IL2 mutant protein

Abstract

The invention relates to an IL2 mutant protein. The invention relates to IL2 mutant protein and application thereof in treating human diseases.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202180009469.X.

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 960,847, filed January 14, 2020, and is incorporated herein by reference in its entirety for all purposes.

[0004] Statement regarding government funding

[0005] No funds from the U.S. government were used in conceiving or implementing the subject of this disclosure.

[0006] sequence list

[0007] This application contains a sequence list that has been electronically submitted in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy created on March 1, 2021, is named 1218661_SL.txt and has a size of 37,939 bytes. Background Technology

[0008] Interleukin-2 (IL-2) is a pluripotent cytokine primarily produced by activated CD4+ T cells, participating in the generation of normal immune responses. IL-2 has a wide range of effects on the immune system, playing an important role in regulating immune activation, suppression, and homeostasis. IL-2 promotes the proliferation and expansion of activated T lymphocytes, enhances B cell growth, and activates monocytes and natural killer cells. The amino acid sequence of human IL-2 (SEQ ID NO:1) can be found in GenBank, accession number NP_000577.2.

[0009] As an immune system stimulant, IL-2 has been used to treat cancer and chronic viral infections. However, the role of IL-2 is also associated with the regulation of autoimmunity and transplant rejection. IL-2 therapy, especially high-dose IL-2 therapy, has been associated with significant toxicity in human subjects. Therefore, the goal of treatment is to maintain the optimal function of IL-2 while minimizing associated autoimmune or immunosuppressive responses. Due to its role in immune regulation and disease, the search for new IL-2 analogues and variants remains an active area of ​​research.

[0010] IL-2 functions on mammalian immune cells by interacting with three different cell surface proteins: (1) CD25 (also known as IL2 receptor α, IL-2Rα, p55), CD122 (also known as interleukin-2 receptor β, IL2Rβ, IL15Rβ and p70-75) and CD132 (also known as interleukin-2 receptor γ, IL-2Rγ; or sharing a γ chain, which is a component of other multimeric receptors in this family).

[0011] CD25 is a 55 kDa polypeptide that is constitutively expressed in Treg cells and inducibly expressed on other T cells in response to activation (e.g., by CD3). The kDa of hIL-2 binding to hCD25 is approximately 10. -8 M. CD25 is also referred to in the literature as a "low-affinity" IL-2 receptor. Human CD25 is expressed as a 272-amino acid preprotein, which includes a 21-amino acid signal sequence that is removed post-translation to form a 251-amino acid mature protein. Amino acids 22-240 (amino acids 1-219 of the mature protein) correspond to the extracellular domain. Amino acids 241-259 (amino acids 220-238 of the mature protein) correspond to the transmembrane domain. Amino acids 260-272 (amino acids 239-251 of the mature protein) correspond to the intracellular domain. The intracellular domain of CD25 is relatively small (13 amino acids) and is not associated with any independent signal transduction activity. No detectable intracellular signal transduction response has been observed from the IL2 / CD25 complex. The nucleic acid and protein sequences of human CD25 can be found in Genbank accessions NM__000417 and NP_0004Q8, respectively.

[0012] CD122 is a single-channel type I transmembrane protein. Human CD122 (hCD122) is expressed as a 551-amino acid protein, with the first 26 amino acids comprising a signal sequence that is cleaved post-translational in the mature 525-amino acid protein. Amino acids 27-240 (amino acids 1-214 of the mature protein) correspond to the extracellular domain, amino acids 241-265 (amino acids 225-239 of the mature protein) correspond to the transmembrane domain, and amino acids 266-551 (amino acids 240-525 of the mature protein) correspond to the intracellular domain. As used herein, the term CD122 includes naturally occurring variants of the CD122 protein, including S57F and D365E (according to the designation of the mature hCD122 protein). hCD122 is cited in the UniProtKB database as entry P14784. The nucleic acid and protein sequences of human CD122 can be found in Genbank accession numbers NM_000878 and NP_000869, respectively.

