural killer cells, a selective Treg stimulant composition (including RUR provided herein) 20kD -IL-2 embodiments and related compositions) is administered at a dose, such as a dose less than or equal to 500 μg / kg. One preferred dosing regimen of the present disclosure is where RUR is administered at a dose between 3 - 24 μg / kg every two weeks 20kD -IL-2 and related compositions, and particularly those of Formulations A - E. Another preferred dosing regimen of the present disclosure is where RUR is administered at a dose between 3 - 18 μg / kg every two weeks 20kD -IL-2 and related compositions, and particularly those of Formulations A - E. Another preferred dosing regimen of the present disclosure is where RUR is administered at a dose between 3 - 12 μg / kg every two weeks 20kD -IL-2 and related compositions, and particularly those of Formulations A - E. Another preferred dosing regimen of the present disclosure is where RUR is administered at a dose between 3 - 6 μg / kg every two weeks 20kD -IL-2 and related compositions, and particularly those of Formulations A - E. Another preferred dosing regimen of the present disclosure is where RUR is administered at a dose of 3 μg / kg every two weeks 20kD -IL-2 and related compositions, and particularly those of Formulations A - E.

[0131] The compositions provided herein effectively restore the homeostatic capacity of the immune system, such as the ability to have a positive impact on diseases (such as autoimmune diseases, allergies, and transplant rejection) in which Treg dysfunction plays a role. In one embodiment, provided herein is a method of treating a subject having a disease or disorder 20kD-Methods for selectively expanding endogenous Tregs in vivo with IL-2 embodiments and related compositions. Illustrative dosing ranges include, for example, from about 100 μg / kg to about 500 μg / kg, or from about 150 μg / kg to about 450 μg / kg, or from about 175 μg / kg to about 400 μg / kg, or even from about 175 μg / kg to about 350 μg / kg. Preferred doses and dosing regimens are described in the examples provided herein. A suitable dose effectively achieves maximal expansion of Treg cells with minimal stimulation of Teff cells and NK cells; this can be monitored by collecting peripheral blood for flow cytometry analysis to identify the incidence of Treg cells, effector CD4+ and CD8+ T cells, and NK cells. Based on these numbers, the dose can be adjusted appropriately.

[0132] The dosing regimen can be adjusted to provide an optimal desired response (e.g., therapeutic effect). For intravenous (i.v.) or non-intravenous administration, local or systemic or a combination thereof, the dosing schedule generally ranges from a single bolus dose or continuous infusion to multiple administrations per day (e.g., every 4 - 6 hours), or as indicated by the attending physician and the patient's condition. Regarding the administration of selective Treg stimulant compositions (including RUR provided herein) 20kD-IL-2 embodiments and related compositions), a person of ordinary skill in the art can determine an appropriate dosing regimen. For example, during a treatment cycle, a clinician can decide to administer the composition as a single dose or a series of doses, such as over the course of several days or weeks. Based on the long-acting nature of the composition, it is preferably administered relatively infrequently (e.g., once every three weeks, once every two weeks, once every 8 - 10 days, once a week, etc.). Exemplary lengths of time associated with the course of therapy include about one week; about two weeks; about three weeks; about four weeks; about five weeks; about six weeks; about seven weeks; about eight weeks; about nine weeks; about ten weeks; about eleven weeks; about twelve weeks; about thirteen weeks; about fourteen weeks; about fifteen weeks; about sixteen weeks; about seventeen weeks; about eighteen weeks; about nineteen weeks; about twenty weeks; about twenty-one weeks; about twenty-two weeks; about twenty-three weeks; about twenty-four weeks; about seven months; about eight months; about nine months; about ten months; about eleven months; about twelve months; about thirteen months; about fourteen months; about fifteen months; about sixteen months; about seventeen months; about eighteen months; about nineteen months; about twenty months; about twenty-one months; about twenty-two months; about twenty-three months; about twenty-four months; about thirty months; about three years; about four years and about five years. Generally, the treatment methods described herein are continued as long as the clinician overseeing the patient's care deems the treatment method effective, i.e., the patient responds to the treatment, or until the relevant symptoms of the condition abate. Non-limiting parameters indicating the effectiveness of the treatment method can include one or more of the following: an increase in regulatory T cells, such as the number of CD25+ Tregs and FoxP3+ Tregs, and / or a decrease in the number of NK cells and CD4+ and CD8+ effector cells.

[0133] When administered to a subject at a therapeutically effective dose, the compositions provided herein can be used to increase the ratio of regulatory T cells (such as Foxp3+ and CD25+ cells) to effector T cells (such as CD4+ and CD8+ cells). For example, when compared to baseline and evaluated in an in vivo mouse model (e.g., as described herein), a selective Treg stimulant composition (including the RUR provided herein) 20kD -IL-2 and related compositions) can effectively result in at least a two-fold increase in regulatory T cells. In some embodiments, when compared to baseline and evaluated in an in vivo mouse model (e.g., as described herein), the method can also effectively result in at least a four-fold increase in regulatory T cells. In some cases, the increase in the number of regulatory T cells is maintained above the baseline level and persists for at least 3 days after administration, or even persists for at least 5 days after administration.

[0134] As shown in the appended examples, when administered within a suitable dose range, a selective Treg stimulant composition (including the RUR provided herein) 20kD-IL-2 embodiments and related compositions) effectively and preferentially increase the cell population and immunosuppressive function of regulatory T cells with minimal stimulation of effector T cells. In certain embodiments, the selective Treg stimulant compositions (including the RUR provided herein) 20kD -IL-2 embodiments and related compositions) enable sustained exposure to provide the magnitude, duration, and specificity of Treg responses to Teff that cannot be achieved with equivalent doses of native IL-2. Examples

[0135] It should be understood that the foregoing description and the following examples are intended to illustrate rather than limit the scope of the disclosure provided herein. Other aspects, advantages, and modifications within the scope of this disclosure will be apparent to those skilled in the art to which this disclosure pertains.

[0136] Materials and Methods Clone and express recombinant human IL-2 having an amino acid sequence identical to that of aldesleukin (des-alanyl-1, serine-125 human interleukin-2, see Figure 2 ) and use it to prepare the exemplary selective Treg stimulant composition referred to herein as RUR 20kD -IL-2. This sequence excludes amino acid #1 (alanine) from the native mature human IL-2 sequence and has a cysteine-to-serine amino acid mutation at amino acid #125. The first amino acid in this sequence is methionine for direct bacterial expression (no encoded signal peptide). After expression, the N-terminal methionine is removed by host methionine aminopeptidase. A single disulfide bond is formed between the cysteines at amino acid positions #58 and #105. The protein is not glycosylated as it is derived from Escherichia coli. In some descriptions, the conjugated IL-2 composition can be described in some aspects as (1,3-bis(methoxypoly(ethylene glycol)carbamoyl)-2-propoxy)-4-butanamide) interleukin-2), noting that this nomenclature does not fully describe the PEGylation pattern or mixture.

[0137] Polyethylene glycol reagent, mPEG2(20kD)-butyric acid, N-hydroxysuccinimide ester (1,3-bis(methoxypoly(ethylene glycol) 10kD carbamoyl)-2-propoxy)-4-succinimidyl butyrate (also referred to herein as mPEG2-ru-20KNHS) was prepared as described in Example 2 of U.S. Patent No. 7,887,789. Appearance: white to off-white granular powder; molecular weight (Mn) 18 - 22 kDa (due to polymer polydispersity). The structure of 1,3-bis(methoxypoly(ethylene glycol) 10kD carbamoyl)-2-propoxy)-4-succinimidyl butyrate is shown below.

[0138] Unless otherwise specified, the concentrations, amounts, and dosing levels of the selective Treg stimulant compositions (including the RUR 20kD -IL-2 embodiments and related compositions herein) are reported on a protein basis, which only counts the mass contributed by the protein component and not the mass contributed by the PEG moiety. By using a protein basis, even for a mixture of conjugated rIL-2 molecules with various degrees of PEGylation, the effective RUR 20kD -IL-2 composition molecular weight is also 15.3 kDa because only the rIL-2 protein is counted.

[0139] RUR 20kD -IL-2 related compositions are mixtures of PEGylated conjugate compositions consisting of rhIL-2 (aldesleukin sequence) conjugated with multiple polyethylene glycol (PEG) moieties covalently bound at lysine groups. The number of PEG moieties per rhIL-2 molecule (degree of PEGylation) is a distribution of predominantly 2 and 3 PEG moieties per molecule (di- or tri-PEGylated), with minor species containing 1 PEG (mono-PEGylated) and 4 PEGs (tetra-PEGylated) and / or higher PEGylated molecules, resulting in an average of about 2.5 PEG moieties per rhIL-2. Each PEG moiety has a nominal molecular weight of 20 kDa, and rhIL-2 has a molecular weight of 15.3 kDa, resulting in a nominal molecular weight of RUR 20kD -IL-2 of 65 kDa.

[0140] Example 1 RUR 20kD Preparation of RUR Prepare a stock solution (100 mg / mL) of mPEG2-ru-20K NHS in 2 mM HCl. A typical IL-2 PEGylation reaction was carried out as follows: Transfer 115 mL of an IL-2 (aldesleukin) stock solution (1.3 mg / mL) to a 250 mL plastic bottle, and add 15 mL of 0.5 M Bicine (N,N-bis(2-hydroxyethyl)glycine), pH 9.2 and 0.5 mL of water to the IL-2 solution. PEGylation was initiated by dropwise addition of 19.5 mL of the mPEG2-ru-20K NHS stock solution to the solution containing IL-2. The resulting reaction mixture contained 1 mg / mL IL-2, 50 mM Bicine, and 10 molar equivalents of mPEG2-ru-20K NHS (relative to the protein), and had a pH of 8.7. The reaction was allowed to proceed at ambient temperature with gentle stirring for 40 min. The reaction was terminated by adding 2.2 mL of acetic acid to lower the reaction pH to 4.1.

[0141] The resulting IL-2 conjugate product was purified by cation exchange chromatography using SP FF Sepharose. After the conjugation reaction was complete, the reaction mixture was dialyzed against 20 volumes of 10 mM sodium acetate buffer (pH 4.0). The dialyzed sample was diluted 1:4 with water and loaded onto a column packed with SP FF Sepharose resin. The buffers used for cation exchange chromatography were as follows: Buffer A: 10 mM sodium acetate (pH 4.0), and Buffer B: 10 mM sodium acetate, 1.0 M sodium chloride (pH 4.0). Before sample loading, the resin was washed with Buffer B and equilibrated with Buffer A. After loading, the resin was washed with 3 column volumes of Buffer A. Conjugated and unconjugated IL-2 were eluted using a four-step gradient consisting of: 0 to 50% Buffer B over 5 column volumes, 25% to 50% Buffer B over 1 column volume, 50% Buffer B over 1 column volume, 50% to 100% Buffer B over 1 column volume, and 100% Buffer B over 1 column volume, with a flow rate of 28 cm / h. Fractions containing IL-2 conjugates with a degree of PEGylation (dP) of 2 and 3 (i.e., dimers and trimers) were identified by SDS-PAGE and pooled.

