Selective TREG stimulator and methods of uses thereof

Abstract

The instant disclosure provides improved pegylated IL-2 compositions that are selective Treg stimulator and methods of using these compositions for treating autoimmune diseases that provide a selective increase in numbers and activation of regulatory T cells over effector T cells.

Description

PHAR 00072 / PEC 029-PCT / P00139WO SELECTIVE TREG STIMULATOR AND METHODS OF USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit [[under 35 U.S.C. § 119]] to U.S. Provisional Patent Applications Serial Number 63 / 655,446 filed on June 03, 2024, and to U.S. Provisional Patent Applications Serial Number 63 / 717,753 filed on November 07, 2024. The content of all of which are incorporated herein by reference in their entirety. SEQUENCE LISTING

[0002] This application is being filed electronically via EFS-Web and includes an electronically submitted sequence listing in .xml format in ST.26 format. The .xml file contains a sequence listing entitled “029-PCT-SEQ-Listing.xml” created on April 10, 2025, and having a size of 10,810 bytes and 12,288 bytes on disk. The sequence listing contained in this .xml file is part of the specification and is herein incorporated by reference in its entirety. TECHNICAL FIELD

[0003] The disclosure relates to the field of selective Treg stimulator compositions, including IL-2- mPEG embodiments and related compositions, preparation methods and applications thereof, which increase the number and activation of regulatory T cells, and to methods of using these Treg stimulator compositions in the treatment of autoimmune and inflammatory diseases, and / or other conditions responsive to Treg stimulatory therapy. BACKGROUND

[0004] Typically, the immune system does not attack the body's own tissues, a phenomenon known as self-tolerance. However, autoimmune diseases arise when this self-tolerance breaks down, causing the immune system to target and destroy the body's own cells. In individuals with autoimmune diseases, antigen-specific cytotoxic T cells or auto-antibodies attack body tissues, leading to inflammation that can result in functional impairment and, in severe cases, death. Autoimmune diseases encompass a group of conditions with a range of symptoms. Despite the varied clinical presentations, these diseases share a common feature: an adaptive immune response directed against the body's own antigens.

[0005] Conventional treatments for autoimmune diseases, such as corticosteroids, cyclophosphamide, azathioprine, and methotrexate, have shown limited effectiveness and are associated with significant side effects and toxicity. These treatments do not address the root causePHAR 00072 / PEC 029-PCT / P00139WO of the autoreactive immune response. Recent advances in understanding the pathophysiology of autoimmune diseases have led to the development and evaluation of new therapies targeting specific cellular or molecular mechanisms. The etiology of autoimmune diseases is believed to involve a complex interplay of genetic factors, improper immune regulation, and hormonal and environmental influences. Proposed mechanisms for the onset of autoimmune diseases include sequestered antigens, molecular mimicry, abnormal expression of MHC class II molecules, cytokine imbalances, idiotype network dysfunction, regulatory T cell defects, lack of regulatory T cell or sufficient number of regulatory T cell, and polyclonal B cell activation. Approaches such as B cell depletion, anti-cytokine therapy, and stem cell therapy have been explored, but they often fall short in terms of efficacy, safety, and strong side effects. In addition, conventional therapies suppress the entire immune system, increasing the risk of infection and other serious side effects. Therefore, there is a need for new treatments that offer better efficacy, safety, and tolerability.

[0006] IL-2 was understood to act as a pro-inflammatory cytokine in autoimmune responses. However, studies indicate that IL-2 can have a protective role in chronic autoimmune inflammation under certain conditions. Administering IL-2, or PEGylated multi-isomer IL-2 (e.g., NKTR-358) for autoimmune therapy requires frequent dosing, leading to painful injections and poor patient compliance, or inconclusive clinical results. Long-term use of IL-2 carries risks of unwanted systemic effects due to its narrow therapeutic window, necessitating very low doses that can limit efficacy. At the time of the filing of this disclosure, there are no IL-2 related composition that is approved by the U.S. Food and Drug Administration, nor any composition entered phase 3 clinical trial for autoimmune diseases, chronic inflammatory conditions or transplant rejection according to www.clinicaltrials.gov.

[0007] Thus, there is a need for alternative and more effective, improved therapeutic compositions and treatment regimens that can effectively reduce autoimmune symptoms, improve quality of life, and provide prolonged remission in various autoimmune diseases. The present disclosure addresses the limited availability and associated shortcomings of current options for treating chronic autoimmune diseases. SUMMARY

[0008] In an aspect, the present disclosure relates to a composition comprising methoxy PEGylated IL-2 conjugates including Formula (I):PHAR 00072 / PEC 029-PCT / P00139WOwherein the IL-2 in or 5; and wherein each mPEG includes independently methoxy polyethylene glycol having a nominal average molecular weight between about 1,000 daltons to 60,000 daltons. In one embodiment, the present disclosure includes a mixture of compositions with molecules having Formula I with IL-2 including SEQ ID No: 1 and molecules having Formula I with IL-2 having SEQ ID NO:5. Either SEQ ID No: 1 or SEQ ID NO: 5 is conjugated to the proline as recited in Formula I. In one embodiment, the present disclosure includes a mixture of compositions with molecules having Formula I with IL-2 including SEQ ID No: 1 and molecules having Formula II (below) with IL-2 having SEQ ID NO: 6, wherein SEQ ID No: 1 is conjugated to the proline as recited in Formula I and SEQ ID NO: 6 is conjugated to the structure of mPEG linker. In some embodiments, any of the above compositions and / or mixtures thereof can have each of the mPEG having a nominal average molecular weight in a range from about 10,000 daltons to about 40,000 daltons. In other embodiments each of the mPEG has a nominal average molecular weight in a range from about 10,000 daltons to about 25,000 daltons. In yet other embodiments each of the mPEG has a nominal average molecular weight in a range from about 19,000 daltons to about 23,000 daltons.

[0009] In another aspect, the present disclosure relates to a composition comprising methoxy PEGylated IL-2 conjugates including formula (II):PHAR 00072 / PEC 029-PCT / P00139WO wherein the IL-2 in Formula II includes SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or any combinations thereof; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 1,000 daltons to 60,000 daltons. . Alternative, or additionally, the present disclosure relates to a mixture of composition of Formula II, wherein the composition include molecules having Formula II with IL-2 comprises both composition of Formula II with IL-2 comprising SEQ ID No: 6 and a second composition having Formula II with IL-2 comprising SEQ ID NO: 8. In another aspect, the present disclosure relates to a mixture of composition of Formula II, wherein the composition includes a second composition having Formula II with IL-2 comprising SEQ ID NO: 7 and molecules with IL-2 comprising with SEQ ID NO: 9. In some embodiments, any of the above compositions an / or mixtures thereof can have each of the mPEG in Formula II having a nominal average molecular weight in a range from between about 10,000 daltons to about 40,000 daltons. In other embodiments, each of the mPEG has a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons. In yet other embodiments each of the mPEG has a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.

[0010] Yet in another aspect, the present disclosure relates to a composition comprising PEGylated IL-2 conjugates including Formula (I):(Formula I) wherein the IL-2 in Formula I includes SEQ ID NO:1 and / or SEQ ID NO: 5; and wherein any of the above compositions and / or mixture can have each of the mPEG in Formula I with a nominal average molecular weight in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons; and a second composition comprising PEGylated IL-2 conjugates including Formula (II)PHAR 00072 / PEC 029-PCT / P00139WO (Formula II)NO: 6, SEQ ID NO: 7, SEQ ID 8, SEQ ID NO: 9, or any combinations thereof; wherein any of the above compositions and / or mixture can have each of the mPEG in Formula II with a nominal average molecular weight in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.

[0011] In another aspect, the present disclosure relates to a composition as disclosed herein made into a formulation that further comprises a formulation including one or more pharmaceutically acceptable excipients, an aqueous diluent, sodium acetate, sodium chloride, sucrose, methionine, and / or polysorbate. In some embodiments the formulation has a pH between about 4.0 to about 6.0 and is in a form for parenteral administration or in a form for subcutaneous administration. In another embodiment the formulation comprises between about 0.01-10.0 mg / mL, 0.01-1.0 mg / mL, 0.01-3.0 mg / mL, or 0.01-5.0 mg / mL of the composition or mixture thereof, between about 5.0-35.0 mM sodium acetate, between about 10.0-30.0 mM sodium chloride, between about 0.1-3.5 mM methionine, and / or between about 0.005% to 0.15 polysorbate 80, and wherein the formulation comprises a pH between about pH 4.0 to 6.0.

[0012] In another aspect, the present disclosure relates to any of the compositions or mixture thereof as disclosed herein that further comprises Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

[0013] In yet another aspect, the present disclosure relates to a method of increasing the ratio of regulatory T cells to effector T cells in a subject or subject in need comprising administering to the subject a therapeutically effective amount of the composition or mixture thereof comprising PEGylated IL-2 conjugates including Formula (I):PHAR 00072 / PEC 029-PCT / P00139WOwherein the IL-2 in 5; and wherein each mPEG is independently a methylated polyethylene glycol can have a nominal average molecular weight in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.

[0014] In another aspect, the present disclosure relates to a method of increasing the ratio of regulatory T cells to effector T cells in a subject or subject in need comprising administering to the subject a therapeutically effective amount of the composition or mixture thereof comprising PEGylated IL-2 conjugates including formula (II):wherein the IL-2 in Formula II includes SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or any combinations thereof; and wherein each mPEG is independently a methylated polyethylene glycol can have a nominal average molecular weight in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons..

[0015] In yet another aspect, the present disclosure relates to a method of increasing the ratio of regulatory T cells to effector T cells in a subject or subject in need comprising administering to thePHAR 00072 / PEC 029-PCT / P00139WO subject a therapeutically effective amount of a first composition and / or mixture thereof comprising PEGylated IL-2 conjugates including Formula (I):wherein the IL-2 in Formula I includes SEQ ID NO:1 and / or SEQ ID NO: 5; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.; and a therapeutically effective amount of a second composition comprising PEGylated IL-2 conjugates including formula (II):wherein the IL-2 in Formula II includes SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or any combinations thereof; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.

[0016] Yet in another embodiment, the present disclosure relates to a pharmaceutical formulation including a composition and / or mixtures thereof denoted in Formula I having IL-2 comprising SEQ ID NO: 1 and / or 5, or a pharmaceutical formulation including Formula II having IL-2 comprisingPHAR 00072 / PEC 029-PCT / P00139WO SEQ ID NO: 6, 7, 8, 9, or any combinations thereof, wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons. Alternatively or additionally, the present disclosure relates to pharmaceutical formation including a mixture having a first composition with Formula I having IL-2 comprising SEQ ID NO: 1 and a second composition with Formula II having IL-2 comprising SEQ ID NO: 6, wherein each mPEG can have a nominal average molecular weight in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons. In another aspect, the present disclosure relates to a pharmaceutical formulation including a mixture having a first composition with Formula I having IL-2 comprising SEQ ID NO: 5 and a second composition with Formula II having IL-2 comprising SEQ ID NO: 7, wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.

[0017] In an embodiment, the present disclosure relates to compositions or mixtures thereof, or pharmaceutical formulation including a composition and / or mixtures thereof denoted in Formula II having IL-2 with SEQ ID NO: 6, 7, 8, 9, or any combinations thereof, wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.

[0018] In an aspect, the present disclosure relates to a vector comprising a promoter operatively linked to SED ID NO: 2, 3, or 4.

[0019] In one embodiment, any of the present disclosure’s compositions and / or mixtures thereof can change the amount of the regulatory T cells including CD4+, Foxp3+and / or CD25+cells and / or the effector T cells comprising CD4+Foxp3- and / or CD25- cells in a subject or subject in need. In another embodiment, the increase in regulatory T cells in a subject or subject in need when compared to a baseline, reaches a value of at least about 2, 3, 4, 5, 10, 12, 15, 20, 25, 30, 31.5, 35, 40, 45, 50, 55,PHAR 00072 / PEC 029-PCT / P00139WO 60, 70, 100-fold, or higher. The baseline can comprise number or count of regulatory T cells from a naïve subject or subject in need , and / or obtain from subject or subject in need after the subject or subject in need is administered with Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

[0020] In one embodiment, the increase in regulatory T cells, when compared to a baseline, reaches a value of at least about 2, 3, 4, 5, 10, 12, 15, 20, 25, 30, 31.5, 35, 40, 45, 55, 60, 70, 100 fold or higher when evaluated in an in-vivo animal and / or non-human primate model. The baseline comprising number or count of regulatory T cells from the animal and / or non-human primate and can be obtained from the naïve animal and / or non-human primate model , and / or after the animal and / or non-human primate is administered with Cyclosporin A, Anti-IL-4Ra antibodies, Rituximab, BMS- 986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG- 592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

[0021] In another embodiment, the increase in regulatory T cell numbers is sustained above a baseline level for at least 1, 3, 5, 8, 10, 14, 15, 20, 30, 35, 40, 45, 50, or longer days in the subject or subject in need post-administration, wherein the post administration includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more administrations. The baseline levels comprise regulatory T cells numbers from the naïve subject or subject in need , and / or obtain from the subject or subject in need after administered with Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

[0022] In another embodiment, the increase in regulatory T cell numbers is sustained at least 1, 3, 5, 8, 10, 14, 15, 20, 30, 35, 40, 45, 50, or longer days in the subject or subject in need post-administration above a baseline level as compared to regulatory T cell from a naïve subject or subject in need, and / or subject or subject in need after administered with Cyclosporin A, Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

[0023] In one embodiment the therapeutically effective amount comprises a dose between about 0.01 to 3μg / kg, between about 0.01 to 5μg / kg, between about 0.01 to 10 μg / kg, between about 0.01 to 20μg / kg, between about 3 to 30μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 toPHAR 00072 / PEC 029-PCT / P00139WO 150 μg / kg. The therapeutically effective amount of any of the above dosage can be carried out at a frequency comprising once about every 1, 2, 3, 4, 5 days or 1, 2, 3, 4 or 5 weeks.

[0024] In another aspect, the present disclosure relates to a method of treating a subject having an immune-mediated disease, comprising administering to a subject or subject in need a therapeutically effective amount of the composition or mixture thereof comprising PEGylated IL-2 conjugates including Formula (I):wherein the IL-2 in Formula I includes SEQ ID NO:1 and / or SEQ ID NO: 5; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.

[0025] In another aspect, the present disclosure relates to a method of treating a subject having an immune-mediated disease, comprising administering to a subject or subject in need a therapeutically effective amount of the composition or mixture thereof comprising PEGylated IL-2 conjugates including formula (II):wherein the IL-2 in Formula II includes SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or any combinations thereof; and wherein each mPEG is independently a methylated polyethylenePHAR 00072 / PEC 029-PCT / P00139WO glycol having a nominal average in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.

[0026] A therapeutically effective amount can be by parenteral administration and / or by subcutaneous injection. In one embodiment the therapeutically effective amount is carried out at a frequency comprising once about every 1, 2, 3, 4, 5 days or 1, 2, 3, 4 or 5 weeks. In one embodiment the therapeutically effective amount comprises a dose between about 0.01 to 10μg / kg, between about 0.01 to 20 μg / kg, between about 3 to 30μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 to 150 μg / kg.

[0027] In another aspect, the present disclosure relates to a method of treating a subject having an immune-mediated disease, comprising administering to a subject or subject in need a therapeutically effective amount of the composition comprising PEGylated IL-2 including a mixture of molecules having Formula (I) with each molecule’s IL-2 comprising SEQ ID NO: 1 or 5, and / or Formula (II) with each molecule’s IL-2 comprising SEQ ID NO: 6, 7 or 8, wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight of in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.

[0028] In one embodiment, the immune-mediated disease comprises systemic lupus erythematosus and / or immune aplastic anemia. In one embodiment, the immune-mediated disease comprises atopic dermatitis. In another embodiment, the immune-mediated disease comprises ulcerative colitis. In yet another embodiment, the immune-mediated disease comprises Crohn's disease.

[0029] In one embodiment, any of the present disclosure’s compositions and / or mixtures thereof can further comprise Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

[0030] In another aspect, the present disclosure relates to a method of treating a subject or subject in need having an allergic disease, comprising administering to the subject or subject in need a therapeutically effective amount of the composition comprising PEGylated IL-2 conjugates and / or mixtures of related compositions thereof including formula (II):PHAR 00072 / PEC 029-PCT / P00139WOwherein the IL-2 in Formula II includes SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID 8, SEQ ID NO: 9, or any combinations thereof; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.

[0031] In one embodiment, any of the above composition and / or mixture thereof can further comprise Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

[0032] In one embodiment, the allergic disease comprises peanut allergy. The compositions and / or mixtures of the present disclosure can further comprise Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR- 809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

[0033] In another aspect, the present disclosure relates to a pharmaceutical formulation for treating, suppressing and / or inhibiting an immune-mediated disease comprising administering the composition and / or mixtures thereof comprising PEGylated IL-2 conjugates including formula (II):PHAR 00072 / PEC 029-PCT / P00139WO (Formula II) wherein the IL-2 in Formula II includes SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or any combinations thereof; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons, and with a pharmaceutically accepted carrier.

[0034] In one embodiment, the formulation can further comprise Cyclosporin A, one or more Anti- IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

[0035] In one embodiment, the immune-mediated disease comprises systemic lupus erythematosus, atopic dermatitis, ulcerative colitis, immune aplastic anemia, and / or Crohn's disease. In one embodiment, the administration regimen comprises every 1, 2, 3 , 4 or 5 days, or 1, 2, 3, 4 or 5 weeks. In one embodiment, the formulation comprises a dose between about 0.01 to 20 μg / kg, between about 0.01 to 3 μg / kg, between about 0.01 to 6 μg / kg, between about 0.01 to 10 μg / kg, between about 3 to 30μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 to 150 μg / kg.

[0036] In another aspect, the present disclosure relates to a composition comprising PEGylated IL-2 conjugates including Formula (I):wherein the IL-2 in Formula I includes SEQ ID NO:1 or SEQ ID NO: 5; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons for use in preventing, treatingPHAR 00072 / PEC 029-PCT / P00139WO and / or suppressing an immune-mediated disease, wherein an effective amount of the composition is administered to a subject or subject in need.

[0037] In another aspect, the present disclosure relates to a composition comprising PEGylated IL-2 conjugates including formula (II):wherein the IL-2 in Formula II includes SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or any combinations thereof; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons for use in preventing, treating and / or suppressing an immune-mediated disease, wherein an effective amount of the composition is administered to a subject or subject in need.

[0038] In another embodiment, the present disclosure relates to a first composition and / or mixture thereof comprising PEGylated IL-2 conjugates as denoted in Formula I for treating, suppressing and / or inhibiting an immune-mediated disease, comprising administering to a subject or subject in need a therapeutically effective amount of the first composition comprising PEGylated IL-2 including a mixture having molecules with Formula I with IL-2 comprising SEQ ID NO: 1 and molecules with Formula II having IL-2 comprising SEQ ID NO: 6 for use in preventing, treating and / or suppressing an immune-mediated disease, wherein an effective amount of the composition is administered to a subject or subject in need, wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons, and a therapeutically effective amount of a second composition and / or mixture thereof comprising PEGylated IL-2 as denoted in Formula II having IL-2 comprising SEQ ID NO: 5 and molecules with Formula II having IL-2 comprising SEQ ID NO: 7 for use in preventing, treatingPHAR 00072 / PEC 029-PCT / P00139WO and / or suppressing an immune-mediated disease, wherein an effective amount of the composition and / or mixture thereof is administered to a subject or subject in need, wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.

[0039] In one embodiment the immune-mediated disease comprises systemic lupus erythematosus, atopic dermatitis, ulcerative colitis, immune aplastic anemia, and / or Crohn's disease.

[0040] In one embodiment the administration regimen of any of the composition, formulation, and / or mixtures comprises every 1, 2, 3, 4, or 5 days, or 1, 2, 3, 4 or 5 weeks. In one embodiment the therapeutically effective amount comprises a dose between about 0.01 to 10 μg / kg, between about 0.01 to 20μg / kg, between about 0.01 to 3μg / kg, between about 0.01 to 6μg / kg , between about 3 to 30μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 to 150 μg / kg.

[0041] In another aspect, the present disclosure relates to a composition, formulation, and / or mixtures thereof for use in preventing, treating and / or suppressing an immune-mediated disease, wherein the composition can further comprises Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

[0042] In another aspect, the present disclosure relates to a use of the composition, formulation, and / or mixtures thereof comprising PEGylated IL-2 conjugates including Formula (I):wherein the IL-2 in Formula I includes SEQ ID NO:1 and / or SEQ ID NO: 5; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a rangePHAR 00072 / PEC 029-PCT / P00139WO from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltonsfor the preparation of a medicament for the treatment of a subject or a subject in need having an immune-mediated disease.

[0043] In another aspect, the present disclosure relates to a use of the composition, formulation, and / or mixtures thereof comprising PEGylated IL-2 conjugates including formula (II):wherein the IL-2 in Formula II includes SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or any combinations thereof; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight f between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons for the preparation of a medicament for the treatment of a subject or a subject in need having an immune-mediated disease.

[0044] In another embodiment, the present disclosure relates to a use of a first composition, mixtures, and / or formulation thereof comprising PEGylated IL-2 conjugates including Formula I with IL-2 comprising SEQ ID NO: 1 and molecules with Formula II having IL-2 comprising SEQ ID NO: 6, wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons and / or, a second composition, mixture, and / or formulation thereof comprising PEGylated IL-2 conjugates including a mixture having molecules with Formula I having IL-2 comprising SEQ ID NO: 5 and molecules with Formula II having IL-2 comprising SEQ ID NO: 7, wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in aPHAR 00072 / PEC 029-PCT / P00139WO range from between about 19,000 daltons to about 23,000 daltons, for the preparation of a medicament for the treatment of a subject or a subject in need having an immune-mediated disease.

[0045] In one embodiment, the immune-mediated disease comprises systemic lupus erythematosus, atopic dermatitis, ulcerative colitis, immune aplastic anemia, and / or Crohn's disease. The use of the composition wherein the composition, mixture, and / or formulation thereof can further comprise Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

[0046] In one embodiment, the treatment comprises an administration regimen to the subject or subject in need with the composition, mixture, and / or formulation thereof once every 3 or 5 days, or 1, 2, 3, 4 or 5 weeks. In one embodiment of the use of the composition, the treatment comprises administration of a dosage to the subject or subject in need between about 0.01 to 10μg / kg, between about 0.01 to 3μg / kg, between about 0.01 to 6μg / kg, between about 0.01 to 20μg / kg, between about 3 to 30μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 to 150 μg / kg.

[0047] In another aspect, the present disclosure relates to a use of the composition, mixture, and / or formulation thereof comprising PEGylated IL-2 conjugates including Formula (I):wherein the IL-2 in Formula I includes SEQ ID NO:1 or SEQ ID NO: 5; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons, for the preparation of a medicament to increase the ratio of regulatory T cells to effector T cells in a subject or subject in need by administering to the subject a therapeutically effective dose of the composition as disclosed herein.PHAR 00072 / PEC 029-PCT / P00139WO

[0048] In another aspect, the present disclosure relates to a use of the composition, mixture, and / or formulation thereof comprising PEGylated IL-2 conjugates including formula (II):wherein the IL-2 in Formula II includes SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or any combinations thereof; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons, for the preparation of a medicament to increase the ratio of regulatory T cells to effector T cells in a subject or subject in need by administering to the subject a therapeutically effective dose of the composition, mixture, and / or formulation thereof as disclosed herein.

[0049] In another aspect, the present disclosure relates to a use of first composition, mixture, and / or formulation thereof comprising PEGylated IL-2 conjugates including Formula I with IL-2 comprising SEQ ID NO: 1 and a second, mixture, and / or formulation thereof having molecules with Formula II having IL-2 comprising SEQ ID NO: 6 and / or, a use of the composition, mixture, and / or formulation thereof comprising PEGylated IL-2 conjugates including molecules with Formula I having IL-2 comprising SEQ ID NO: 5 and molecules with Formula II having IL-2 comprising SEQ ID NO: 7, wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons, for the preparation of a medicament to increase the ratio of regulatory T cells to effector T cells in a subject or subject in need by administering to the subject a therapeutically effective dose of the composition and / or mixture thereof as disclosed herein.

[0050] In one embodiment the preparation comprises an administration regimen of once every 1, 2, 3, 4 or 5 days, or 1, 2, 3, 4 or 5 weeks. In one embodiment the therapeutically effective dose comprisesPHAR 00072 / PEC 029-PCT / P00139WO between about 0.01 to 10 μg / kg, between about 0.01 to 20μg / kg, between about 0.01 to 3μg / kg, between about 0.01 to 6μg / kg, between about 3 to 30 μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30-150 μg / kg.

[0051] In one embodiment any of the present disclosure’s composition, mixture, or formulation thereof can change the amount of the regulatory T cells which comprise CD4+, Foxp3+and / or CD25+cells. In one embodiment the effector T cells comprise CD4+, Foxp3-, and / or CD25- cells. In one embodiment the increase in regulatory T cells, when compared to a baseline, reaches a value of at least about 2, 3, 4, 5, 10, 12, 15, 20, 25, 30, 31.5, 35, 40, 45, 50, 55, 60, 70, 100-fold, or higher when evaluated in the subject or subject in need. The baseline can be compared with naïve subject or subject in need, or subject or subject in need after administered with compositions comprising Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

[0052] In one embodiment, the compositions, mixtures, or formulation thereof can further comprise Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

[0053] In another aspect, the present disclosure relates to a composition, mixture, or formulation thereof comprising PEGylated IL-2 conjugates including Formula (I):wherein the IL-2 in Formula I includes SEQ ID NO:1 or SEQ ID NO: 5; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in aPHAR 00072 / PEC 029-PCT / P00139WO range from between about 19,000 daltons to about 23,000 daltons characterized for use in treating a subject or a subject in need having an immune-mediated disease.

[0054] In another aspect, the present disclosure relates to the composition, mixtures, and / or formulation comprising PEGylated IL-2 conjugates including formula (II):wherein the IL-2 in Formula II includes SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, or any combinations thereof; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons characterized that for use in treating a subject or a subject in need having an immune-mediated disease.

[0055] In one embodiment, the immune-mediated disease is characterized to include systemic lupus erythematosus, atopic dermatitis, ulcerative colitis, immune aplastic anemia, and / or Crohn's disease.

[0056] In one embodiment, the composition is characterized to further include Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

[0057] In one embodiment, the composition is characterized to be administered to the subject or subject in need once every 1, 2, 3, 4, or 5 days, or 1, 2, 3, 4 or 5 weeks. In one embodiment the composition is characterized to be administered at a dosage to the subject or subject in need between about 0.01 to 10 μg / kg, between about 0.01 to 20μg / kg, between about 3 to 30 μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 to 150 μg / kg.

[0058] In another aspect, the present disclosure relates to a method of increasing the ratio of regulatory T cells to effector T cells in a subject or subject in need characterized by administering toPHAR 00072 / PEC 029-PCT / P00139WO a subject or subject in need a therapeutically effective amount of the composition, mixture, and / or formulation thereof comprising PEGylated IL-2 conjugates including Formula (I):wherein the IL-2 in Formula I includes SEQ ID NO:1 or SEQ ID NO: 5; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.

[0059] In another aspect, the present disclosure relates to a method of increasing the ratio of regulatory T cells to effector T cells in a subject or subject in need characterized by administering to a subject or subject in need a therapeutically effective amount of the composition, mixture, and / or formulation thereof comprising PEGylated IL-2 conjugates including formula (II):wherein the IL-2 in Formula II includes SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or any combination thereof; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.PHAR 00072 / PEC 029-PCT / P00139WO

[0060] In one embodiment, the regulatory T cells are characterized to include CD4+, Foxp3+and / or CD25+cells and / or the effector T cells are characterized to include CD4+Foxp3- and / or CD25- cells.

[0061] In one embodiment, any of the present disclosure’s composition and / or mixtures thereof can increase regulatory T cells when compared to a baseline, which is characterized to reach a value of at least about 2, 3, 4, 5, 10, 12, 15, 20, 25, 30, 31.5, 35, 40, 45, 50, 55, 60, 70, 100 fold, or higher when evaluated in an in-vivo animal and / or non-human primate model. The baseline is characterized to include one or more numbers of regulatory and / or effector T cells from the in-vivo animal and / or non- human primate model that can be obtained after administered with Cyclosporin A, one or more Anti- IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin. The increase in regulatory T cells when compared to a baseline, is characterized to reach a value of at least about 2, 3, 4, 5, 10, 12, 15, 20, 25, 30, 31.5, 35, 40, 45, 50, 55, 60, 70, 100 fold, or higher in the subject or subject in need. The baseline is characterized to include one or more numbers of regulatory and / or effector T cells from naive subject or subject in need, or the subject or subject in need that can be obtained after administered with Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin. The increase in regulatory T cell numbers is characterized to sustained above a baseline level for at least 1, 3, 5, 8, 10, 14, 15, 20, 30, 35, 40, 45, 50, or longer days in the subject or subject in need post-administration.

[0062] In one embodiment, the baseline levels is characterized to include one or more regulatory and / or effector T cells numbers that can be obtained from naive subject or subject in need, or the subject or subject in need after administered with Cyclosporin A, Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

[0063] The therapeutically effective amount is characterized to include a dose between about 0.01 to 20μg / kg, between about 0.01 to 10 μg / kg, between about 0.01 to 6μg / kg, between about 0.01 to 3μg / kg, between about 3 to 30 μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 to 150 μg / kg. The amount is characterized to be carried out once about every 1, 2, 3, 4, 5 days or 1, 2, 3, 4 or 5 weeks.PHAR 00072 / PEC 029-PCT / P00139WO

[0064] Yet one more aspect of this disclosure is directed to a method of treating a subject having an allergic disease, comprising administering to the subject a therapeutically effective amount of a PEGylated IL-2 N2 composition, mixture, and / or formulation thereof.

[0065] Yet one more aspect of this disclosure is directed to a method of treating a subject having an allergy, comprising administering to the subject a therapeutically effective amount of a PEGylated IL-2 N2 composition, mixture, and / or formulation thereof.