[0013] CD132 is a type 1 cytokine receptor shared by the receptor complexes of IL-4, IL-7, IL-9, IL-15, and IL-21, hence the term "shared" γ-chain. Human CD132 (hCD132) is expressed as a 369-amino acid preprotein, including a 22-amino acid N-terminal signal sequence. Amino acids 23-262 (amino acids 1-240 of the mature protein) correspond to the extracellular domain, amino acids 263-283 (amino acids 241-262 of the mature protein) correspond to the 21-amino acid transmembrane domain, and amino acids 284-369 (amino acids 262-347 of the mature protein) correspond to the intracellular domain. hCD132 is cited in the UniProtKB database as entry P31785. The nucleic acid and protein sequences of human CD132 can be found in GenBank accessions NM_000206 and NP_000197, respectively.

[0014] The IL-2 receptor proteins combine to produce two other IL-2 receptor complexes: (a) a “moderate affinity” IL-2 receptor comprising CD122 and CD132 (also known as IL2Rβγ) and (b) a “high affinity” IL-2 receptor complex comprising CD25, CD122, and CD132 proteins (also known as IL2Rαβγ). The Kd of hIL-2 for the moderate affinity CD122 / CD132 (IL2βγ) receptor complex is approximately 10e-9M. The moderate affinity CD122 / CD132 (IL2βγ) The receptor complex is primarily expressed on resting T cells and NK cells. The Kd of hIL-2 for the high-affinity receptor complex is approximately 10e-11M. Most cells, such as resting T cells, are poorly responsive to IL-2 because they express only CD122 and CD132, resulting in relatively low affinity for IL-2 compared to the CD25 / CD122 / CD132 high-affinity receptor complex. The high-affinity receptor complex is primarily identified on activated lymphocytes that express inducible CD25 and on Treg cells that constitutively express CD25.

[0015] Given the pluripotency of the IL-2 molecule and its proven ability to regulate the activity of multiple cell types associated with human diseases, mutant IL-2 proteins that retain some of the desired features of the natural molecule while minimizing unwanted features according to the therapeutic context could be used to treat human diseases.

[0016] Garcia et al. (International application number PCT / 2018 / 062122, PCT international publication number WO2019 / 104092 A1 published on May 31, 2019, hereinafter referred to as "Garcia '092") described certain IL2 mutant proteins with modifications including positions 18, 22, and 126, which exhibited weakened binding to CD132 while retaining some IL2 activity.

[0017] This invention provides a mutant IL-2 protein with partial IL-2 agonist and antagonist functions. Summary of the Invention

[0018] The present invention provides methods and compositions for treating and / or preventing inflammatory, infectious or autoimmune diseases, disorders or conditions, which involve administering a therapeutically effective amount of a human IL-2 mutant protein, which has a reduced binding affinity for CD132 while retaining a significant binding affinity for CD122 and / or CD25 comparable to that of wild-type human IL-2.

[0019] In some embodiments, the present invention provides methods and compositions for treating and / or preventing inflammatory, infectious, or autoimmune diseases, disorders, or conditions by administering a therapeutically effective amount of a human IL-2 mutant protein in combination with a supplementary agent, the IL-2 mutant protein having reduced binding affinity for CD132 while retaining significant binding affinity for CD122 and / or CD25 comparable to the activity of wild-type human IL-2, said supplementary agent including, but not limited to, one or more of chemotherapy, immune checkpoint modulators, radiotherapy, and / or physical interventions (such as surgery).

[0020] In some embodiments, the present invention provides methods and compositions for treating and / or preventing inflammatory, infectious, or autoimmune diseases, disorders, or conditions, which involve administering a therapeutically effective amount of a human IL-2 mutant protein, which has a reduced binding affinity for CD132 but retains a significant binding affinity for CD122 and / or CD25 comparable to the activity of wild-type human IL-2, wherein the serum concentration of the IL-2 mutant protein is maintained for a period of time at or above an effective concentration of the IL-2 mutant protein sufficient to promote the proliferation of CD3-activated primary T cells associated with such IL-2 mutant protein, but at or below an effective concentration of the IL-2 mutant protein sufficient to induce T cell activation associated with such IL-2 mutant protein.