[0142] The combined fractions containing dimers and trimers were concentrated using a stirred ultrafiltration cell (Amicon) and nitrogen. The composition of the final product was determined by RP-HPLC using the following mobile phases: A, 0.09% TFA / water, and B, 0.04% TFA / acetonitrile. An Intrada WP-RP C18 column (3 x 150 mm) was used, with a flow rate of 0.5 ml / min and a column temperature of 50 °C. The purified conjugate mixture was determined to contain approximately 4.6% (mol) of mono-PEGylated rIL-2, approximately 47.7% (mol) of di-PEGylated rIL-2, approximately 42.9% (mol) of tri-PEGylated rIL-2, and approximately 4.8% (mol) of tetra-PEGylated IL-2. See Figure 1, where the elution time is provided on the x-axis. The average degree of PEGylation of the final product mixture was determined to be 2.48 (i.e., approximately 2.5). Free IL-2 was not detected in the final product mixture. The formulation was the RUR of Formulation A 20kD - An example of a composition of -IL-2.

[0143] Example 1 - A RUR 20kD - Alternative preparation of -IL-2 and related compositions Desired RUR 20kD - The preparation of -IL-2 and related compositions consists of: fermenting and purifying the rhIL-2 protein process intermediate, conjugating rhIL-2 with the PEG reagent starting material mPEG2-ru-20K NHS, purifying the IL-2 conjugate fractions with the specified degree of PEGylation, and finally formulating the PEGylated rhIL-2 conjugate to generate the desired distribution of RUR according to the embodiments described herein 20kD - IL-2 composition.

[0144] Desired RUR 20kD - The IL-2 composition is prepared by: reacting 1,3-bis(methoxypoly(ethylene glycol) 10kD carbamoyl)-2-propoxy)-4-succinimidyl butyrate (also referred to herein as mPEG2-ru-20K NHS) with lysine residues on the interleukin 2 (IL-2) protein (aldesleukin sequence), resulting in a distribution of PEGylated IL-2 conjugates. The product mainly contains di-PEGylated and tri-PEGylated species, with smaller amounts of mono-PEGylated and / or tetra-PEGylated species.

[0145] Thaw the frozen IL-2 starting material (purified recombinant IL-2 (aldesleukin sequence) in 10 mM acetate, 5% trehalose, pH 4.5 buffer that has been stored at -70 °C) to room temperature. The PEG reactant mPEG2-ru-20K NHS (powder) is dissolved by adding it to 2 mM HCl solution at ~90 g / L at room temperature and stirring for at least 15 minutes. The solution is then loaded into a reaction vessel. The thawed IL-2 is added to the reaction vessel, appropriately diluted with water, and then 0.75 M bicine pH 9.7 buffer is added. The final IL-2 concentration in the reaction mixture is approximately 1.0 g / L, and the bicine concentration is approximately 50 mM to achieve a target pH of 8.7. Typically, in a bicine buffer solution at pH 8.5 to 9.5, the PEG:rhIL-2 mass ratio is about 10:1 to 13:1 to PEGylate the protein. The reactants are incubated at 22 °C with continuous stirring for 40 minutes, as measured from the completion of the addition of the mPEG2-ru-20K NHS solution. At the end of the incubation period, the reaction is quenched by adding 1 N acetic acid to rapidly lower the pH, and then immediately further stepwise titrated to pH 4.0 using additional 1 N acetic acid. The quenched reactant is diluted 10-fold by adding water. The diluted quenched reactant is filtered through a 0.22 μm filter to provide the crude product.

[0146] The crude product is then subjected to fast-flow cation exchange chromatography to partially separate the PEGylated reaction fractions. The fast-flow cation exchange chromatography column is equilibrated and loaded with the feed at room temperature with a residence time of ~5 minutes, followed by washing with the loading buffer for 5 CV (column volume). The PEGylated rhIL-2 binds to the resin while the free PEG is washed away. The product is then eluted using a linear gradient with 0 - 500 mM sodium chloride in a 10 mM sodium acetate pH 4.0 buffer background. Fractions of 0.15 CV each are collected, starting ~1 CV into the elution. Fraction collection is ended when the absorbance at 280 nm is <5% of the maximum peak. The concentration of the PEGylated fractions in each fraction (i.e., mono-PEGylated IL-2 (monomer), di-PEGylated IL-2 (dimer), tri-PEGylated IL-2 (trimer), tetra-PEGylated IL-2 (tetramer), etc.) is measured by absorbance at a wavelength of 280 nm. The distribution of the PEGylated fractions is measured by RP-HPLC as described herein, and the fractions containing mono-PEG, di-PEG, tri-PEG, and higher components are identified and used to determine the generation of a fraction distribution profile with the target PEGylation (such as the RUR provided herein) 20kD-Recombination of fractions necessary for the compositions described in the -IL-2 compositions and especially in Formulations A - E). Aliquots of the selected fractions of the identified compositions (e.g., di-PEG-IL-2 and tri-PEG-IL-2, and / or mono-PEG or higher PEG) are calculated in order to achieve the target profile as provided herein and then recombined as needed to obtain the RUR of the product with the desired PEGylation fraction distribution 20kD -IL-2 compositions. Alternatively, a purification protocol can be designed whereby, according to the embodiments described herein, elution and collection can provide the desired profile without recombination. The desired (and / or recombined) chromatographically purified formulation is then concentrated and diafiltered using tangential flow filtration (TFF) into 10 mM sodium acetate, 150 mM sodium chloride, 2% w / v sucrose, pH 5.0 to achieve an RUR of 1 mg / mL (based on protein) 20kD -The final target concentration of the -IL-2 composition drug substance

[0147] Analyze the recombined and / or target product and verify the composition distribution by the methods described herein (including RP-HPLC) to evaluate the profile of the PEG fractions. Illustrative product batches of Examples 1 - 4 listed in Table 1 below demonstrate the RUR of Formulas A - E according to the present specification 20kD -Preparation of the compositions of the -IL-2 compositions. Determination of properties is known to those skilled in the art and / or described in Examples 1 - B to 1 - I or elsewhere herein. Establish appropriate historical reference sample compositions and use them for comparison in subsequent preparations

[0148] Table 1. RUR by RP-HPLC and SEC-HPLC 20kD -Overview of the illustrative analysis of samples of different batches of the -IL-2 composition ND Not detected, NMT Not more than

[0149] In some embodiments, on a molar basis, the RUR 20kD -IL-2 composition product will contain less than 1% free, un-conjugated IL-2 (more preferably no detectable free IL-2), 5% or less mono-PEGylated IL-2, about 28% to about 60% di-PEGylated IL-2, about 24% to about 65% tri-PEGylated IL-2, 12% or less of higher-PEGylated IL-2 species, and 80% or more of the combined di- and tri-PEGylated IL-2 species

[0150] In some embodiments, the RUR 20kD-The IL-2 composition product will contain, for example, less than 0.5 mol% of free IL-2, about 2.5 to about 4.5 mol% of mono-PEGylated IL-2, about 35 to about 50 mol% of di-PEGylated IL-2, about 38 to about 46 mol% of tri-PEGylated IL-2, about 3 to about 10 mol% of higher PEGylated IL-2 species, and about 80 to about 95 mol% of the combined total of di-PEGylated and tri-PEGylated IL-2.

[0151] In some embodiments, on a molar basis, the RUR 20kD -The IL-2 composition product will contain, for example, 5% or less of mono-PEGylated IL-2, and 28% to about 60% of di-PEGylated IL-2, and about 24% to about 65% of tri-PEGylated IL-2, and about 12% or less of higher PEGylated IL-2 species. Preferably, the composition comprises 80% or more of the combined di- and tri-PEGylated IL-2 species.

[0152] In some embodiments, the RUR 20kD -The IL-2 composition product will contain, for example, about 2.5 to about 4.5 mol% of mono-PEGylated IL-2, and about 35 to about 50 mol% of di-PEGylated IL-2, and about 38 to about 46 mol% of tri-PEGylated IL-2, and about 3 to about 10 mol% of higher PEGylated IL-2 species. Preferably, the composition comprises about 80 to 95 mol% of the combined total of di-PEGylated and tri-PEGylated IL-2.

[0153] In some embodiments, the RUR 20kD -The IL-2 composition product will contain, for example, about 2.8 to about 3.8 mol% of mono-PEGylated IL-2, and about 44 to about 48 mol% of di-PEGylated IL-2, and about 41 to about 44 mol% of tri-PEGylated IL-2, and about 7 to about 9 mol% of higher PEGylated IL-2 species. Preferably, the composition comprises about 87 to 90 mol% of the combined total of di-PEGylated and tri-PEGylated IL-2.

[0154] In some embodiments, the RUR 20kD-The IL-2 composition product will contain, for example, from about 2.8 to about 3.8 mol% of mono-PEGylated IL-2, and from about 44 to about 48 mol% of di-PEGylated IL-2, and from about 41 to about 44 mol% of tri-PEGylated IL-2, and from about 7 to about 9 mol% of higher PEGylated IL-2 species, and wherein the composition comprises a mixture of mono-PEGylated IL-2 conjugates having a PEG moiety attached at one of lysines K7 or K8 or K31 or K75. Preferably, the composition comprises from about 87 to 90 mol% of the combined total of di-PEGylated and tri-PEGylated IL-2.

[0155] In some embodiments, the RUR 20kD -The IL-2 composition product will contain, for example, from about 2.8 to about 3.8 mol% of mono-PEGylated IL-2, and from about 44 to about 48 mol% of di-PEGylated IL-2, and from about 41 to about 44 mol% of tri-PEGylated IL-2, and from about 7 to about 9 mol% of higher PEGylated IL-2 species, and wherein the composition comprises a mono-PEGylated IL-2 conjugate having a PEG moiety attached at lysine K7. Preferably, the composition comprises from about 87 to 90 mol% of the combined total of di-PEGylated and tri-PEGylated IL-2.