[0066] Yet one more aspect of this disclosure is directed to a pharmaceutical composition for treating, suppressing and / or inhibiting an autoimmune disease comprising a PEGylated IL-2 N2 composition, mixture, and / or formulation thereof in a pharmaceutically accepted carrier.

[0067] Yet one more aspect of this disclosure is directed to a method of treating a subject having an allergic disease, comprising administering to the subject a therapeutically effective amount of a PEGylated IL-2 N88D composition, mixture, and / or formulation thereof.

[0068] Yet one more aspect of this disclosure is directed to a method of treating a subject having an allergy, comprising administering to the subject a therapeutically effective amount of a PEGylated IL-2 N88D composition, mixture, and / or formulation thereof.

[0069] Yet one more aspect of this disclosure is directed to a pharmaceutical composition for treating, suppressing and / or inhibiting an autoimmune disease comprising a PEGylated IL-2 N88D composition, mixture, and / or formulation thereof in a pharmaceutically accepted carrier. BRIEF DESCRIPTION OF FIGURES

[0070] For a more complete understanding of the features and advantages of the present disclosure, reference is now made to the detailed description of the disclosure along with the accompanying figures and in which: FIGURE 1A shows the amino acid sequence of aldesleukin without the N terminal proline which is denoted in SEQ ID NO: 1. FIGURE 1B shows the nucleic acid sequence for aldesleukin (SEQ ID No: 2). FIGURE 1C shows the nucleic acid sequence for IL-2 N2 (SEQ ID No: 3). FIGURE 1D shows the nucleic acid sequence for IL-2 N88D (SEQ ID No: 4). FIGURE 1E shows the amino acid sequence for IL-2 N88D without methionine and proline denoted (SEQ ID No: 5). Amino acid at position 88 has been modified from N to D. The position 88 is counted from the first Methionine of SEQ ID NO: 6.PHAR 00072 / PEC 029-PCT / P00139WO FIGURE 1F shows the amino acid sequence for IL-2 N2 where a methionine and proline are included at the N terminal (SEQ ID No: 6). FIGURE 1G shows the amino acid sequence for IL-2 N88D with methionine and proline denoted (SEQ ID No: 7). Amino acid at position 88 has been modified from N to D. FIGURE 1H shows the amino acid sequence for aldesleukin (SEQ ID NO: 8). FIGURE 1I shows the amino acid sequence for aldesleukin IL-2 N88D without methionine (SEQ ID NO: 9) FIGURE 2A and FIGURE 2B show the reduced Tryptic Peptide mapping of IL-2 and PEG- IL-2 respectively. FIGURE 3 shows the intact mass analysis of IL-2 N2. FIGURE 4A shows the RP-HPLC analysis of PEG-IL-2 N2. FIGURE 4B shows a MALDI-TOF MS analysis of PEG-IL-2 N2. FIGURE 5A and FIGURE 5B show an image of an SDS-PAGE analysis of PEG-IL-2 N2 with non-reduced silver stain and reduced silver stain. FIGURE 6A shows the percentage of P-STAT5-positive cells in Nonhuman Primate cells in response to IL-2 N2. FIGURE 6B shows the percentage of P-STAT5-positive cells in Nonhuman Primate cells in response to 40K PEG-IL-2 N2. FIGURE 7A shows the percentage of P-STAT5-positive cells in human cells in response to IL-2 N2. FIGURE 7B shows the percentage of P-STAT5-positive cells in human cells in response to PEG-IL-2 N2. FIGURE 7C shows the percentage of P-STAT5-positive cells in human cells in response to PEG-IL-2 N88D. FIGURE 8A denotes percentage of P-STAT5-positive cells in CD3⁺ T cells in humans in response to increasing concentrations of rhIL-2 and NKTR-358. FIGURE 8B denotes percentage of P-STAT5-positive cells in CD3⁺CD4⁺CD25⁺ Tregs in humans in response to increasing concentrations of rhIL-2 and NKTR-358. FIGURE 8C denotes percentage of P-STAT5-positive cells in CD3⁺CD8⁺ T cells in humans in response to increasing concentrations of rhIL-2 and NKTR-358. FIGURE 8D is a table of data from PBMC P-STAT5 assay with NKTR-358 for human and non-human primates comparing rhIL-2 and NKTR-358. FIGURE 9A shows a mouse splenocyte P-STAT5 assay for IL-2 N2.PHAR 00072 / PEC 029-PCT / P00139WO FIGURE 9B shows a mouse splenocyte P-STAT5 assay PEG IL-2 N2. FIGURE 9C shows a mouse splenocyte P-STAT5 assay PEG IL-2 N88D. FIGURE 9D shows a rat splenocyte P-STAT5 assay for IL-2 N2. FIGURE 9E shows a rat splenocyte P-STAT5 assay PEG IL-2 N2. FIGURE 9F shows a rat splenocyte P-STAT5 assay PEG IL-2 N88D. FIGURE 10A shows a Human Frozen PBMC Cytokine Release assay- IFN-gamma. The data contains comparisons of IL-2 N2, PEGylated IL2 N2, PEGylated IL-2 N2 with N88D mutation, and ProLeukin. FIGURE 10B shows a Human Frozen PBMC Cytokine Release assay- TNFα. The data contains comparisons of IL-2 N2, PEGylated IL2 N2, PEGylated IL-2 N2 with N88D mutation, and ProLeukin. FIGURE 10C shows a Human Frozen PBMC Cytokine Release assay IL-1β. The data contains comparisons of IL-2 N2, PEGylated IL2 N2, PEGylated IL-2 N2 with N88D mutation, and ProLeukin. FIGURE 10D shows a Human Frozen PBMC Cytokine Release assay IL-6. The data contains comparisons of IL-2 N2, PEGylated IL2 N2, PEGylated IL-2 N2 with N88D mutation, and ProLeukin. FIGURE 11A shows PEGylation increases the half-life, Tmax and AUC∞ of 40K PEG-IL-2 N2 and 40K PEG-IL-2 N88D as compared to IL-2 N2. FIGURE 11B and FIGURE 11C show Treg proliferative and functional marker (Ki67, Foxp3, CD25, ICOS) levels and NKTR-358 plasma concentration following administration of NKTR-358 in mice. FIGURES 12A-12C shows the expansion of regulatory T cells versus other immune cells in mice dosed with non-PEGylated IL-2 N2, PEG IL-2 N2 or PEG IL-2 N88D. FIGURES 12D-12G compare IL-2 N2, PEG-IL-2 N2, and PEG-IL-2 N88D dose-dependently and selectively increasing Treg functional markers CD25 and FOXP3 on blood Treg in mice three days following a single subcutaneous dose. FIGURES 12H-12J compare IL-2 N2, PEG-IL-2 N2, and PEG-IL-2 N88D in increasing blood Treg proliferation and functional marker expression in mice three days following a single subcutaneous dose. FIGURES 12K-12N show mouse PD time course data for NKTR-358 with various dose levels.PHAR 00072 / PEC 029-PCT / P00139WO FIGURES 13A-13E shows a mouse PD time course that compares the change in cell count in various immune cells in mice after a single subcutaneous dose of IL-2 N2 or PEG IL-2 N2. FIGURES 13F-13H shows a mouse PD time course study that compares the levels of Foxp3 on Treg and CD25 on Treg versus Teff in mice after a single subcutaneous dose of IL-2 N2 or PEG IL-2 N2. FIGURE 14A is a plot demonstrating the results of a pharmacodynamic analysis of mouse Tregs following administration of NKTR-358 FIGURE 14B is a plot demonstrating the results of a pharmacodynamic analysis of mouse Tregs following administration of aldesleukin in mice. FIGURE 15A shows the in vivo mouse delayed-type hypersensitivity model study design. FIGURE 15B shows PEG-IL-2 N2 dose-dependently inhibits ear swelling following KLH challenge in the mouse DTH model. FIGURES 15C and 15D show PEG-IL-2 N2 dose-dependently inhibits ear histopathology. FIGURES 15E – 15G show PEG-IL-2 N2 dose-dependently inhibits ear cytokines and serum anti-KLH antibodies in the mouse DTH model. FIGURES 15H – 15K show PEG-IL-2 N2 dose-dependently expands splenic Treg with minimal effects on other immune cell subsets in the mouse DTH model. FIGURES 15L – 15O show PEG-IL-2 N2 expands ear Treg with minimal effects on other immune cell subsets in the mouse DTH model. FIGURE 16A shows the in vivo mouse MC-903 model study design. FIGURE 16B shows IL-2 N2 and PEG-IL-2 N2 inhibition on ear swelling in the mouse MC- 903 model. FIGURE 16C shows IL-2 N2 and PEG-IL-2 N2 dose-dependently inhibit ear histopathology. FIGURES 16D – 16E show IL-2 N2 and PEG-IL-2 N2 inhibition on ear Th2 cytokines in the mouse MC-903 model. FIGURES 16F – 16I show PEG-IL-2 N2 selectively expands splenic Treg in the mouse MC- 903 model, while IL-2 N2 has minimal effects. FIGURES 16J – 16M show PEG-IL-2 N2 dose-dependently expands auricular lymph nodes Treg with minimal effects on other immune cells. FIGURE 17 shows PK profile for IL-2 N2 and PEG-IL-2 N2. PEG-IL-2 N2 shows (Cmax and AUC0-last) proportionally increased exposure as dose levels increased within the dose range from 0.0125 mg / kg to 0.09 mg / kg in non-human primates. FIGURE 18A shows the results of pharmacodynamic analysis of total CD3+, CD4+, CD8-, CD25+, and FoxP3+ Tregs in Cynomolgus monkeys' peripheral blood samples over time (days)PHAR 00072 / PEC 029-PCT / P00139WO following a single administration of varying dosage amounts of PEG-IL2 N2 or five subcutaneous injections of IL-2 (N2). FIGURES 19A and FIGURE 19B show the results of pharmacodynamic analysis (cell counts and fold change in cell counts) of total CD3+, CD4+, CD8-, CD25+, and FoxP3+ T cells in Cynomolgus monkeys' peripheral blood samples over time (days) following a single administration of varying dosage amounts of PEG-IL2 N2 or five subcutaneous injections of IL-2 (N2). FIGURE 19C and FIGURE 19D show the results of pharmacodynamic analysis (cell counts and fold change in cell counts) of total CD3+, CD4-, and CD8+ T cells in Cynomolgus monkeys' peripheral blood samples over time (days) following a single administration of varying dosage amounts of PEG-IL2 N2 or five subcutaneous injections of IL-2 (N2). FIGURES 20A – 20B show that PEG IL-2 N2 selectively expands Treg in non-human primates as compared to N2-IL-2. FIGURES 20C and 20D show a single dose of PEG IL-2 N2 induces a greater frequency and fold change in the percent of proliferative (Ki67+) Treg compared to five daily doses of IL-2 N2 in non-human primates. FIGURES 20E-20H show Treg activation and expansion in non-human primates for NKTR- 358. FIGURE 21A and FIGURE 21B show PEG IL-2 N2 / P11838 led to low-level increases of NK cells at 90 ug / kg as compared to N2 IL-2 in non-human primates. FIGURES 22A-22B show PEG IL-2 N2 / P11838 induces greater levels of plasma IL-5 but lower levels of eosinophils compared to IL-2 N2 / P10748 in non-human primates. . FIGURES 22C-22D show the NKTR-358 induces higher levels of serum IL-5 but lower levels of eosinophils compared to aldesleukin in non-human primates. FIGURES 22E shows that P11838 induced a limited increase of eosinophil on Day 14 at 12.5, 25, and 90 ug / kg dose levels in non-human primates as compared to P10748. FIGURE 22F shows that P11838 dose-dependently increased plasma IL-5 at 48 hrs post- dosing compared to P10748 in non-human primates. FIGURE 23A and FIGURE 23B shows the PK profile for P11838 at day 1 and day 15 respectively showing the exposure (Cmax and AUC0-last) of P11838 increased proportionally as dose levels increased within the dose range from 0.06 mg / kg to 0.15 mg / kg in non-human primates.PHAR 00072 / PEC 029-PCT / P00139WO FIGURE 24A shows the results of pharmacodynamic analysis (fold change in cell counts) of CD45+, CD3+, CD4+, CD25+, and FoxP3+ Tregs in Cynomolgus monkeys' peripheral blood samples over time (days) following repeated dosing of varying dosage amounts of PEG-IL2. FIGURE 24B shows the results of pharmacodynamic analysis (fold change in cell counts) of CD45+, CD3+, and CD4- T cells in Cynomolgus monkeys' peripheral blood samples over time (days) following repeated dosing of varying dosage amounts of PEG-IL2. FIGURE 24C shows the results of pharmacodynamic analysis (fold change in cell counts) of CD45+, CD3-, CD4+, and CD159+ NK cells in Cynomolgus monkeys' peripheral blood samples over time (days) following repeated dosing of varying dosage amounts of PEG-IL2. FIGURE 24D shows the results of pharmacodynamic analysis (cell counts) of CD45+, CD3+, CD4+, CD25+, and FoxP3+ Tregs in Cynomolgus monkeys' peripheral blood samples over time (days) following repeated dosing of varying dosage amounts of PEG-IL2. FIGURE 24E shows the expansion of blood CD4+ Treg in M / F Cynomolgus monkeys following biweekly (once every other week) subcutaneous dosing with NKTR-358 at 0 (vehicle control), 30, 90, or 150 μg / kg. FIGURE 25 shows the dextran sodium sulfate (DSS)-IBD model study to evaluate efficacy comparison of PEG- IL-2 N2 and IL-2 N2. FIGURE 26A shows P11838 and P10748 efficacy in alleviating body weight loss. FIGURE 26B shows P11838 better efficacy in reducing the disease activity index (DAI) score than P10748 or CsA.. FIGURE 26C shows the colon length in IBD model. FIGURE 26D and FIGURE 26E are plots showing P11838 reduced the IL-6 and MPO (serum) at the endpoint in IBD model. FIGURE 27A shows P11838 efficacy in alleviating body weight loss. FIGURE 27B shows a dose dependent effect of P11838 in reducing the disease activity index (DAI) AUEC (area under the effect curve). FIGURE 27C shows the effect of P11838 on colon length restoration in DSS-IBD model. FIGURE 27D shows a dose dependent effect of P11838 in reducing the colitis index in DSS- IBD model. FIGURE 28A is a PK profile graph showing the plasma concentration of P11838 in ng / mL over time (days) following a single administration on Day 2 in DSS-induced colitis mice.PHAR 00072 / PEC 029-PCT / P00139WO FIGURE 28B and FIGURE 28C show the results of pharmacodynamic analysis of CD45+, CD3+, CD4+, CD8-, FoxP3+, and CD25+ Tregs in blood samples over time (days) following a single administration of varying dosage amounts of P11838 or five subcutaneous injections of P10748. DETAIL DESCRIPTIONS

[0071] While the making and using of various embodiments of the present disclosure are discussed in detail below, it should be appreciated that the present disclosure provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the disclosure and do not delimit the scope of the disclosure.

[0072] To facilitate the understanding of this disclosure, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present disclosure. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the disclosure, but their usage does not delimit the disclosure, except as outlined in the claims.

[0073] In describing and claiming certain features of this disclosure, the following terminology will be used in accordance with the definitions described below unless indicated otherwise.

[0074] As used herein, unless otherwise specified, the terms “interleukin-2,” “IL-2”, “IL-2 N2”, “aldesleukin”, “N2 IL-2”, “N2-IL-2”, and“P10748” are used interchangeably to denote non- PEGylated IL-2 having the sequence recited in SEQ ID NO: 8. In an embodiment, if IL-2 is expressed in an E. Coli system, an extra methionine is present at the N-terminus as denoted in SEQ ID NO: 6, all of which are without PEGylation.

[0075] As used herein, unless otherwise specified, the terms “IL-2 N88” and “IL-2 N88D,” are used interchangeably, and denote the amino acid recited in SEQ ID NO: 9. As compared to IL-2 N2, the Asparagine (N) amino acid at position 88 has changed to Aspartate (D). The position 88 is counted from the first Methionine of SEQ ID NO: 6. Alternatively or additionally, the terms “IL-2 N88” and “IL-2 N88D” can be presented as using with IL-2 being SEQ ID NO: 5, wherein a proline is added at the N terminus, all of which are without PEGylation.

[0076] As used herein, “wild type human IL2”, which refers to a peptide which as has alanine in front of the proline at the N terminus, and a cysteine at position 125, as compared to SEQ ID NO: 8.PHAR 00072 / PEC 029-PCT / P00139WO

[0077] As used herein, aldesleukin can be expressed in E. coli known in the art. Aldesleukin can also be expressed in FIGURE 1F (SEQ ID NO: 6) denotes the aldesleukin sequence with an initial methionine because aldesleukin can be expressed in E. coli, wherein a methionine is in front of the proline after protein expression. The methionine can be removed by methods known by one skilled in the art, leaving proline as the initial amino acid at the N terminus. It will be understood that when the aldesleukin can be attached to a polymer such as methoxy polyethylene glycol, the aldesleukin resulted is altered due to the presence of one or more covalent bonds associated with linkage to the (methoxy) polyethylene glycol. This altered form of the aldesleukin attached to another molecule such as a branched mPEG may be referred to in some instances as a “residue”, with the understanding that the aldesleukin comprised in such polymer conjugate is altered due to the presence of one or more covalent bonds, each linking a branched PEG to the aldesleukin. International Patent Publication No. WO 2012 / 065086 described making of aldesleukin, and the entire contents of which are incorporated by reference in their entirety. It will be understood that when the other types of altered IL-2 do exist as compared to the present disclosure. In addition, these altered IL-2s can be attached to a polymer such as polyethylene glycol, the altered IL-2 can be due to the presence of one or more covalent bonds associated with linkage to the polymer(s). This altered form of the IL-2 attached to another molecule such as a branched PEG may be referred to in some instances as a “residue” of the other types of IL-2, with the understanding that the altered IL-2 comprised in such polymer conjugate can be due to the presence of one or more covalent bonds, each linking a branched PEG to the IL-2. Example of other types of altered IL-2 moieties are described in the literature, and in for example, U.S. Pat. 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. Other types of IL-2 residue such as multi-isomer PEGylated aldesleukin with different kinds of (methoxy) polyethylene glycol than the present disclosure are known in the art. For example, see U.S. Patent Number US 10,960,079 B2 or U.S. Patent Application Number US 20210205413 A1. The contents of all of which are incorporated by reference in their entirety.

[0078] As used herein, IL-2 N2 or IL-2 N88D can also be presented as sequences having additions / mutation / substitution such as for example SEQ ID NOs: 6 and / or 7where a methionine is present if expressed in an E. Coli system. In addition, IL-2 can be presented in a truncated form with one or more amino acids residues removed from the sequence, for example the 2 N-terminal amino acids (MP if made in E. Coli, or just P is not made in E. Coli) removed from SEQ ID NOs: 6 and / or 7 to achieve SEQ ID NOs: 1 and / or 5, respectively. Other modifications to IL-2 can include, but are not limited to, analogs having from 1 to 6 additional glycosylation sites, analogs having at leastPHAR 00072 / PEC 029-PCT / P00139WO one additional amino acid at the carboxy terminal end of the protein wherein the additional amino acid(s) includes at least one glycosylation site, and analogs having an amino acid sequence which includes at least one glycosylation site.

[0079] As used herein, unless otherwise specified, “P11838,” “PEG-IL-2 N2,” “IL-2-PEG N2,” “PEG-IL-2 (N2)”, “PEGylated IL-2 N2”, “40K PEG-IL2 (N2)”, “PEG-IL2”, and “PEGylated IL-2 (N2)” are all used interchangeably to denote a composition with the general structure of Formula II below with IL-2 as recited in SEQ ID NO: 8 attached to a unique branched methoxy polyethylene glycol . One other exemplary way to present this composition is by the general structure Formula I below, where SEQ ID No: 1 is conjugated to the Proline1already drawn, and the proline is linked to the linker that comprises branched polyethylene glycol .(Formula II) and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 1,000 daltons to 60,000 daltons. In another embodiment, PEGylated IL-2 N2 can be presented using Formula II with the IL-2 sequences recited in SEQ ID NO: 6, whereinPHAR 00072 / PEC 029-PCT / P00139WO the N terminus of methionine of SEQ ID NO: 6 can be removed to allow proline to conjugate to the unique branched polyethylene glycol; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight of in a range from between about 1,000 daltons to about 60,000 daltons.

[0080] As used herein, the terms “PEGylated IL-2 N88D” “40K PEG-IL-2 N88D”, and “PEG-IL-2 N88” are used interchangeably, to denote aldesleukin IL-2 wherein the Asparagine (N) amino acid at position 88 has changed to Aspartate (D). An exemplary way to present this composition is using Formula I with IL-2 as recited in SEQ ID NO: 5, wherein the attachment of SEQ ID NO: 5 to the unique branched methoxy polyethylene glycol is via the already drawn proline in Formula I. The designation 88 is derived from counting the amino acid numbers from the first methionine of SEQ ID NO: 6, and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight of in a range from between about 1,000 daltons to about 60,000 daltons. Alternatively, PEGylated IL-2 N88D can be presented using Formula II with the IL-2 sequences recited in SEQ ID NO: 9, wherein the proline at the N-terminus of SEQ ID NO: 9 is conjugated to the linker having the unique branched polyethylene glycol ; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight of in a range from between about 1,000 daltons to about 60,000 daltons.

[0081] In another embodiment, PEGylated IL-2 N88D can be presented using Formula II with the IL-2 sequences recited in SEQ ID NO: 7, wherein the N terminus of methionine of SEQ ID NO: 7 can be removed to allow proline to conjugate to the unique branched polyethylene glycol; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight of in a range from between about 1,000 daltons to about 60,000 daltons.

[0082] The PEG-IL-2 N2 and PEG IL-2 N88D compositions described herein are in one respect long- acting agents. Long-acting, in reference to other types of altered IL-2, altered PEG-IL-2 (e.g., NKTR- 358), or aldesleukin, and refers to such composition having a circulating half-life in plasma that is extended over time.

[0083] As used herein, the terms “NKTR-358” (also known as LY3471851 available from Nektar Therapeutics) is a heterogenous mixture of mono, di-, tri- and tetra-PEGylated IL-2 molecules based on the aldesleukin sequence. NKTR-358 is disclosed in United Stated Patent Application Number 17 / 056,050 (See U.S, Patent Publication Number 2021 / 0205413, the contents of which are incorporated in reference in their entirety).PHAR 00072 / PEC 029-PCT / P00139WO

[0084] The term “PEG,” “polyethylene glycol moiety,” or “polyethylene glycol,” as used herein, is meant to encompass any water-soluble poly(ethylene oxide). Unless otherwise indicated, a “PEG polymer” or a polyethylene glycol is one in which substantially all (or all) monomeric subunits are ethylene oxide subunits, though, the polymer may contain distinct end capping moieties or functional groups, e.g., for conjugation. PEG polymers for use in the present disclosure will comprise one of the two following structures: “—(CH2CH2O)n—” or “—(CH2CH2O)n-1CH2CH2—,” depending upon whether or not the terminal oxygen(s) has been displaced, e.g., during a synthetic transformation. As stated above, for the PEG polymers, the variable (n) ranges from about 3 to 4000, and the terminal groups and architecture of the overall PEG can vary. mPEG refers to PEG with methoxy ends on the PEG.

[0085] The term “Branched,” as used herein, refers 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, an illustrative PEG reagent, the branched polyethylene glycol moiety comprised of two linear PEG chains, each covalently attached via a carbamate linkage (˜NHC(O)O˜).

[0086] Molecular weight in the context of a water-soluble polymer, such as PEG, can be expressed as either a number (nominal) average molecular weight or a weight average molecular weight. Unless otherwise indicated, all references to molecular weight herein refer 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 the use of end-group analysis or the measurement of colligative properties (e.g., freezing-point depression, boiling-point elevation, or osmotic pressure) to determine number average molecular weight or the use of light scattering techniques, ultracentrifugation, or viscometry to determine weight average molecular weight. Gel filtration chromatography is often used to determine the average molecular weight of branched polymers. PEG polymers are typically polydispersed (i.e., number average molecular weight and weight average molecular weight of the polymers are not equal), possessing low polydispersity values of less than about 1.2, less than about 1.15, less than about 1.10, less than about 1.05, or less than about 1.03.

[0087] A “stable” linkage or bond refers to a chemical bond that is substantially stable in water, that is to say, does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time. Examples of hydrolytically stable linkages generally include but are notPHAR 00072 / PEC 029-PCT / P00139WO limited to the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, amines, and the like. Generally, a stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks.

[0088] The term “selective” or “selectivity” are as used and described herein, refers to an in vivo immunological response which embodies characteristics of induced immune cell, or immunological signal responses, in some respects, but not in others. In particular, “selective” with respect to Treg induction and / or activation refers to an immune response presenting an increase in Treg cell numbers (CD25 high and total by flow cytometry), and / or an increase in the Treg activation state, as indicated by one or more markers of activation, such as ICOS or Ki67 or STAT5, and / or activation refers to downstream induced immuno-suppression responses, and / or induced immunological tolerance responses, while lacking certain other immune responses. In this context, “selective Treg induction” refers to an immune response of Tregs as described, while at the same time, lacking significant and / or clinically material effector T cell and associated immunological activation responses. Significant and / or clinically material effector T cell and associated immunological activation responses include for example CD4 positive T effector cell, and / or CD8 positive T effector cell proliferation, and / or markers of activation, such as ICOS or Ki67, or other well-known effector immune responses. Other effector immune response signals may include elevation of certain pro-inflammatory cytokines, such as those known involved in “cytokine syndrome”, and / or such as IL-5, IFN gamma, IL-6, IFN alpha, IL-17, IL-22, IL-19. Selective Treg stimulation can also be reflected in the mean Treg:Tcon ratio.

[0089] The term “substantially homologous” means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. For the purposes herein, sequences having greater than 95 percent, 96 percent, 97 percent, 98 percent, 98.5 percent, 99 percent, or 99.5 percent homology, equivalent biological activity (although not necessarily equivalent strength of biological activity), and equivalent expression characteristics are considered substantially homologous. For purposes of determining homology, truncation of the mature sequence should be disregarded.

[0090] The term “regulatory T cells” or “Tregs” refer to T cells such CD4+, FoxP3+, or CD25 bright phenotypes as known in the art. The term “T cons” or “conventional T cells” refer to T lymphocytes that express an αβ T cell receptor (TCR), as well as a co-receptor CD4 or CD8, and carry out well- established adaptive immunity effector functions, such as T helper cell functions and cytotoxic T cellPHAR 00072 / PEC 029-PCT / P00139WO effector functions. For example, Tcon can refer to CD4+, CD25− naive conventional T cells. “Effector T cells (Teff)” refers to CD4+ and CD8+ cellular effector phenotypes, such as helper T cell, Cytotoxic T cells, and others, as known to the skilled artisan. “NK cells”, also known as “natural killer cells”, “K cells”, or “killer cells” are 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 tumors and virally infected cells.

[0091] As used herein, the term “isolated molecule” as referring to a molecule (where the molecule is, for example, a polypeptide, a polynucleotide, or an antibody) that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same source, e.g., species, cell from which it is expressed, library, etc., (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized, or expressed in a cellular system different from the system from which it naturally originates, will be “isolated” from its naturally associated components. A molecule also can be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art. Molecule purity or homogeneity may be assayed by a number of means known in the art. For example, the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art of purification.

[0092] Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” all refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder and / or (2) prophylactic or preventative measures that prevent and / or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. In certain aspects, a subject is successfully “treated” for cancer according to the methods of the present disclosure if the patient shows, e.g., total, partial, or transient remission of a certain type of immune and / or autoimmune disorder, or cancer.

[0093] The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” are used interchangeably herein to refer to chains of amino acids of any length. The chain may be linear or branched, it may comprise modified amino acids, and / or may be interrupted by non-amino acids. The terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any otherPHAR 00072 / PEC 029-PCT / P00139WO manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that the polypeptides can occur as single chains or associated chains.

[0094] As known in the art, “polynucleotide,” or “nucleic acid,” are used interchangeably herein, refer to chains of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the chain. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L- lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5’ and 3’ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2’-O-methyl-, 2’-O- allyl, 2’-fluoro- or 2’-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2(“amidate”), P(O)R, P(O)OR’, CO or CH2(“formacetal”),PHAR 00072 / PEC 029-PCT / P00139WO in which each R or R’ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0095] As used herein, “substantially pure” refers to material which is at least about 50% pure, at least about 90% pure, at least about 92.5% pure, at least 95% pure, at least 98% pure, at least about 99% pure, or at least 99.9% pure.

[0096] A “host cell” includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of this disclosure. One example is E. Coli.

[0097] As used herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0098] As used herein, an “effective dosage” or “effective amount” of drug, compound, or pharmaceutical composition is an amount sufficient to affect any one or more beneficial or desired results. In more specific aspects, an effective amount prevents, alleviates, ameliorates symptoms of disease, and / or prolongs the survival of the subject being treated. For prophylactic use, beneficial or desired results include, but not limited to: eliminating or reducing the risk, lessening the severity, or delaying the outset of the disease, including biochemical, histological and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include, but not limited to clinical results such as reducing one or more symptoms of a disease such as, for example, cancer including, for example without limitation, solid tumors, decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication, and / or delaying the progression of the cancer in patients. An effective dosage can be administered in one or more administrations. For purposes of this disclosure, an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction withPHAR 00072 / PEC 029-PCT / P00139WO another drug, compound, or pharmaceutical composition. Thus, an “effective dosage” or “effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

[0099] An “individual” or a “subject” is a mammal, non-limiting example is a human. Mammals also include, but are not limited to, farm animals (e.g., cows, pigs, horses, chickens, etc.), sport animals, pets, primates, horses, dogs, cats, mice and rats.