[0021] In some embodiments, the present invention provides a human interleukin-2 (IL-2) mutant protein that provides binding properties to modifications of one or more IL2 receptors for the treatment of inflammatory, infectious, or autoimmune diseases, disorders, or conditions. In some embodiments, the IL-2 mutant protein exhibits decreased binding affinity to the extracellular domain of hCD132.

[0022] In some embodiments, the IL-2 mutant protein exhibits decreased binding affinity to the extracellular domain of hCD132 while retaining significant binding to the hCD25 / hCD122 receptor complex and / or activation of the hCD25 / hCD122 / hCD132 receptor complex.

[0023] In some implementations, the IL-2 mutant protein has reduced binding affinity for CD132 while retaining minimal binding affinity for hCD25.

[0024] In one aspect, the present invention provides an hIL-2 mutant protein that, compared to wild-type human IL-2 (hIL-2), exhibits a significant or enhanced binding affinity for hCD25 and a reduced binding affinity for the extracellular domain of the hCD132 receptor. In some embodiments, the IL-2 mutant protein comprises one or more amino acid substitutions that reduce the binding affinity for the CD132 receptor, said substitutions being selected from amino acids 18, 22, and 126, numbered according to mature wild-type hIL-2.

[0025] On the other hand, the present invention provides a polypeptide comprising the amino acid sequence of formula (SEQ ID NO: 97):

[0026] (AA1) a –(AA2) b -(AA3) c -(AA4) d -(AA5) e -(AA6) f -(AA7) g -(AA8) h -(AA9) i-T10-Q11-L12-Q13-L14-E15-H16-L17-(AA18)-L19-D20-L21-(AA22)-M23-I24-L25-N26 -G27-I28-N29-N30-Y31-K32-N33-P34-(AA35)-L36-T37-(AA38)-(AA39)-L40-T41-F42- K43-F44-Y45-M46-P47-K48-K49-A50-T51-E52-L53-K54-(AA55)-L56-Q57-C58-L59-E60 -E61-E62-L63-K64-P65-L66-E67-E68-(AA69)-L70-N71-L72-A73-(AA74)-S75-K76-N77- F78-H79-(AA80-(AA81)-P82-R83-D84-(AA85)-(AA86)-S87-N88-(AA89)-N90-(AA91)-( AA92)-V93-L94-E95-L96-(AA97)-G98-S99-E100-T101-T102-F103-(AA104)-C105-E106- Y107-A108-(AA109)-E110-T111-A112-(AA113)-I114-V115-E116-F117-L118-N119-R12 0-W121-I122-T123-F124-(AA125)-(AA126)-S127-I128-I129-(AA130)-T131-L132-T133

[0027] in:

[0028] • a, b, c, d, e, f, g, h, and i are each individually selected from 0 or 1;

[0029] •AA1 is A (wild-type, a=1) or missing (a=0);

[0030] •AA2 is P (wild type, b=1) or missing (b=0);

[0031] •AA3 is T (wild-type, c=1), C, A, G, Q, E, N, D, R, K, P or deleted (c=0);

[0032] •AA4 is S (wild type, d=1) or missing (d=0);

[0033] •AA5 is S (wild-type, e=1) or missing (e=0);

[0034] •AA6 is S (wild-type, f=1) or missing (f=0);

[0035] •AA7 is T (wild type, g=1) or deleted (g=0);

[0036] •AA8 is K (wild-type, h=1) or missing (h=0);

[0037] •AA9 is K (wild type, i=1) or missing (i=0);

[0038] •AA18 is L (wild type) or R, L, G, M, F, E, H, W, K, Q, S, V, I, Y, H, D or T;

[0039] •AA22 is Q (wild type) or F, E, G, A, L, M, F, W, K, S, V, I, Y, H, R, N, D, T or F;

[0040] •AA35 is K (wild type) or E;

[0041] •AA38 is R (wild type), W, or G;

[0042] •AA39 is M (wild type), L, or V;

[0043] •AA55 is either H (wild type) or Y;

[0044] •AA69 is either V (wild type) or A;