[0156] Example 1-B RUR via reverse-phase high performance liquid chromatography 20kD -Purity and characterization of the IL-2 composition Using an Agilent 1200 series instrument equipped with a diode array detector (UV at 215 nm), reverse-phase high performance liquid chromatography (RP-HPLC) was used to evaluate the chromatographic purity and identity of samples of the RUR 20kD -IL-2 composition. The column used can be an ACE 3Phenyl-300 column (Mac-Mod Analytical Inc.) (or other suitable column), with an eluent flow rate of 0.6 mL / min. RP-HPLC was carried out using a gradient of a mixture of the following two mobile phases: (1) mobile phase A, a solution of 0.1% formic acid in water, and (2) mobile phase B, a solution of 0.1% formic acid in acetonitrile. The linear gradient ranges from 60% mobile phase A / 40% mobile phase B to 40% mobile phase A / 60% mobile phase B, to 20% mobile phase A / 80% mobile phase B, to 60% mobile phase A / 40% mobile phase B. The components of the diluent / formulation buffer are 10 mM sodium acetate, 200 mM sodium chloride, 2% sucrose, pH 5.0.

[0157] The frozen RUR 20kD-The IL-2 composition reference material and samples were thawed and diluted to 1.0 mg / mL with the formulation buffer. First, at least one blank control of the formulation buffer was subjected to RP-HPLC via injection to ensure no interference with the RUR 20kD -Analysis of IL-2-composition related peaks. Next, the RUR 20kD -IL-2 composition reference material or control was injected five times. Next, the RUR 20kD -IL-2 composition sample was injected. After every six sample injections and at the end of the injection sequence, the RUR 20kD -IL-2 composition reference material / control was injected.

[0158] Containing di-PEGylated (di-PEG) and tri-PEGylated (tri-PEG) RUR 20kD -The % relative standard deviation (RSD) of the retention times of the first five reference material injections of the IL-2 composition did not exceed 2.0%. All reference material RUR of the di-PEG and tri-PEG components 20kD -The % RSD area percentage of the IL-2 composition injections did not exceed 5.0%. Integrate all RUR from the reference and sample injections 20kD -IL-2 composition peaks. Specifically, for the RUR at a concentration of 1.0 mg / mL 20kD -IL-2 composition, the di-PEG and tri-PEG RUR above 0.5% of the detection limit (LOD) were respectively 20kD -IL-2 composition species and the rhIL-2 peak above 0.3% LOD were integrated. For the RUR at a concentration of 1.0 mg / mL 20kD -IL-2, the limit of quantification (LOQ) was 1.0% for the di-PEG and tri-PEG RUR 20kD -IL-2 species and 0.5% for rhIL-2. The results from the analysis are shown in Tables 2 (6 samples) and 3 (12 samples) below.

[0159] Table 2: Area percentages of the mono-PEG, di-PEG, tri-PEG, tetra-PEG, and penta-PEG fractions of six RUR 20kD -IL-2 composition replicate samples

[0160] Table 3: Area percentages of the mono-PEG, di-PEG, tri-PEG, tetra-PEG, and penta-PEG fractions of twelve RUR 20kD -IL-2 composition replicate samples

[0161] Example 1-C Purity and Characterization of RUR 20kD -IL-2 Composition Size-exclusion high performance liquid chromatography (SEC-HPLC) can also be used to determine the purity and characterize the RUR 20kD -IL-2 composition using an Agilent 1200 series instrument equipped with a diode array detector (UV at 280 nm) and a Yarra SEC-2000 column (Phenomenex), and the eluent flow rate is 0.225 mL / min. The mobile phase is 0.2 M ammonium acetate (pH 5.5), and its volume ratio with acetonitrile is 80:20. The diluent / formulation buffer contains 10 mM sodium acetate, 200 mM sodium chloride, 2% sucrose, and the pH is 5.0. The frozen RUR 20kD -IL-2 composition reference material and the analytical sample are thawed and diluted to 1.0 mg / mL with the formulation buffer. The sample is stable in solution at 5 °C for up to 5 days.

[0162] Procedurally, first at least one blank control of the formulation buffer is subjected to RP-HPLC via injection to ensure no interference with the analysis of RUR 20kD -IL-2-related peaks. Next, the RUR 20kD -IL-2 composition, the system suitability solution, is injected to ensure resolution of aggregates or higher molecular weight species from the tetra-PEG RUR 20kD -IL-2 fraction. Subsequently, the RUR 20kD -IL-2 composition reference material or control is injected five times. Next, the RUR 20kD -IL-2 composition sample is injected. After every six sample injections and at the end of the injection sequence, the RUR 20kD -IL-2 composition reference material / control is injected.

[0163] The %RSD of the retention times of the di-PEG and tri-PEG RUR 20kD -IL-2 fractions in the first five reference material injections does not exceed 2.0%. The %RSD of the area percentages of the di-PEG and tri-PEG RUR 20kD -IL-2 in all reference material injections does not exceed 5.0%. All RUR 20kD -IL-2 fraction peaks from the reference and sample injections are integrated. Specifically, for the RUR 20kD -IL-2 composition at a concentration of 1.0 mg / mL, the di-PEG and tri-PEG RUR 20kD -IL-2 fractions above the 1.0% detection limit of detection (LOD) are integrated. For the RUR at a concentration of 1.0 mg / mL20kD -IL-2, reporting only di-PEG and tri-PEG RUR above 3.0% LOQ 20kD -IL-2.

[0164] RUR 20kD Analysis of replicate samples of the -IL-2 composition is shown in Tables 4 and 5 below, which provide RUR 20kD Peak areas of the mono-PEG, di-PEG, tri-PEG, tetra-PEG, and penta-PEG fractions of the -IL-2 composition.

[0165] Table 4: RUR by SEC-HPLC 20kD Peak area % of the mono-PEG, di-PEG, tri-PEG, tetra-PEG, and penta-PEG components of -IL-2

[0166] Table 5: RUR by SEC-HPLC 20kD Peak area % of the mono-PEG, di-PEG, tri-PEG, tetra-PEG, and penta-PEG fractions of the -IL-2 composition samples

[0167] RUR by both RP-HPLC and SEC-HPLC 20kD An overview of representative analyses of different samples of the -IL-2 composition is shown in Table 1. As can be seen, RUR 20kD -IL-2 composition formulations demonstrate good inter-batch consistency with respect to the mixture of PEGylated fractions (i.e., mono-PEGylated, di-PEGylated, tri-PEGylated, tetra-PEGylated, penta-PEGylated, etc.).

[0168] Example 1-D SDS-Page SDS-PAGE was used to confirm the RUR 20kD -IL-2 composition identity. The RUR 20kD -IL-2 composition, molecular weight marker, and a sample of the appropriate RUR 20kD -IL-2 composition reference material were loaded onto a NuPAGE Bis-Tris gel and migrated through the gel. After electrophoresis, the gel was stained using GelCodeTM Blue Safe Protein Stain. Comparison of the gel migration banding pattern to the reference material and confirmation that no new bands were present in the sample confirmed the identity of the sample. The two strongest bands will correspond to the tri-PEGylated and di-PEGylated fractions. The topmost band in the lane corresponds to the more highly PEGylated variants, and the lowest band corresponds to the mono-PEGylated variant.

[0169] Example 1-E Affinity for IL-2Rαβ using surface plasmon resonance (SPR), and efficacy in U-2OS cells expressing the human IL-2Rαβα complex.

[0170] The binding affinity of the RUR 20kD -IL-2 composition was determined using a Biacore X-100 surface plasmon resonance with polarized light detection. The technique involves activating the surface of a Biacore CM5 sensor chip with a 1:1 complex of N-hydroxysuccinimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (NHS EDC) to generate reactive NHS esters. Goat anti-human Fc antibody in sodium acetate, pH 4.0 buffer was covalently attached to the surface of the chip. Residual NHS esters were quenched with 1M ethanolamine. A 1:1 mixture of IL-2-Rα-Fc (human IL-2Rα-Fc chimera; Symansis) and IL-2Rβ-Fc (human IL-2Rβ-Fc chimera; Symansis) was captured on the chip using HBS-EP buffer (1 mM HEPES, pH 7.4, 15 mM NaCl, 0.3 mM EDTA, 0.0005% v / v surfactant P20) with 0.1% BSA. The RUR 20kD -IL-2 composition was serially diluted in HBS-EP buffer with 0.1% BSA and injected onto the sensor chip. Kinetic binding affinity was measured by applying the solution for 3 minutes (kon), followed by washing for 3 minutes (koff). The ratio between koff and kon was used to calculate the kinetic binding affinity KD. Results from triplicate analyses of two batches of RUR 20kD -IL-2 composition are presented in Table 6. The binding affinities and rates for the two API batches were consistent.

[0171] Table 6: Binding affinity of RUR 20kD -IL-2 composition for IL-2Rαβ

[0172] Alternatively, a platform, cryopreserved ready-to-use cell assay format, provides a more robust and consistent cellular response than that of cultured cells. An enzyme (β-galactosidase) fragment complementation assay (by the platform of DiscoverX Corporation, CA) was used to measure drug / ligand-receptor interactions. RUR was measured in U-2OS cells expressing the human IL-2Rαβα complex. 20kD-Efficacy of the IL-2 composition. The assay is based on the use of isolated enzyme fragments that are inactive. Two enzyme fragments are fused to the intracellular domain of the IL-2Rβ or IL-2Rγ subunit, and after ligand-receptor interaction, the receptor subunits are brought into close proximity to restore enzyme activity. In the presence of added substrate, the enzyme functions and generates a luminescence signal. Receptor activation via enzyme fragment complementation is measured after incubating samples and reference with cells for ~6 hours. RUR 20kD -The IL-2 composition provides low-dose signaling through the high-affinity heterotrimeric αβγ IL-2 receptor (IL-2R).

[0173] Example 1-F RUR 20kD -PEGylation site occupancy of the IL-2 composition By peptide mapping by direct comparison of RUR 20kD -The IL-2 composition with rhIL-2 to characterize two batches of RUR 20kD -PEGylation site occupancy of the IL-2 composition. In RUR 20kD -The IL-2 digest, peptides containing lysine can be PEGylated and are reflected by their corresponding native lysine-containing peptides with lower abundance (compared to the same peptides in the reference rhIL-2 digest). Thus, the PEGylation site occupancy can be calculated based on the reduced abundance of native peptides in the analyzed RUR 20kD-IL-2 digest. Additionally, peptide mapping of alternative materials can be used to further confirm site occupancy.