[0100] As used herein, “vector” or “expression vector” means a construct, which is capable of delivering, and expressing one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0101] As used herein, “expression control sequence” means a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or an inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[0102] As used herein, the terms “operative linkage” and “operatively linked” (or “operably linked”) are used interchangeably with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. By way of illustration, a transcriptional regulatory sequence, such as a promoter, is operatively linked to a coding sequence if the transcriptional regulatory sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcriptional regulatory sequence is generally operatively linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is operatively linked to a coding sequence, even though they are not contiguous. A nucleic acid is “operably linked” to a regulatory sequence when the nucleic acid molecule is linked to the regulatory sequence in a manner which allows expression of the nucleic acid sequence.PHAR 00072 / PEC 029-PCT / P00139WO

[0103] The term “immune modulator” refers to a substance capable of altering (e.g., inhibiting, decreasing, increasing, enhancing, or stimulating) the immune response (as defined herein) or the working of any component of the innate, humoral or cellular immune system of a host mammal. Thus, the term “immune modulator” encompasses the “immune-effector-cell enhancer” as defined herein and the “immune-suppressive-cell inhibitor” as defined herein, as well as substance that affects other components of the immune system of a mammal.

[0104] The term “immune response” refers to any detectable response to a particular substance (such as an antigen or immunogen) by the immune system of a host mammal, such as innate immune responses (e.g., activation of Toll receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells, such as antigen-specific T cells, and non-specific cells of the immune system), and humoral immune responses (e.g., responses mediated by B cells, such as generation and secretion of antibodies into the plasma, lymph, and / or tissue fluids).

[0105] The term “immunogenic” refers to the ability of a substance to cause, elicit, stimulate, or induce an immune response, or to improve, enhance, increase or prolong a pre-existing immune response, against a particular antigen, whether alone or when linked to a carrier, in the presence or absence of an adjuvant.

[0106] The term “immune-suppressive-cell inhibitor” or “ISC inhibitor” refers to a substance capable of reducing or suppressing the number or function of immune suppressive cells of a mammal. Examples of immune suppressive cells include regulatory T cells (“T regs”), myeloid-derived suppressor cells, and tumor-associated macrophages.

[0107] The term “intradermal administration”, “i.d.”, or “administered intradermally,” in the context of administering a substance to a mammal including a human, refers to the delivery of the substance into the dermis layer of the skin of the mammal. The skin of a mammal is composed of an epidermis layer, a dermis layer, and a subcutaneous layer. The epidermis is the outer layer of the skin. The dermis, which is the middle layer of the skin, contains nerve endings, sweat glands and oil (sebaceous) glands, hair follicles, and blood vessels. The subcutaneous layer is made up of fat and connective tissue that houses larger blood vessels and nerves. In contrast in intradermal administration, “subcutaneous administration” refers to the administration of a substance into the subcutaneous layer and “topical administration” refers to the administration of a substance onto the surface of the skin.PHAR 00072 / PEC 029-PCT / P00139WO

[0108] The term “preventing” or “prevent” refers to (a) keeping a disorder from occurring or (b) delaying the onset of a disorder or onset of symptoms of a disorder.

[0109] The term “tumor-associated antigen” or “TAA” refers to an antigen which is specifically expressed by tumor cells or expressed at a higher frequency or density by tumor cells than by non- tumor cells of the same tissue type. Tumor-associated antigens may be antigens not normally expressed by the host; they may be mutated, truncated, misfolded, or otherwise abnormal manifestations of molecules normally expressed by the host; they may be identical to molecules normally expressed but expressed at abnormally high levels; or they may be expressed in a context or milieu that is abnormal. Tumor-associated antigens may be, for example, proteins or protein fragments, complex carbohydrates, gangliosides, haptens, nucleic acids, or any combination of these or other biological molecules.

[0110] As used herein, “about” mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, “about” can mean a range of up to ±5%, ±10%, ±15%, or ±20%. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to ±3-fold of a value. When particular values are provided in the application and claims, unless otherwise stated, the meaning of “about” should be assumed to be within an acceptable error range for that particular value. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”. Numeric ranges are inclusive of the numbers defining the range. In additionally or alternatively, throughout this disclosure, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range and can be up to two decimals for such number. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.PHAR 00072 / PEC 029-PCT / P00139WO

[0111] As used herein, the term “conservative sequence modifications” is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR- mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of the disclosure can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the functions set forth in (c) through (I) above) using the functional assays described herein.

[0112] The term “cytotoxic agents” refers to substances that inhibit or block cell expression activity, cell function and / or result in cell destruction. The term includes radioisotopes, chemotherapeutics, and toxins, such as small-molecular toxins or enzymatically active toxins (including fragments and / or variants thereof) derived from bacteria, fungi, plants or animals. Examples of cytotoxic agents include, but are not limited to: Auristatins (for example, Auristatin E, Auristatin F, MMAE and MMAF), chlortetracycline, metotanol, ricin, ricin A-chain, cobustatin, dokamicin, Dorastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracnose diketone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, α- Sarcina, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crocotin, calicheamicins, Sapaonaria officinalis inhibitor, as well as glucocorticoid and other chemotherapy agents, as well as radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or Bi213, P32 and Lu (including Lu177). Antibodies can also be conjugated to anticancer prodrug activating enzymes that can convert a prodrug into the active form.PHAR 00072 / PEC 029-PCT / P00139WO

[0113] “Pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” refers to a component that may be included in the compositions described herein and causes no significant adverse toxicological effects to a subject. The compositions of the present disclosure are formulated as pharmaceutical compositions administered by any route that makes the composition bioavailable, such as parenteral administration, including intravenous, intramuscular or subcutaneous. Such pharmaceutical compositions and processes for preparing same are known in the art. Optionally, the compositions provided herein may further comprise a pharmaceutically acceptable excipient, and exemplary excipients include, without limitation, those selected from the group consisting of 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 particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects. A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and / or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, cyclodextrins, and the like. The excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof. The composition can also include an antimicrobial agent for preventing or deterring microbial growth. 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, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof. An antioxidant can be present in the composition as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the conjugate or other components of the preparation. Suitable antioxidants for use in 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.PHAR 00072 / PEC 029-PCT / P00139WO A surfactant can be present as an excipient. 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, N.J.); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; and chelating agents, such as EDTA; zinc and other such suitable cations. Acids or bases can be present as an excipient in the composition. Non- limiting examples of acids that can be used include those acids selected from the group consisting of 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, without limitation, bases selected from the group consisting of 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 fumerate, and combinations thereof. One or more amino acids can be present as an excipient in the compositions described herein. Exemplary amino acids in this regard include arginine, lysine and glycine. Additional pharmaceutically acceptable excipients include those described in the art.

[0114] In an embodiment, any formulation of the selective Treg stimulator compositions disclosed herein, including but not limited to IL2-N2, PEG-IL-2 N2 and / or PEG-IL-2 N88D embodiments and related compositions provided herein, can be between about 0.1 to 4.0 mg / ml protein equivalent, with about 8, 10, 12, 15, 20, or 25 mM sodium acetate, with about 8, 10, 12, 15, 20, or 25 mM acetic acid, about 110, 120, 130, 140, 150, 160, or 170 mM sodium chloride, about 0.001, 0.01, 0.1, or 1.0 % polysorbate 80, at between about pH 5.0 to 6.0, or optionally with between about 2 to 4% sucrose (w / v). Additionally, methionine can be added to the formulation to reduce impurity formation, for example, at between about 0.1-3.5 mM methionine. For storage, for example, PEG-IL-2 N2 or PEG- IL-2 N88D composition can be stored in sterile single-use polycarbonate or glass bottles of appropriate volume with a cap with a liner, supplied sterile and ready-to-use.

[0115] The term “patient,” or “subject in need” can be used interchangeably herein and refers to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a composition as provided herein, such as an autoimmune disease, and includes both humans and animals. Patients or subjects in need include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and can be human. In certain embodiments, the patient, such a human, is further characterized with a disease, disorder orPHAR 00072 / PEC 029-PCT / P00139WO condition, such as an autoimmune condition, that would benefit from administration of a composition of the present disclosure.

[0116] The term “treatment”, “therapy”, or “treating” as used herein are used interchangeably and refer to the management and care of a patient having a condition for which administration of a composition of the present disclosure is indicated for the purpose of combating or alleviating symptoms and complications of those conditions. Treating includes administering a composition of the present disclosure to a patient in need thereof to prevent the onset of symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder. For example, an autoimmune disorder. Treating includes administering a composition 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 or a human being. Administering as used herein includes either when the patient consumes the composition and / or when the patient is directed to consume the composition.

[0117] The phrases “pharmaceutically effective amount”, “pharmacologically effective amount”, “therapeutically effective amount” and “physiologically effective amount” are used interchangeably herein. In one embodiment, it refers to the amount of a PEG-IL-2 N2 or PEG-IL-2 N88D composition, mixture, and / or formulation thereof provided herein that is needed to achieve a desired level of the substance in the bloodstream or target tissue. The precise amount will depend upon numerous factors, such as for example, the particular condition being treated, the intended patient population, individual patient considerations, the components and physical characteristics of the therapeutic composition to be administered, and the like.

[0118] Pharmaceutical compositions comprising the compound, mixture and / or formulation thereof of the present disclosure that can be administered parenterally to patients in need of such treatment. Parenteral administration can be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe, optionally a pen-like syringe, or mechanical driven injector. Alternatively, parenteral administration can be performed by means of an infusion pump. Embodiments of the present disclosure provide pharmaceutical compositions for administration to a patient 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 for pharmaceutical products which are known in the art.PHAR 00072 / PEC 029-PCT / P00139WO

[0119] In an embodiment, the doses of the selective Treg stimulator compositions, including but not limited to PEG-IL-2 N2 or PEG-IL-2 N88D compositions provided herein, as well as the dosing regimen associated with the methods and compositions will vary depending upon the age, weight, and general condition of the subject, as well as the type and status of the condition being treated, the judgment of the health care professional, and the particular selective Treg stimulator composition to be administered.

[0120] As used herein, the term “per dose”, “dosage”, or “dose” means administering a given numeric amount of the composition, mixture, and / or formulation thereof of the present disclosure to a subject. Per dose can be administered in separate injections or tablet at about the same time and / or different time, so long as a subject receives the denoted drug amount.

[0121] As used herein, the term “per injection” means administering the entire denoted amount of the composition, mixture, and / or formulation thereof of the present disclosure to a subject for a given dose in a single injection or tablet.

[0122] In an embodiment, the present disclosure relates to a composition comprising methoxy PEGylated IL-2 conjugates including Formula (I):(Formula I) wherein the IL-2 in Formula I includes SEQ ID NO:1 or SEQ ID NO: 5; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight of in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons. In one embodiment, the present disclosure includes a mixture of compositions with molecules having Formula I with IL-2 including SEQ ID No: 1 and molecules having Formula I with IL-2 having SEQ ID NO:5. Either SEQ ID No: 1 or SEQ ID NO: 5 is conjugated to the proline as recited in Formula I.PHAR 00072 / PEC 029-PCT / P00139WO

[0123] In In one embodiment, the present disclosure includes a mixture of compositions with molecules having Formula I with IL-2 including SEQ ID No: 1 and molecules having Formula II (below) with IL-2 having SEQ ID NO: 6, wherein SEQ ID No: 1 is conjugated to the proline as recited in Formula I and SEQ ID NO: 6 is conjugated to the structure of mPEG linker, wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight of in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.

[0124] In some embodiments, any of the above compositions, mixtures, and / or formulations thereof can have each of the mPEG having a nominal average molecular weight in a range from about 10,000 daltons to about 40,000 daltons. In other embodiments each of the mPEG has a nominal average molecular weight in a range from about 10,000 daltons to about 25,000 daltons. In yet other embodiments each of the mPEG has a nominal average molecular weight in a range from about 19,000 daltons to about 23,000 daltons.

[0125] In another embodiment, the present disclosure relates to a composition, mixture, and / or formulation comprising methoxy PEGylated IL-2 conjugates including formula (II):wherein the IL-2 in Formula II includes SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or any combinations thereof; and wherein each mPEG is independently a methylated polyethylene glycol moiety having a nominal average molecular weight of in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons. Alternative, or additionally, the present disclosure relates to a mixture of composition of Formula II, wherein the composition include molecules having Formula II with IL-2 comprises both SEQ ID No: 6 and molecules having Formula II with IL-2 comprises SEQ ID NO: 8. In another aspect, the present disclosure relates to a mixture of composition of Formula II, wherein the composition include molecules with IL-2 comprising SEQPHAR 00072 / PEC 029-PCT / P00139WO ID NO: 7 and molecules with IL-2 comprising with SEQ ID NO: 9. In some embodiments, any of the above compositions can have each of the mPEG in Formula II has a nominal average molecular weight in a range from about 10,000 daltons to about 40,000 daltons. In other embodiments, each of the mPEG has a nominal average molecular weight in a range from about 10,000 daltons to about 25,000 daltons. In yet other embodiments each of the mPEG has a nominal average molecular weight in a range from about 19,000 daltons to about 23,000 daltons.

[0126] Yet in another aspect, the present disclosure relates to a composition, mixture, and / or formulation thereof comprising PEGylated IL-2 including Formula (I):wherein the IL-2 in Formula I includes SEQ ID NO:1 or SEQ ID NO: 5; and wherein each mPEG is independently a methylated polyethylene glycol moiety having a nominal average molecular weight of in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons; and a composition comprising, mixture, and / or formulations thereof of PEGylated IL-2 including formula (II)wherein the IL-2 in Formula II includes SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID 8; and wherein each mPEG is independently a methylated polyethylene glycol moiety having a nominal average molecular weight of in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, orPHAR 00072 / PEC 029-PCT / P00139WO a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.

[0127] In an embodiment, the present disclosure relates to a composition comprising, mixture, and / or formulations thereof of PEGylated IL-2 N2 including formula (II)wherein the IL-2 in Formula II includes SEQ ID NO: 6 and / or SEQ ID 8; and wherein each mPEG is independently a methylated polyethylene glycol moiety having a nominal average molecular weight of in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.

[0128] In an embodiment, the present disclosure relates to a composition comprising, mixture, and / or formulations thereof of PEGylated IL-2 N88D including formula (II)(Formula II)

[0129] wherein the IL-2 in Formula II includes SEQ ID NO: 7 and / or SEQ ID 9; and wherein each mPEG is independently a methylated polyethylene glycol moiety having a nominal average molecular weight of in a range from between about 10,000 daltons to about 40,000 daltons, a nominal average molecular weight in a range from between about 10,000 daltons to about 25,000 daltons, or a nominal average molecular weight in a range from between about 19,000 daltons to about 23,000 daltons.

[0130] FIGURE 1A shows the protein sequence for IL-2 referred to as aldesleukin without the initial proline, nor the possible methionine if it is expressed in the E. Coli (SEQ ID No: 1). FIGURE 1BPHAR 00072 / PEC 029-PCT / P00139WO shows the nucleic acid sequence for aldesleukin that is not codon optimized (SEQ ID No: 2). FIGURE 1C shows the nucleic acid sequence for IL-2 N2 with codon optimized (SEQ ID No: 3). FIGURE 1D shows the nucleic acid sequence for IL-2 N88D (SEQ ID NO: 4).

[0131] FIGURE 1E shows the amino acid sequence of IL-2 N88D without the initial proline, nor the possible methionine if it is expressed in the E. Coli (SEQ ID No: 5). FIGURE 1F shows the amino acid sequence for IL-2 N2 with the initial proline, and the possible methionine if it is expressed in the E. Coli and not further processed to chop off the “M” (SEQ ID No: 6). FIGURE 1G shows the amino acid sequence for IL-2 N88D with methionine and proline denoted at the N-terminus (SEQ ID No: 7). Amino acid at position 88 has been modified from N to D, where the position 88 is counted from the first methionine of SEQ ID No: 6. FIGURE 1H shows the full amino acid sequence of aldesleukin (SEQ ID NO: 8). FIGURE 1I is the amino acid sequence of IL-2 N88D (SEQ ID NO: 9).

[0132] In an embodiment, the present disclosure provides selective Treg stimulator compositions, including PEG-IL-2 embodiments and related compositions. Generally, the chemically modified IL- 2 conjugate compositions provided herein are characterized by having a branched methoxy polyethylene glycol moiety having two methoxy polyethylene glycol moieties stably covalently linked to IL-2N2. In a particular embodiment the PEG-IL-2 composition includes aldesleukin as the IL-2, wherein Formula I below already denoted the first proline of the aldesleukin. Alternatively, or additionally, the IL-2 in Formula I can also have an amino acid mutation at position 88 form “N” to “D” as referenced PEGylated IL-2 N88D. The position 88 is counted from the first Methionine and the proline at the N-terminus as denoted in SEQ ID NO: 6. Compositions provided herein comprise PEG-IL-2 N2 or PEG IL-2 N88D conjugates having a branched polyethylene glycol moiety with two polyethylene glycol moieties as seen below, where PEG-IL-2 N2 includes SEQ ID NO: 1 or 5 and PEG IL-2 N88D includes SEQ ID NO: 6 or 7.PHAR 00072 / PEC 029-PCT / P00139WO

[0133] 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 of the branched methoxy polyethylene glycol moiety is in a range from about 250 daltons to about 100,000 daltons. In some other embodiments, the nominal average molecular weight of each branched methoxy polyethylene glycol moiety is in a range from about 1000 daltons to about 90,000 daltons. In yet further embodiments, the nominal average molecular weight of each branched methoxy polyethylene glycol moiety is in a range from about 10,000 daltons to about 80,000 daltons. In some other embodiments, the nominal average molecular weight of each branched methoxy polyethylene glycol moiety is in a range from about 30,000 daltons to about 85,000 daltons.

[0134] In yet some additional embodiments, the nominal average molecular weight of each methoxy polyethylene glycol moiety is in a range from about 15,000 daltons to about 25,000 daltons. In yet one or more further embodiments, the nominal average molecular weight of each branched methoxy polyethylene glycol moiety is in a range from about 18,000 daltons to about 22,000 daltons. In yet some further embodiments, the nominal average molecular weight of each branched methoxy polyethylene glycol moiety is about 20,000 daltons.

[0135] In another embodiment, the PEG-IL-2 N2 or PEG-IL2 N88D composition can include a linker which includes two methoxy polyethylene glycol moieties each having a nominal average molecular weight between about 10,000 and about 80,000 dalton. For example, the PEG-IL-2 N2 or PEG-IL2 N88D composition includes two methoxy polyethylene glycol moieties each having a nominal average molecular weight of about 10,000 dalton. In another example the PEG-IL-2 N2 or PEG-IL2 N88D composition includes two methoxy polyethylene glycol moieties each having a nominal average molecular weight of about 18,000 dalton. In another example the PEG-IL-2 composition includes two methoxy polyethylene glycol moieties each having a nominal average molecular weight of about 19,000 dalton. In another example the PEG-IL-2 N2 or PEG-IL2 N88D composition includes two methoxy polyethylene glycol moieties each having a nominal average molecular weight of about 20,000 dalton. In another example the PEG-IL-2 N2 or PEG-IL2 N88D composition includes two methoxy polyethylene glycol moieties each having a nominal average molecular weight of about 21,000 dalton. In another example the PEG-IL-2 N2 or PEG-IL2 N88D composition includes two methoxy polyethylene glycol moieties each having a nominal average molecular weight of about 22,000 dalton. In another example the PEG-IL-2 N2 or PEG-IL2 N88D composition includes two methoxy polyethylene glycol m moieties oiety each having a nominal average molecular weight of about 23,000 dalton. In another example the PEG-IL-2 N2 or PEG-IL2 N88D composition includesPHAR 00072 / PEC 029-PCT / P00139WO two methoxy polyethylene glycol moieties each having a nominal average molecular weight of about 25,000 dalton. In another example the PEG-IL-2 N2 or PEG-IL2 N88D composition includes two methoxy polyethylene glycol moieties each having a nominal average molecular weight of about 40,000 dalton.

[0136] Additional exemplary compositions comprise compositions in accordance with the above formula wherein the overall polymer portion of the molecule has a nominal average molecular weight in a range from about 10,000 daltons to about 90,000 daltons. Additional suitable ranges for the polymer portion of the molecule include nominal average molecular weights in a range selected from about 20,000 daltons to about 85,000 daltons, in a range from about 15,000 daltons to about 80,000 daltons, in a range of about 10,000 daltons to about 75,000 daltons, in a range from about 15,000 daltons to about 70,000 daltons, and in a range from about 20,000 daltons to about 60,000 daltons.

[0137] Yet in another embodiment, compositions provided herein comprise PEG-IL-2 N2 or PEG- IL2 N88D conjugates having a branched methoxy polyethylene glycol moiety with two methoxy polyethylene glycol moieties as seen below:wherein the branched polyethylene glycol moiety includes two methoxy polyethylene glycol moieties where each polyethylene glycol moiety has a different nominal average molecular weight with each polyethylene glycol moiety independently being between about 10,000 daltons and about 80,000 daltons.

[0138] In an embodiment, the branched polyethylene glycol polymer nominal average molecular weight can be described as the sum of the two methoxy polyethylene glycol moieties. In the embodiments each of the two methoxy polyethylene glycol moieties may have a different nominal average molecular weight of independently about 10,000 daltons; about 15,000 daltons; about 20,000PHAR 00072 / PEC 029-PCT / P00139WO 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; about 75,000 daltons; about 80,000 daltons; about 85,000 daltons; about 90,000 daltons; about 95,000 daltons; or about 100,000 or more daltons.

[0139] For example, in one embodiment the PEG-IL-2 N2 or PEG-IL2 N88D composition includes a branched methoxy polyethylene glycol polymer having two methoxy polyethylene glycol moieties the first having a nominal average molecular weight from about 5,000 dalton to about 80,000 dalton and the second having a nominal average molecular weight from about 5,000 dalton to about 80,000 dalton. With the total nominal average molecular weight ranging from about 10,000 dalton to about 160,000 dalton.

[0140] In another embodiment, the present disclosure provides selective Treg stimulator compositions, including PEG-IL-2 N2 or PEG-IL-2 N88D embodiments and related compositions, mixtures and / or formulation thereof. Generally, the chemically modified IL-2 conjugate compositions provided herein are characterized by having a branched polyethylene glycol moiety with two methoxy polyethylene glycol moieties stably covalently linked to IL-2. The total nominal average molecular weight of the branched polyethylene glycol moiety is about 40,000 daltons. In a further particular embodiment of the compositions, PEGylated IL-2 conjugates of the composition have mPEG attached at proline.

[0141] In some embodiments, the selective Treg stimulator composition of formula I comprises IL- 2 N2 stably covalently-linked with a branched polyethylene glycol moiety. For example, in an embodiment the selective Treg stimulator composition is encompassed by the following structure:PHAR 00072 / PEC 029-PCT / P00139WO wherein IL-2 is one of the amino acid residues of IL-2; wherein IL-2 can include SEQ ID Nos: 1 or 5, and wherein each of the methoxy polyethylene glycol moieties range independently from about 10,000 to about 50,000 daltons.

[0142] In some embodiments, the selective Treg stimulator composition of formula II comprises IL- 2 N88D stably covalently-linked with a branched polyethylene glycol moiety. For example, in an embodiment the selective Treg stimulator composition is encompassed by the following structure:Formula II wherein IL-2 in Formula II include SEQ ID Nos: 7 or 9, and wherein each of the methoxy polyethylene glycol moieties range independently from about 10,000 to about 60,000 daltons.

[0143] Further embodiments of the selective Treg stimulator compositions provided herein comprise pharmaceutically acceptable salts thereof. As described above, the PEG-IL-2 N2 or PEG-IL-2 N88D composition, mixture and / or formulation thereof may be in the form of a pharmaceutically acceptable salt. Typically, such salts are formed by reaction with a pharmaceutically acceptable acid or an acid equivalent. The term “pharmaceutically acceptable salt” in this respect, will generally refer to the relatively non-toxic, inorganic and organic acid addition salts. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a long- acting interleukin-2 composition as described herein with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, oxylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci.66:1-19). Thus, salts as described may be derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; or prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic,PHAR 00072 / PEC 029-PCT / P00139WO sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like. As used herein, the term “composition” or “compositions”, including the PEG-IL-2 composition embodiments and related compositions described herein, comprise any and / or all pharmaceutically acceptable salts of PEG-IL-2 composition. This description applies whether the term “or pharmaceutically acceptable salt thereof” is added to the description of the composition or not.

[0144] Methods of Use Embodiments: In contrast to other types of altered IL-2 or other types of altered IL-2 that is PEGylated, the selective Treg stimulator compositions, including PEG-IL-2 composition and related compositions described herein, address the underlying pathology associated with autoreactive immunity, as well as target specific mechanisms for producing beneficial T cell functions, and provide significant improvements over administration of other types of IL-2 or other types of altered IL-2 that are PEGylated. To address deficiencies in existing autoimmune disease therapies, the instant compositions provide sustained exposure upon administration and have a unique pharmacological profile. The instant compositions selectively expand and activate endogenous Tregs in vivo, with limited expansion of conventional T cells and / or natural killer cells, and thereby provide a superior approach for the treatment of autoimmune diseases.

[0145] More particularly, the selective Treg stimulator compositions, including PEG-IL-2 N2 or PEG-IL-2 N88D compositions and related compositions, mixture and / or formulation thereof, provided herein, having branched polyethylene glycol moieties stably covalently linked to IL-2 N2 or IL-2 N88D via its amino groups, have been discovered to be particularly effective when administered at a low doses. The instant compositions, mixture and / or formulation thereof are effective in binding and activating the IL-2 receptor to increase the cell population and immune- suppressive function of regulatory T cells (Treg), while having minimal stimulatory effect on T effector cells (Teff). Sustained exposure to the present compositions (generally referred to herein as PEG-IL-2 N2 or PEG-IL-2 N88D compositions) in rodent, non-human primate studies, and human clinical studies, are effective to provide a magnitude, duration, and specificity of Treg to Teff responses that could not be achieved with equivalent doses of other types of modified IL-2 or modified PEGylated IL-2.

[0146] Administration of a single low ascending subcutaneous dose of the selective Treg stimulator PEG-IL-2 N2 or PEG-IL-2 N88D compositions, mixture and / or formulation thereof (as described in the supporting examples) to humans result in no dose-limiting toxicities, or serious adverse events or clinically significant abnormalities. Pharmacokinetic analysis shows that the composition can reachPHAR 00072 / PEC 029-PCT / P00139WO maximum concentrations around about 4-6 days post-dose, with little change in concentrations up to approximately 2 weeks post-dose. Pharmacodynamic assessment reveals that administration of the selective long-acting IL-2 N2 or PEG-IL2-N2 receptor agonist Treg stimulator composition lead to a dose-dependent increase in circulating CD4+FoxP3+CD25 bright Tregs, i.e., there is a sustained increase in the absolute numbers of circulating CD4+FoxP3+CD25 bright Tregs, with levels not returning to baseline until approximately 20 to 25 days following administration. In an embodiment, there can be a mean increase in the numbers of CD4+FoxP3+CD25 bright Tregs of several fold (with magnitude depending upon dose), compared to pre-dose. There can an increase in the total CD4+FoxP3+CD25+ Treg population. For the lowest doses, there can be no change in the numbers of Tregs in the treated subjects versus placebo subjects. The primary effect can be seen on Tregs, as no changes in percentage or numbers of T cell populations (CD4+, CD8+) can be observed with a PEG-IL-2 N2 or PEG-IL-2 N88D composition at dose of the present disclosure. Thus, the instant compositions, mixture and / or formulation thereof, and methods are surprisingly effective to increase the suppressive capacity of Treg in in vivo / ex vivo bioassays (even when compared to alternative chemically- modified IL-2, such as with other multi-isomer PEGylated IL-2 compounds, e.g., NKTR-358) and in human studies as well.

[0147] Yet in another embodiment, the selective Treg stimulator compositions, mixture and / or formulation thereof, including PEG-IL-2 N2 or PEG-IL-2 N88D compositions and related compositions, mixture and / or formulation thereof provided herein, are useful for (among other things) treating autoimmune diseases and disorders. Exemplary autoimmune diseases that can be treated by administration of a PEG-IL-2 N2, PEG-IL-2 N88D composition, mixture and / or formulation thereof as described herein 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 include type 1 diabetes, Addison's disease, Hashimoto thyroiditis, Graves' disease, Sjogren's syndrome, vitiligo, pernicious anemia, glomerulonephritis, myasthenia gravis, Goodpasture's syndrome, autoimmune hemolytic anemia, idiopathic thrombocytopenia purpura, peanut allergy, and pulmonary fibrosis. In another embodiment, the compositions disclosed herein can treat IMD-related disease / disorder with defective barrier function and achieve unmet clinical need to provide robust efficacy with durable response, for example, by reduce CD122 binding / engineer CD25 bias for preferential proliferation and activation of Tregs without corresponding effects on conventional T- cells or NK cells.PHAR 00072 / PEC 029-PCT / P00139WO

[0148] In some embodiments, the condition being treated is systemic lupus erythematosus (SLE). Systemic Lupus Erythematosus (SLE) is an autoimmune inflammatory disease that affects mostly middle-aged women. Characteristics of SLE include, for example, skin eruptions, joint pain, recurrent pleurisy, and kidney disease. A progressive homeostatic imbalance of Tregs relative to Tcons is shared by many autoimmune diseases, including SLE. Taken together, the activity of low-dose IL-2 N2 or IL-2 N88D in SLE patients, and the superior Treg inducing properties of the PEG-IL-2 N2 composition and related compositions, mixture and / or formulation thereof described herein, relative to IL-2, provide ample support for the use of the instant PEG-IL-2 N2 or PEG-IL-2 N88D composition and related compositions, mixture and / or formulation thereof in treating SLE and other autoimmune diseases and conditions. In one or more further embodiments, provided herein is a method of treating a condition by administering a PEG-IL-2 N2 or PEG-IL-2 N88D composition related composition, mixture and / or formulation thereof as described herein, wherein the condition is selected from the group consisting of, for example, allergy, GVHD, Crohn's disease, ulcerative colitis, rheumatoid arthritis, type-1 diabetes, multiple sclerosis, and psoriasis.

[0149] In yet some further embodiments, the PEG-IL-2 N2 or PEG-IL-2 N88D compositions, mixture and / or formulation thereof can be effective when administered at a therapeutically effective dose to a subject to preferentially expand and activate regulatory T cells over conventional T cells and natural killer cells.

[0150] 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 a PEG- IL-2 N2 or PEG-IL-2 N88D composition, mixture and / or formulation thereof. In an embodiment, the mean Treg:Tcon ratio achieved in response to a PEG-IL-2 N2 or N88D composition described herein is at least about 5, 7, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 fold.