[0045] •AA74 is Q (wild type), P, N, H, S;

[0046] •AA80 is L (wild type), F, or V;

[0047] •AA81 is R (wild type), I, D, or T;

[0048] •AA85 is either L (wild type) or V;

[0049] •AA86 is either I (wild type) or V;

[0050] •AA89 is either I (wild type) or V;

[0051] •AA91 is V (wild type), R, or K;

[0052] •AA92 is either I (wild type) or F;

[0053] •AA97 is either K (wild type) or Q;

[0054] •AA104 is either M (wild type) or A;

[0055] •AA109 is a non-natural amino acid, either D (wild type), C, or with an activated side chain;

[0056] •AA113 is either T (wild type) or N;

[0057] •AA125 is C (wild type), A, or S;

[0058] •AA126 is Q (wild type) or H, M, K, C, D, E, G, I, R, S, or T; and

[0059] •AA130 is S (wild type), T, G, or R; and

[0060] The premise is that if AA18 is R and AA22 is E, then AA126 is not H, M, K, C, D, E, G, I, R, S or T.

[0061] In some embodiments of this aspect,

[0062] •AA18 is selected from the following group: L (wild type) or R, L, G, M, F, E, H, W, K, Q, S, V, I, Y, H, D or T;

[0063] •AA22 is selected from the following groups: Q (wild type) or F, E, G, A, L, M, F, W, K, S, V, I, Y, H, R, N, D, T, or F; and

[0064] •AA126 is selected from the following groups: Q (wild type) or H, M, K, C, D, E, G, I, R, S or T.

[0065] In some embodiments, the polypeptide includes a group of mutations selected from the group consisting of: L18R, Q22E, and Q126M; L18R, Q22E Q126T; L18R; Q22E; Q126H; L18R and Q126H; Q22E and Q126H; L18G, Q22E and Q126H; L18A, Q22E and Q126H; L18M, Q22E and Q126H; L18F, Q22E and Q126H; L18W, Q22E and Q126H; L18K, Q22E and Q126H; L18Q, Q22E and Q126H; L18E, Q22E and Q126H; L18S, Q22E and Q126H; L18V, Q22E and Q126H; L18I, Q22E and Q126H; L18Y, Q22E and Q1 26H; L18H, Q22E and Q126H; L18N, Q22E and Q126H; L18D, Q22E and Q126H; L18T, Q22E and Q126H; L18R, Q22G and Q126H; L18R, Q22A and Q126H; L18R, Q22L and Q126H; L18R, Q22M and Q126H; L18R, Q22F and Q126H; L18R, Q22W and Q126H; L18R, Q22K and Q126H; L18R, Q22S and Q126H; L18R, Q22V and Q126H; L18R, Q22I and Q126H; L18R Q22Y and Q126H; L18R Q22H and Q126H; L18R Q22R and Q126H; L18R Q22N and Q126H; L18R Q22D and Q126H; and L18R Q22T and Q126H.

[0066] In some embodiments, the polypeptide is PEGylated. In some embodiments, the polypeptide is PEGylated and the molecular weight of the PEG component of the PEGylated polypeptide is from about 10 kD to about 70 kD.

[0067] In some embodiments, the polypeptide is a fusion protein. In some embodiments, the fusion protein includes an Fc domain.

[0068] On the other hand, the present invention provides nucleic acids encoding the polypeptides described herein. In some embodiments, the nucleic acid is DNA.

[0069] On the other hand, the present invention provides a recombinant expression vector comprising the nucleic acid described herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector.

[0070] On the other hand, the present invention provides host cells transformed using the vector described herein.

[0071] On the other hand, the present invention provides pharmaceutical formulations comprising the polypeptides, nucleic acids or carriers described herein.

[0072] On the other hand, the present invention provides a method for treating mammalian subjects suffering from autoimmune or inflammatory diseases, disorders or symptoms or viral infections, the method comprising administering a therapeutically effective amount of the pharmaceutical preparation described herein.