[0174] Typically, the analysis can be performed as follows. In a direct peptide mapping comparison study, RUR 20kD -The IL-2 composition and the rhIL-2 reference control samples are digested simultaneously with GluC and GluC / trypsin, followed by LC-UV / MS / MS analysis to provide peptide identification and abundance. RUR 20kD -Peptide mapping comparison of the IL-2 composition and rhIL-2 is used to determine PEGylation site occupancy.

[0175] In short, in RUR 20kD -One or more common peptides without lysine are selected as one or more references in the analysis of both the IL-2 composition and rhIL-2. The relative intensity of the peptide is normalized to one or more of its references. The reduced relative abundance of the native peptide with lysine (RR) is calculated by the relative intensity of the peptide (Formula 1). The PEGylation site occupancy at lysine is the average RR from the lysine-containing peptides. Formula 1: Peptide relative intensity (Pep / Ref) = UV peak area (peptide) / UV peak area (reference peptide).

[0176] Used as RUR 20kD The material used as an alternative to RUR-IL-2 is a product generated by conjugating monodisperse 4kD PEG with the lysines of rhIL-2. To mimic the PEGylation profile of RUR-IL-2, the alternative is prepared using the same conjugation linker and the conjugation reaction is carried out under the same reaction conditions used for preparing RUR-IL-2. LCMS / MS-based GluC mapping and trypsin mapping of the alternative identify the PEGylated lysines and provide supporting information for RUR-IL-2. 20kD -IL-2 composition, the conjugation reaction is carried out under the same reaction conditions used for preparing RUR-IL-2. LCMS / MS-based GluC mapping and trypsin mapping of the alternative identify the PEGylated lysines and provide supporting information for RUR-IL-2. 20kD -IL-2. LCMS / MS-based GluC mapping and trypsin mapping of the alternative identify the PEGylated lysines and provide supporting information for RUR-IL-2. 20kD -IL-2.

[0177] RUR 20kD The GluC map of RUR-IL-2 (GMP batch) has 95% rhIL-2 sequence coverage. A direct comparison of the GluC maps of RUR-IL-2 and rhIL-2 provides the relative quantification of 4 out of 11 lysines in RUR-IL-2 (see Table 7), where lysines 7 and 8 are counted as one site in the peptide map. Peptides containing the remaining lysines in the GluC map show evidence of PEGylation with no site differences. Additional trypsin cleavage of the lysine-containing peptides in the GluC / trypsin map provides PEG occupancy at K31, K34, K42, and K47. Comparison of the GluC / trypsin mapping chromatograms of RUR-IL-2 and rhIL-2 shows a significant reduction in those peptides (see Table 7). Due to enzymatic miscleavage, no PEG occupancy at the K48 site is available (N / A) in the trypsin / GluC map. 20kD -IL-2 and rhIL-2 provides 20kD -IL-2. Four out of 11 lysines in RUR-IL-2 are relatively quantified (see Table 7), where lysines 7 and 8 are counted as one site in the peptide map. Peptides containing the remaining lysines in the GluC map show evidence of PEGylation with no site differences. Additional trypsin cleavage of the lysine-containing peptides in the GluC / trypsin map provides PEG occupancy at K31, K34, K42, and K47. Comparison of the GluC / trypsin mapping chromatograms of RUR-IL-2 and rhIL-2 shows a significant reduction in those peptides (see Table 7). Due to enzymatic miscleavage, no PEG occupancy at the K48 site is available (N / A) in the trypsin / GluC map. 20kD -IL-2 and rhIL-2 shows a significant reduction in those peptides (see Table 7). Due to enzymatic miscleavage, no PEG occupancy at the K48 site is available (N / A) in the trypsin / GluC map.

[0178] Peptide mapping of 4k PEGylated rhIL-2 alternative identifies peptides with 4k PEG-labeled lysines with high mass accuracy (<5 ppm). The combined results of direct peptide mapping of RUR-IL-2 and 4k PEGylated rhIL-2 alternative show that K7, K31, and K75 are the major PEGylation sites (see Table 8). The less major PEGylation sites of the RUR-IL-2 composition can be K8, K34, K42, K47, K53, and K63. K48 can be PEGylated while K96 is undetermined. 20kD -IL-2 and 4k PEGylated rhIL-2 alternative show that K7, K31, and K75 are the major PEGylation sites (see Table 8). 20kD -IL-2 composition can be K8, K34, K42, K47, K53, and K63. K48 can be PEGylated while K96 is undetermined.

[0179] In the second RUR 20kD- In the GMP formulation of the IL-2 composition and in the development batches, the PEGylation site occupancy is comparable (see Table 7). The combined method of GluC mapping and trypsin / GluC mapping provides information on the batches for some of the major PEGylation sites of the conjugates in the IL-2 composition. 20kD - Some of the major PEGylation sites of the conjugates in the IL-2 composition provide inter-batch information.

[0180] Table 7. RUR 20kD - PEGylation site occupancy in the IL-2 composition: GMP batches and DEMO batches DEMO batches refer to the preparations made to demonstrate the operability of the production process.

[0181] Table 8. RUR 20kD - Overview of PEGylation site occupancy in the IL-2 composition and 4k PEGylated rhIL-2 substitute

[0182] Example 1 - G RUR 20kD - Solution-phase stability of the IL-2 composition The solution of the IL-2 composition (~1 mg / mL rhIL-2 equivalent in 10 mM sodium acetate, 200 mM sodium chloride (pH 5) containing 2% (w / v) sucrose) was evaluated at 1, 3, 5, and 7-day time points at three different storage conditions (room temperature (ambient laboratory conditions), 5 °C (refrigerated), and -20 °C) by RP-HPLC as described previously. 20kD - Stability of the solution of the IL-2 composition.

[0183] For the control, freshly prepared sample solution of the IL-2 composition 20kD - The differences between the IL-2 composition samples were evaluated. The di-PEG and tri-PEG species of the IL-2 composition samples stored at room temperature, 5 °C, and -20 °C for up to 7 days showed a relative difference of up to 1% compared to the nominal -70 °C sample storage. 20kD - The relative difference (Rel. Diff.) of the smaller percentage components of IL-2 PEGylation species (mono-PEG, tetra-PEG, and penta-PEG species) was up to 8%. This indicates that the solution samples stored under these representative storage conditions are stable. 20kD - The di-PEG and tri-PEG species of the IL-2 composition samples showed a relative difference of up to 1% compared to the nominal -70 °C sample storage. 20kD - The relative difference (Rel. Diff.) of the smaller percentage components of IL-2 PEGylation species (mono-PEG, tetra-PEG, and penta-PEG species) was up to 8%. This indicates that the solution samples stored under these representative storage conditions are stable.

[0184] In vitro bioassay: In vitro methods can be used to further measure RUR 20kD-Biological effects and biological characterization of IL-2 compositions, including cell-based assays to characterize bioactivity after activation of receptors (representative of the IL-2 receptor complex): Biological assay methods

[0185] In all three assays, data from dose-response curves (response versus concentration) were evaluated using a non-linear regression model. RUR 20kD -The efficacy of IL-2 composition samples was measured by the ratio of the half-maximal effective concentration (EC 50 ) relative to a reference material.

[0186] Example 1-H CTLL-2 cell proliferation assay In the cell proliferation assay, cell growth was measured in vitro using CTLL-2 cells after incubating the samples and reference for ~26 hours, where cell proliferation was measured via a luminescence-based adenosine triphosphate assay ( Promega, WI). For example, this cell-based proliferation assay uses the CTLL-2 cell line, which exhibits a dose-dependent proliferative response to rhIL-2 protein. rhIL-2 was used as an assay control and was prepared from RUR 20kD -IL-2 compositions at different concentration ranges. The assay was performed in 96-well plates. CTLL-2 cells were starved for rhIL-2 in starvation medium and incubated overnight at 37 °C and 5% CO 2 in an incubator for 20 ± 3 hours. The starved cells were plated in 96-well plates and a RUR 20kD -dilution series of IL-2 compositions was fed to the cells and incubated for an additional 25 ± 3 hours at 37 °C and 5% CO 2 in an incubator. The cell growth induced by the RUR -IL-2 compositions in each well was measured using a detection kit from Promega 20kD -IL-2 compositions. A luminescence signal proportional to the amount of ATP present in each well was generated, which was directly proportional to the live cells present. The luminescence signal was read on a SpectraMax M5 plate reader. Dose-response curves for the RUR 20kD -IL-2 composition reference material and each sample were generated by plotting the fluorescence signal (y-axis) against concentration (x-axis). The graph was fitted to a 4-parameter logistic non-linear regression model. Parallel line analysis (PLA) software was used to evaluate the equivalence test for slope differences (parallelism), the significance of the regression, and to calculate the efficacy ratio of the samples relative to the reference material in the same plate.

[0187] Example 1-I Phosphorylated-STAT5 activation In the phosphorylated-STAT5 assay following receptor binding, downstream cellular signaling can then promote gene expression to induce cell proliferation by phosphorylating the Signal Transducer and Activator of Transcription 5 (STAT5). In CTLL-2 cells (an IL-2-dependent murine T lymphocyte cell line), the activation of phosphorylated-STAT5 in response to sample and reference treatments for ~10 minutes was measured using a phosphorylated-STAT5 / total STAT5 multiplex assay (Meso Scale Discovery, MD).

[0188] Example 2 In vivo study: Single-dose PK / PD study in mice The selective stimulation of Tregs by RUR 20kD -IL-2 composition can be demonstrated in mice. RUR 20kD -IL-2 composition was administered subcutaneously as a single dose to C57BL / 6 mice (n = 4 / group) at doses of 0.03, 0.1, and 0.3 mg / kg. After administration, blood and spleen samples were collected on days 1 - 7 and 10 after administration. More specifically, at each time point, blood and spleen samples were collected; the samples were pooled and evaluated by flow cytometry for the drug effect on lymphocyte cell populations for pharmacodynamic analysis (see, for example, Example 5), expressed as fold change relative to the vehicle control. In addition to changes in cell numbers, functional markers and markers of activity were quantified. Finally, plasma drug concentrations were also evaluated.