[0151] In some embodiments related to the foregoing method, the regulatory T cells are selected from Foxp3+and CD25+cells. In one or more embodiments related to the former embodiment or method, the effector T cells are selected from CD4+and CD8+cells.

[0152] In some further embodiments related to the method or related embodiments above, the fold- increase in regulatory T cells when compared to baseline reaches a value of at least about 2, or at least about 4, or even at least about 6, when evaluated in an in-vivo mouse model.PHAR 00072 / PEC 029-PCT / P00139WO

[0153] In some embodiments of the method, the increase in regulatory T cell numbers is sustained above baseline levels for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days post-administration. In some additional embodiments, the increase in regulatory T cell numbers is sustained above baseline levels for at least about 3, 4, 5, 6, 7, 8, 9, 10 or more days post-administration. The increase in regulatory T cell numbers can also be sustained above baseline levels for at least about 3, 4, 5, 6, 7, 8, 9, 10 or more days.

[0154] In yet a further aspect, provided herein is a method of treating a subject having an autoimmune disease, comprising administering to the subject a therapeutically effective amount of a selective Treg stimulator composition, including PEG-IL-2 N2 or PEG-IL-2 N88D composition, mixture and / or formulation thereof embodiments as described above or elsewhere herein.

[0155] In yet a further aspect, provided herein is a method of treating a subject having an autoimmune disease, comprising administering to the subject a therapeutically effective amount of a PEG-IL-2 N2 or PEG-IL-2 N88D composition, mixture and / or formulation thereof.

[0156] In yet a further aspect, provided herein is the use of a selective Treg stimulator PEG-IL-2 N2 or PEG-IL-2 N88D composition, mixture and / or formulation thereof for the manufacture of a medicament for treating autoimmune disease.

[0157] In a more particular embodiment, treatment of systemic lupus erythematosus (SLE) comprises subcutaneous administration of a formulation comprising a therapeutically effective amount of a PEG-IL-2 N2 or PEG-IL-2 N88D composition, mixture and / or formulation thereof, which illustrate the effect of PEG-IL-2 N2 or PEG-IL-2 N88D composition, mixture and / or formulation thereof induced Tregs on control of the physiological immune response and disease progression in a representative animal model of SLE. As described therein, a PEG-IL-2 N2 or PEG-IL-2 N88D composition, mixture and / or formulation thereof are effective to suppress the biomarker of kidney damage (one of the characteristics of patients having SLE) to nearly the same levels as observed in normal mice.

[0158] In embodiments that refer to a method of treatment as described herein, such embodiments are also further embodiments for use in that treatment, or alternatively for the use in the manufacture of a medicament for use in that treatment. The present disclosure further provides a composition, mixture and / or formulation thereof according to any one of the embodiments of a composition as described herein for use in therapy. The present disclosure further provides a composition accordingPHAR 00072 / PEC 029-PCT / P00139WO to any one of the embodiments of a composition, including formulations thereof, as described herein, for use in the treatment of an autoimmune disease.

[0159] Preparations of PEG-IL2-N2 or PEG-IL-2 N88D. It should be understood that the Preparations and Examples are set forth by way of illustration and not limitation, and various modifications may be made by one of ordinary skill in the art. Methods of preparing the selective Treg stimulator compositions, including PEG-IL-2 N2 or PEG-IL-2 N88D compositions and related compositions, mixture and / or formulation thereof are described herein and / or known to the skilled artisan. The reagents and starting materials are readily available or may be readily synthesized by one of ordinary skill in the art. Conditions for the steps of these methods are known, and appropriate substitutions of buffers and reagents are within the skill of the art. Furthermore, the skilled artisan will appreciate that in some circumstances the steps and order by which compositions are produced may be modified and are well appreciated by the skilled biochemist. Likewise, it will be appreciated that preparations may be isolated and / or purified by various known techniques as needed or desired.

[0160] Methods for producing and expressing recombinant polypeptides in vitro and in prokaryotic and eukaryotic host cells are known to those of ordinary skill in the art. See, for example, U.S. Pat. No.5,614,185. The IL-2 N2 or IL-2 N88D can be expressed in bacterial, mammalian, yeast, and plant expression systems. Although recombinant based methods for preparing proteins can differ, recombinant methods typically involve constructing the nucleic acid encoding the desired polypeptide or fragment, cloning the nucleic acid into an expression vector, transforming a host cell (e.g., plant, bacteria, yeast, transgenic animal cell, or mammalian cell such as Chinese hamster ovary cell or baby hamster kidney cell), and expressing the nucleic acid to produce the desired polypeptide or fragment. Various methods of protein purification may be employed to purify a composition of the present disclosure, and such methods are known in the art and described. To facilitate identification and purification of the recombinant polypeptide, nucleic acid sequences that encode the recombinant protein can be codon optimized, and nucleic acid sequences for an epitope tag or other affinity binding sequence can be inserted or added in-frame with the coding sequence, thereby producing a protein comprised of the desired polypeptide.

[0161] Depending on the system used to express proteins having IL-2 activity, the IL-2 N2 or IL-2 N88D can be unglycosylated or glycosylated and either may be used. That is, the IL-2 N2 or IL-2 N88D may be unglycosylated or the IL-2 N2 or IL-2 N88D may be glycosylated, and in one or more embodiments the IL-2 N2 or IL-2 N88D is unglycosylated. The IL-2 N2 or N88 can also be modified to include and / or substitute one or more amino acid residues such as, for example, lysine, cysteinePHAR 00072 / PEC 029-PCT / P00139WO and / or arginine, in order to provide facile attachment of the polymer to an atom within the side chain of the amino acid.

[0162] In addition, the IL-2 N2 or IL-2 N88D can be modified to include attachment of a functional group (other than through addition of a functional group-containing amino acid residue). For example, the IL-2 N2 or IL-2 N88D can be modified to include a thiol group. In addition, the IL-2 N2 or IL-2 N88D can be modified to include an N-terminal alpha carbon. In addition, the IL-2 N2 or IL-2 N88D can be modified to include one or more carbohydrate moieties. In addition, the IL-2 N2 or IL-2 N88D can be modified to include an aldehyde group. In addition, the IL-2 N2 or IL-2 N88D can be modified to include a ketone group. In some embodiments of the disclosure, IL-2 N2 or IL-2 N88D is not modified to include one or more of a thiol group, an N-terminal alpha carbon, carbohydrate, aldehyde group and / or ketone group.

[0163] For any given peptide or protein moiety, or composition, mixture and / or formulation thereof, the protein activity can be determined. Various methods for determining the in vitro IL-2 N2 or IL-2 N88D activity are described in the art and herein. An exemplary approach is the CTTL-2 cell proliferation assay described herein. Briefly, in a non-specific binding assay, a proposed IL-2 N2 or IL-2 N88D or composition and / or mixture is allowed to pre-incubate for one hour at 4° or 37° C. in the presence of a cell line bearing a receptor of IL-2. Thereafter, 125I-labelled IL-2 is allowed to incubate in the system for three hours at 4° C. Data is expressed as % inhibitory capacity of the proposed IL-2 N2 or IL-2 N88D activity versus wild-type IL-2. Other methodologies known in the art can also be used to assess IL-2 function, including electrometry, spectrophotometry, chromatography, and radiometric methodologies.

[0164] For preparation of Selective Treg Stimulator Compositions, mixture and / or formulation thereof, an exemplary selective Treg stimulator composition of PEG-IL-2 N2 or PEG-IL-2 N88D is generally prepared by reacting purified IL-2 N2 or N88 with a molar excess of mPEG reagent in a bicine solution at high pH between about 6.0 to 9.0. The reactants are mixed for between about 30 minutes to about 5 hours, or from between about 30 minutes to 4 hours, or from between about 30 minutes to 2 hours, or from between about 30 minutes to 1 hour, generally under mild conditions, e.g., from about 20° C to about 65° C, or from about 20° C to about 40° C, or at ambient or room temperature. The reaction is quenched by acidification to low pH by addition of a suitable acid such as acetic acid.PHAR 00072 / PEC 029-PCT / P00139WO

[0165] The reaction product can then be purified by a method such as ion exchange chromatography. For example, when employing ion exchange chromatography, the PEG-IL-2 N2 or PEG-IL-2 N88D composition binds to the resin and then is eluted with a suitable gradient, such as a sodium chloride gradient. The chromatography product pool is then concentrated and diafiltered into suitable formulation buffer (for example, sodium acetate buffer with sucrose) using, for example, tangential flow filtration (TFF).

[0166] In one embodiment, the product can be pooled and may be further separated to detect positional isomers by reverse phase chromatography using a reverse phase-high performance liquid chromatography (RP-HPLC) with a column (e.g., a C18 column or C3 column, available commercially from companies, or by ion exchange chromatography using an ion exchange column, e.g., a Sepharos (trade name) ion exchange column available from Amersham Biosciences. Either approach can be used to separate polymer-active agent isomers having the same or similar molecular weight (i.e., positional isoforms).

[0167] Selective Treg stimulator compositions, including PEG-IL-2 N2 or PEG-IL-2 N88D embodiments and related compositions, mixture and / or formulation thereof, can be characterized by various analytical and bioassay techniques described herein and / or known to the skilled artisan, including analytical HPLC, SDS-Page, LCMS, and bioassays such as CTLL-2 proliferation, and Treg induction in-vivo.

[0168] Formulations: In yet one or more embodiments provided herein is a selective Treg stimulator composition, including PEG-IL-2 N2 or PEG-IL-2 N88D embodiments and related compositions, comprising an IL-2 conjugate composition as described herein, and a pharmaceutically acceptable excipient.

[0169] In an embodiment, dosage amounts are low dosage amounts that are effective to expand and activate regulatory T cells over conventional T cells and natural killer cells in a subject, as compared to a subject not being treated, or treated with other drugs, such as Cyclosporin A, Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR- 809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin. Activation of regulatory T cells can be measured by a number of different approaches. For example, given the integral role of STAT5 in IL-2-dependent T cell processes, the detection of increased STAT5 in lymphocytes can be utilized as a key marker of Treg activation. Phenotypically, activation of Treg can also be measured by flow cytometry throughPHAR 00072 / PEC 029-PCT / P00139WO increased cell surface IL-2Ra (CD25), and / or increased intracellular expression of the protein forkhead box P3 (Foxp3), a master regulator of the Treg lineage, and / or increased expression of the protein Ki67 which is associated with cell proliferation. Collectively, these markers are linked with the functionality of Treg cells and are often dysregulated in such cells in autoimmune diseases. Herein a detection of Treg cell induction and activation is by flow cytometry. The functionality of Treg can also be assessed through an ex vivo suppression assay, which measures their ability to inhibit the proliferation of conventional T cells. The consequence of Treg mobilization and activation can also be directly measured in vivo using antigen-driven inflammation models.

[0170] Administration of the PEG-IL-2 N2 or PEG-IL-2 N88D embodiments and related compositions, mixture and / or formulation thereof provided herein are typically 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, intra- arterial, intratumoral, intralymphatic, intraperitoneal, intracardiac, intrathecal, and intramuscular injection, as well as infusion injections. In a particular embodiment, injection is subcutaneous. For example, administration to a patient can be achieved through injection of a composition comprising PEG-IL-2 N2 or PEG-IL-2 N88D embodiments and related compositions, mixture and / or formulation thereof provided herein and a diluent. With respect to possible diluents, a diluent can be selected from, for example, bacteriostatic water for injection, dextrose 5% in water, 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 through testing whether two given pharmacological components are compatible together in a given formulation. An exemplary composition for administration to a patient, e.g., a subcutaneous formulation, comprises, e.g., a therapeutically effective dose of PEG-IL-2 N2 or PEG-IL-2 N88D embodiments and related compositions, mixture and / or formulation thereof provided herein, water, sodium acetate, sodium chloride and sucrose. The liquid composition will have a pH in a range of about 4.5-7.5; or from about 4.5-6.0.

[0171] In certain embodiments, the selective Treg stimulator compositions, including PEG-IL-2 N2 or PEG-IL-2 N88D embodiments and related compositions, mixture and / or formulation thereof provided herein, are in solid form. Solid forms are those that are solid dry forms, e.g., containing less than 5 percent by weight water, or less than 2 percent by weight water. The solid forms are generally suitable for reconstitution in an aqueous diluent. Solid formulations are stable for at least about 12, 24, 36, 52 months or longer when stored in sealed containers at temperatures from about 0-10° C.PHAR 00072 / PEC 029-PCT / P00139WO

[0172] In an embodiment, the present disclosure’s compositions, mixture and / or formulation thereof can include an effective amount or dose which upon single or multiple administration to the patient or subject will induce a selective Treg cell increase of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more fold over pre dose levels. In an embodiment, irrespective for use of increasing T regulatory cell and / or treating for immune and / or autoimmune disease / disorder, the dosage can include an initial priming dose to initiate physiological activation, followed by dose escalation, and a subsequent maintenance phase at the target therapeutic dose. Alternatively, or additionally, the dosage can include an initial priming dose to initiate physiological activation, followed by dose escalation or decrease in dosage and a subsequent maintenance dose, which the maintenance dose can also be adjusted upward or downward depending on the subject or subject in need’s physical condition. For example, a dose can include a higher initial loading dose, followed by a lower dose. In one or more instances a therapeutically effective amount of the selective Treg stimulator compositions, including PEG-IL-2 N2 or PEG-IL-2 N88D compositions, mixture and / or formulation thereof provided herein, is an amount encompassed by one or more of the following ranges expressed in amount of PEG-IL-2 N2 or PEG-IL-2 N88D: between about 0.01-10.0 mg / mL; between about 0.05-10.0 mg / mL; between about 0.1-10.0 mg / mL; between about 0.2-10.0 mg / mL; between about 0.3-10.0 mg / mL; between about 0.4-10.0 mg / mL; between about 0.5-10.0 mg / mL; between about 0.6-10.0 mg / mL; between about 0.7-10.0 mg / mL; between about 0.8-10.0 mg / mL; between about 0.01-30.0 mg / mL; between about 0.1-30.0 mg / mL; between about 0.1-25.0 mg / mL; between about 0.1-20.0 mg / mL; between about 0.1-15.0 mg / mL; between about 1.0-10.0 mg / mL; between about 1.0-15.0 mg / mL; between about 1.0-30.0 mg / mL of said composition, including any and all combinations of the foregoing beginning and ending values from each and every of the foregoing ranges. Alternatively, or additionally, the dosage can be expressed as μg / kg, for example, between about 0.01 μg / kg to 300 μg / kg, encompassing various subranges to ensure comprehensive coverage. For instance, in certain embodiments, the range may be between about 0.01 μg / kg to 15 μg / kg, between about 0.01 to 10μg / kg, between about 0.01 to 20μg / kg, between about 0.01-30 μg / kg, between about 3 to 30 μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 to 150 μg / kg including any and all combinations of the foregoing beginning and ending values from each and every of the foregoing ranges. Additionally, ranges include about 0.01 μg / kg to 250 μg / kg, about 0.05 μg / kg to 200 μg / kg, or about 1 μg / kg to 150 μg / kg. Further, ranges may include about 5 μg / kg to 100 μg / kg, about 10 μg / kg to 75 μg / kg, or about 20 μg / kg to 50 μg / kg.

[0173] In some embodiments, for example, for treating an autoimmune disease, or a disease or condition which can benefit from the preferential expansion and activation of regulatory T cells overPHAR 00072 / PEC 029-PCT / P00139WO conventional T cells and natural killer cells in a subject, the selective Treg stimulator compositions, including PEG-IL-2 N2 or PEG-IL-2 N88D embodiments and related compositions, mixture and / or formulation thereof provided herein, is administered at a dose, for example, a dose between about 0.01-10.0 mg / mL; between about 0.05-10.0 mg / mL; between about 0.1-10.0 mg / mL; between about 0.2-10.0 mg / mL; between about 0.3-10.0 mg / mL; between about 0.4-10.0 mg / mL; between about 0.5-10.0 mg / mL; between about 0.6-10.0 mg / mL; between about 0.7-10.0 mg / mL; between about 0.8-10.0 mg / mL; between about 0.01-6.0 mg / mL; between about 0.01-3.0 mg / mL; between about 0.1-25.0 mg / mL; between about 0.1-20.0 mg / mL; between about 0.1-15.0 mg / mL; between about 1.0-10.0 mg / mL; between about 1.0-15.0 mg / mL; between about 1.0-30.0 mg / mL of said composition, including any and all combinations of the foregoing beginning and ending values from each and every of the foregoing ranges. Alternatively, or additionally, the dosage can be less than or equal to 300 μg / kg. A dose regimen of the present disclosure is wherein a PEG-IL-2 N2 or PEG-IL- 2 N88D, mixture and / or formulation thereof can also be administered at a dose between about 0.01- 30 μg / kg once every one, two, three, four, five or more weeks. Another dose regimen of the present disclosure wherein a PEG-IL-2 N2 or PEG-IL-2 N88D composition and related composition and / or mixture thereof is administered at a dose between about 0.01-18 μg / kg once every one, two, three, four, five or more weeks. Another dose regimen of the present disclosure is wherein a PEG-IL-2 N2 or PEG-IL-2 N88D composition, mixture and / or formulation thereof, is administered at a dose between about 0.01-12 μg / kg once about every 1, 2, 3, 4, or 5 weeks. Another dose regimen of the present disclosure is wherein a PEG-IL-2 N2 or PEG-IL-2 N88D composition, is administered at a dose between 0.01-6 μg / kg once every two weeks. Another dose regimen of the present disclosure is wherein a PEG-IL-2 N2 or PEG-IL-2 N88D composition, is administered at a dose of 3 μg / kg once about 1, 2, 3, 4, 5 days, or once about every 1, 2, 3, 4, or 5 weeks.

[0174] In another embodiment, the present disclosure includes administration dosage and regimen of PEG-IL-2 N2 or PEG-IL-2 N88D and related composition mixture and / or formulation thereof once every two weeks at doses between about 0.01-10 μg / kg, between about 0.01-6 μg / kg, and between about 0.01-10 μg / kg. Yet in another embodiment, administration dosage and regimen of PEG-IL-2 N2 or PEG-IL-2 N88D and related composition and / or mixture thereof can be once every 1, 2, 3, 4, or 5 weeks at doses between about 0.01-12 μg / kg. In an embodiment, administration dosage and regimen of PEG-IL-2 N2 or PEG-IL-2 N88D and related composition and / or mixture thereof once every 1, 2, 3, 4, 5 days, or once every 1, 2, 3, 4, or 5 weeks at a dose of about 3 μg / kg.PHAR 00072 / PEC 029-PCT / P00139WO

[0175] Yet in another embodiment, the dosing amount of the selective Treg stimulator compositions, including PEG-IL-2 N2 or PEG-IL-2 N88D and related composition, mixture and / or formulation thereof embodiments and related compositions provided herein, will vary depending on a number of factors, but will optimally be a therapeutically effective dose when the composition, mixture and / or formulation thereof is stored in a unit dose container (e.g., a vial). For example, the dose may be between about 0.01 μg / kg to 300 μg / kg, encompassing various subranges. The dose may be between about 0.01 to 10μg / kg, between about 0.01 to 20μg / kg, between about 0.01-30 μg / kg, between about 3 to 30 μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 to 150 μg / kg. In addition, pharmaceutical preparations can be housed in a syringe. A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the selective Treg stimulator compositions, including PEG-IL-2 N2 or PEG-IL-2 N88D and related composition, mixture and / or formulation thereof embodiments and related compositions provided herein, in order to determine an amount that produces a clinically desired endpoint as described herein, such as relief of autoimmune symptoms and / or immunosuppression, and / or induction of tolerance. The dose may be between about 0.01 μg / kg to 300 μg / kg, encompassing various subranges to ensure comprehensive coverage. For instance, in certain embodiments, the dose may be between about 0.01 μg / kg to about 250 μg / kg, about 0.2 μg / kg to about 225 μg / kg, about 0.5 μg / kg to about 200 μg / kg, about 1 μg / kg to about 175 μg / kg, about 2 μg / kg to about 150 μg / kg, about 3 μg / kg to about 125 μg / kg, about 5 μg / kg to about 100 μg / kg, about 7 μg / kg to about 75 μg / kg, about 10 μg / kg to about 50 μg / kg, about 15 μg / kg to about 40 μg / kg, about 20 μg / kg to about 35 μg / kg, about 25 μg / kg to about 30 μg / kg, about 30 μg / kg to about 60 μg / kg, about 40 μg / kg to about 80 μg / kg, about 50 μg / kg to about 90 μg / kg, about 60 μg / kg to about 110 μg / kg, about 70 μg / kg to about 130 μg / kg, about 80 μg / kg to about 150 μg / kg, about 90 μg / kg to about 175 μg / kg, about 100 μg / kg to about 200 μg / kg, about 125 μg / kg to about 225 μg / kg, about 150 μg / kg to about 250 μg / kg, about 175 μg / kg to about 275 μg / kg, or about 200 μg / kg to about 300 μg / kg. Further specific subranges may include between about 0.01 μg / kg to about 1 μg / kg, between about 0.1 μg / kg to about 2 μg / kg, between about 0.2 μg / kg to about 3 μg / kg, about 0.3 μg / kg to about 4 μg / kg, about 0.4 μg / kg to about 5 μg / kg, about 0.5 μg / kg to about 6 μg / kg, about 0.75 μg / kg to about 7 μg / kg, about 1 μg / kg to about 8 μg / kg, about 1.5 μg / kg to about 9 μg / kg, about 2 μg / kg to about 10 μg / kg, about 3 μg / kg to about 12 μg / kg, about 4 μg / kg to about 15 μg / kg, about 5 μg / kg to about 18 μg / kg, about 6 μg / kg to about 20 μg / kg, about 7 μg / kg to about 22 μg / kg, about 8 μg / kg to about 25 μg / kg, about 9 μg / kg to about 28 μg / kg, about 10 μg / kg to about 30 μg / kg, about 12 μg / kg to about 35 μg / kg, about 14 μg / kg to about 40 μg / kg, about 16 μg / kg to about 45 μg / kg, about 18 μg / kg to about 50 μg / kg, about 20 μg / kg to about 55 μg / kg, about 25 μg / kg to aboutPHAR 00072 / PEC 029-PCT / P00139WO 60 μg / kg, about 30 μg / kg to about 70 μg / kg, about 40 μg / kg to about 85 μg / kg, about 50 μg / kg to about 100 μg / kg, about 60 μg / kg to about 115 μg / kg, about 70 μg / kg to about 130 μg / kg, about 80 μg / kg to about 145 μg / kg, about 90 μg / kg to about 160 μg / kg, about 100 μg / kg to about 180 μg / kg, about 120 μg / kg to about 200 μg / kg, about 140 μg / kg to about 220 μg / kg, about 160 μg / kg to about 240 μg / kg, about 180 μg / kg to about 260 μg / kg, about 200 μg / kg to about 280 μg / kg, or about 220 μg / kg to about 300 μg / kg. Even more intervals may include about 0.05 μg / kg to about 0.1 μg / kg, about 0.1 μg / kg to about 0.2 μg / kg, about 0.2 μg / kg to about 0.3 μg / kg, about 0.3 μg / kg to about 0.5 μg / kg, about 0.5 μg / kg to about 0.75 μg / kg, about 0.75 μg / kg to about 1 μg / kg, about 1 μg / kg to about 1.5 μg / kg, about 1.5 μg / kg to about 2 μg / kg, about 2 μg / kg to about 3 μg / kg, about 3 μg / kg to about 4 μg / kg, about 4 μg / kg to about 5 μg / kg, about 5 μg / kg to about 6 μg / kg, about 6 μg / kg to about 7 μg / kg, about 7 μg / kg to about 8 μg / kg, about 8 μg / kg to about 9 μg / kg, about 9 μg / kg to about 10 μg / kg, about 10 μg / kg to about 11 μg / kg, about 11 μg / kg to about 12 μg / kg, about 12 μg / kg to about 13 μg / kg, about 13 μg / kg to about 14 μg / kg, about 14 μg / kg to about 15 μg / kg, about 15 μg / kg to about 16 μg / kg, about 16 μg / kg to about 17 μg / kg, about 17 μg / kg to about 18 μg / kg, about 18 μg / kg to about 19 μg / kg, about 19 μg / kg to about 20 μg / kg, about 20 μg / kg to about 21 μg / kg, about 21 μg / kg to about 22 μg / kg, or continuing in 1 μg / kg increments up to about 300 μg / kg. Further, narrower ranges may include 5 μg / kg to 100 μg / kg, 10 μg / kg to 75 μg / kg, or 20 μg / kg to 50 μg / kg. Additionally, the dose may range from 0.05 μg / kg to 5 μg / kg, while higher efficacy doses may be between 100 μg / kg to 300 μg / kg. Any of the dosage amount above can be administered in the frequency of once every 1, 2, 3, 4, 5 days, or once every 1, 2, 3, 4, or 5 weeks.

[0176] In another example, the dose of the compound may range between about 0.01 to 10 μg / kg, between about 0.01 to 20 μg / kg, between about 3 to 30 μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 to 150 μg / kg. The dose of the compound may range from about 0.01 μg / kg to about 15 μg / kg, covering various subranges to accommodate different therapeutic applications. In some embodiments, the dose may be between about 0.01 μg / kg to about 14 μg / kg, about 0.02 μg / kg to about 13 μg / kg, about 0.03 μg / kg to about 12 μg / kg, about 0.4 μg / kg to about 11 μg / kg, about 0.5 μg / kg to about 10 μg / kg, about 0.75 μg / kg to about 9 μg / kg, about 1 μg / kg to about 8 μg / kg, about 1.5 μg / kg to about 7 μg / kg, about 2 μg / kg to about 6 μg / kg, about 2.5 μg / kg to about 5 μg / kg, about 3 μg / kg to about 4.5 μg / kg, about 3.5 μg / kg to about 4 μg / kg, and about 4 μg / kg to about 15 μg / kg. Further specific subranges may include about 0.05 μg / kg to about 0.1 μg / kg, about 0.1 μg / kg to about 0.2 μg / kg, about 0.2 μg / kg to about 0.3 μg / kg, about 0.3 μg / kg to about 0.4 μg / kg, about 0.4 μg / kg to about 0.5 μg / kg, about 0.5 μg / kg to about 0.6 μg / kg, about 0.6 μg / kg to about 0.7 μg / kg, about 0.7 μg / kg to about 0.8 μg / kg, about 0.8 μg / kg to about 0.9 μg / kg, about 0.9 μg / kg to about 1.0 μg / kg,PHAR 00072 / PEC 029-PCT / P00139WO about 1.0 μg / kg to about 1.1 μg / kg, about 1.1 μg / kg to about 1.2 μg / kg, about 1.2 μg / kg to about 1.3 μg / kg, about 1.3 μg / kg to about 1.4 μg / kg, about 1.4 μg / kg to about 1.5 μg / kg, about 1.5 μg / kg to about 1.6 μg / kg, about 1.6 μg / kg to about 1.7 μg / kg, about 1.7 μg / kg to about 1.8 μg / kg, about 1.8 μg / kg to about 1.9 μg / kg, about 1.9 μg / kg to about 2.0 μg / kg, about 2.0 μg / kg to about 2.1 μg / kg, about 2.1 μg / kg to about 2.2 μg / kg, about 2.2 μg / kg to about 2.3 μg / kg, about 2.3 μg / kg to about 2.4 μg / kg, about 2.4 μg / kg to about 2.5 μg / kg, about 2.5 μg / kg to about 2.6 μg / kg, about 2.6 μg / kg to about 2.7 μg / kg, about 2.7 μg / kg to about 2.8 μg / kg, about 2.8 μg / kg to about 2.9 μg / kg, about 2.9 μg / kg to about 3.0 μg / kg, about 3.0 μg / kg to about 3.1 μg / kg, about 3.1 μg / kg to about 3.2 μg / kg, about 3.2 μg / kg to about 3.3 μg / kg, about 3.3 μg / kg to about 3.4 μg / kg, about 3.4 μg / kg to about 3.5 μg / kg, about 3.5 μg / kg to about 3.6 μg / kg, about 3.6 μg / kg to about 3.7 μg / kg, about 3.7 μg / kg to about 3.8 μg / kg, about 3.8 μg / kg to about 3.9 μg / kg, about 3.9 μg / kg to about 4.0 μg / kg, about 4.0 μg / kg to about 4.2 μg / kg, about 4.2 μg / kg to about 4.4 μg / kg, about 4.4 μg / kg to about 4.6 μg / kg, about 4.6 μg / kg to about 4.8 μg / kg, about 4.8 μg / kg to about 5.0 μg / kg, about 5.0 μg / kg to about 6.0 μg / kg, about 6.0 μg / kg to about 7.0 μg / kg, about 7.0 μg / kg to about 8.0 μg / kg, about 8.0 μg / kg to about 9.0 μg / kg, about 9.0 μg / kg to about 10.0 μg / kg, about 10.0 μg / kg to about 11.0 μg / kg, about 11.0 μg / kg to about 12.0 μg / kg, about 12.0 μg / kg to about 13.0 μg / kg, about 13.0 μg / kg to about 14.0 μg / kg, or about 14.0 μg / kg to about 15.0 μg / kg. The dose of the compound may range from about 0.1 μg / kg to about 40 μg / kg, covering various subranges to accommodate different therapeutic applications. Any or all of the dosage amounts above can be administered in the frequency of once every 1, 2, 3, 4, 5 days, or once every 1, 2, 3, 4, or 5 weeks.

[0177] In some embodiments, the dose may be between about 0.01 μg / kg to about 39 μg / kg, about 0.1 μg / kg to about 38 μg / kg, about 0.5 μg / kg to about 35 μg / kg, about 0.8 μg / kg to about 30 μg / kg, about 3 μg / kg to about 25 μg / kg, or about 4 μg / kg to about 20 μg / kg. Additional refined ranges may include about 5 μg / kg to about 15 μg / kg, about 10 μg / kg to about 12 μg / kg, or about 0.1 μg / kg to about 5 μg / kg. Further subranges may include about 0.1 μg / kg to about 1 μg / kg, about 1 μg / kg to about 10 μg / kg, about 2 μg / kg to about 8 μg / kg, about 5 μg / kg to about 10 μg / kg, about 10 μg / kg to about 20 μg / kg, about 15 μg / kg to about 25 μg / kg, about 20 μg / kg to about 30 μg / kg, and about 30 μg / kg to about 40 μg / kg. Even more specific dosing intervals may include about 0.1 μg / kg to about 0.5 μg / kg, about 0.5 μg / kg to about 2 μg / kg, about 2 μg / kg to about 4 μg / kg, about 4 μg / kg to about 6 μg / kg, about 6 μg / kg to about 8 μg / kg, about 8 μg / kg to about 12 μg / kg, about 12 μg / kg to about 18 μg / kg, about 18 μg / kg to about 24 μg / kg, about 24 μg / kg to about 32 μg / kg, or about 32 μg / kg to about 38 μg / kg. Any of the dosage amount above can be administered in the frequency of once every 1, 2, 3, 4, 5 days, or once every 1, 2, 3, 4, or 5 weeks.PHAR 00072 / PEC 029-PCT / P00139WO

[0178] Yet in another embodiment, the dose may be between about 0.01 μg / kg to about 6 μg / kg, between about 0.03 μg / kg to about 6 μg / kg, between about 0.05 μg / kg to about 6 μg / kg, between about 0.1 μg / kg to about 6 μg / kg, between about 1.0 μg / kg to about 6 μg / kg, between about 0.01 μg / kg to about 5.5 μg / kg, between about 0.01 μg / kg to about 5.0 μg / kg, between about 0.01 μg / kg to about 4.5 μg / kg, between about 0.01 μg / kg to about 4.0 μg / kg, between about 0.05 μg / kg to about 4 μg / kg, between about 0.05 μg / kg to about 3.5 μg / kg, between about 0.05 μg / kg to about 3.0 μg / kg, between about 1.0 μg / kg to about 3.0 μg / kg, between about 1.0 μg / kg to about 2.5 μg / kg, between about 1.0 μg / kg to about 2.0 μg / kg, or between about 0.01 μg / kg to about 6 μg / kg. Any of the dosage amount above can be administered in the frequency of once every 1, 2, 3, 4, 5 days, or once every 1, 2, 3, 4, or 5 weeks.

[0179] In addition, the individual dose range may be from 0.01 μg / kg to 45 μg / kg. as a result the individual dose may be about 0.01, 0.04, 0.07, 0.1, 0.4, 0.7, 1.0, 1.3, 1.6, 1.9, 2.2, 2.5, 2.8, 3.1, 3.4, 3.7, 4.0, 4.3, 4.6, 4.9, 5.2, 5.5, 5.8, 6.1, 6.4, 6.7, 7.0, 7.3, 7.6, 7.9, 8.2, 8.5, 8.8, 9.1, 9.4, 9.7, 10.0, 10.3, 10.6, 10.9, 11.2, 11.5, 11.8, 12.1, 12.4, 12.7, 13.0, 13.3, 13.6, 13.9, 14.2, 14.5, 14.8, 15.1, 15.4, 15.7, 16.0, 16.3, 16.6, 16.9, 17.2, 17.5, 17.8, 18.1, 18.4, 18.7, 19.0, 19.3, 19.6, 19.9, 20.2, 20.5, 20.8,21.1, 21.4, 21.7, 22.0, 22.3, 22.6, 22.9, 23.2, 23.5, 23.8, 24.1, 24.4, 24.7, 25.0, 25.3, 25.6, 25.9, 26.2, 26.5, 26.8, 27.1, 27.4, 27.7, 28.0, 28.3, 28.6, 28.9, 29.2, 29.5, 29.8, 30.1, 30.4, 30.7, 31.0, 31.3, 31.6, 31.9, 32.2, 32.5, 32.8, 33.1, 33.4, 33.7, 34.0, 34.3, 34.6, 34.9, 35.2, 35.5, 35.8, 36.1, 36.4, 36.7, 37.0, 37.3, 37.6, 37.9, 38.2, 38.5, 38.8, 39.1, 39.4, 39.7, 40.0, 40.3, 40.6, 40.9, 41.2, 41.5, 41.8, 42.1, 42.4, 42.7, 43.0, 43.3, 43.6, 43.9, 44.2, 44.5, or 44.8 μg / kg. Although other individual dose range may be used, e.g., 45 μg / kg to 150 μg / kg, 150 μg / kg to 300 μg / kg or greater. Any of the dosage amount above can be administered in the frequency of once every 1, 2, 3, 4, 5 days, or once every 1, 2, 3, 4, or 5 weeks.

[0180] The compositions, mixture, and / or formulation provided herein are effective to restore homeostatic capacity of the immune system, e.g., have the ability to positively affect diseases in which Treg dysfunction plays a role such as autoimmune diseases, allergy, and graft rejection. In one embodiment, provided herein is a method for selectively expanding endogenous Treg in vivo by administering the selective Treg stimulator compositions, including PEG-IL-2 N2 or PEG-IL-2 N88D embodiments and related compositions, mixture, and / or formulation provided herein. Illustrative dosing ranges include for example, from about 0.01 μg / kg to 300 μg / kg, encompassing various subranges to ensure comprehensive coverage. For instance, in certain embodiments, the range may be about 0.01 μg / kg to 15 μg / kg, about 0.01 to 10μg / kg, between about 0.01 to 20μg / kg, betweenPHAR 00072 / PEC 029-PCT / P00139WO about 0.01-30 μg / kg, between about 3 to 30 μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 to 150 μg / kg, including any and all combinations of the foregoing beginning and ending values from each and every of the foregoing ranges. Additionally, ranges include between about 0.01 μg / kg to 250 μg / kg, about 0.5 μg / kg to 200 μg / kg, or about 1.0 μg / kg to 150 μg / kg. Further, ranges may include about 5 μg / kg to 100 μg / kg, about 10 μg / kg to 75 μg / kg, or about 20 μg / kg to 50 μg / kg. Other doses and dosing regimens are described in the examples provided herein. Suitable doses are effective to achieve a maximal amplification of Treg cells, with a minimal stimulation of Teff cells and NK cells; such can be monitored by collection of peripheral blood for flow cytometric analysis to identify the prevalence of Treg cells, effector CD4+ and CD8+ T cells, and NK cells. Based upon these numbers, dosages can be adjusted accordingly.

[0181] In an embodiment, dosage regimens and / or length may be adjusted to provide the optimum desired response (e.g., a therapeutic effect). Dosing schedules, for intravenous (i.v.) or non- intravenous administration, localized or systemic, or combinations thereof, typically range from a single bolus dosage or continuous infusion to multiple administrations per day (e.g., every 4-6 hours), or as indicated by a treating physician and the patient's condition. With regard to the frequency and schedule of administering the selective Treg stimulator compositions, including PEG-IL-2 N2 or PEG-IL-2 N88D and related composition, mixture, and / or formulation thereof embodiments and related compositions provided herein, one of ordinary skill in the art is able to determine an appropriate dosing regimen. For example, in a treatment cycle (frequency and / or total length), a clinician can decide to administer the composition, mixture, and / or formulation provided herein, either as a single dose or in a series of doses, e.g., over the course of several days or weeks). Based upon the long-acting nature of the composition, mixture, and / or formulation thereof, it can be typically administered relatively infrequently (e.g., once every three weeks, once every two weeks, once every 8-10 days, once every 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; aboutPHAR 00072 / PEC 029-PCT / P00139WO twenty-three months; about twenty-four months; about thirty months; about three years; about four years and about five years. The treatment methods described herein are typically continued for as long as the clinician overseeing the patient's care deems the treatment method to be effective, i.e., that the patient is responding to treatment, or until related symptoms of the condition subside. Non- limiting parameters that indicate the treatment method is effective may include one or more of the following: increased numbers of regulatory T cells such as CD25+ Treg and FoxP3+ Treg, and / or decreased numbers of NK cells and CD4+ and CD8+ effector cells.

[0182] The compositions, mixture, and / or formulation thereof provided herein increase the ratio of regulatory T cells, such as Foxp3+ and CD25+ cells, to effector T cells, such as CD4+ and CD8+ cells, when administered to a subject at a therapeutically effective dose. For example, administration of the selective Treg stimulator compositions, including PEG-IL-2 N2 or PEG-IL-2 N88D and related compositions, mixture, and / or formulation thereof provided herein, may be effective to result in at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100-fold or more increase in regulatory T cells, when compared to baseline and evaluated in an in-vivo mouse model, e.g., such as described herein. The method may also, in some embodiments, be effective to result in at least a four- fold-increase in regulatory T cells, when compared to baseline and evaluated in an in-vivo mouse model and / or subject or subject in need, e.g., such as described herein. In some instances, the increase in regulatory T cell numbers is sustained above baseline levels for at least about 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14,15 days or longer post-administration.

[0183] As shown in the accompanying examples, the selective Treg stimulator compositions, including PEG-IL-2 N2 or PEG-IL-2 N88D embodiments and related compositions, mixture, and / or formulation thereof provided herein, when administered within a suitable dosage range, are effective to preferentially increase the cell population and immune-suppressive function of regulatory T cells while having minimal stimulatory effect on T effector cells. In certain embodiments, the selective Treg stimulator compositions, including PEG-IL-2 N2 or PEG-IL-2 N88D embodiments and related compositions, mixture, and / or formulation thereof provided herein, are capable of achieving a sustained exposure for providing a magnitude, duration and specificity of Treg to Teff responses that cannot be attained with equivalent doses of IL-2.

[0184] In an embodiment, the present disclosure also includes using PEG-IL-2 N2 or PEG-IL-2 N88D and related composition, mixture, and / or formulation thereof in combination with Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR- 214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194),PHAR 00072 / PEC 029-PCT / P00139WO CUG252, ALM223 (SIM0278), and / or aldesleukin. Furthermore, any of the Treg expansion or other comparative value can be measured using a subject in need post administration of Cyclosporin A, Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin as a baseline.

[0185] Surprising and unexpected Results

[0186] At the time of the filing of this disclosure, there is only one altered IL-2 molecules used and approved for oncology, and none for immune related diseases. A brief survey showed there are about 7 altered IL-2 (that are different than the present disclosure’s compositions and / or mixture thereof) related molecules in clinical trials undertaken by various companies in various stages of development for immune therapy, with the most advanced being NKTR-358 (REZPEG, trademarked). Others such as SAR444336, also known as THOR-809 by Sanofi, AMG-592 by Amgen (clinical trial terminated), XmAb564, PT101 / MK-6194, CUG252, ALM223, RG7835 (trial terminated), DEL106 (trial terminated), or BMS-986326 are either terminated due to lack of efficacy or have safety concerns, with NKTR-358 being the most advanced in phase 2; however, the data according to public records, are inconclusive relating to treating immune related disorders, based on dispute between two collaborated companies.

[0187] The present disclosure, as denoted in below examples and throughout the disclosure, on the other hand, demonstrates superiority against the most advanced NKTR-358. NKTR-358 is a heterogenous mixture of mono, di-, tri- and tetra-PEGylated IL-2 molecules based on the aldesleukin sequence. In contrast, PEG IL-2 N2 and PEG IL-2 N88D are homogenous, mono-PEGylated IL-2 molecules. PEG IL-2 N2 has a surprisingly small reduction in potency while maintaining an incredibly high selectivity for Treg over CD8 T cells. For example, the potency of PEG IL-2 N2 on Treg is less than 100-fold reduced compared to IL-2 N2 and the selectivity for Treg over CD8 T cells is 3,365-fold.

[0188] These surprising and unexpected results are also seen in non-human primate (NHP) PBMCs. For example, in non-human primate, NKTR-358 exhibits 4655-fold decreased potency on Treg compared to IL-2 with only 108-fold selectivity for Treg over CD8 T cells. In contrast, PEG IL-2 N2 exhibits only 44-fold decreased potency on Treg compared to IL-2 and 1,114-fold selectivity for Treg over CD8-positive T cells. Again PEG IL-2 N2 has a surprisingly small reduction in potency while maintaining an incredibly high selectivity for Treg over CD8 T cells. Similar superiority can be observed in non-human primate where PEG IL-2 N2 selectively expanded Treg (12-fold)PHAR 00072 / PEC 029-PCT / P00139WO (comparable to NKTR-385 at the same dose, 25 μg / kg) with minimal effects on CD8 T cells or NK cells. Moreover, in an NHP dose-range finding study, PEG IL-2 N2 unexpectedly induced greater numbers of blood Treg compared to NKTR-358 when dosed at a similar level.

[0189] Overall, PEG IL-2 is a homogenous, mono-PEGylated IL-2 molecule with surprisingly and unexpectedly superior potency and selectivity on human Treg and Treg expansion compared to NKTR-358.

[0190] Examples

[0191] It is to be understood that the foregoing description as well as the examples that follow are intended to illustrate and not limit the scope of the disclosure(s) provided herein. Other aspects, advantages and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.

[0192] Example 1 Preparation of PEG-IL2 N2 or PEG-IL2 N88D protein

[0193] Cloning of IL2 (aldesleukin (N2) or with N88D mutation) protein was performed in E. Coli BLR cells in pET24 expression vector encoding DNA sequences expressing IL2 protein. The cells were used in fermentation, in brief: IL-2 N2 or IL2 N88D was prepared based on recombinant E.coli cellular fermentation to obtain the IL-2s protein then followed a PEGylation reaction to obtain PEGylated protein. Briefly, the IL-2 expressing cells were inoculated in medium and incubated for between about 15-17 hours, then the seed culture was transferred to a 5L fermenter for cell growth. Before cell growing to stationary phase, an IPTG solution was added to induce the target protein expression. At the end of fermentation, the cells were harvested, centrifuged, and stored at -20°C. To purify protein, the cells were thawed and homogenized to collect inclusion bodies which were then solubilized and refolded. The crude solution was flowed through the depth filtration, then concentrated and buffer exchanged. The CM chromatography column was used to purify the IL-2 proteins. The protein then was filtered through 0.2μm filter. The reaction mixture was then purified through a Sepharose cationic column. The purified fractions, containing mono-pegylated IL-2, were collected and buffer exchanged to formulation buffer by a Tangential Flow Filtration system.

[0194] Example 2 Methionine Removal

[0195] Protein synthesis is initiated by Methionine in eukaryotes and formylmethionine in prokaryotes. N-terminal methionine can be co-translationally cleaved by the enzyme methionine aminopeptidase (MAP). When recombinant proteins are expressed in bacterial cells and mammalianPHAR 00072 / PEC 029-PCT / P00139WO expression systems there is a simple universal rule that predicts whether the initiating methionine will be processed by MAP based on the size of the residue adjacent (penultimate) to the N-terminal methionine.

[0196] There are various methods of removing the N-terminal methionine known in the art. Examples include Chemical methods (CNBr); In-vivo cleavage of Met : (1) Signal peptides: potential heterogenicity in N-terminal (examples: Nutropin and Genotropin). (2) MAP (methionine amino peptidase): it is requisite to rationally design the amino acid adjacent to the N terminal-Met (gyration radius dependent); In-vitro cleavage of Met: (1) Factor Xa: issues of non-specific cleavage. (2) DAPI: such as Humatrope and Norditropin. (3) MAP / AP: such as Zomacton.

[0197] For example, U.S. Patent number 5,565,349, entitled “Dictyostelium dipeptidylaminopeptidase” discloses methods for using the dDAP enzyme to remove dipeptides from the N-terminus of recombinantly produced precursor proteins or peptides. U.S. Patent number 4,755,465, entitled “Secretion of correctly processed human growth hormone in E. coli and Pseudomonas,” discloses production of mature hGH in E. coli and Pseudomonas strains transformed by a plasmid which encodes pre hGH and processing the pre-hGH to cleave the signal sequence and, thereby, production of mature hGH. U.S. Patent number 8,765,411, entitled “Process for production of recombinant human growth hormone from E. coli cells” discloses an improved process for the production of human growth hormone.

[0198] Example 3 Physicochemical characteristics

[0199] The physicochemical characteristics of PEG-IL-2 N2 and PEG-IL-2 N88D were disclosed herein. This includes the primary sequence and peptide mapping (Tryptic) and intact mass analysis for N-terminal heterogeneity (for IL-2 N2 protein). Peptide mapping was used to identify the PEG- IL-2 protein sample. Peptides were generated by digesting with endoproteinase Tryptic and separated in an RP-HPLC C18 column to generate Tryptic map which was analyzed by on-line LC-MS / MS. FIGURE 2A shows the reduced Tryptic Peptide mapping of IL-2. FIGURE 2B shows the reduced Tryptic Peptide mapping of PEG-IL-2. Two cystine residues (residue 58 &105) of the IL-2 form a disulfide bond. After PEGylation the disulfide bond was maintained, as indicated by signals of the T10a‐s‐s‐T13, T10‐s‐s‐T13, T10‐s‐s‐T13‐14, etc. After PEGylation, only the T1 signal significantly reduced, indicating that PEGylation was attached onto the N terminus, as indicated by the signal of the PEG‐peptide. IL-2 maintains alpha helix in secondary structure (far UV CD) and was folded intoPHAR 00072 / PEC 029-PCT / P00139WO a tertiary structure, as indicated in the near UV CD spectra (not shown). The structure of IL2 was maintained in PEG-IL-2 N2 and PEG-IL-2 N88D.

[0200] Peptides identified by peptide full mass analysis followed by sequence verification with MS / MS of the respective peptides, served as a peptide map for confirmation of PEG-IL-2 identity. The method of peptide mapping is known in the art. Table 1 below showed the masses and peptide assignment from reduced the Tryptic peptide map of IL-2 and PEG-IL-2. Table 1:

[0201] Molecular mass analysis was performed via MALDI-TOF MS Analysis. FIGURE 3 showed the intact mass analysis of IL-2 N2. FIGURE 4A denoted RP-HPLC results while determining molecular mass for PEG-IL-2 N2. The m / z and the number of charges were indicated. The PEG-IL- 2 N2 was analyzed by an MALDI-TOF MS FIGURE 4B. The transformed mass spectrum showed a predominant product centered at about 57,356 m / z. Since the mass of IL-2 N2 protein was determined to be 15,328.9 Da, this result indicated that PEG-IL-2 was mono-PEGylated.PHAR 00072 / PEC 029-PCT / P00139WO

[0202] FIGURE 5A and FIGURE 5B showed an image of an SDS-PAGE analysis of PEG-IL-2 N2 with non-reduced silver stain and reduced silver stain. SDS-PAGE followed by silver staining were performed using methods known in the art on PEG-IL-2 N2 compared with marker proteins. SDS- PAGE gel showing PEG-IL-2 samples under reducing and non-reducing conditions, visualized using silver staining. Lane 1 contains molecular weight marker proteins (30X, 2 μL). Lanes 2–4 and 6-7 represent PEG-IL-2 N2 samples in non-reducing / reducing conditions at concentrations of 100% (Lane 2), 1% (Lane 3), 1% (Lane 6), and 400% (Lane 7). The electrophoretic analyses of PEG-IL-2 N2 showed a major band with a molecular size close to 110 kDa instead of 59 kDa determined by MALDI-TOF MS. A larger molecular weight observed during SDS-PAGE due to the contribution of the PEG moiety. The bulky, hydrated PEG can lead to slower migration of the conjugated product compared to the non-PEGylated molecule on SDS-PAGE.

[0203] Example 4 In-Vitro NHP P-STAT5 Assay

[0204] Non-human Primate PBMC P-STAT5 assay. This example examined signaling downstream of the IL-2 receptor (P-STAT5) in non-human primate (NHP) PBMCs treated with IL-2 and PEG-IL-2 N2. A custom lymphocyte flow cytometry was employed to delineate specific lymphocyte populations including Treg, CD8, CD4 and NK cells. The potencies (EC50s) of IL-2, PEG-IL-2 N2 and PEG-IL-2 N88D were then determined in each of these immune cell types. For IL-2 N2 and PEG IL-2 N2, multiple batches of each test article were tested using PBMCs from up to 6 different healthy donors.

[0205] FIGURE 6A showed IL-2 N2 induced STAT5 phosphorylation in Treg cells with 316.1-fold selectivity (greater potency) over Tcon, 257-fold selectivity over CD8 T cells and 18-fold selectivity over NK cells. Table 2 showed Ec50 and IL2-N2 selectivity. EC50s (ng / mL) IL-2 N2 Selectivity Ratios Treg 0.032

[0206] FIGURE 6B shows PEG-IL-2 N2 exhibited improved selectivity for Treg over other immune cell types compared to IL-2 (FIGURE 6A). PEG-IL-2 N2 is 2,939-fold more selective (greater potency) for Treg over Tcon, 1,114-fold more selective for Treg over CD8 T cells and 164-fold more selective for Treg over NK cells. The potency of PEG-IL-2 N2 on Treg is only 44-fold decreased compared to IL-2 N2 (FIGURE 6A). Table 3 showed EC50 and PEG IL2 N2 selectivity.PHAR 00072 / PEC 029-PCT / P00139WO Table 3 EC50s (ng / mL) PEG IL-2 N2 Selectivity Ratios Treg 1.40 T 41147 T T 2939

[0207] In primary NHP cells, PEG-IL-2 N2 exhibited increased selectivity for Treg over other immune cell types compared to IL-2, but with 44-fold decreased potency on Treg as compared to IL- 2.

[0208] Example 5 In-Vitro human PBMC P-STAT5 assay

[0209] IL-2 human PBMC P-STAT5 assay. This example examined signaling downstream of the IL-2 receptor (P-STAT5) in healthy volunteer human PBMCs treated with IL-2 N2, PEG-IL-2 N2, and PEG-IL-2 N88D. A custom lymphocyte flow cytometry was employed to delineate specific lymphocyte populations including Treg, CD8, CD4 and NK cells. The potencies (EC50s) of IL-2 N2, PEG-IL-2 N2, and PEG-IL-2 N88D were then determined in each of these immune cell types.

[0210] FIGURE 7A shows IL-2 N2 induced STAT5 phosphorylation in Tregs with 150-fold selectivity (greater potency) over Tcon, 1,682-fold selectivity over CD8-positive T cells and 796-fold selectivity over NK cells. Table 4 showed the details: EC50s (ng / mL) IL-2 N2 Selectivity Ratios Treg 0.003

[0211] FIGURE 7B shows the P-STAT5 assay for PEG-IL-2 N2. PEG-IL-2 N2 exhibited improved selectivity for Treg over other immune cell types compared to IL-2 (FIGURE 7A). PEG-IL-2 N2 was 318-fold more selective (greater potency) for Treg over Tcon, 3,365-fold more selective for Treg over CD8-positive T cells and 1,917-fold more selective for Treg over NK cells. The potency of PEG- IL-2 N2 on Treg was only 87.2 fold decreased compared to IL-2 N2 (FIGURE 7A). Table 5 EC50s (ng / mL) PEG IL-2 N2 Selectivity RatiosPHAR 00072 / PEC 029-PCT / P00139WO Tcon 75.007 Treg / Tcon 318 CD8pos 794.076 Treg / CD8 3,365

[0212] FIGURE 7C shows the P-STAT5 assay for PEG-IL-2 N88D. PEG-IL-2 N88D exhibited improved selectivity for Treg over other immune cell types compared to IL-2 N2 (FIGURE 7A). PEG-IL-2 N88D had minimal activity / potency on Tcon, CD8pos T cells or NK cells. However, the potency of PEGylated IL-2 N88D on Treg was more than 15,705 fold reduced compared to IL-2 N2 (FIGURE 7A). Treg EC50 was 42.5ng / mL.

[0213] In primary human cells, PEG-IL-2 N2 exhibited increased selectivity for Treg over other immune cell types compared to IL-2, but with 87.2- fold decreased potency on Treg compared to IL- 2 N2. PEG-IL-2 N88D exhibited even greater selectivity for Treg over other immune cell types, but with 15,705 fold decreased potency on Treg compared to IL-2 N2.

[0214] Example 6 In-Vitro Non-human primate PBMC P-STAT5 Assay with NKTR-358

[0215] For FIGURES 8A-8C, the term rhIL-2 is defined as Aldesleukin by Nektar’s publication. More details can be found in U.S. Patent Application Serial Number US20210205413A1, which is incorporated herein by reference in its entirety. FIGURE 8A denoted percentage of P-STAT5-positive cells in CD3⁺ T cell in humans in response to increasing concentrations of rhIL-2 (recombinant human IL-2) and NKTR-358. FIGURE 8B denoted percentage of P-STAT5-positive cells in CD3⁺CD4⁺CD25⁺ Tregs (Regulatory T Cells) in humans in response to increasing concentrations of rhIL-2and NKTR-358. FIGURE 8C denoted percentage of P-STAT5-positive cells in CD3⁺CD8⁺ T Cells in humans in response to increasing concentrations of rhIL-2 (aldesleukin) and NKTR-358. FIGURE 8D is a table of data from PBMC P-STAT5 Assay with NKTR-358 for human and Non- human Primate.

[0216] In primary NHP cells, NKTR-358 exhibited 4655-fold decreased potency on Treg compared to aldesleukin (NKTR published data below). In comparison PEG-IL-2 N2 only exhibited 44-fold decreased potency on Treg compared to IL-2 and therefore has superior Treg potency compared to NKTR-358. NKTR-358 only exhibited 108-fold selectivity for Treg over CD8-positive T cells, while PEG-IL-2 N2 exhibited 1,114-fold selectivity for Treg over CD8-positive T cells. PEG-IL-2 N2 therefore exhibits superior Treg potency and selectivity on primary NHP cells in vitro.

[0217] Example 7 In-Vitro Human PBMC P-STAT5 Assay with NKTR-358PHAR 00072 / PEC 029-PCT / P00139WO

[0218] In primary human cells, NKTR-358 exhibited 7475-fold decreased potency on Treg compared to IL-2 (NKTR published data below). In comparison PEG-IL-2 N2 only exhibited 87.2-fold decreased potency on Treg compared to IL-2 and therefore has superior Treg potency compared to NKTR-358. NKTR-358 only exhibited 32-fold selectivity for Treg over CD8-positive T cells, while PEG-IL-2 N2 exhibited 3,365-fold selectivity for Treg over CD8-positive T cells. PEG-IL-2 N2 therefore exhibited superior Treg potency and selectivity on primary human cells in vitro.

[0219] Example 8 In-Vitro mouse splenocyte P-STAT5 assay.

[0220] IL-2 mouse splenocyte P-STAT5 assay. One purpose of this example was to examine signaling downstream of the IL-2 receptor (P-STAT5) in freshly isolated mouse splenocytes treated with IL-2, PEG-IL-2 N2 and PEG-IL-2 N88D. A custom lymphocyte flow cytometry panel was employed to delineate specific lymphocyte populations including Treg, CD8, CD4 and NK cells. The potencies (EC50s) of IL-2, PEG-IL-2 N2 and PEG-IL-2 N88D were then determined in each of these immune cell types.

[0221] FIGURE 9A shows the P-STAT5 assay for IL-2 N2. IL-2 N2 induced STAT5 phosphorylation in mouse splenic Tregs with 46,303-fold selectivity (greater potency) over CD8-positive T cells, 9,044- fold selectivity over NK cells and minimal activity on Tcon as shown in Table 6. Table 6 EC50s (ng / mL) IL-2 N2 Selectivity Ratios Treg 0.005

[0222] FIGURE 9B shows the P-STAT5 assay for PEG-IL-2 N2. PEG-IL-2 N2 exhibited improved selectivity for mouse splenic Treg over other immune cell types compared to IL-2 (FIGURE 9A). PEG-IL-2 N2 had minimal activity on Tcon, CD8-positive T cells or NK cells. The potency of PEG- IL-2 N2 on mouse splenic Treg (0.628ng / mL) is 131-fold decreased compared to IL-2 N2 (FIGURE 9A).

[0223] FIGURE 9C shows the P-STAT5 assay for PEG-IL-2 N88D. PEG-IL-2 N88D exhibits improved selectivity for mouse splenic Treg over other immune cell types compared to IL-2 (FIGURE 9A). PEG-IL-2 N88D has minimal activity / potency on Tcon, CD8pos T cells or NK cells.PHAR 00072 / PEC 029-PCT / P00139WO However, the potency of PEG IL-2 N88D on mouse splenic Treg is 1,884-fold reduced compared to IL-2 N2 (FIGURE 9A). Table 7 EC50s (ng / mL) Treg 0.003 T 44

[0224] In primary mouse splenocytes, PEG-IL-2 N2 exhibited increased selectivity for Treg over other immune cell types compared to IL-2, but with 131-fold decreased potency on Treg compared to IL-2. PEG-IL-2 N88D exhibited even greater selectivity for Treg over other immune cell types, but with 1,884-fold decreased potency on Treg compared to IL-2.

[0225] Example 9 In-Vitro rat splenocyte P-STAT5 assay

[0226] IL-2 rat splenocyte P-STAT5 assay. This example examines signaling downstream of the IL- 2 receptor (P-STAT5) in freshly isolated rat splenocytes treated with IL-2 N2, PEG IL-2 N2, and PEG IL-2 N88D. A custom lymphocyte flow cytometry was employed to delineate specific lymphocyte populations including Treg, CD8, CD4 and NK cells. The potencies (EC50s) of IL-2, PEG-IL-2 N2 and PEG-IL-2 N88D were determined in each of these immune cell types. The concentrations of the tested materials were as follows: IL-2 (N2) at 4.30 mg / mL, PEG IL-2 N88D at 1.38 mg / mL, and PEG IL-2 (N2) at 2.70 mg / mL.

[0227] FIGURE 9D shows IL-2 N2 induced STAT5 phosphorylation in rat splenic Tregs with 11,294-fold selectivity (greater potency) over CD8-positive T cells, 26,753-fold selectivity over NK cells and minimal activity on Tcon.

[0228] FIGURE 9E shows PEG IL-2 N2 exhibited improved selectivity for rat splenic Treg over other immune cell types compared to non-PEGylated IL-2 as seen in FIGURE 9D. PEG IL-2 N2 had minimal activity on Tcon, CD8-positive T cells or NK cells. The potency of PEG IL-2 N2 on rat splenic Treg was 524-fold decreased compared to non-PEG IL-2 N2 as seen in FIGURE 9D.