[0189] As Figure 3A and 3B shown, RUR 20kD -IL-2 composition administration resulted in a dose-dependent increase in CD4 + Tregs in both blood and spleen, with the cell number increase reaching a peak four days after administration. At the highest dose tested (0.3 mg / kg), a sustained effect on Treg mobilization was achieved, where Treg levels did not return to baseline until 7 - 10 days after administration. In blood, after administration of the highest dose tested, NK cells increased, while the change in CD4 T cells was moderate, and CD8 T cells decreased slightly ( Figure 4A -C). After administration of the highest dose tested (also a dose that resulted in an increase in NK cells of less than 2-fold), B cells and CD8 T cells decreased slightly. Markers of Treg function and activity ( Figure 5A and5B ) showed that at the highest dose tested, administration of the RUR 20kD -IL-2 composition resulted in an increase in Treg activation, as measured by the mean fluorescence intensity (MFI) of CD25 and Foxp3. Although the number of Tregs did not reach its maximum until four days after administration, these activation markers reached their maximum within the first two days after administration and slowly decreased according to the plasma exposure of RUR 20kD -IL-2. The percentage of rapidly proliferating Tregs, as measured by Ki67, increased rapidly two days after administration and this percentage remained maintained until day 6 and then returned to baseline levels. Additionally, the percentage of Tregs expressing the cell surface marker inducible T cell co-stimulator (ICOS) also increased, a notable finding as ICOS expression is associated with increased inhibitory activity of Tregs in an autoimmune setting. Although the increases in Ki67 and ICOS appeared somewhat delayed relative to the peak RUR 20kD -IL-2 composition concentration, in this preclinical mouse study, their return to baseline levels did coincide with a decrease in plasma concentration.

[0190] Example 3 In vitro Treg suppression assay The goal of this study was to evaluate the inhibitory function of regulatory T cells. On days 1 - 7 and 10 after subcutaneous administration, Tregs were magnetically isolated from untreated and RUR 20kD -IL-2 composition - treated C57BL / 6 mice. Tregs and Tcons were co - cultured for three days at a ratio range of 1:2 to 1:512. Cell proliferation was assessed by incorporation of 3 H - thymidine during the last 16 hours of the assay and the % of proliferating cells relative to the plate control was calculated.

[0191] Briefly, at the indicated times after dose administration from mice treated with various dose levels (0.03, 0.1, and 0.3 mg / kg) of RUR 20kDFemale C57BL / 6 mice treated with the -IL-2 composition or vehicle were sacrificed to collect spleens (n = 4 mice / treatment group / time point). Single cell isolates were prepared from each spleen, and the resulting splenocyte mixtures were pooled for each dose at each time point. The pooled samples were aliquoted to be equivalent to a fraction of a spleen for immunocyte profiling. The remaining splenocyte preparations were used to isolate regulatory T cells (Tregs). CD4+CD25+ Tregs were isolated from mouse spleens by magnetic-activated cell sorting (MACS) using the CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's recommendations. CD4+ T cells were negatively selected and then separated into CD4+CD25- T cells and CD4+CD25+ Tregs. Untreated conventional CD4+CD25- T cells (Tcons) were isolated from mouse spleens harvested from untreated animals by MACS using the Untreated CD4+ T Cell Isolation Kit (Miltenyi Biotec) following the procedure recommended by the manufacturer.

[0192] In vitro suppression assays were performed in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.5 μM β-mercaptoethanol, and 1X antibiotic / antimycotic (100 units / mL penicillin, 100 μg / mL streptomycin, and 250 ng / mL amphotericin B). In 96-well round-bottom plates, 5 x 10 4 Tcons were stimulated with beads coated with anti-CD3 and anti-CD28 (T Cell Activation / Expansion Kit, mouse, Miltenyi Biotec) at a ratio of 2 beads:per Tcon in 100 μL of medium. The inhibitory capacity of Tregs was evaluated by adding Tregs to Tcons at different ratios (Treg:Tcon ratios from 2:1 to 1:512). Each Treg:Tcon ratio was tested in triplicate. The cells were co-cultured for 72 h at 37 °C and 5% CO 2 in a humidified atmosphere; 0.5 μCi [3H]-thymidine was added to the wells 16 h before the end of the assay. After washing the cells to remove unincorporated [3H]-thymidine, thymidine uptake was measured as counts per minute (CPM) using a microplate scintillation counter (TopCount NXT, Perkin Elmer). Individual CPM values were normalized to maximum proliferation by dividing by the mean CPM recorded for the four lowest Treg:Tcon dilutions. At Concentration-response curves were plotted using four-parameter non-linear regression and 1 / y2 weighting in Prism 6.03 (GraphPad Software, San Diego, California).

[0193] As shown in Figures 6A-D, Tregs from spleens isolated from vehicle-treated mice at 1 and 4 days after the start of the study exhibited inhibitory capacity, with maximal inhibition occurring at a ratio of 1:2. However, Tregs isolated at these time points after administration of the RUR 20kD -IL-2 composition exhibited a greatly increased inhibitory capacity, as demonstrated by reduced Tcon proliferation, especially at ratios greater than 1:8. The relative inhibitory capacity of isolated Tregs cultured with Tcons at a ratio of 1:2 was also evaluated over time ( Figure 7 ). After administration of RUR 20kD -IL-2, the increased Treg inhibitory activity was maintained for four days before returning to the baseline activity exhibited by the vehicle control treatment group.

[0194] Example 4 Evaluation of RUR 20kD -IL-2 composition in a murine KLH DTH potency model To evaluate the ability of Treg induction by administration of the RUR 20kD -IL-2 composition to inhibit T-cell antigen-driven inflammation, Balb / c mice (n = 6-10 / group) were utilized in a delayed-type hypersensitivity (DTH) model. Mice were subcutaneously sensitized in their dorsal region with 100 μl of a subcutaneous injection containing 100 μg of keyhole hemocyanin (KLH) in an emulsion containing complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant at a ratio of 1:1:1, respectively. Five days later, baseline ear thickness was measured prior to subcutaneous challenge with 10 μg of KLH in the left ear, with the right ear remaining untreated. In all groups, ear thickness measurements were taken with calipers at 24, 48, 72, and 96 hours after KLH challenge. The RUR 20kD -IL-2 composition was administered at day 0, at the time of sensitization, at a subcutaneous dose range of 0.003 mg / kg to 0.3 mg / kg every three days. A positive control consisting of cyclosporine (10 mg / kg, single dose) was administered at day 0.

[0195] As Figure 8A 、 8B shown, ear swelling was induced after antigen challenge, with the mean increase in ear thickness reaching a maximum of over 14 mm at 48 hours. During the course of the study, untreated, unchallenged ears did not exhibit a change in thickness. Throughout the sensitization and challenge periods in this study, the RUR20kD -Administration of the IL-2 composition resulted in a significant dose-dependent decrease in ear swelling, as demonstrated by the reduction in inflammation relative to the vehicle control at each time point. To more quantitatively evaluate the effect after challenge, the AUC of the change in thickness was calculated for each treatment group (AUC 0-96h ). As Figure 8A and 8B shown, the minimum effective dose was 0.01 mg / kg q3d, while the maximum effect was achieved with 0.3 mg / kg q3d. Statistically significant AUC values from the vehicle group are noted with an asterisk (p < 0.05; ANOVA, Tukey’s). In summary, this data indicates that the enhanced mobilization and activation of Tregs achieved after administration can inhibit antigen-driven inflammatory mechanisms in vivo.

[0196] In rodents and cynomolgus monkeys, the activity of the RUR 20kD -IL-2 composition of Example 1 was evaluated after in vivo administration. In mice, the RUR 20kD -IL-2 composition resulted in a dose-dependent increase in Tregs, which reached a maximum four days after administration. Flow cytometry analysis of Tregs induced by the RUR 20kD -IL-2 composition in mice showed that the mean fluorescence intensity (MFI) of markers of Treg activation, such as Foxp3 and CD25, reached their maximum within the first two days of administration and that the plasma exposure of the RUR 20kD -IL-2 composition gradually decreased over time. The percentage of actively proliferating Tregs also reached its maximum within 2 days after administration and was maintained up to day 6. The expression of the Treg functional marker ICOS peaked on day 3 and then returned to baseline by day 7. Tregs isolated from the spleens of treated mice greatly increased their inhibitory capacity within the first four days after administration and then returned to baseline activity levels. When administered every three days, the RUR 20kD -IL-2 composition of Example 1 inhibited the antigen-driven inflammatory response in a delayed-type hypersensitivity (DTH) mouse model.

[0197] Example 5 Single-dose study in cynomolgus monkeys In this study, 25 μg / kg of the RUR 20kD -IL-2 composition was administered subcutaneously to cynomolgus monkeys (one female and one male). A series of blood samples were taken from each animal at multiple intervals before treatment (days -6 and -1) and after treatment for evaluation of Treg cell number and activation status by flow cytometry.

[0198] For immunophenotyping, blood samples (approx. 1.0 mL) were collected from each monkey at the following time points: before treatment (Day -6 and Day -1), and on Days 2, 3, 4, 5, 6, 7, 10, 14, and 21 after treatment. Venipuncture samples were collected into tubes containing anticoagulant K 2 EDTA. Tubes were placed on wet ice awaiting transfer. Whole blood samples were analyzed by flow cytometry using the following panels and analyzed for the following: T cell panel: CD45 / CD3 / CD4 / CD8 / ICOS T / B / NK panel: CD45 / CD3 / CD16 / CD20 pSTAT5 panel: CD3 / CD4 / CD8 / CD25 / CD127 / pSTAT5 Treg panel 1: CD3 / CD4 / CD8 / CD25 / FoxP3 / Ki67 Treg panel 2: CD3 / CD4 / CD8 / CD25 / FoxP3 / Helios A computerized system was used for the conduct of the study. For example, flow cytometry data acquisition could be performed using BDFACSCanto II / FACSDiva LEGENDPlex data analysis software, and flow cytometry data analysis could be performed using DeNovo FCS Express software.

[0199] Values from male and female were averaged and the magnitude of change relative to the d-1 value was shown, marked with a dashed line. As shown in Figure 9, the number of Treg cells increased significantly after administration, reaching its maximum level at 7 days after administration, and returning to near d-1 level by Day 14-21. As Figure 9A shown by the open triangles in 20kD almost all Treg induced by the RUR

[0200] -IL-2 composition were proliferative, as measured by Ki67. 20kD The relative activation status of Treg stimulated by administration of the RUR-IL-2 composition was further measured by the mean fluorescence intensity (MFI) of FoxP3 and CD25. CD25 MFI reached its maximum value at Day 6, and then plateaued by Day 10, and then returned to near pre-dose level by Day 21. FoxP3 MFI also reached its maximum value 6 days after administration, and then nearly returned to pre-dose level between Days 14-21. Collectively, these data suggest that the findings in mice are translatable to cynomolgus monkeys, as a similar magnitude of Treg induction in blood was seen, accompanied by increased Treg activation. However, compared to the findings in mice, the effect in cynomolgus monkeys is naturally more persistent.