[0229] FIGURE 9F shows PEG IL-2 N88D exhibited improved selectivity for rat splenic Treg over other immune cell types compared to IL-2 as seen in FIGURE 9D. PEG IL-2 N88D had minimal activity / potency on Tcon, CD8-positive T cells or NK cells. However, the potency of PEG IL-2PHAR 00072 / PEC 029-PCT / P00139WO N88D on rat splenic Treg was 27,197-fold reduced compared to non-PEGylated IL-2 N2 as seen in FIGURE 9D.

[0230] In primary rat splenocytes, PEG IL-2 N2 exhibited increased selectivity for Treg over other immune cell types compared to IL-2 N2, but with 524-fold decreased potency on Treg compared to IL-2 N2. PEG IL-2 N88D exhibited even greater selectivity for Treg over other immune cell types, but with 27,197-fold decreased potency on Treg compared to non-PEG IL-2 N2.

[0231] Example 10 In-Vitro Treg Suppression Assay

[0232] In-vitro suppression assays were carried out in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.5 μM β-mercaptoethanol, and 1× antibiotic / antimycotic.5×104Tcon were stimulated with beads coated with anti-CD3 and anti-CD28 (T Cell Activation / Expansion kit, mouse, Miltenyi Biotec) at a ratio of about 2 beads to each Tcon in 100 μL culture medium in 96-well round-bottom plates. The suppressive capacity of Tregs was assessed by the addition of Tregs to Tcon at different ratios (Treg:Tcon ratios of 2:1 to 1:512). Each Treg:Tcon ratio was tested in triplicate. Cells were co-cultured for between about 48-72 hours at between about 36-37° C. and between about 4-5% CO2in a humidified atmosphere; between about 12-16 hours prior to the termination of the assay, between about 0.3-0.5 μCi [3H]-thymidine was added to wells. After washing cells free from 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 maximal proliferation by dividing by the mean CPM recorded for the four lowest Treg:Tcon dilutions. Concentration-response curves were graphed using four-parameter non-linear regression and 1 / y2 weighting in Prism (trademarked) 6.03 (GraphPad Software, San Diego, Calif.).

[0233] Splenic Treg's isolated from vehicle treated mice at 1 and 4 days following the study initiation exhibited suppressive capacity with the greatest suppression occurring at a ratio of 1:2. However, Treg's isolated at these time points following administration of a PEG-IL-2 N2 or PEG-IL-2 N88D composition exhibited a greatly increased suppressive capacity, as evidenced by decreased Tcon proliferation, particularly at ratios greater than 1:8. The relative suppressive capacity of isolated Treg's cultured with Tcon at a ratio of 1:2 was also assessed over time. Following PEG-IL-2 N2 or PEG-IL-2 N88D administration, increased Treg suppressive activity was maintained for four days before returning to baseline activity exhibited by the vehicle control-treated group.PHAR 00072 / PEC 029-PCT / P00139WO

[0234] Example 11 In-Vitro Human Frozen PBMC Cytokine Release assay

[0235] Human Frozen PBMC Cytokine Release assay. This example examined the levels of pro- inflammatory cytokines produced by cryo-preserved human PBMCs from four different donors treated with non-PEGylated IL-2, PEG-IL-2 N2, PEG-IL-2 N88D, and the FDA-approved IL-2 therapeutic, ProLeukin. A custom cytokine release assay was employed to measure pro-inflammatory cytokine release elicited by the candidates / therapeutics 48 hours post-treatment. PHA was used as positive control to stimulate T cell cytokine production downstream of the T cell receptor along with total human IgG as a respective isotype negative control.

[0236] FIGURE 10A shows a Human Frozen PBMC Cytokine Release assay-IFN-gamma. Dose- dependent IFN-gamma release was observed across all treatment groups, IL-2 N2, PEG-IL-2 N2, PEG-IL-2 N88D, and ProLeukin. PEG-IL-2 N2 elicited less IFN-gamma release at the three lower concentrations tested (1000, 100 and 10 pg / mL) compared to IL-2 N2, IL-2 N88D, and ProLeukin. In addition, PEG-IL-2 N88D elicited lower IFN-gamma release compared to IL-N2 at all concentrations tested.

[0237] FIGURE 10B shows a Human Frozen PBMC Cytokine Release assay TNFα. FIGURE 10B PEG IL-2 N2, and PEG IL-2 N88D induced low < 10 pg / mL, dose-dependent TNFα release. TNFα release elicited by both molecules is comparable or slightly lower than levels induced by IL-2 N2 and ProLeukin.

[0238] FIGURE 10C shows a Human Frozen PBMC Cytokine Release assay IL-1β. Dose-dependent IL-1β release was observed across all treatment groups. PEG-IL-2 N2 and PEG-IL-2 N88D elicited lower levels of IL-1β release compared to IL-2 N2 and ProLeukin. At the highest concentration tested, IL-2 N2 and ProLeukin induced IL-1β more strongly than PHA positive control.

[0239] FIGURE 10D shows a Human Frozen PBMC Cytokine Release assay IL-6. PEG-IL-2 N2 and PEG-IL-2 N88D had minimal effects on IL-6 release, similar to IL-2 N2 and ProLeukin. At the highest concentration tested, ProLeukinincluded IL-6 production by PBMCs from 2 / 4 donors.

[0240] In primary human PBMCs from four separate donors, PEG-IL-2 N2 and PEG-IL-2 N88D elicited comparable (TNFα, IL-6) or lower (IFNγ, IL-1β) levels of cytokine production after 48 hours of treatment. For some cytokines (IL-6), there was donor-to-donor variability with cytokine production only elicited by the highest concentration of ProLeukinin PBMCs from 2 / 4 donors.PHAR 00072 / PEC 029-PCT / P00139WO

[0241] Example 12 In-Vivo Mouse PK / PD Study

[0242] The example measured the pharmacokinetics of PEG-IL-2 N2 and PEG-IL-2 N88D compared to IL-2 in Balb / c mice (N = 6 per dose group) following a single subcutaneous dose. Control mice (N = 4) were administered as a vehicle alone. At the indicated time points, blood was collected via tail nick (in-life) or via cardiac puncture at the end of the study (7 days) following humane euthanasia. Heparinized whole blood was then spun down to plasma and stored at -80°C until later measurement of test article concentrations using a human IL-2 V-plex MSD assay per the manufacturer’s instructions. In the example IL-2 (N2) had a stock concentration of 4.30 mg / mL, PEG IL-2 (N2) had a stock concentration of 2.70 mg / mL, and PEG IL-2 N88D had a stock concentration of 1.38 mg / mL.

[0243] FIGURE 11A showed PEGylation increased the half-life, Tmax and AUC∞ of 40K PEG-IL- 2 N2 and 40K PEG-IL-2 N88D compared to IL-2 N2 following a single subcutaneous dose in Balb / c mice. PEG IL-2 N2 had a half-life of 25 hours, Tmax of 18 hours and AUC∞ of 74014.5 hr*ng / mL. PEG IL-2 N88D had a half-life of 11.9 hours, Tmax of 8 hours and AUC∞ of 95428.0 hr*ng / mL. The data is summarized in Table 8 below: Parameters Half_life Tmax Cmax AUC∞ Vd / F Cl / F (hr*ng kg) 6

[0244] Example 13 In Vivo Mouse PK Comparison of NKTR-358

[0245] NKTR-358 Comparison to PEG-IL-2 N2 and PEG-IL-2 N88D: FIGURE 11B and FIGURE 11C are plots of Treg function and activity as measured by the mean fluorescence intensity (MFI) of CD25 and Foxp3 following administration of a single-dose of NKTR-358 in mice. FIGURE 11B and FIGURE 11C are FIGs. 5A and 5B in United Stated Patent Application Number 17 / 056,050 (Publication Number 2021 / 0205413), the content of which are incorporated herein by reference in their entirety. The Table 9 below (Table 11 in United Stated Patent Application Number 17 / 056,050 (Publication Number 2021 / 0205413, wherein RUR IL-2 is NKTR-385)) summarizes the NKTR-358 pharmacokinetic parameters. Table 9PHAR 00072 / PEC 029-PCT / P00139WO, ug / mL), Tmax (18 hours versus 24 hours) and AUC∞ (74.1 hr*ug / mL versus 245 hr*ug / mL) compared to NKTR-358 following a single subcutaneous 0.3 mpk dose in mice as seen in FIGURE 11B and FIGURE 11C.

[0247] Example 14 In-Vivo Mouse PD Dose Response

[0248] This example was to measure the expansion and activation of regulatory T cells in Balb / c mice (N = 5 per dose group) administered a single subcutaneous dose of PEG-IL-2 N2 and PEG-IL- 2 N88D. Control mice were either administered as a single dose of vehicle or three daily doses of IL-2. Three days after administration of PEG-IL-2 N2 and PEG-IL-2 N88D, mice were sacrificed and whole blood was collected for downstream assessment of immune cell subsets by flow cytometry. Immune cell subsets were identified by a combination of surface and intracellular markers. The stock concentrations of the tested materials are as follows: IL-2 (N2) at 4.30 mg / mL, PEG IL-2 N88D at 1.38 mg / mL, and PEG IL-2 (N2) at 2.70 mg / mL.

[0249] FIGURES 12A-12C showed the expansion and activation of regulatory T cells in mice. Compared to IL-2, PEG-IL-2 N2 and PEG-IL-2 N88D increased the frequency of blood Treg in mice three days after a single subcutaneous dose with limited effects on other immune cell subsets.

[0250] FIGURES 12D-12G compared to IL-2, PEG-IL-2 N2 and PEG-IL-2 N88D dose-dependently and selectively increased Treg functional markers CD25 and FOXP3 on blood Treg in mice three days following a single subcutaneous dose.PHAR 00072 / PEC 029-PCT / P00139WO

[0251] FIGURES 12H-12J compared to IL-2, PEG-IL-2 N2 and PEG-IL-2 N88D increased blood Treg proliferation and functional marker expression in mice three days following a single subcutaneous dose. IL-2 increased PD-1 expression on blood Treg.

[0252] PEG-IL-2 N2 and PEG-IL-2 N88D selectively increased the frequency and number of mouse blood Treg up to (at the 0.3 mpk dose level) 14- to 17-fold three days after a single subcutaneous dose compared to only 3-fold after 3 daily doses of IL-2. PEG-IL-2 N2 also dose-dependently and selectively induced blood Treg when tested at 0.3, 0.1 and 0.03 mpk (data not shown). Blood Treg in mice dosed with PEG-IL-2 N2 and PEG-IL-2 N88D were also highly proliferative as indicated by Ki67 staining and exhibit increased expression of Treg functional markers including CD25, FOXP3, ICOS and PD- 1. With the exception of PD-1 expression, PEG-IL-2 N2 and PEG-IL-2 N88D elicited greater effects on Treg proliferation and functional markers compared to IL-2. PEGylated IL-2 effects on the critical Treg marker such as CD25 were also largely restricted to Treg versus CD25pos CD4 and CD8 T cells, further demonstrating the selectivity of these molecules.

[0253] Example 15 In-Vivo NKTR-358 Mouse PD Dose Response

[0254] NKTR-358 Comparison: PEG-IL-2 N2 selectively increased the number (FIGURE 12A-12C) and frequency (data not shown) of blood Treg in mice following a single subcutaneous dose. The magnitude of Treg expansion with PEG-IL-2 N2 and PEG-IL-2 N88D (14- to 17-fold at the 0.3 mpk dose level) was similar to that reported using the highest dose of NKTR-358 (0.3 mpk - 22-fold). PEG-IL-2 N2 and PEG-IL-2 N88D induced critical Treg markers CD25 and FOXP3 (FIGURES 12D- 12G) and the effects on CD25 were largely selective for Treg compared to other CD25pos immune cells. PEG-IL-2 N2 and PEG-IL-2 N88D robustly induced Treg proliferation (>90% Ki67pos) (FIGURES 12H-12J). While NKTR-358 induces ICOS staining in ~40% of Treg, PEG-IL-2 N2 and PEG-IL-2 N88D induced ICOS in ~60 – 70% of Treg (FIGURES 12H-12J).

[0255] FIGURES 12K-12N, 11B and 11C, 14A and 14B showed Mouse PD Time Course data for NKTR-358.

[0256] FIGURE 12K is a plot demonstrating the results of a pharmacodynamic analysis of mouse Tregs in blood following administration of a single-dose of NKTR-358 in mice. FIGURE 12K is FIG. 3A in United Stated Patent Application Number 17 / 056,050 (Publication Number 2021 / 0205413).PHAR 00072 / PEC 029-PCT / P00139WO

[0257] FIGURES 12L-12N are plots showing levels of NK cells, CD4 T cells, and CD8 T cells, respectively, in blood, following administration of a single-dose of NKTR-358 in mice. FIGURE 12L-12N are FIGS. 4A-4C in United Stated Patent Application Number 17 / 056,050 (Publication Number 2021 / 0205413).

[0258] FIGURES 11B and 11C are plots of Treg function and activity as measured by the mean fluorescence intensity (MFI) of CD25 and Foxp3 following administration of a single-dose of NKTR- 358 in mice.

[0259] Example 16 In-Vivo PEG-IL-2 N2 Mouse PD Time Course

[0260] PEG-IL-2 N2 Mouse PD Time Course. The example measured the longitudinal expansion and activation of regulatory T cells in Balb / c mice (N = 4 per group) administered a single subcutaneous dose of PEGylated IL-2 molecule as described in the present disclosure. Control mice were either administered a single dose of vehicle or five daily doses of non-PEGylated IL-2. At the indicated time points, mice were sacrificed and whole blood was collected for downstream assessment of immune cell subsets by flow cytometry. Immune cell subsets were identified by a combination of surface and intracellular markers. The stock concentrations of the tested materials are as follows: IL-2 (N2) at 4.30 mg / mL and PEG IL-2 N2 at 2.70 mg / mL.

[0261] FIGURE 13A showed the fold change in Treg cell counts after treatment with IL-2 N2 and PEG-IL-2 N2. Compared to IL-2, PEG IL-2 N2 increased the number of blood Treg in mice after a single subcutaneous dose with limited effects on other immune cell subsets. The peak Treg expansion was at 3 days following dosing and Treg number were elevated until day 5.

[0262] FIGURE 13B showed the fold change in NK cell counts after treatment with IL-2 N2 and PEG-IL-2 N2.

[0263] FIGURE 13C showed the fold change in Teff cell counts after treatment with IL-2 N2 and PEG-IL-2 N2. FIGURE 13D showed the fold change in CD4pos cell counts after treatment with IL- 2 N2 and PEG-IL-2 N2. FIGURE 13E showed the fold change in CD8pos cell counts after treatment with IL-2 N2 and PEG-IL-2 N2.

[0264] FIGURES 13F-13H showed a mouse PD time course that compared IL-2 with PEG-IL-2 N2 with PEG-IL-2 N2. PEG IL-2 N2 selectively increased the Treg functional marker CD25 on blood Treg in mice following a single subcutaneous dose administration. In particular, FIGURE 13FPHAR 00072 / PEC 029-PCT / P00139WO showed the increase in CD25 geometric mean on Treg after treatment with IL-2 N2 and PEG-IL-2 N2. FIGURE 13G showed the increase in FOXP3 geometric mean on in Treg after treatment with IL- 2 N2 and PEG-IL-2 N2. FIGURE 13H showed minimal change in CD25 geometric mean on Teff cell after treatment with IL-2 N2 and PEG-IL-2 N2.

[0265] PEG-IL-2 N2 selectively increased the frequency and number of mouse blood Treg 39-fold three days after a single subcutaneous dose compared to only 7-fold after 5 daily doses of IL-2. Blood Treg in mice dosed with PEG-IL-2 N2 exhibited increased expression of Treg functional markers including CD25 and FOXP3. PEG-IL-2 N2 elicited greater effects on Treg CD25 levels compared to IL-2. PEG-IL-2 N2 effects on the critical Treg marker CD25 are also restricted to Treg versus effector T cells, further demonstrating the Treg selectivity.

[0266] FIGURE 14A and 14B are plots demonstrating the results of a pharmacodynamic analysis of mouse Tregs following administration of either NKTR-358 (labeled as RUR-IL-2, as defined in U.S. Patent Application Serial Number US20210205413A1, which is incorporated herein by reference in its entirety), or IL-2 (which is aldesleukin, also defined in U.S. Patent Application Serial Number US20210205413A1) in mice. FIGURE 14A and FIGURE 14B are FIGS. 10A and 10B in United Stated Patent Application Number 17 / 056,050 (Publication Number 2021 / 0205413).

[0267] Example 17 In-Vivo NKTR-358 Mouse PD Time Course

[0268] FIGURES 11B, 11C, 14A and 14B show mouse PD time course data for NKTR-358.

[0269] FIGURE 11B is a plot of Treg function and activity as measured by the mean fluorescence intensity (MFI) of CD25 and Foxp3 following administration of a single-dose of NKTR-358 in mice.

[0270] NKTR-358 Comparison: PEG-IL-2 N2 selectively increased the frequency and number (e.g., see FIGUREs 13A-13E) of blood Treg in mice following a single subcutaneous dose. The magnitude of Treg expansion with PEG-IL-2 N2 (39-fold) was greater than that reported using the highest dose of NKTR-358 (0.3 mpk - 22-fold). The kinetics of Treg expansion were similar, with Treg expansion peaking 3 days following PEG-IL-2 N2 administration versus 4 days following administration with NKTR-358. Also similar to NKTR-358, PEG-IL-2 N2 induced critical Treg markers CD25 and FOXP3 (FIGURES 13F-13H) and the effects on CD25 were selective for Treg compared to effector T cells.

[0271] Example 18 In-Vivo Mouse Delayed-type Hypersensitivity ModelPHAR 00072 / PEC 029-PCT / P00139WO

[0272] Evaluation of PEG-IL-2 N2 in a mouse keyhole limpet hemocyanin (KLH) KLH delayed type hypersensitivity (DTH) efficacy design model was performed to determine the efficacy and pharmacodynamic effects of PEG-IL-2 N2 compared to a reference cytokine receptor blocking antibody in a mouse model of T cell-dependent skin inflammation. The example design was shown in FIGURE 15A. In this design, female Balb / c mice (N = 7 per group) were sensitized to the antigen KLH and then challenged with the same antigen one week later in both ears to provoke antigen-dependent T cell and B cell responses including ear swelling and pro-inflammatory cytokine production. PEG IL-2 N2 was administered s.c. every 3 days at doses ranging from 0.3 mpk to 0.003 mpk. The immunosuppressive calcineurin inhibitor Cyclosporin A (administered daily p.o.) and anti-IL-4Rα antagonistic antibody (surrogate for Dupixent, administered once i.p on day 8) were included as positive controls. Efficacy was determined by measuring ear swelling (daily after KLH challenge on day 7) and by histologic measurement of epidermal thickness and semi-quantitative assessment of epidermal inflammation, epidermal hyperplasia, dermal inflammation and epidermal erosion / ulceration to produce a sum score. Pharmacodynamics effects were determined by measuring the levels of pro-inflammatory cytokines in ear lysates, immunophenotyping of cells in spleens and ears by flow cytometry and measuring serum anti- KLH antibodies. For immunophenotyping, regulatory T cells (Treg) and other immune cell subsets were identified by a combination of surface and intracellular markers.

[0273] Following the antigen challenge, ear swelling was induced with a mean increase in ear thickness reaching a maximum at 48 hours. Naive, non-challenged ears exhibited no changes in thickness during the course of study. The administration of a PEG-IL-2 N2 through the sensitization and challenge period in this example leads to significant dose-dependent decreases in ear swelling as evidenced by reduced inflammation at each time point relative to the vehicle control.

[0274] FIGURE 15B showed PEG-IL-2 N2 dose-dependently inhibited ear swelling following KLH challenge in the mouse DTH model. The inhibition of ear swelling by PEG IL-2 N2 was statistically significant at the 0.3, 0.1 and 0.01 mpk dose levels, and PEG IL-2 had greater efficacy (p<0.05) than the reference IL-4Rα cytokine receptor blocking antibody at the 0.3 and 0.1 mpk dose levels.

[0275] FIGURES 15C and 15D showed PEG-IL-2 N2 dose-dependently inhibited ear histopathology. The reductions in epidermal thickness (Left) and sum score (Right) were significant (p<0.05) at the 0.3 mpk dose level. In comparison, the anti-IL-4Rα blocking antibody did not significantly affect either parameter.

[0276] FIGURES 15E – 15G showed PEG-IL-2 N2 inhibited ear cytokines and serum anti-KLH antibodies in the mouse DTH model. PEG IL-2 significantly inhibited (p<0.05) levels of IFNγ andPHAR 00072 / PEC 029-PCT / P00139WO IL-4 at all dose levels tested, with significantly (p<0.05) lower levels of Il-4 compared to the reference anti-IL-4Rα antibody at the 0.1, 0.03 and 0.01 mpk dose levels. PEG IL-2 N2 dose-dependently and significantly (p<0.05) inhibited anti-KLH antibodies in serum at all dose levels tested. PEG IL-2 N2 inhibited serum KLH to a greater degree than the reference anti-IL-4Rα antibody at the 0.3 mpk dose level (p<0.05).

[0277] FIGURES 15H – 15K showed PEG-IL-2 N2 dose-dependently expands splenic Treg with minimal effects on other immune cell subsets in the DTH mouse model. In comparison, the reference anti-IL-4Rα antibody did not affect the frequency of splenic Treg. In DTH model, while Treg number was increased, no increase in NK cell numbers were seen and cytokine release syndrome (CRS) was not observed. In fact, cytokine levels (in ears) were reduced. In the DTH model, at 0.1 mpk, robust and durable efficacy weight loss was observed.

[0278] FIGURES 15L – 15O show PEG-IL-2 N2 expanded ear Treg with minimal effects on other immune cell subsets in the mouse DTH model. In comparison, the reference anti-IL-4Rα antibody did not affect the frequency of ear Treg.

[0279] PEG-IL-2 N2 showed dose-dependently decreased in clinical parameters (ear swelling, epidermal thickness and histopathology sum score) in the mouse KLH-induced DTH model. This was accompanied by reductions in pro-inflammatory cytokines and serum anti-KLH antibodies at all doses tested. PEG-IL-2 N2 was superior to a reference anti-IL-4Rα antibody (surrogate for Dupixent) in terms of reducing ear swelling (0.3 to 0.03 mpk dose levels), ear IFNγ production (0.1 to 0.003 mpk dose levels) reflecting its distinct mechanism of action in selectively inducing peripheral and ear skin Treg to inhibit activated T and B cells in this model.

[0280] Example 19 In-vivo Mouse MC-903 Atopic Dermatitis Model

[0281] Evaluation of a PEG-IL-2 N2 in a mouse MC-903 atopic dermatitis model was used to determine the efficacy and pharmacodynamic effects of PEG-IL-2 N2 compared to IL-2 N2 in a mouse model of skin inflammation and tissue damage. The example design is shown in FIGURE 16A. In this 10-day model, MC-903 was applied daily to one ear of female Balb / c mice (N = 10 per group) from days 0 - 8. MC-903 is a vitamin D3 analog which, when topically applied to mouse skin, induces inflammatory cellular infiltration, Th2-dominated cytokine response, scaling and erythema analogous to human atopic dermatitis. In this example, IL-2 N2 and PEG IL-2 N2 were administered s.c. every 3 days at doses ranging from 0.3 mpk to 0.03 mpk. The corticosteroid clobetasol (topicallyPHAR 00072 / PEC 029-PCT / P00139WO administered daily) was included as a positive control. Efficacy was determined by measuring ear swelling (Days 0, 3, 7, 8,9 and 10) and by semi-quantitative histopathologic assessment of keratinocyte death, acanthosis (thickened, velvety and hyperpigmented patches of skin), spongiosis (accumulation of fluid between the cells of the epidermis), parakeratosis (retention of nuclei in the outermost layer of skin), hyperkeratosis (excessive thickening of the outer layer of the skin), inflammation, eosinophils and pustules in hematoxylin / eosin-stained ear skin sections to produce a sum score. Pharmacodynamics effects were determined by measuring the levels of pro-inflammatory Th2 cytokines in ear lysates, and immunophenotyping of cells in spleens and auricular lymph nodes by flow cytometry. For immunophenotyping, regulatory T cells (Treg) and other immune cell subsets were identified by a combination of surface and intracellular markers.

[0282] FIGURE 16B showed PEG-IL-2 N2 inhibited ear swelling in the mouse MC-903 model. The inhibition of ear swelling by PEG IL-2 N2 was statistically significant at the 0.3 and 0.03 mpk dose levels, and PEG IL-2 had greater efficacy (p<0.05) than non-PEGylated IL-2 N2 at the 0.3 and 0.03 mpk dose levels.

[0283] FIGURE 16C shows PEG-IL-2 N2 dose-dependently inhibited ear histopathology. The reduction in sum score was significant (p<0.05) at the 0.3 mpk dose level. In comparison, the anti- IL-4Rα blocking antibody did not significantly affect either parameter.

[0284] FIGURES 16D – 16E show PEG-IL-2 N2 inhibited ear Th2 cytokines in the mouse MC-903 model. PEG IL-2 significantly inhibited (p<0.05) levels of IL-4 in ear lysates at all dose levels tested, with significantly (p<0.05) lower levels of IL-4 as compared to IL-2 N2 at the 0.3 mpk dose level. PEG IL-2 N2 also significantly inhibited (p<0.05) levels of IL-13 in ear lysates at the 0.1 and 0.03 mpk dose levels.

[0285] FIGURES 16F – 16I show PEG-IL-2 N2 selectively expanded splenic Treg in the mouse MC- 903 model, while IL-2 N2 had minimal effects. Both molecules increased the frequency of splenic Treg producing the immune-modulatory cytokine IL-10, though PEG IL-2 N2 had a significantly greater effect (p<0.05) at the 0.03 and 0.1 mpk dose levels. PEG IL-2 N2 also significantly decreased the frequency of splenic CD8 T cells, while IL-2 N2 has no effect.

[0286] FIGURES 16J – 16M showed PEG-IL-2 N2 dose-dependently expanded auricular lymph nodes Treg with minimal effects on other immune cells. PEG IL-2 N2 modestly increased lymphPHAR 00072 / PEC 029-PCT / P00139WO node NK and CD8 T cells at higher dose levels tested. In comparison, IL-2 N2 did not increase any immune cell type in lymph nodes.

[0287] PEG-IL-2 N2 dose-dependently inhibited ear swelling (0.3 and 0.03 mpk dose levels) and ear skin histopathology (0.1 and 0.3 mpk dose levels) in the mouse MC-903 model of dermatitis. These efficacy measurements correlate with decreased ear Th2 cytokines (IL-4 and IL-13) as well as selective expansion of Treg in the spleen and auricular lymph nodes. Comparatively, IL-2 N2 did not significantly affect either clinical parameters or Treg levels, indicating minimal activity in this model. PEG-IL-2 N2 therefore has superior efficacy via robust, selective Treg expansion and restoration of immune homeostasis in (skin) tissues.

[0288] Example 20 PEG IL-2 Cynomolgus Monkey PK PD Study

[0289] This example assessed the pharmacokinetics (PK) and pharmacodynamics (PD) of PEG-IL-2 (N2), at 0.0125, 0.025 and 0.09 mg / kg when administered to cynomolgus monkeys following a single subcutaneous administration with a 21-day follow-on period was compared with IL-2 (N2) at 0.005 mg / kg following five days of consecutive subcutaneous administration (1 male and 1 female per dose group).

[0290] For PK analysis, blood samples (approximately 0.3 mL / time point) were collected via femoral vein from each monkey at the following time points of IL-2 (N2) animals: Pre-dose, 0.083, 0.25, 0.5, 1, 4, 8hr post dose on day 0 and day 4, and PEG-IL-2 (N2) animals: Pre-dose, 0.083, 0.25, 0.5, 1, 4, 8, 12, 24, 48, 72, 96, 144, 168, 336, and 504 hr post treatment. PK blood samples were collected into serum separation tubes (SST). Then, the blood was held at room temperature for at least 30 minutes and allowed to clot prior to centrifugation. Samples were centrifuged at 2 to 8°C for approximately 10 minutes at approximately 2700 g within 2 hours of collection, and serum was harvested and stored at -60 to -80°C until later measurement of test article concentrations using a human IL-2 V-plex MSD assay method.

[0291] For PD assessment (immunophenotyping analysis), blood samples (approximately 0.3 mL / time point) were collected from each monkey at the following time points: pre-dose, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 240 hr, 336 hr, and 504 hr after the first dosing. Blood samples were collected into tubes containing the anticoagulant K2EDTA. The blood samples were analyzed by flow cytometry for the following lymphocyte subsets (phenotypes), and absolute counts and relativePHAR 00072 / PEC 029-PCT / P00139WO percentages (from lymphocytes) were reported for each phenotype. Total T / Helper T / Cytotoxic T cells: CD3 / CD4 / CD8 / Ki67 B / NK / NKT cells: CD3 / CD20 / CD159a / CD56 / Ki67.

[0292] After five subcutaneous injections of IL-2 (N2) at 0.005 mg / kg in male and female monkeys, the mean Cmax were 6.46 ng / mL and 7.68 ng / mL, the mean Tmax were 1.00 hr and 1.00 hr, the mean AUC0-last were 35.8 hr*ng / mL and 38.8 hr*ng / mL, the mean AUC0-inf were 53.4 hr*ng / mL and 44.5 hr*ng / mL, the mean MRTinf were 7.62 hr and 4.37 hr, and the mean T1 / 2 were 5.15 hr and 2.90 hr, respectively, on day 0 and day 4.

[0293] After single subcutaneous injection of PEG-IL-2 (N2) at 0.0125 mg / kg in male and female monkeys, the mean Cmax was 114 ng / mL, the mean Tmax was 36.0 hr, the mean AUC0-last was 6050 hr*ng / mL, the mean AUC0-inf was 6070 hr*ng / mL, the mean MRTinf was 39.1 hr, and the mean T1 / 2 was 8.37 hr.

[0294] After single subcutaneous injection of PEG-IL-2 (N2) at 0.025 mg / kg in male and female monkeys, the mean Cmax was 300 ng / mL, the mean Tmax was 24.0 hr, the mean AUC0-last was 15000 hr*ng / mL, the mean AUC0-inf was 15000 hr*ng / mL, the mean MRTinf was 30.8 hr, and the mean T1 / 2 was 8.68 hr. After single subcutaneous injection of PEG-IL-2 (N2) at 0.09 mg / kg in male and female monkeys, the mean Cmax was 962 ng / mL, the mean Tmax was 18.0 hr, the mean AUC0- last was 51600 hr*ng / mL, the mean AUC0-inf was 51600 hr*ng / mL, the mean MRTinf was 33.9 hr, and the mean T1 / 2 was 16.9 hr.