[0201] Example 6 Single-dose pharmacokinetics and toxicokinetics in mice, rats, and monkeys Overview of RUR 20kD - Results of the single-dose pharmacokinetics / toxicokinetics of the RUR

[0202] Table 10: RUR 20kD - Overview of the single-dose pharmacokinetics and toxicokinetics study of the RUR

[0203] For the mouse study, the vehicle for the RUR 20kD -IL-2 composition was 10 mM sodium acetate, 200 mM sodium chloride, and 2% sucrose (pH 5). For the rat and monkey studies, the vehicle for the RUR 20kD -IL-2 composition was 50 mM sodium acetate, 200 mM sodium chloride, and 2% sucrose (pH 5).

[0204] After subcutaneous administration, the RUR 20kD -IL-2 composition was slowly absorbed in mice, rats, and monkeys, with T max max values of 0.33 - 1.0, 1.0 - 2.3, and 2.0 days, respectively (Table 11). The RUR 20kD -IL-2 plasma exposure increased more or less dose-proportionally in mice and rats. The bioavailability in rats was in the range of 29.8 - 46.0%, and the bioavailability in monkeys was 86.2%.

[0205] In rats, the RUR 20kD -IL-2 composition's volume of distribution at steady state (V ss ) appeared to increase with dose and ranged between 25.1 (0.01 mg / kg) and 52.6 mL / kg (1.0 mg / kg) (Table 12). Overall, in rats and monkeys, V ss was 1 - 2 times and 2 - 4 times larger than the species-specific plasma volume, respectively, indicating that the RUR 20kD -IL-2 mainly remained in the vascular space.

[0206] Plasma clearance (CL) was very low (0.560 - 1.14 mL / hr / kg in rats and 0.245 mL / hr / kg in monkeys) (Table). After intravenous or subcutaneous administration, the RUR 20kD- The concentration of the RUR-IL-2 composition appeared to exhibit mono-exponential decay, with a half-life of 1.85 - 2.24 days in mice, 1.25 - 2.44 days in rats, and 10.4 - 12.9 days in monkeys (Tables 11 and 12 and Figure 10A and 10B ). Renal excretion of RUR 20kD -IL-2 was expected to be low due to its average molecular weight of 63 kDa, which is close to the molecular weight cut-off of the glomerular filtration membrane.

[0207] Table 11: Mean ± SE plasma pharmacokinetic / toxicokinetic parameters after administration of a single subcutaneous dose of RUR 20kD -IL-2 composition to C57BL / 6 mice, Sprague-Dawley rats, or cynomolgus monkeys AUCinf: Area under the plasma concentration-time curve from time zero to infinite time; AUClast: Area under the plasma concentration-time curve from time zero to the last measurable concentration; Cmax: Observed maximum plasma concentration; MRTinf: Mean residence time; Tmax: Time at which the maximum plasma concentration was observed 1. PK parameters are based on the mean of three rats at each time point.

[0208] 2. Mean of male and female monkeys.

[0209] Table 12: Mean ± SE plasma pharmacokinetic parameters after administration of a single intravenous dose of RUR 20kD -IL-2 composition to Sprague-Dawley rats or cynomolgus monkeys ND: Not determined; AUCinf: Area under the plasma concentration-time curve from time zero to infinite time; AUClast: Area under the plasma concentration-time curve from time zero to the last measurable concentration; CL: Clearance; MRTinf: Mean residence time; Vss: Apparent volume of distribution at steady state. * Mean of male and female monkeys.

[0210] Example 7 Comparative study in mice A study was conducted that was substantially similar to the study described in Example 2, in which single subcutaneous doses of 0.03, 0.1, and 0.3 mg / kg of RUR were administered to C57BL / 6 mice 20kD- An IL-2 composition or unmodified IL-2 (aldesleukin) administered at doses of 0.03 mg / kg (qddx5), 0.1 mg / kg (qdx5), and 1 mg / kg (qdx5). After administration, blood and spleen samples were collected and analyzed by flow cytometry for pharmacodynamic analysis of the drug effect on lymphocyte cell populations, expressed as fold change relative to the vehicle control. The results are shown in Figure 10A and 10B (RUR 20kD - The IL-2 composition was labeled "RUR-IL-2" and aldesleukin was labeled "IL-2").

[0211] Example 8 RUR 20kD - Study of the efficacy of the RUR -IL-2 composition in a murine model of systemic lupus erythematosus (SLE) 20kD This study was conducted to determine the efficacy of the RUR 20kD-The stock solution of the IL-2 composition was used as the test article (1.58 mg / mL) provided in a vehicle (clear liquid; 10 mM sodium acetate / 200 mM sodium chloride / 2% (w / v) sucrose) and prepared in sterile water for injection (SWFI), USP; pH 5.0 ± 0.1). On the day of dosing, an appropriate amount of the test article was removed and diluted with the vehicle to achieve the desired dosing concentrations (0.03 mg / kg dose and 0.3 mg / kg dose); the dose volume was 5 mL / kg. The animals used in this study were MRL / MpJ-Faslpr mice and MRL / MpJ untreated female mice, 6 - 8 weeks of age. The animals were randomly assigned to treatment groups. The treatment groups are described in Table 13 below. Based on the body weight before starting the experiment and the protein content level in urine, 45 MRL / MpJ-Faslpr mice were randomly divided into 3 groups (15 mice in each group for groups 2 - 4). The animals in groups 2 - 4 received the vehicle or the test article delivered subcutaneously as described in Table 6. Group 1 - MRL / MpJ mice received the vehicle as a negative control. Three (3) days after the first dose administration at week 8, 3 mice from groups 2 - 4 were humanely sacrificed, and blood samples were collected and processed. Body weight was measured twice a week starting from the beginning of the study and throughout the course. Skin lesion photographs were taken when first observed and then at one-week intervals. Urine was obtained on the day before dosing (at baseline) and then collected weekly thereafter. The protein level in urine was measured using a Siemens Clinitek Status analyzer. On the day of sacrifice (3 days after the last dose at the end of week 20), all mice were anesthetized by intraperitoneal injection of chloral hydrate (50 mg / kg). Blood samples were collected and centrifuged at 10,000 r / min for 10 min to obtain serum samples. The serum was stored at -80 °C until clinical biochemical tests. The anti-dsDNA level of the serum samples (100 μl) was analyzed by ELISA (mouse anti-dsDNA IgG-specific ELISA kit, Alpha Diagnostic International, catalog number 5120), and the BUN concentration of the serum was tested using a Hitachi 7020 automated biochemistry analyzer. For lymphocyte analysis, blood samples were collected in EDTA-K tubes and tested for CD3 / CD4 / CD8 / Treg / NK / B cell % by flow cytometry. The results are shown in Figure 11 as shown therein. RUR was administered at a dose of 0.3 mg / kg 20kD-The IL-2 composition effectively inhibits the biomarker of kidney damage (i.e., the protein level in urine) to a level close to that observed in normal mice. This study further elucidated the role of RUR-IL-2-induced Tregs in the control of physiological immune responses and disease progression in a representative animal model of SLE.

[0212] Table 13 - Treatment groups

[0213] a: Vehicle for the test article.

[0214] Example 9 RUR 20kD -Study of the IL-2 composition in an antigen-dependent T cell-mediated delayed-type hypersensitivity (DTH) model This study simulated how in a food allergy model (where a high degree of allergic reaction is established), the in vivo Treg stimulation and expansion by the RUR 20kD -IL-2 composition can downregulate the T cell-mediated delayed-type hypersensitivity (DTH) response in an antigen-dependent manner.

[0215] To develop the DTH model, Balb / c mice were sensitized by subcutaneous administration of the model antigen keyhole hemocyanin (KLH) emulsified in complete and incomplete Freund's adjuvant. The RUR 20kD -IL-2 composition (0.003, 0.01, 0.3, 0.1, or 0.3 mg / kg, q3d) or cyclosporin A (10 mg / kg, qd) was administered subcutaneously starting on day 0 and continued until day 8. On day 5, an intradermal challenge with the administered KLH was performed, and ear swelling was measured for four days. Immunohistochemistry (IHC) was performed on the inflamed ears to quantify the percentage of FoxP3+ Treg cells after the KLH challenge. After an additional 3 - 4 weeks, the specificity of the response was evaluated without treatment by re-challenge with KLH or by sensitization and challenge with an unrelated antigen ovalbumin (OVA). To understand the effect of the Tregs expanded by the RUR 20kD -IL-2 composition on food allergens, Balb / C mice were sensitized intraperitoneally twice with OVA emulsified in alum twice within one week. Ten days after the second sensitization, the mice were orally challenged with OVA eight times every other day. The RUR 20kD- Subcutaneous administration of the -IL-2 composition (0.1 mg / kg, q3dx3) or cyclosporin A (10 mg / kg, qd) was initiated on day 0 and continued until day 8. The severity of the allergic response was evaluated by clinical scoring within 30 - 45 min after the 8th challenge. In addition, serum mast cell protease 1 (MCPT 1) and OVA-specific IgE titers were quantified. The % Treg in peripheral blood and spleen was determined by flow cytometry.

[0216] In this murine model of DTH, RUR 20kD - Administration of the -IL-2 composition inhibited the inflammatory response to KLH rechallenge in a dose-dependent manner. IHC analysis of the inflamed ear showed marked infiltration of FoxP3+ Treg cells. The inhibitory effect on inflammation was persistent and antigen-specific, as exemplified by rechallenge with the same antigen 3 - 4 weeks later and rechallenge with an unrelated antigen after sensitization (without further administration of RUR 20kD -IL-2 composition). Finally, it was found that RUR 20kD - Administration of the -IL-2 composition was effective in reducing the symptoms of hypersensitivity reactions caused by repeated administration of the model food allergen OVA. The decrease in the clinical score of the allergic reaction was associated with a significant decrease in the levels of MCPT1 and anti-OVA-specific IgE titers and a significant increase in Treg. RUR 20kD - The -IL-2 composition demonstrated antigen-specific and persistent Treg expansion and a therapeutic response in this KLH-hypersensitive murine model. In addition, it was found that RUR 20kD - The -IL-2 composition was effective in the food allergy model. This data supports the use of RUR 20kD -IL-2 composition for antigen-specific inflammation, as is the case in autoimmune and / or inflammatory diseases.