[0295] The mean pharmacokinetic parameters of all groups are summarized in Table 10 using the combined value of male and female. Table 10PHAR 00072 / PEC 029-PCT / P00139WO

[0296] FIGURE 17 showed the PK profile for PEG-IL-2 N2 showing the exposure (Cmax and AUC0-last) of PEG-IL-2 (N2) increased proportionally as dose levels increased within the dose range from 0.0125 mg / kg to 0.09 mg / kg in non-human primates. After single subcutaneous injection of PEG-IL-2 (N2) at three dose levels (0.0125 mg / kg, 0.025 mg / kg and 0.09 mg / kg), the sex combined mean Cmax of PEG-IL-2 (N2) were 114 ng / mL, 300 ng / mL and 962 ng / mL, the sex combined mean AUC0-last were 6050 hr*ng / mL, 15000 hr*ng / mL and 51600 hr*ng / mL, the T1 / 2 were 8.37 hr, 8.68 hr and 16.9 hr, respectively.

[0297] After five subcutaneous injections of IL-2 (N2) at 0.005 mg / kg, the sex combined mean Cmax and AUC0-last of IL-2 (N2) were 6.46 ng / mL and 35.8 hr*ng / mL, 7.68 ng / mL and 38.8 hr*ng / mL respectively, on day 0 and day 4. The exposure after multiple dosing of IL-2 (N2) was similar with that of a single dose.

[0298] In brief, PEGylation of the present disclosure’s IL-2 N2 increased the half-life, Tmax and AUC following a single subcutaneous dose in Cynomolgus monkeys.

[0299] Example 21 Pharmacodynamics (PD) in Cynomolgus monkeys

[0300] FIGURE 18A showed the results of pharmacodynamic analysis of total CD3+CD4+CD8- CD25+FoxP3+ Tregs in Cynomolgus monkeys' peripheral blood samples over time (days) following a single administration of varying dosage amounts of PEG-IL2 N2 or five subcutaneous injections of IL-2 (N2) at 5 μg / kg. An increase in the fold changes in percentage and cell numbers of Treg cells (compared to baseline) was observed at 12.5, 25, and 90 μg / kg of PEG IL-2 N2. The results showed PEG IL-2 N2 had a higher potency on Treg expansion than IL-2 N2. PEG IL-2 N2 sustained Treg expansion for at least 14 to 21 days.

[0301] FIGURES 19A-19D showed PEG-IL-2 N2 activated more CD4+ Treg versus CD8 positive cytotoxic T cells in non-human primates.

[0302] FIGURES 20A – 20H showed PEG IL-2 N2 selectively expands Treg in non-human primates (comparable to NKTR-385 at the same dose, 25 μg / kg).

[0303] FIGURE 21A and FIGURE 21B showed PEG IL-2 N2 led to a low-level increases of NK cells and CD3+CD4+CD8-FoxP3+CD25+ Tregs at 90 ug / kg in non-human primates.PHAR 00072 / PEC 029-PCT / P00139WO

[0304] FIGURES 22A-22B and FIGURES 22C-22D showed PK / PD for PEG-IL-2 N2 and NKTR- 358 in non-human primates respectively. The example showed PEG IL-2 N2 induced a limited increase of IL-5 or Eosinophil at 25 ug / kg in non-human primates.

[0305] FIGURE 22E and FIGURE 22F showed that PEG IL-2 N2 induced a limited increase in eosinophil on Day 14 at 12.5, 25, and 90 ug / kg in non-human primates. A transient increase of IL-5 was observed on Day 2 post PEG IL-2 N2 treatment and didn’t affect the cell number of eosinophils on Day 14.

[0306] Example 22 PEG IL-2 Cynomolgus Monkey Dose Range Assessment

[0307] This example assessed the pharmacokinetics (PK) and pharmacodynamics (PD) of PEG-IL-2 N2 at 0.06, 0.09, and 0.15 mg / kg when administered once every 2 weeks via subcutaneous injection to cynomolgus monkeys (2 males and 2 females per group). Control animals received the formulation buffer only. For Toxicokinetics (TK) analysis, blood samples (approximately 1 mL / time point) were collected via femoral vein from each monkey at the following time points of PEG IL-2 animals: Pre- dose, 4, 12, 24, 48, 72, 96, 144, 168, 240, and 336 hours post-dose. Blood samples were collected into serum separation tubes (SST). Then, the blood was held at room temperature for at least 30 minutes and allowed to clot prior to centrifugation. Samples were centrifuged at 2 to 8°C for approximately 10 minutes at approximately 2700 g within 2 hours of collection, and serum was harvested and stored at -60 to -80°C until later measurement of test article concentrations using a human IL-2 V-plex MSD assay method.

[0308] For PD assessment (immunophenotyping analysis), blood samples (approximately 0.5 mL / time point) were collected from each monkey at the following time points: pre-dose; Days 2, 4, 6, 8, 15, 16, 18, 20, 22, and 29. Blood samples were collected into tubes containing the anticoagulant K2EDTA. The blood samples were analyzed by flow cytometry for the following lymphocyte subsets (phenotypes), and absolute counts and relative percentages (from lymphocytes) were reported for each phenotype. Total T / Helper T / Cytotoxic T cells: CD3 / CD4 / Ki67. B / NK / NKT cells: CD3 / CD20 / CD159a / CD56 / Ki67. Treg panel: CD3 / CD4 / CD25 / FoxP3 / CD127 / Ki67

[0309] The toxicokinetics parameters were presented using the combined value of male and female as shown in Table 11 because the drug exposure showed no significant sex differences in each group. Table 11PHAR 00072 / PEC 029-PCT / P00139WO PEG-IL-2 N2 DoseDay Cmax(ng / mL) AUC (h*ng / mL) T1 / 2(h) Tmax(h)y 1 and day 15respectively showing that the exposure (Cmax and AUC0-last) of PEG-IL-2 (N2) increased proportionally as dose levels increased within the dose range from 0.06 mg / kg to 0.15 mg / kg. Results showed that mean concentrations of PEG-IL-2 N2 were similar after multiple doses when compared to a single dose.

[0311] After subcutaneous injection administration, PEG-IL-2 N2 was absorbed, and the Tmax value range was 12.0 to 24.0 hour on Day 1 and 18.0 to 24.0 on Day 15. After reaching Cmax, PEG-IL-2 N2 concentrations declined, with half-life (t1 / 2) values ranging from 17.5 to 34.9 hours on Day 1 and from 72.8 to 88.1 hours on Day 15. No accumulation of PEG-IL-2 N2 was observed after multiple doses of PEG-IL-2 N2 in cynomolgus monkeys.

[0312] Example 23 Additional Pharmacodynamics (PD) in Cynomolgus monkeys

[0313] FIGURE 24A showed the results of pharmacodynamic analysis of CD45+CD3+CD4+CD25+FoxP3+ Tregs in Cynomolgus monkeys' peripheral blood samples over time (days) following repeated dosing of varying dosage amounts of PEG-IL2 N2. An increase in the fold changes in cell numbers of Treg cells (compared to the control group) was observed at 60, 90, and 150 μg / kg of PEG IL-2 N2. The results showed PEG IL-2 N2 has a higher fold induction on Treg expansion after the second dosing. PEG IL-2 N2 sustained Treg expansion for at least 14 days.

[0314] FIGURE 24B showed the results of pharmacodynamic analysis of CD45+CD3+CD4- T cells in Cynomolgus monkeys' peripheral blood samples over time (days) following repeated dosing of varying dosage amounts of PEG-IL2 N2.PHAR 00072 / PEC 029-PCT / P00139WO

[0315] FIGURE 24C showed the results of pharmacodynamic analysis of CD45+CD3- CD4+CD159+ NK cells in Cynomolgus monkeys' peripheral blood samples over time (days) following repeated dosing of varying dosage amounts of PEG-IL2 N2.

[0316] FIGURE 24D showed the results of pharmacodynamic analysis of CD45+CD3+CD4+CD25+FoxP3+ Tregs in Cynomolgus monkeys' peripheral blood samples over time (days) following repeated dosing of varying dosage amounts of PEG-IL2 N2.

[0317] FIGURE 24E showed the expansion of blood CD4+ Treg in M / F Cynomolgus monkeys following biweekly (once every other week) subcutaneous dosing with NKTR-358 at 0 (vehicle control), 30, 90, or 150 μg / kg.

[0318] Example 24 PEG- IL-2 N2 and IL-2 N2 DSS-IBD Mouse Efficacy

[0319] FIGURE 25 showed the DSS-IBD model to compare efficacy of PEG- IL-2 N2 and IL-2 N2 and to assess the pharmacokinetics (PK) and pharmacodynamics (PD) of PEG- IL-2 N2.

[0320] A DSS-induced colitis (IBD) mice model is a widely used experimental model for studying inflammatory bowel disease (IBD), particularly ulcerative colitis (UC). It is created by administering dextran sulfate sodium (DSS) in the drinking water of mice, which induces acute or chronic colonic inflammation resembling human IBD. DSS damages the intestinal epithelial barrier, allowing gut microbiota and immune cells to trigger an inflammatory response.

[0321] Here, the DSS-induced colitis (IBD) mice model, was used to evaluate the efficacy of IL-2 N2 and PEG IL-2 N2 to determine the effective doses of PEG IL-2 N2 and to assess the pharmacokinetics (PK) and pharmacodynamics (PD) of PEG IL-2 N2.

[0322] C57BL / 6JNarl female mice received water for injection (WFI) or dextran sulphate sodium (DSS) solution (2.5% DSS dissolved in WFI) for one week (from Day 1 to Day 8) as the DSS- induction period and then receive normal drinking water from Day 8 for five days during the recovery period (Days 8-12). All animals were sacrificed on Day 12 (or a few days earlier).

[0323] In the example, groups included: 1. Water control: Animals were given normal drinking water during the entire study period.2. DSS control: Animals received 2.5% DSS solution via water bottles for one week and then received normal drinking water for five days. 3. Positive control: Animals received the immunosuppressive calcineurin inhibitor Cyclosporin A (p.o.) daily from Day 1 to Day 11 at 25 mg / kg.4. IL-2 N2 treatment groups: Animals were administered PEG IL-2 N2 via s.c at 0.1PHAR 00072 / PEC 029-PCT / P00139WO mg / kg once daily from Day 2 to Day 6 or on Day 2 and Day 6. 5. PEG IL-2 N2 treatment groups: Animals were administered a single s.c. dose of PEG IL-2 N2 (0.1 mg / kg) on Day 2 or two doses on Days 2 and 6. Efficacy was determined by measuring the disease activity index (DAI) score, which was used to evaluate the DSS-induced colitis. DAI index was calculated as total score (body weight decrease + stool consistency + rectal bleeding). At necropsy, colons were removed for length measurement, and blood samples were collected for cytokine or MPO (myeloperoxidase) analyses. Concentrations included CsA at 2.5 mg / mL, IL-2 N2 at 3.74 mg / mL and PEG IL-2 N2 at 1.05 mg / mL.

[0324] PEG IL-2 N2 (once on Day 2 or two doses on Days 2 and 6) and IL-2 N2 (IL-2, QDx5days) showed efficacy in alleviating body weight loss, decreasing the DAI and reversing the colon shortening in DSS-IBD mice (at 0.1 mg / kg). PEG IL-2 had greater efficacy than CsA control article and IL-2 N2. FIGURE 26B showed a plot of the DAI score. FIGURE 26C showed the colon length in IBD model. FIGURE 26D and FIGURE 26E were plots showing PEG IL-2 N2 reduced the IL-6 and MPO (serum) at the endpoint in the IBD model. PEG IL-2 N2 (once on Day 2 or two doses on Days 2 and 6) and IL-2 N2 (QDx5days) showed their efficacy in alleviating body weight loss, decreasing the DAI and suppressing the colon shortening on the DSS-IBD mice (at 0.1 mg / kg). PEG IL-2 had greater efficacy than CsA control article and IL-2 N2. FIGURE 26D showed PEG IL-2 N2 reversed colon shortening, reduced colitis (FIGURE 26C) and decreased serum IL-6 and MPO (FIGURES 26D-26E) in the IBD model.

[0325] Example 25 PEG- IL-2 N2 and IL-2 N2 DSS-IBD Mouse Efficacy Study for Dose Range Finding

[0326] The DSS-induced colitis (IBD) mice model, was again used to evaluate the efficacy of IL-2 N2 and PEG IL-2 N2 to determine the effective doses of PEG IL-2 N2 and to assess the pharmacokinetics (PK) and pharmacodynamics (PD) of PEG IL-2 N2.

[0327] In the example, groups included: 1. Water control: Animals were given normal drinking water during the entire study period. 2. Animals received 2.5% DSS solution via water bottles for one week and then received normal drinking water for five days. 3. Positive control: Animals received the immunosuppressive calcineurin inhibitor Cyclosporin A (p.o.) daily from Day 1 to Day 11 at 25 mg / kg.4. PEG IL-2 N2 treatment groups: Animals were administered a single dose of PEG IL-2 N2 via s.c at doses ranging from 0.003 mg / kg to 0.3 mg / kg on Day 2. 5. An anti-IL-12 (p40 / p70) antibody (administered three times i.p on days 2, 5, and 8) was included. Stock concentrations included CsA at 2.5 mg / mL, anti-IL-12 N2 at 1.00 mg / mL and PEG IL-2 N2 at 1.05 mg / mL.PHAR 00072 / PEC 029-PCT / P00139WO

[0328] Efficacy was determined by measuring the disease activity index (DAI) score, which was used to evaluate the DSS-induced colitis. DAI index was calculated as total score (body weight decrease + stool consistency + rectal bleeding). In addition, colons were removed, measured for length, and fixed for histological examination (H&E or IHC).

[0329] FIGURE 27A showed PEG IL-2 N2 efficacy in alleviating body weight loss. FIGURE 27B showed a plot of the DAI AUEC (area under the effect curve). FIGURE 27C showed the colon length in the IBD model. PEG IL-2 N2 dose-dependently reduced body weight loss and DAI AUEC (area under the effect curve) values and restored colon lengths. PEG IL-2 N2 demonstrated statistically significant efficacy in reducing body weight loss, improved DAI, and restored colon length at doses of 0.03, 0.1, and 0.3 mg / kg. PEG IL-2 has greater efficacy than CsA control.

[0330] FIGURE 27D showed the colitis index (H & E) for the DSS-IBD model study to evaluate the efficacy of PEG-IL-2 (N2). PEG IL-2 N2 demonstrated statistically significant efficacy in reducing the colitis index after histological evaluation at doses of 0.003, 0.01, 0.1, and 0.3 mg / kg.

[0331] Example 26 PEG- IL-2 N2 and IL-2 N2 DSS-IBD Mouse Efficacy Study for PK and PD

[0332] The DSS-induced colitis (IBD) mice model, was used to evaluate the efficacy of IL-2 N2 and PEG IL-2 N2, determine the effective doses of PEG IL-2 N2 and to assess the pharmacokinetics (PK) and pharmacodynamics (PD) of PEG IL-2 N2. In the study groups included: 1. Water control: Animals were given normal drinking water during the entire study period. 2. DSS control: Animals received 2.5% DSS solution via water bottles for one week and then received normal drinking water for several days. 3. Positive control: Animals received the immunosuppressive calcineurin inhibitor Cyclosporin A (p.o.) daily at 25 mg / kg. 4. PEG IL-2 N2 treatment groups: Animals were administered a single s.c. dose of P11838 at doses ranging from 0.003 mg / kg to 0.3 mg / kg on Day 2. Stock concentrations included CsA at 2.5 mg / mL, IL-12 N2 at 3.74 mg / mL and PEG IL-2 N2 at 1.05 mg / mL. At the indicated time points, blood was collected via facial vein (in-life) or via cardiac puncture at the end of the study following human euthanasia. Serum sample was collected and stored at -80°C until later measurement of test article concentrations using a human IL-2 V-plex MSD assay. In addition, whole blood was also collected for downstream assessment of immune cell subsets by flow cytometry.

[0333] FIGURE 28A is a PK profile graph representing the concentration of PEG-IL-2 in ng / mL over time (days) following a single administration on Day 2 in DSS-induced colitis mice. PEGylationPHAR 00072 / PEC 029-PCT / P00139WO increases the half-life, Tmax and AUC∞ of PEG IL-2 N2 compared to IL-2 N2 following a single subcutaneous dose in DSS-IBD mice. Table 14 Test Article PEG IL-2 N2 PEG IL-2 N2 PEG IL-2 N2 PEG IL-2 N2 Parameters 10 µg / kg 30 µg / kg 100 µg / kg 300 µg / kg

[0334] FIGURE 28B and FIGURE 28C showed the results of pharmacodynamic analysis of CD45+CD3+CD4+CD8-FoxP3+CD25+ Tregs in blood samples over time (days) following a single administration of varying dosage amounts of PEG-IL2 N2 or five subcutaneous injections of IL-2 (N2) at 100 μg / kg. An increase in the fold change in percentage and numbers of Treg cells (compared to control animals) was observed at 10, 30, and 100 μg / kg of PEG IL-2 N2. The results showed PEG IL-2 N2 has a higher potency on Treg expansion than IL-2 N2.

[0335] Example 27 Efficacy of a PEG-IL-2 N2 in a Mouse Model of Systemic Lupus Erythematosus (SLE).

[0336] This example was conducted to determine the efficacy of a PEG-IL-2 N2 or IL-2 N2 composition on the development and progression of SLE and its associated characteristics using a MRL / MpJ-Faslpr mouse model, a commonly studied mouse model of this disease. The MRL / MpJ- Faslpr mouse model develops an autoimmune disease that reflects pathologies of human SLE, including lymph node enlargement, increased IgG levels, antinuclear antibody production, proteinuria, and kidney failure caused by inflammation of the glomeruli. A stock solution of a PEG- IL-2 N2 or IL-2 N2 composition as described herein was used as the test article (1.58 mg / mL) supplied in vehicle (transparent liquid; 10 mM sodium acetate / 200 mM sodium chloride / 2% (w / v) sucrose), prepared in sterile water for injection (SWFI), USP; pH 5.0±0.1). On dosing days, of test article was withdrawn and diluted with vehicle to arrive at the desired dosing concentration (0.03 mg / kg dose and 0.3 mg / kg dose); dose volume was 5 mL / kg. Animals used for the study were MRL / MpJ-Faslpr mice and MRL / MpJ naive, female mice, aged from 6-8 weeks. Animals were assigned to treatment groups by randomization. 45 MRL / MpJ-Faslpr mice were randomized into 3 groups (15 each for Groups 2-4) based on body weight and level of protein content in urine beforePHAR 00072 / PEC 029-PCT / P00139WO the commencement of the experiment. Animals in Groups 2-4 received vehicle or test article delivered subcutaneously. Group 1-MRL / MpJ mice received the vehicle as a negative control. Three (3) days after the first dose administration on Week 8, 3 mice from Groups 2-4 were humanely sacrificed and blood samples were collected and processed. Body weights were measured twice a week from the commencement of the study and continued throughout. Skin lesion pictures were taken when first observed and then at one week intervals. Urine was obtained the day before dosing (at baseline) and then collected weekly thereafter. Protein levels in the urine were measured using a Siemens Clinitek Status Analyzer. On sacrifice day (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 about 10000 r / min for about 10 min to obtain serum samples. The serum was stored at −80° C until clinical biochemistry testing. Serum samples (100 μl) were analyzed for anti-dsDNA level by ELISA (Mouse Anti-dsDNA IgG-specific ELISA Kit, Alpha Diagnostic International, Cat. No.5120) and the serum tested for BUN concentration using a Hitachi 7020 Automatic 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. Results showed administration of a PEG-IL-2 N2 or IL-2 N2 composition at a dose of 0.3 mg / kg was effective to suppress the biomarker of kidney damage (i.e., protein levels in urine) to nearly the same levels as observed in normal mice. This example further elucidates the effect of RUR-IL-2-induced Tregs on control of the physiological immune response and disease progression in a representative animal model of SLE.

[0337] Example 28 Evaluate the Safety, Tolerability, Pharmacokinetics and Pharmacodynamics Single Dose of a PEG-IL-2 N2

[0338] Single-arm, open label study to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of a single ascending subcutaneous dose of a PEG-IL-2 N2 or IL-2 N2 composition in healthy volunteers. A Single-arm, open label study is conducted to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of single ascending low sub-cutaneous doses of a PEG-IL-2 N2 or IL-2 N2 composition in healthy volunteers. The study is divided into seven cohorts, in which subjects received about 0.1, 0.3, 1.0, 3.0, 6.0, 9.0, 10, 13.5 or 20.0 μg / kg PEG-IL- 2 N2 or IL-2 N2. Twelve subjects are randomized to each dose cohort, nine of whom received a single subcutaneous dose of PEG-IL-2 N2 or IL-2 N2 while three receive placebo. PEG-IL-2 N2 or IL-2 N2 is formulated as a sterile liquid for subcutaneous injection that is diluted with sterile 0.9% sodium chloride solution. The drug product is supplied in single-use glass vials or pre-filled containers and stored at 2-8° C. Each vial of the drug product contained about 0.75±0.1 mg of PEG-IL-2. PEG-IL-2PHAR 00072 / PEC 029-PCT / P00139WO N2 or IL-2 N2 that is formulated at a concentration of approximately 1.0 mg / mL protein. Placebo is a commercially available 0.9% sodium chloride solution. A starting dose of about 0.1, 0.3, or 1.0 μg / kg is chosen using the minimally anticipated biological effect level (MABEL) approach and is supported by the no observed adverse effect level (NOAEL) in the most sensitive species from nonclinical toxicology studies. The starting dose is set at about 0.1, 0.3 or 1.0 μg / kg to allow for evaluation of PEG-IL-2 N2 or IL-2 N2 pharmacokinetics and safety. Two subjects, one receiving PEG-IL-2 N2 or IL-2 N2 and one placebo, are dosed and monitored for possible side-effects for a period of at least 7 days prior to initiation of the study.

[0339] One objective of this example evaluates the safety and tolerability of PEG-IL-2 N2 or IL-2 N2 administered as a single subcutaneous dose. Other objectives of the example are to (1) observe the time course and extent of changes in the number and / or activity of regulatory T cells (Tregs), (2) characterize the pharmacokinetic (PK) profile of PEG-IL-2 N2 or IL-2 N2 administered as a single subcutaneous dose, and (3) assess the immunologic effects of PEG-IL-2 N2 or IL-2 N2 in blood, including effects on cytokines, T cells, other peripheral blood populations, other serum proteins, changes in gene expression, and anti-drug antibodies. In a first phase, immune markers are tested using pre-dose up to 20 hours post-dose. Specifically, Tregs, CD4 +T cells, CD8 +T cells, natural killer (NK) cells, cytokines, soluble CD25, and RNA are tested in PEG-IL-2- N2 or IL-2 N2 and placebo-receiving cohorts. In subsequent phases, the same immune markers are also tested at 1, 3, 4- , 5-, 6-, 7-, 8-, 10-, 12-, 15-, 18-, 20-, 25-, 30-, 40, and 50-days post-dose.

[0340] No dose-limiting toxicities (DLTs), serious adverse events (SAEs), deaths, or clinically significant abnormalities are reported. Adverse events (AEs) are limited to mild (Grade 1) injection site reactions, and no evidence of AEs known to be associated with high dose IL-2 N2 and / or PEG IL-2 N2.

[0341] Preliminary PK analysis shows that PEG-IL-2 N2 or IL-2 N2 can reach maximum concentrations around 1-6 days post-dose in most subjects, with little change in concentrations up to approximately 2 weeks post-dose, after which concentrations declined with a half-life of approximately 1-6 days.

[0342] Pharmacodynamic (PD) assessment reveals that PEG-IL-2 N2 or IL-2 N2 leads to a dose- dependent increase in circulating CD4+, FoxP3+, CD25+, and / or CD25bright Tregs. In the 0.1, 0.3, 1.0, 3.0, 6.0, 9.0, 10, 13.5 or 20.0 μg / kg single-dose cohorts, there is a sustained increase in the absolute numbers of circulating CD4+, FoxP3+, CD25+, and / or CD25bright Tregs, with levels notPHAR 00072 / PEC 029-PCT / P00139WO returning to baseline until approximately 20 to 25 days following administration. There is a mean increase in the numbers of CD4+, FoxP3+, CD25+, and / or CD25bright Tregs of about 3-, 3.5-, 4.1-, 5-fold, and 8.1-fold, compared to pre-dose at about 0.1, 0.3, 1.0, 3.0, 6.0, 9.0, 10, 13.5 or 20.0 μg / kg doses. There is also an increase in the total CD4+, FoxP3+, and / or CD25+, Treg population at about 0.1, 0.3, 1.0, 3.0, 6.0, 9.0, 10, 13.5 or 20.0 μg / kg doses, but the magnitude of the change is smaller than observed for the CD4+, FoxP3+, CD25+, and / or CD25bright Tregs. There is no change in the numbers of Tregs in the PEG-IL-2-treated subjects versus placebo subjects at about 0.1 / 0.3 and / or 1.0 μg / kg doses compared with those receiving placebo. The primary effect of PEG-IL-2 N2 or IL-2 N2 is seen on Tregs, as no changes in percentage or numbers of other T cell populations (CD4+, CD8+) are observed with PEG-IL-2 N2 or IL-2 N2 at any dose. There is a small increase in the percentage and absolute numbers of NK cells at 13.5 and 20.0 μg / kg without evidence of AEs associated with high-dose IL-2.

[0343] PEG-IL-2 N2 or IL-2 N2 composition lead to a dose-dependent increase in CD4+, FoxP3+, CD25+, and / or CD25bright Tregs. At about 0.1, 0.3, 1.0, 3.0, 6.0, 9.0, 10, 13.5 or 20.0 μg / kg, there is a sustained increase in the absolute numbers of CD4+, FoxP3+, CD25+, and / or CD25bright Tregs, with levels not returning to baseline until 20-25 days following administration. There is a mean increase in the numbers of CD4+, FoxP3+, CD25+, and / or CD25bright Tregs of about 3.0-fold, 3.5- fold, 4.1-fold, and 5.0-fold compared to placebo at about 0.1, 0.3, 1.0, 3.0, 6.0, 9.0, 10, 13.5 or 20.0 μg / kg dose respectively, with a maximal response shifting from a peak at 84 hours at about 1.0 or 3.0 μg / kg to a more extended peak response lasting from 7 to 12 days at 13.5 μg / kg, before returning to baseline levels by day 20-25. There also are a dose-dependent increase in the total CD4+, FoxP3+, and / or CD25+ Treg population at the about 0.1, 0.3, 1.0, 3.0, 6.0, 9.0, 10, 13.5 or 20.0 μg / kg doses, but the magnitude of the change are smaller than observed for the CD4+, FoxP3+, CD25+, and / or CD25bright Tregs. No changes in total CD4+ Tregs in the PEG-IL-2 treated subjects versus placebo subjects are observed at about 0.1, 0.3, and 1 μg / kg doses.

[0344] The primary effect of PEG-IL-2 N2 or IL-2 N2 are seen on Tregs, as no changes in percentage of other T cell populations (CD4+, CD8+) are observed in either the PEG-IL-2 N2 or IL-2 N2 or placebo-treated subjects. However, small increases in the absolute number of CD8+ T cells and the percentage of Ki67+ CD8+ T cells are observed at about 13.5 μg / kg in the PEG-IL-2 N2 or IL-2 N2 subjects. There are no changes in CD4+ T cell absolute numbers at any dose level.

[0345] The CD56+ NK cell population is also analyzed. An increase is noted in the absolute numbers of circulating NK cells with a similar increase in the percentage of this cell subset at about 13.5 μg / kgPHAR 00072 / PEC 029-PCT / P00139WO dose level but not at the lower dose levels. Also noted at about 3.0, 6.0, 9.0 and 13.5 μg / kg, there is a dose-dependent increase in the percentage of CD56+ NK cells expressing Ki67, a marker of proliferation and therefore a marker of activation. At about 3.0, 6.0, and 9.0 μg / kg, the percentage expressing Ki67 approximately 10%, 20-30%, and 30-40%, respectively, after PEG-IL-2 N2 or IL-2 N2 administration. There are no further increase in the percentage expressing Ki67 at the about 13.5 μg / kg dose, which remained at 30-40%.

[0346] PEG-IL-2 treatment according to the SAD study leads to an increase in the numbers of CD4+,FoxP3+, CD25+, and / or CD25bright Tregs, with levels not returning to baseline until 20-25 days following administration. There is also an increase in the total CD4+, FoxP3+, and / or CD25+ Treg population. Increases in the numbers of CD8+ T cells and NK cells are observed at about 13.5 μg / kg.

[0347] Additional cohorts of PEG-IL-2 N2 or IL-2 N2, 20.0 μg / kg (n=13); Placebo (n=3), and PEG- IL-2, N2 or IL-2 N2, 28.0 μg / kg (n=9); Placebo (n=3) are also conducted. Each cohort is followed for 50 days to assess the effects of subcutaneous administration of single ascending doses of PEG- IL-2 N2 or IL-2 N2 in healthy volunteers on safety and tolerability in subjects as evaluated by adverse events, vital signs, and clinical laboratory assessments, as well as the time course and extent of the changes in the numbers and activity of Tregs, Tcons, and NK cells and subsets, pharmacokinetics of PEG-IL-2 N2 or IL-2 N2, and other immunological effects such as cytokine levels, peripheral blood cell populations, serum proteins and gene expression.

[0348] Generally, safety results find no dose-limiting toxicities, deaths, or adverse events leading to study discontinuation, no clinically significant vital sign, ECG, or physical examination abnormalities. Adverse events are primarily limited to mild or moderate (Grade 1 or 2) injection site reactions, 4 subjects who experience Grade 1 events of headache, 1 subject at the highest dose (about 28.0 μg / kg) who experience mild (Grade 1) signs and symptoms of pyrexia, anorexia, vomiting, diarrhea, tachycardia, and myalgia (all Grade 1 in severity) attributes to elevated cytokine levels, and no elicitation of anti-drug antibodies.

[0349] Generally, a sustained, dose-dependent increase in CD25+ and / or CD25-bright Tregs are observed in response to PEG-IL-2 N2 or IL-2 N2. At about 28 μg / kg of a PEG-IL-2 N2 or IL-2 N2 composition, a 17-fold mean peak increase are observed in numbers of CD25-bright Tregs above pre- dose values. Treg levels peak at days 10-12, and do not return to baseline until days 20-25 followingPHAR 00072 / PEC 029-PCT / P00139WO administration. Increases in Treg activation markers ICOS and CTLA4 are observed at doses ≥ about 13.5 μg / kg.

[0350] No substantial changes are observed in the percentage of Tcon cells, and minimal increases are observed in CD56+ NK cells in response to PEG-IL-2 N2 or IL-2 N2. (CD16+, and / or CD56+ NK cells are also enumerated, data not shown). Increases in NK cells are not dose-dependent. A 2- fold increase in NK cells at highest concentration of PEG-IL-2 N2 or IL-2 N2 are observed. PEG-IL- 2 N2 or IL-2 N2 induces dose-dependent increases in Tregs with no induction of CD8+ T cells up to about 28 μg / kg. PEG-IL-2 N2 or IL-2 N2 administration leads to at least about 15-fold increase in mean peak Treg:CD8 ratio over baseline at about 28 μg / kg.

[0351] Example 29 safety and tolerability of Single Dose of PEG-IL-2 N2 in humans

[0352] This example assesses the safety and tolerability of PEG-IL-2 N2 or IL-2 N2 in humans receiving single ascending doses subcutaneously (SC). In addition, time course and extent of changes in the numbers and percentages of Tregs, conventional CD4+ and CD8+ T cells, NK cells, cytokine levels, and the pharmacokinetics (PK) of a PEG-IL-2 N2 or IL-2 N2 composition in peripheral blood are investigated. In this in-human study, healthy volunteers receive SC doses ranging from about 0.1 to 28 μg / kg (9 active:3 placebo per cohort) and subjects are followed for 50 days. All 8 planned cohorts completed dosing. There are no dose-limiting toxicities, serious adverse events, deaths, or clinically significant abnormalities in vital signs, electrocardiograms, or laboratory test values. Adverse events attribute to PEG-IL-2 N2 or IL-2 N2 are primarily limited to mild (grade 1) injection site reactions. One subject at the highest dose demonstrates transient and mild (grade 1) symptoms of elevated cytokine levels and lymphopenia. No other individual at any dose level had systemic signs or symptoms known to be associated with IL-2 therapy. The first 6 cohorts are test for anti-drug antibodies and none can been detected. PEG-IL-2 N2 or IL-2 N2 reaches maximum plasma levels 4- 6 days after administration, with little change for 2 weeks, and then can decrease with a half-life of ˜8-9 days. The primary effect of PEG-IL-2 N2 or IL-2 N2 are seen on Tregs. In the about 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+, CD25+, and / or CD25bright Tregs are observed. The elevated levels peaked at Days 10-12 and not return to baseline until ˜20 to 25 days following administration. At about 28.0 μg / kg, the mean peak increase in numbers of these CD25bright Tregs are 17-fold above baseline. In addition, there is an increase in Treg activation markers at doses ≥about 10.0 to 13.5 μg / kg. There is a mean increase of 3.5-fold in the percentages and numbers of NK cells at the highest dose tested, but no changes in percentages or numbers of conventional CD4+ or CD8+ T cells arePHAR 00072 / PEC 029-PCT / P00139WO observed. PEG-IL-2 N2 or IL-2 N2 composition selectively induced Tregs, evident by a 15-fold increase in the mean peak Treg:CD8 ratio over baseline in the 28.0 μg / kg group. In conclusion, single doses of the PEG-IL-2 N2 or IL-2 N2 in the dose range tested are well tolerated and safe. PEG-IL-2 N2 or IL-2 N2 leads to a striking and selective dose-dependent increase in circulating CD25+ and / or CD25bright Tregs with minimal effects on conventional T cells and with relatively small effects on NK cells. These results extend previous animal studies showing the prolonged and Treg selective action of PEG-IL-2 N2 or IL-2 N2, and provide strong support for testing PEG-IL-2 as a new therapeutic in autoimmune diseases, such as systemic lupus, among other immune diseases.

[0353] PEG-IL-2 N2 or IL-2 N2 composition is safe and well tolerated in this in human single ascending dose study, and leads to a striking and selective dose-dependent increase in circulating CD25+ and / or CD25-bright Treg cells. There is minimal effect on Tcons and NK cells, and this data provides support for testing PEG-IL-2 N2 or IL-2 N2 in autoimmune and inflammatory diseases.

[0354] Example 30 Evaluate the Safety, Tolerability, PK PD of Multiple-Dose PEG-IL-2 N2

[0355] Double-Blind, Randomized Placebo-Controlled Ascending Multiple-Dose Study to Evaluate the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Subcutaneous PEG-IL-2 N2 or IL-2 N2 in Patients with Systemic Lupus Erythematosus. A double-blind, randomized, placebo- controlled study to evaluate the safety, tolerability, PK, and immunologic effects of ascending multiple doses of PEG-IL-2 N2 or IL-2 N2 in four dose cohorts of patients with minimal to moderate systemic lupus erythematosus (SLE) is performed. The effects on SLE disease activity are also evaluated. Twelve SLE patients with minimal to moderate disease activity are randomized to each of four dose cohorts, nine of whom receive multiple 1.0 mg / mL aqueous solution sub-cutaneous doses of PEG-IL-2 N2 or IL-2 N2, while three receive placebo. PEG-IL-2 N2 or IL-2 N2 drug and placebo are prepared as described herein. Active clinical SLE disease activity is not required as an inclusion criterion. In Cohort 1, a starting dose of about 0.1, 1.0 or 3.0 μg / kg is administered three times at two- week intervals (Days 1, 15, and 29). This starting dose is based on the favorable safety and PD profile of single sub-cutaneous doses of PEG-IL-2 N2 or IL-2 N2. The subsequent dose levels in Cohorts 2, 3, and 4, are up to two-fold that of the previous dose cohort. Patients in Cohorts 1-3 receive three doses of study drug at two-week intervals over a total of four weeks. Doses to be evaluated over the course of the study range from about 1.0 μg / kg to 24 μg / kg. Patients in Cohort 4 receive twelve weeks of treatment with PEG-IL-2 N2 or IL-2 N2, administered on Days 1, 3,6, 15, 29, 43, 57, 71 and 85. This cohort provides data on the safety of administration and PK and PD profiles over a longer duration of PEG-IL-2 N2 or IL-2 N2 treatment. After receiving the final dose of PEG-IL-2 N2 or IL-PHAR 00072 / PEC 029-PCT / P00139WO 2 N2 or placebo, patients are followed for an additional fifty days to evaluate safety, PK, PD, and preliminary efficacy. Eight of twelve subjects in each cohort are evaluated two weeks after the third dose of the final patient by the Safety Review Committee for possible safety issues. In addition, all patients in Cohort 4 are evaluated by the Safety Review Committee twice: (1) two weeks after the first eight subjects receive their third dose and (2) two weeks after all subjects receive all doses of study drug. Immunologic changes, including Tregs, CD4 + T cells, CD8 + T cells, and NK cell responses, cytokine levels, and available PK data, in addition to safety findings, are used to determine dose levels. One objective of the study is to evaluate the safety and tolerability of PEG-IL-2 N2 or IL-2 N2 administered as multiple ascending subcutaneous doses to patients with SLE. Other objectives of the example are to (1) characterize the PK profile of PEG-IL-2 N2 or IL-2 N2 following multiple sub-cutaneous doses in patients with SLE, (2) assess the effects of PEG-IL-2 N2 or IL-2 N2 on the time course and extent of changes in PD biomarkers, including number and function of Tregs and Treg subsets, CD4 + T cells, CD8 + T cells, NK cells, and cytokine levels in patients with SLE, (3) assess the effects of PEG-IL-2 on the presence and levels of antibodies against double-stranded DNA, and levels of complement C3 and C4 in patients with SLE, and (4) assess effects of PEG-IL-2 N2 or IL-2 N2 on disease activity in SLE patients.

[0356] Example 31 PEG IL-2 N2Mouse PK Data.

[0357] This example measured the pharmacokinetics data for mice. The example showed in mouse, at about 0.3 mpk, the plasma levels of PEG-IL-2 N2 can vary from about <1 to ~ 2000 ng / ml. At this dose, it was observed that robust Treg expansion with minimal effect on cytokine levels for the compositions of the present disclosure. In mouse DTH model, it is shown PEG-IL-2 N2, dosed from about 0.03-0.3 mpk, reduced (not increased) ear inflammatory cytokines. The data showed no or limited cytokine induction in both Balb / c and BL6 mice (up to single dose of about 0.3 mpk). PEG- IL-2 N2 reduced ear inflammatory cytokine levels in DTH model; in IBD model, PEG-IL-2 N2 at about 0.1 mpk did not induce inflammatory cytokine levels; and in NHP, single dose of about12.5 or about 25 ug / kg, with no or little IL-5 was seen.

[0358] The data showed significant Treg expansion with no or lower NK expansion in mice (Balb / c) and NHP. In addition, the data showed significant Treg expansion with no or lower NK expansion in mouse DTH model. Another example shows PK / PD data for PEG-IL-2 N2 in non-human primates (in life of 21 days).PHAR 00072 / PEC 029-PCT / P00139WO

[0359] In mice, PEG-IL-2 N2 induced an earlier peak Treg expansion (Day 3) compared to NKTR- 358 (Day 4), the maximal effect (peak) and the AUC based on fold change in Treg counts is greater for PEG-IL-2 N2. In non-human primates, a single dose of PEG-IL-2 N2 at about 25 μg / kg induces 10X more Treg (counts) than NKTR-358 at 30 μg / kg (3000 vs 300 cells / μL). Thus, the molecules of the present disclosure have robust effects on Treg expansion. Based on these data, PEG-IL-2 N2 is superior to NKTR-358. In addition, data showed PEG-IL-2 N2 has superior potency and selectivity on human Treg compared to NKTR-358 (0.236 vs 148 ng / ml for PEG-IL-2 N2 and NKTR-358 respectively). PEG-IL-2 N2 requires lower exposure levels to achieve Treg expansion due to its strong potency in vitro. This is similar to the low dose IL-2 therapy where the exposure was maintained at a much lower levels than NKTR-358 (1 ng / ml vs 140 ng / ml), consistent with its Treg potency in vitro. The comparison of IL-2 N2, PEG-IL-2 N2, and NKTR-358 showed that PEG-IL-2 N2 is superior potency and selectivity as well as Treg expansion in mice and non-human primates, shown in Table 12 below.

[0360] Example 32 Surprising and unexpected results for PEG- IL-2 N2 and PEG- IL-2 N2 compared to NKTR-358

[0361] NKTR-358 is a heterogenous mixture of mono, di-, tri- and tetra-PEGylated IL-2 molecules based on the aldesleukin sequence. In contrast, PEG IL-2 N2 and PEG IL-2 N88D are homogenous, mono-PEGylated IL-2 molecule based on the partial aldesleukin sequence.

[0362] In terms of in-vitro potency, NKTR-358 exhibits dramatically reduced potency on human regulatory T cells (Treg) (7475-fold versus IL-2) with minimal selectivity for Treg over CD8 T cells (32-fold). On the other hand, PEG IL-2 N2 had a surprisingly small reduction in potency while maintaining an incredibly high selectivity for Treg over CD8 T cells. The potency of PEG IL-2 N2 on Treg is less than 100-fold reduced compared to IL-2 N2 and the selectivity for Treg over CD8 T cells is 3,365-fold.

[0363] These surprising and unexpected results are also seen in non-human primate (NHP) PBMCs. In non-human primate, NKTR-358 exhibiting 4655-fold decreased potency on Treg compared to IL- 2 with only 108-fold selectivity for Treg over CD8 T cells. In contrast, PEG IL-2 N2 exhibited only 44-fold decreased potency on Treg compared to IL-2 and 1,114-fold selectivity for Treg over CD8- positive T cells. Again PEG IL-2 N2 has a surprisingly small reduction in potency while maintaining an incredibly high selectivity for Treg over CD8 T cells. PEG IL-2 N2 and / or PEG IL-2 N88D therefore have superior potency and selectivity compared to NKTR-358.PHAR 00072 / PEC 029-PCT / P00139WO

[0364] In terms of PK parameters, PEG IL-2 N2 had a half-life of 25 hours versus 44.4 hours, Cmax of 2.01 ug / mL versus 3.33 ug / mL, Tmax of 18 hours versus 24 hours and AUC∞ of 74.1 hr*ug / mL versus 245 hr*ug / mL comparable to NKTR-358 following a single subcutaneous 0.3 mpk dose in mice. Surprisingly, the magnitude of Treg expansion with PEG IL-2 N2 (39-fold) at lower doses was greater than that reported using a highest dose of NKTR-358 (0.3 mpk - 22-fold). Similar to NKTR- 358, PEG IL-2 N2 induced critical Treg markers CD25 and FOXP3 selectively on Treg versus effector T cells (Teff). In a separate dose response study in mice, PEG IL-2 N2 dose-dependently and selectively increased Treg numbers, Treg activation markers and Treg proliferation. However, while NKTR-358 induced inducible T cell co-stimulator (ICOS) staining in ~40% of Treg, PEG IL- 2 N2 surprisingly induced ICOS in ~60 – 70% of Treg, demonstrating that PEG IL-2 N2 is superior at promoting Treg function that NKTR-358.

[0365] In non-human primate following a single subcutaneous dose, PEG IL-2 N2 selectively expanded Treg (12-fold) with minimal effects on CD8 T cells or NK cells. At the same dose level, PEG IL-2 N2 increases serum IL-5 and transiently increases circulating Eosinophils. In an NHP dose- range finding study, PEG IL-2 N2 unexpectedly induced greater numbers of blood Treg compared to NKTR-358 when dosed at a similar level.

[0366] Overall, PEG IL-2 N2 is a homogenous, mono-PEGylated IL-2 molecule with surprisingly and unexpectedly superior potency and selectivity on human Treg and Treg expansion compared to NKTR-358. An overall comparison is shown in Table 12. Table 12. IL-2 N2 PEG-IL2-N2 NKTR-358 KPHAR 00072 / PEC 029-PCT / P00139WO Mouse PD (Treg phenotype) Ki67 Ki67, ICOS, CD25,Ki67, ICOS, PD-1, CD25, FOXP3, function#FOXP3, function#As seen in Table 12 PEG-IL-2 N2 provided unexpectedly superior PBMC potency, selectivity and Treg expansion when compared to IL-2 N2 and NKTR-383.

[0367] Following subcutaneous administration, the PEG-IL-2 N2 or PEG-IL-2 N88D composition were slowly absorbed with Tmax of 0.33-1.0, 1.0-2.3, and 2.0 days in mice, rats, and monkeys, respectively. PEG-IL-2 N2 or PEG-IL-2 N88D composition plasma exposures increase more or less dose proportionally in mice and rats. Bioavailability is in the range of 29.8-46.0% in rats and 86.2% in monkeys.

[0368] Plasma clearance (CL) is low (0.560-1.14 mL / hr / kg in rats and 0.245 mL / hr / kg in monkeys). Following intravenous or subcutaneous dosing, the PEG-IL-2 N2 or PEG-IL-2 N88D composition concentrations exhibited a mono-exponential decay with half-lives of 1.85-2.24 days in mice, 1.25- 2.44 days in rats, and 10.4-12.9 days in monkeys. Renal excretion of PEG-IL-2 N2 or PEG-IL-2 N88D was low due to its average molecular weight of 63 kDa which is near the molecular weight cut-off for the glomerulus filter.

[0369] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure.

[0370] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the disclosure. The principal features of this disclosure can be employed in various embodiments without departing from the scope of the disclosure. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this disclosure and are covered by the claims.PHAR 00072 / PEC 029-PCT / P00139WO

[0371] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0372] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0373] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0374] The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0375] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. All such similar substitutes and modifications apparent to thosePHAR 00072 / PEC 029-PCT / P00139WO skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

Claims

PHAR 00072 / PEC 029-PCT / P00139WO CLAIMS 1. A composition comprising PEGylated IL-2 conjugates including Formula (I):wherein said IL-2 in Formula I includes SEQ ID NO:1 or 5; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 1,000 daltons to 60,000 daltons.

2. The composition of claim 1, wherein the IL-2 comprises the amino acid sequences as recited in SEQ ID NO:

5.

3. A composition comprising PEGylated IL-2 conjugates including formula (II):wherein said IL-2 in Formula II includes SEQ ID NO: 6, 7, 8, 9, or any combinations thereof, and wherein PEGylation is via N-terminus of said SEQ ID NO: 6, 7, 8, and / or 9; and wherein each mPEG is independently a methylated polyethylene glycol having a nominal average molecular weight between about 1,000 daltons to 60,000 daltons.PHAR 00072 / PEC 029-PCT / P00139WO 4. The composition of claim 3, wherein each of the methylated polyethylene glycol moiety has a nominal average molecular weight in a range from about 10,000 daltons to about 40,000 daltons.

5. The composition of claim 3, wherein of the methylated polyethylene glycol moiety has a nominal average molecular weight in a range from about 15,000 daltons to about 30,000 daltons.

6. The composition of claim 3, wherein each of the methylated polyethylene glycol moiety has a nominal average molecular weight in a range from about 19,000 daltons to about 23,000 daltons.

7. The composition of claim 3, wherein the composition is formulated into a pharmaceutical the composition and further comprising formulation including one or more pharmaceutically acceptable excipient.

8. The composition of claim 7, wherein the formulation comprises in a form for parenteral administration.

9. The composition of claim 7, wherein the formulation comprises in a form for subcutaneous administration.

10. The composition of claim 7, wherein the formulation comprises an aqueous diluent.

11. The composition of claim 7, wherein the formulation comprises a pH between about 4.0 to about 6.

0.

12. The composition of claim 7, wherein the formulation comprises sodium acetate, sodium chloride, sucrose, methionine, and / or polysorbate.

13. The composition of claim 7, wherein the formulation comprises between about 0.01-10.0 mg / mL of said composition, between about 5-30 mM sodium acetate, between about 5-30 mM acetic acid, between about 10-200 mM sodium chloride, between about 0.1-2.5 mM methionine, and / or between about 0.005% to 0.03 polysorbate 80, and wherein the formulation comprises a pH betweenPHAR 00072 / PEC 029-PCT / P00139WO about pH 4.0 to 6.

0.

14. The composition of any of the claims 1-13 further comprising Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

15. A vector comprising a promoter operatively linked to SED ID NO: 2, 3, or 4.

16. A method of increasing the ratio of regulatory T cells to effector T cells in a subject or subject in need comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1-13.

17. The method of claim 16, wherein the regulatory T cells comprise CD4+, Foxp3+and / or CD25+cells.

18. The method of claim 16, wherein the effector T cells comprise CD4+Foxp3- and / or CD25- cells.

19. The method of claim 16, wherein the increase in regulatory T cells in a subject or subject in need when compared to a baseline, reaches a value of at least about 2, 3, 4, 5, 10, 12, 15, 20, 25, 30, 31.5, 35, 40, 45, 50, 55, 60, 70, 100 fold, or higher than 100 fold.

20. The method of claim 19, wherein said baseline comprises one or more regulatory T cell numbers from said subject or subject in need obtained after said subject or subject in need is administered with Cyclosporin A, Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

21. The method of claim 16, wherein the increase in regulatory T cells, when compared to a baseline, reaches a value of at least about 2, 3, 4, 5, 10, 12, 15, 20, 25, 30, 31.5, 35, 40, 45, 55, 60, 70, 100 fold, or higher than 100 fold, when evaluated in an in-vivo animal and / or non-human primate model.PHAR 00072 / PEC 029-PCT / P00139WO 22. The method of claim 21, wherein said baseline comprises one or more regulatory T cell numbers from the animal and / or non-human primate obtained after the animal and / or non-human primate is administered with Cyclosporin A, Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

23. The method of claim 16, wherein the increase in regulatory T cells is sustained above a baseline level for at least 1, 3, 5, 8, 10, 14, 15, 20, 30, 35, 40, 45, 50 days, or longer than 50 days in the subject or subject in need post-administration, wherein said post administration includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and / or 10 administrations.

24. The method of claim 23, wherein said baseline levels comprises one or more regulatory T cells numbers from the subject or subject in need obtained after said subject or subject in need is administered with Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

25. The method of claim 16, wherein the increase in regulatory T cells is sustained at least 1, 3, 5, 8, 10, 14, 15, 20, 30, 35, 40, 45, 50 days, or longer than 50 days in the subject or subject in need post-administration above a baseline level as compared to the subject or subject in need after administration with Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

26. The method of claim 16, wherein said therapeutically effective amount comprises a dose between about 0.01 to 10 μg / kg, between about 0.01 to 20μg / kg, between about 3 to 30μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 to 150 μg / kg.

27. The method of claim 16, wherein said therapeutically effective amount is carried out at a frequency comprising once about every 1, 2, 3, 4, 5 days or 1, 2, 3, 4 or 5 weeks 28. A method of treating a subject having an immune-mediated disease, comprising administeringPHAR 00072 / PEC 029-PCT / P00139WO to a subject or subject in need a therapeutically effective amount of the composition of any one of claims 1-13.

29. The method of claim 28, wherein said therapeutically effective amount is by parenteral administration and / or subcutaneous injection.

30. The method of claim 28, wherein said therapeutically effective amount is carried out at a frequency comprising once about every 1, 2, 3, 4, 5 days or 1, 2, 3, 4 or 5 weeks.

31. The method of claim 28, wherein said therapeutically effective amount comprises a dose between about 0.01 to 10μg / kg, between about 0.01 to 20 μg / kg, between about 3 to 30μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 to 150 μg / kg.

32. The method of claim 28, wherein said immune-mediated disease comprises systemic lupus erythematosus, atopic dermatitis, ulcerative colitis, Crohn's disease, and / or immune aplastic anemia.

33. The method of claim 28, wherein said composition further comprises Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

34. A method of treating a subject or subject in need having an allergic disease, comprising administering to the subject or subject in need a therapeutically effective amount of the composition of any one of claims 1-13.

35. The method of claim 34, wherein said composition further comprises Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

36. The method of claim 34, wherein said allergic disease comprises peanut allergy.PHAR 00072 / PEC 029-PCT / P00139WO 37. The method of claim 36, wherein said composition further comprises Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

38. A pharmaceutical formulation comprising the composition of any one of claims 1-13 for use in preventing, treating and / or suppressing an immune-mediated disease, wherein an effective amount of the composition is administered to a subject or subject in need.

39. The pharmaceutical formulation of claim 38, wherein the composition further comprising Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

40. The pharmaceutical formulation of claim 38, wherein said immune-mediated disease comprises systemic lupus erythematosus, atopic dermatitis, ulcerative colitis, immune aplastic anemia, and / or Crohn's disease.

41. The pharmaceutical formulation of claim 38, wherein said administration comprises a regimen of every 1, 2, 3, 4 or 5 days, or 1, 2, 3, 4 or 5 weeks.

42. The pharmaceutical formulation of claim 38, wherein said pharmaceutical formulation comprises a dose between about 0.01 to 10 μg / kg, between about 0.01 to 20 μg / kg, between about 3 to 30μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 to 150 μg / kg.

43. A composition of any one of claims 1-13 for use in preventing, treating and / or suppressing an immune-mediated disease, wherein an effective amount of the composition is administered to a subject or subject in need.

44. The composition of claim 43, wherein said immune-mediated disease comprises systemic lupus erythematosus, atopic dermatitis, ulcerative colitis, immune aplastic anemia, and / or Crohn's disease.PHAR 00072 / PEC 029-PCT / P00139WO 45. The composition of claim 43, wherein said administration comprises a regimen of every 1, 2, 3, 4, or 5 days, or 1, 2, 3, 4 or 5 weeks.

46. The composition of claim 43, wherein said therapeutically effective amount comprises a dose between about between about 0.01 to 10 μg / kg, 0.01 to 20μg / kg, between about 3 to 30μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 to 150 μg / kg.

47. The composition of claim 43 for use in preventing, treating and / or suppressing an immune- mediated disease, wherein the composition further comprises Cyclosporin A, one or more Anti-IL- 4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR- 809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

48. Use of the composition of any of the claims 1-13 for the preparation of a medicament for the treatment of a subject or a subject in need having an immune-mediated disease.

49. The use of the composition of claim 48, wherein said immune-mediated disease comprises systemic lupus erythematosus, atopic dermatitis, ulcerative colitis, immune aplastic anemia, and / or Crohn's disease.

50. The use of the composition of claim 48, wherein said composition further comprises Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

51. The use of the composition of claim 48, wherein said treatment comprises an administration regimen to the subject or subject in need with the composition once every 1, 2, 3, 4, or 5 days, or 1, 2, 3, 4 or 5 weeks.

52. The use of the composition of claim 48, wherein said treatment comprises administration of a dosage to the subject or subject in need between about 0.01 to 10μg / kg, between about 0.01 to 20 μg / kg, between about 3 to 30μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 to 150 μg / kg.PHAR 00072 / PEC 029-PCT / P00139WO 53. Use of the composition of any of the claims 1-13 for the preparation of a medicament to increase the ratio of regulatory T cells to effector T cells in a subject or subject in need, wherein a therapeutically effective dose of the composition of any one of claims 1-13 is administered.

54. The use of the composition of claim 53, wherein said preparation comprises an administration regimen once every 1, 2, 3, 4, or 5 days, or 1, 2, 3, 4 or 5 weeks.

55. The use of the composition of claim 53, wherein said therapeutically effective dose comprises between about 0.01 to 10μg / kg, between about 0.01 to 20μg / kg, between about 3 to 30 μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30-150 μg / kg.

56. The use of the composition of claim 53, wherein the regulatory T cells comprise CD4+, Foxp3+and / or CD25+cells.

57. The use of the composition of claim 53, wherein the effector T cells comprise CD4+, Foxp3-, and / or CD25- cells.

58. The use of the composition of claim 53, wherein the increase in regulatory T cells, when compared to a baseline, reaches a value of at least about 2, 3, 4, 5, 10, 12, 15, 20, 25, 30, 31.5, 35, 40, 45, 50, 55, 60, 70, 100 fold, or higher than 100 fold, when evaluated in said subject or subject in need.

59. The use of the composition of claim 58, wherein said baseline is compared with said subject or subject in need after administration with one or more compositions comprising Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

60. The use of the composition of claim 53, wherein said compositions further comprises Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.PHAR 00072 / PEC 029-PCT / P00139WO 61. A composition of any of the claims 1-13, characterized for used in treating a subject or a subject in need having an immune-mediated disease.

62. The composition of claim 61, wherein said immune-mediated disease is characterized to include systemic lupus erythematosus, atopic dermatitis, ulcerative colitis, immune aplastic anemia, and / or Crohn's disease.

63. The composition of claim 61, wherein the composition is characterized to further include Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

64. The composition of claim 61, wherein the composition is characterized to be administered to the subject or subject in need once every 1, 2, 3, 4, or 5 days, or 1, 2, 3, 4 or 5 weeks.

65. The composition of claim 61, wherein the composition is characterized to be administered at a dosage to the subject or subject in need between about 0.01 to 10μg / kg, between about 0.01 to 20 μg / kg, between about 3 to 30 μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 to 150 μg / kg.

66. A method of increasing the ratio of regulatory T cells to effector T cells in a subject or subject in need characterized by administering to a subject or subject in need a therapeutically effective amount of the composition of any one of claims 1-13.

67. The method of claim 66, wherein the regulatory T cells is characterized to include CD4+, Foxp3+and / or CD25+cells.

68. The method of claim 66, wherein the effector T cells is characterized to include CD4+Foxp3- and / or CD25- cells.

69. The method of claim 66, wherein the increase in regulatory T cells when compared to a baseline, is characterized to reach a value of at least about 2, 3, 4, 5, 10, 12, 15, 20, 25, 30, 31.5, 35, 40, 45, 50, 55, 60, 70, 100 fold, or higher than 100 fold, when evaluated in an in-vivo animal and / orPHAR 00072 / PEC 029-PCT / P00139WO non-human primate model.

70. The method of claim 69, wherein said baseline is characterized to include one or more regulatory T cell numbers from the in-vivo animal and / or non-human primate model obtained after administration with Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

71. The method of claim 66, wherein the increase in regulatory T cells when compared to a baseline, is characterized to reach a value of at least about 2, 3, 4, 5, 10, 12, 15, 20, 25, 30, 31.5, 35, 40, 45, 50, 55, 60, 70, 100 fold, or higher than 100 fold, in the subject or subject in need.

72. The method of claim 71, wherein said baseline is characterized to include one or more regulatory T cell numbers from the subject or subject in need obtained after administration with Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

73. The method of claim 66, wherein the increase in the ratio of regulatory T cells to effector T cells is characterized to be sustained above a baseline level for at least 1, 3, 5, 8, 10, 14, 15, 20, 30, 35, 40, 45, 50 days, or longer than 50 days in the subject or subject in need post-administration.

74. The method of claim 73, wherein said baseline levels is characterized to include one or more regulatory T cell numbers obtained from the subject or subject in need after administration with Cyclosporin A, one or more Anti-IL-4Ra antibodies, Rituximab, BMS-986326, NKTR-358 (REZPEG), NKTR-214, SAR444336 (THOR-809), Efavaleukin alfa (AMG-592), XmAb27564, PT101 (MK-6194), CUG252, ALM223 (SIM0278), and / or aldesleukin.

75. The method of claim 66, wherein said therapeutically effective amount is characterized to include a dose between about 0.01 to 10μg / kg, between about 0.01 to 20μg / kg, between about 3 to 30 μg / kg, between about 37.5 to 62.5 μg / kg, and / or between about 30 to 150 μg / kg.

76. The method of claim 66, wherein said effective amount is characterized to be administeredPHAR 00072 / PEC 029-PCT / P00139WO once about every 1, 2, 3, 4, 5 days or 1, 2, 3, 4 or 5 weeks.

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