[0217] The preclinical evidence provided herein supports the concept that the IL-2 conjugate Treg stimulator RUR 20kD -IL-2 composition increases the number and inhibitory function of regulatory T cells for the treatment of autoimmune and inflammatory disorders. Impaired IL-2 production and regulatory T cell dysfunction have been implicated as immune mechanisms in a variety of autoimmune diseases. Although low-dose IL-2 can be used to stimulate Treg for clinical benefit, poor pharmacokinetics require daily delivery, adverse events are dose-limiting, and Treg increases are modest and short-lived. RUR 20kD -IL-2 composition provides an IL-2 conjugate Treg stimulator intended for low-dose subcutaneous administration to selectively restore Treg homeostasis with minimal effects on conventional T cell function. Characterization of RUR is provided herein 20kD- The ability of the IL-2 composition to selectively expand the number and activity of Tregs in murine and non-human primate models and to evaluate RUR 20kD - Data on the potency of the IL-2 composition in autoimmune models. Affinity for the IL-2 receptor was evaluated by surface plasmon resonance. Activity in human PBMCs was measured by pSTAT5 induction in multiple lymphocyte populations using flow cytometry and cytometry by time-of-flight (CyToF). In vivo activity after subcutaneous administration in C57BL / 6 mice or cynomolgus monkeys was measured by changes in lymphocyte number and activation by flow cytometry. Ex vivo Treg function was determined by the inhibition of Tcon proliferation by isolated splenic Tregs. Efficacy was evaluated in a model of systemic lupus erythematosus (SLE) using MRL / MpJ-Faslpr mice. RUR with respect to the IL-2Rα and IL-2Rαβ complexes 20kD - The affinity of the IL-2 composition for human IL-2Rβ is greatly diminished, indicating preferential activation of Tregs expressing high-affinity IL-2Rαβγ compared to Tcons expressing low-affinity IL-2Rβγ. In vitro, Tregs are more sensitive to stimulation by the RUR 20kD - IL-2 composition, showing an increase in STAT5 phosphorylation relative to other lymphocyte subsets in human PBMCs. In mice, a single administration results in mobilization of Tregs in blood and spleen for 7 - 10 days without Tcon activation, an effect consistent with the induction of Treg activation markers and increased ex vivo inhibitory capacity. In cynomolgus monkeys, plasma exposure is more prolonged compared to an equivalent total dose of rhIL-2 administered daily for five days, with Treg mobilization and activity persisting for more than 14 days after a single administration – an excellent response in magnitude, duration, and specificity. Finally, RUR 20kD - The IL-2 composition is effective in a murine model of SLE. In the SLE model, RUR is administered repeatedly over 12 weeks 20kD - The IL-2 composition maintains elevated Tregs, reduces blood urea nitrogen, and normalizes urinary protein levels and kidney histopathology. In the cGVHD model, RUR 20kD - Repeated administration of the IL-2 composition increases Tregs and reduces germinal center B cells in the spleen and reverses lung dysfunction. RUR 20kD - The IL-2 composition provides sustained, preferential activation of Tregs and demonstrates efficacy in the SLE model system.

[0218] Example 10 A Phase I, double-blind, randomized, placebo-controlled study to evaluate RUR in healthy volunteers 20kD - The safety, tolerability, pharmacokinetics, and pharmacodynamics of single escalating subcutaneous doses of the IL-2 composition A double-blind, randomized, placebo-controlled study was conducted to evaluate a single ascending low subcutaneous dose of RUR 20kD -IL-2 composition (RUR 20kD -IL-2) for safety, tolerability, pharmacokinetics, and pharmacodynamics. The study was divided into seven cohorts in which subjects received 0.3, 1.0, 3.0, 6.0, 9.0, 13.5, or 20.0 μg / kg of RUR 20kD -IL-2. Twelve subjects were randomized to each dose cohort, of which nine received a single subcutaneous dose of RUR 20kD -IL-2, while three received placebo. RUR 20kD -IL-2 was formulated as a sterile liquid for subcutaneous injection and was diluted with sterile 0.9% sodium chloride solution. The drug product was provided in single-use glass vials and stored at 2 - 8°C. Each vial of drug product contained 0.75 ± 0.1 mg of rhIL-2 (based on RUR 20kD -IL-2). RUR 20kD -IL-2 was formulated in 10 mM sodium acetate, 150 mM sodium chloride, 2% (w / v) sucrose, pH 5.0 at a concentration of approximately 1.0 mg / mL protein. The placebo was a commercially available 0.9% sodium chloride solution. The starting dose of 0.3 μg / kg was selected using the minimum anticipated biological effect level (MABEL) method and was supported by the no-observed-adverse-effect level (NOAEL) in the most sensitive species from non-clinical toxicology studies. The starting dose was set at 0.3 μg / kg to allow evaluation of the pharmacokinetics and safety of RUR 20kD -IL-2. Administration was performed in a double-blind manner to two subjects (one receiving RUR 20kD -IL-2 and one receiving placebo), and possible side effects were monitored for a period of at least 7 days before the study was initiated.

[0219] The primary objective of the study was to evaluate the safety and tolerability of RUR 20kD -IL-2 administered as a single subcutaneous dose. The secondary objectives of the study were (1) to observe the temporal course and magnitude of changes in the number and / or activity of regulatory T cells (Tregs), (2) to characterize the pharmacokinetic (PK) profile of RUR 20kD -IL-2 administered as a single subcutaneous dose, and (3) to evaluate the immunological effects of RUR 20kD -IL-2 in the blood, including effects on cytokines, T cells, other peripheral blood populations, other serum proteins, changes in gene expression, and anti-drug antibodies. In the first phase of the study, immune markers were tested from pre-dose up to 20 hours post-dose. Specifically, in RUR20kD - Tregs, CD4 + - T cells, CD8 + - T cells, natural killer (NK) cells, cytokines, soluble CD25, and RNA were tested in the -IL-2 and placebo recipient groups. In subsequent periods, the same immune markers were also tested at 4-, 5-, 6-, 7-, 8-, 10-, 12-, 15-, 18-, 20-, 25-, 30-, 40-, and 50-days post-dose.

[0220] No dose-limiting toxicity (DLT), serious adverse events (SAE), deaths, or clinically significant abnormalities were reported. Adverse events (AE) were limited to mild (grade 1) injection-site reactions, and no evidence of AEs known to be associated with high-dose IL-2 was observed.

[0221] Preliminary PK analysis showed that in most subjects, RUR 20kD -IL-2 reached its maximum concentration at approximately 4 - 6 days post-dose, with little change in concentration until approximately 2 weeks post-dose, after which the concentration declined, with a half-life of approximately 8 - 9 days.

[0222] Pharmacodynamic (PD) evaluation revealed that RUR 20kD -IL-2 caused a dose-dependent increase in circulating CD4+FoxP3+CD25 亮 Tregs. In the 3.0, 6.0, 9.0, 13.5, and 20.0 μg / kg single-dose groups, the absolute number of circulating CD4+FoxP3+CD25 亮 Tregs continued to increase, with levels not returning to baseline until approximately 20 to 25 days post-administration. At the 3.0, 6.0, 9.0, 13.5, and 20.0 μg / kg doses, the number of CD4+FoxP3+CD25 亮 Tregs increased by an average of 3-fold, 3.5-fold, 4.1-fold, 5-fold, and 8.1-fold, respectively, compared to pre-dose. At the 3.0, 6.0, 9.0, 13.5, and 20.0 μg / kg doses, the total CD4+FoxP3+CD25+Treg population also increased, but the magnitude of change was less than that observed for CD4+FoxP3+CD25 亮 Tregs. At the 0.3 and 1.0 μg / kg doses, compared to subjects receiving placebo, RUR 20kD -the number of Tregs in subjects treated with -IL-2 did not change compared to placebo subjects. It was seen that RUR 20kD -IL-2 had a major effect on Tregs, as at any dose, with RUR 20kD- No change in the percentage or number of T cell populations (CD4+, CD8+) was observed with IL-2. At 13.5 and 20.0 μg / kg, there was a small increase in the percentage and absolute number of NK cells, without evidence of AEs associated with high-dose IL-2.

[0223] As Figure 12 shown in 20kD - The IL-2 composition resulted in a dose-dependent increase in CD4+FoxP3+CD25bright Tregs. At 3.0, 6.0, 9.0, and 13.5 μg / kg, the absolute number of CD4+FoxP3+CD25bright Tregs continued to increase, with levels not returning to baseline until 20 - 25 days after administration. At doses of 3.0, 6.0, 9.0, and 13.5 μg / kg, the number of CD4+FoxP3+CD25bright Tregs increased 3.0-fold, 3.5-fold, 4.1-fold, and 5.0-fold, respectively, compared to placebo, with the maximal response shifting from a peak at 84 hours at 3.0 μg / kg to a more prolonged peak response lasting 7 to 12 days at 13.5 μg / kg, then returning to baseline levels by day 20 - 25. As Figure 13 shown in 20kD - There was also a dose-dependent increase in the total CD4+FoxP3+CD25+Treg population at doses of 3.0, 6.0, 9.0, and 13.5 μg / kg, but the magnitude of the change was less than that observed for CD4+FoxP3+CD25bright Tregs. At doses of 0.3 μg / kg and 1 μg / kg, no Figure 13 ) was observed.

[0224] Importantly, 20kD - the major effect of IL-2 on Tregs was seen, as 20kD - no change in the percentage of Tcon cell populations (CD4+, CD8+) was observed in subjects treated with IL-2 or placebo. However, at 13.5 μg / kg, a small increase in the absolute number of CD8+T and the percentage of Ki67+CD8+T cells was observed in 20kD - IL-2 subjects (Figure 14A - D). At any dose level, there was no change in the absolute number of CD4+T cells.

[0225] The CD56+ NK cell population was also analyzed. An increase in the absolute number of circulating NK cells was noted, where the percentage of this cell subset increased less at the 13.5 μg / kg dose level, but not at lower dose levels. In addition, a dose-dependent increase in the percentage of CD56+ NK cells expressing Ki67 (a marker of proliferation and thus activation) was noted at 3.0, 6.0, 9.0, and 13.5 μg / kg. At 3.0, 6.0, and 9.0 μg / kg, after administration of RUR 20kD -IL-2, the percentages expressing Ki67 were approximately 10%, 20 - 30%, and 30 - 40%, respectively. At the 13.5 μg / kg dose, the percentage expressing Ki67 did not increase further and remained at 30 - 40%.

[0226] RUR according to the SAD study 20kD -IL-2 treatment led to a sustained increase in the number of CD4+FoxP3+CD25bright Tregs, where the levels did not return to baseline until 20 - 25 days after administration. The total CD4+FoxP3+CD25+ Treg population also increased, although the magnitude of the change was less than that observed for CD4+FoxP3+CD25bright Tregs. An increase in the number of CD8+ T cells and NK cells was observed at 13.5 μg / kg.

[0227] RUR was also calculated 20kD -IL-2, 20.0 mg / kg (n = 13); placebo (n = 3), and RUR 20kD -IL-2, 28.0 mg / kg (n = 9); placebo (n = 3) of additional groups. Each group was followed for 50 days to evaluate the effects of single ascending doses of subcutaneous administration of RUR 20kD -IL-2 on safety and tolerability in subjects, as evaluated by adverse events, vital signs, and clinical laboratory evaluations, and the time course and extent of changes in the number and activity of Tregs, Tcons, and NK cells and subsets, RUR 20kD -IL-2 pharmacokinetics and other immunological effects, such as cytokine levels, peripheral blood cell populations, serum proteins, and gene expression.

[0228] Typically, safety results found no dose-limiting toxicity, deaths, or adverse events leading to study discontinuation, no clinically significant vital signs, ECG, or physical examination abnormalities. Adverse events were mainly limited to mild or moderate (grade 1 or 2) injection site reactions. Four subjects experienced grade 1 headache events, and one subject receiving the highest dose (28.0 μg / kg) experienced mild (grade 1) signs and symptoms of fever, anorexia, vomiting, diarrhea, tachycardia, and myalgia (all at grade 1 severity), which were attributed to elevated cytokine levels, and no anti-drug antibodies were induced.

[0229] Typically, a sustained dose-dependent increase in CD25-bright Tregs responsive to RUR 20kD -IL-2 was observed (see Figure 15). At 28 μg / kg of RUR 20kD -IL-2 composition, an average peak increase of 17-fold above the pre-dose value was observed in the number of CD25-bright Tregs. Treg levels peaked on days 10 - 12 and did not return to baseline until 20 - 25 days after administration. At a dose > of 13.5 μg / kg, an increase in the Treg activation markers ICOS and CTLA4 was observed.

[0230] No substantial changes were observed in the percentage of Tcon cells, and a minimal increase in CD56+ NK cells responsive to RUR 20kD -IL-2 was observed (see Figure 16). (CD16+CD56+ NK cells were also enumerated, data not shown). The increase in NK cells was not dose-dependent. At the highest concentration of RUR 20kD -IL-2, a 2-fold increase in NK cells was observed. RUR 20kD -IL-2 induced a dose-dependent increase in Tregs without inducing CD8+ T cells up to 28 μg / kg. At 28 μg / kg, RUR 20kD -IL-2 administration resulted in an average peak Treg:CD8 increase of 15-fold compared to baseline (see Figure 17).

[0231] The study objective was to evaluate RUR 20kD-Safety and tolerability of [IL-2] in humans following single escalating subcutaneous (SC) doses. In addition, study the time course and extent of changes in the numbers and percentages of Tregs, conventional CD4+ and CD8+ T cells, NK cells, cytokine levels, and pharmacokinetics (PK) in peripheral blood. In this first-in-human, double-blind, single escalating dose study, healthy volunteers received SC doses ranging from 0.3 to 28 μg / kg (9 active agents per cohort: 3 placebo), and subjects were followed for 50 days. All 8 planned cohorts completed dosing. There were no dose-limiting toxicities, serious adverse events, deaths, or clinically significant abnormalities in vital signs, electrocardiograms, or laboratory test values. Attributable to RUR 20kD -Adverse events of [IL-2] were mainly limited to mild (grade 1) injection site reactions. One subject tested at the highest dose showed transient and mild (grade 1) symptoms of elevated cytokine levels and lymphopenia, which resolved without treatment. No other individuals at any dose level had known systemic signs or symptoms associated with [IL-2] therapy. To date, the first 6 cohorts have been tested for anti-drug antibodies, and none were detected. RUR 20kD -[IL-2] reached peak plasma levels 4 - 6 days after administration, where it remained nearly unchanged for ~2 weeks and then declined, with a half-life of ~8 - 9 days. Seen RUR 20kD -Principal effects of [IL-2] on Tregs. In the 3.0 to 28.0 μg / kg dose cohorts, a dose-dependent and sustained increase in the absolute numbers and percentages of circulating CD4+FoxP3+CD25bright Tregs was observed. Elevated levels peaked at days 10 - 12 and did not return to baseline until ~20 to 25 days after administration. At 28.0 μg / kg, the mean peak increase in the numbers of these CD25bright Tregs was >17-fold above baseline, and the mean peak percentage increased from 0.5% to 7.4%. Additionally, at doses ≥13.5 μg / kg, Treg activation markers increased. At the highest dose tested, the percentage and number of NK cells increased on average 3.5-fold, but no changes in the percentages or numbers of conventional CD4+ or CD8+ T cells were observed. RUR 20kD -[IL-2] composition selectively induces Tregs, as demonstrated by a >15-fold increase in the mean peak Treg:CD8 ratio compared to baseline in the 28.0 μg / kg group. In summary, within the dose range tested, a single dose of [IL-2] conjugate T-reg stimulator RUR 20kD -[IL-2] was well tolerated and safe. RUR 20kD -[IL-2] resulted in a significant and selective dose-dependent increase in circulating CD25bright Tregs, with minimal effects on conventional T cells and relatively small effects on NK cells. These clinical results extend previous findings showing RUR20kD -Animal studies of the persistent and Treg-selective effects of -IL-2 and for testing RUR 20kD -IL-2 provides strong support as a new therapeutic agent for autoimmune diseases such as systemic lupus.

[0232] RUR 20kD -The -IL-2 composition was safe and well tolerated in this first-in-human single ascending dose study and resulted in a significant and selective dose-dependent increase in circulating CD25-bright Treg cells. The effects on Tcon and NK cells were minimal, and the study data provide support for testing RUR 20kD -IL-2 in autoimmune and inflammatory diseases.

[0233] Example 11 A Phase I, double-blind, randomized, placebo-controlled, escalating multi-dose study to evaluate subcutaneous RUR 20kD -IL-2 safety, tolerability, pharmacokinetics, and pharmacodynamics in patients with systemic lupus erythematosus A double-blind, randomized, placebo-controlled study was conducted to evaluate escalating multi-doses of RUR 20kD -IL-2 in patients with minimal to moderate systemic lupus erythematosus (SLE) in 4 dose cohorts. The safety, tolerability, PK, and immunological effects of -IL-2 were also evaluated. The effect on SLE disease activity was also evaluated. Twelve SLE patients with minimal to moderate disease activity were randomized into each of 4 dose cohorts, 9 of which received multiple subcutaneous doses of 1.0 mg / mL aqueous solution of RUR-IL-2-20kD, while 3 received placebo. The RUR 20kD -IL-2 drug and placebo were prepared as described herein, e.g., as described in Example 1-A. Active clinical SLE disease activity was not required as an inclusion criterion. In cohort 1, a starting dose of 3.0 μg / kg was administered 3 times at two-week intervals (days 1, 15, and 29). This starting dose was based on the favorable safety and PD profile of a single subcutaneous dose of RUR 20kD -IL-2 determined in the previous study. The subsequent dose levels in cohorts 2, 3, and 4 were up to twice the dose levels of the previous dose cohort. Patients in cohorts 1-3 received three doses of the study drug at two-week intervals for a total of four weeks. The dose range to be evaluated during the study was 3.0 μg / kg to 24 μg / kg. Patients in cohort 4 received treatment with RUR 20kD -IL-2 administered at days 1, 15, 29, 43, 57, 71, and 85 for twelve weeks. This cohort provided information on the safety of administration and on RUR 20kD-Data on the PK and PD profiles over a longer duration of IL-2 treatment. Patients were followed for an additional 50 days after receiving the final dose of RUR 20kD -IL-2 or placebo to assess safety, PK, PD, and preliminary efficacy. The Safety Review Committee evaluated eight out of twelve subjects in each cohort for possible safety issues two weeks after the third dose in the last patient. In addition, the Safety Review Committee evaluated all patients in Cohort 4 twice: (1) two weeks after the first eight subjects received their third dose, and (2) two weeks after all subjects received all doses of the study drug. In addition to safety findings, immunological changes, including Treg, CD4 + -T cells, CD8 + -T cells, and NK cell responses, cytokine levels, and available PK data were used to determine the dose levels. The primary objective of the study was to evaluate the safety and tolerability of RUR 20kD -IL-2 administered as multiple escalating subcutaneous doses to patients with SLE. The secondary objectives of the study were (1) to characterize the PK profile of RUR 20kD -IL-2 after multiple subcutaneous administrations in patients with SLE, (2) to evaluate the effect of RUR 20kD -IL-2 on the time course and magnitude of changes in PD biomarkers (including Treg and Treg subsets, CD4 + -T cells, CD8 + -T cells, NK cell numbers and functions, and cytokine levels) in patients with SLE, (3) to evaluate the effect of RUR 20kD -IL-2 on the presence and levels of antibodies against double-stranded DNA and the levels of complement C3 and C4 in patients with SLE, and (4) to evaluate the effect of RUR 20kD -IL-2 on disease activity in patients with SLE. In Table 15 below, the results of the preliminary PK data from the escalating multi-dose study were compared with the data from the single subcutaneous study: Table 15. PK data in single-dose and multi-dose human studies

Claims

1. A composition comprising a PEGylated IL-2 conjugate having the following structure: Wherein: IL-2 is interleukin-2; n is independently an integer from about 3 to about 4000 each time it appears.

2. The composition of claim 1, wherein IL-2 is aldesleukin.

3. The composition of claim 2, wherein the composition comprises no more than about 20 mole % of the PEGylated IL-2 conjugate covered by the following formula when considered together Wherein n' is selected from 1, 4, 5, or an integer greater than 5.

4. The composition of claim 3, which comprises no more than about 10 mol % of the PEGylated IL-2 conjugate with n' equal to 1.

5. The composition of claim 3, which comprises no more than about 10 mol % of the PEGylated IL-2 conjugate with n' equal to 4.

6. The composition of claim 1 comprising a mixture of PEGylated IL-2 conjugates, wherein the composition comprises approximately equimolar amounts of 7. A method of increasing the ratio of regulatory T cells to effector T cells in a subject by administering to the subject a therapeutically effective dose of the composition of any one of claims 1-6.

8. The method of claim 7, wherein the regulatory T cells are selected from Foxp3+ and CD25+ cells.

9. A method of treating a subject having an autoimmune disease, which comprises administering to the subject a therapeutically effective amount of the composition of any one of claims 1-6.

10. Use of the composition according to any one of claims 1-6 for the manufacture of a medicament for the treatment of autoimmune diseases.

Images