[0073] In some embodiments, the method further includes administering one or more supplementary agents selected from the group consisting of: steroids, Janus kinase inhibitors, calcineurin inhibitors, mTor inhibitors, IMDH inhibitors, biologics, vaccines, and therapeutic antibodies. In some embodiments, the therapeutic antibody is an antibody that binds to proteins selected from the group consisting of: BLyS, CD11a, CD20, CD25, CD3, CD52, IgE IL-12 / IL-23, IL-17a, IL-1β, IL-4Rα, IL-5, IL-6R, integrin-α4β7, RANKL, TNFα, VEGF-A, and VLA-4.

[0074] In some implementations, the disease, disorder, or condition is selected from: viral infection, Helicobacter pylori infection, HTLV, organ rejection, graft-versus-host disease, autoimmune thyroid disease, multiple sclerosis, allergy, asthma, neurodegenerative diseases such as Alzheimer's disease, systemic lupus erythematosus (SLE), autoinflammatory diseases, inflammatory bowel disease (IBD), Crohn's disease, diabetes, chondritis, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, generalized juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Ritter syndrome, SEA syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile hemorrhage. Vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Rett syndrome, SEA syndrome, psoriasis, psoriatic arthritis, dermatitis (eczema), exfoliative dermatitis or atopic dermatitis, pityriasis rubra pilaris, pityriasis rosacea, psoriasis-like, lichen planus, lichen luster, ichthyosis-like skin disease, keratosis, skin disease, alopecia areata, pyoderma gangrenosa, vitiligo, bullous pemphigoid, urticaria, keratosis, rheumatoid arthritis; seborrheic dermatitis, photodermatitis; seborrheic keratosis, senile keratosis, actinic keratosis, photoinduced keratosis, follicular keratosis; acne vulgaris; keloids; moles; warts, including warts, condyloma acuminata, or genital warts, and human papillomavirus (HPV) infection. Brief description of the attached figures

[0075] The invention will be better understood through the following detailed description in conjunction with the accompanying drawings. It should be emphasized that, by convention, the various features in the drawings are not to scale. Instead, the dimensions of the various features are arbitrarily enlarged or reduced for clear display. The accompanying drawings include the following figures.

[0076] Figure 1 A graphical representation of pSTAT5 levels measured in NKL cells treated with 293T transfection supernatant containing the indicated IL2 mutant protein (and a control) is provided, as described in the examples. The vertical axis represents the IL2 activity level measured according to the examples, and each column represents the activity level of a specific IL2 peptide associated with the construct, as described in the examples, identified by its 3-letter abbreviation.

[0077] Figure 2 A comparison of pSTAT5 activity in CD25-positive and CD25-negative YT cells treated with 293T transfection supernatant containing the indicated IL2 mutant protein (and a control) is provided, as described in the examples. The vertical axis is a selective measurement calculated as the ratio of the pSTAT5 activity level observed on CD25-positive YT cells to the pSTAT5 activity level measured on CD25-negative YT cells. Each column represents the activity level of a specific IL2 peptide being evaluated, identified by its 3-letter abbreviation, as described in the examples.

[0078] Figures 3A-3F Data related to cell proliferation of 3F8 cells in contact with the hIL2 mutant protein are provided, and are described more fully in the specification and Example 8. Detailed Implementation

[0079] To facilitate understanding of this invention, certain terms and phrases are defined below and throughout the specification. The definitions provided herein are non-limiting and should be interpreted based on the knowledge of someone skilled in the art.

[0080] Before describing the methods and compositions of the present invention, it should be understood that the invention is not limited to the methods or compositions described herein, as they may, of course, vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be limiting.

[0081] When providing a numerical range, it should also be considered as specifically disclosing intermediate values ​​between the upper and lower limits of the range, spaced one-tenth of the lower limit unit, unless the context explicitly states otherwise. This invention also includes smaller ranges between any set value or intermediate value within the set range and any other set value or intermediate value within the set range. Depending on any explicitly excluded limit value within the set range, the range may independently include or exclude the upper and lower limits of these smaller ranges. This invention also includes ranges that do not contain a limit value, or that contain one or both limit values. When a set range contains one or two limit values, this invention also includes ranges that exclude one or both of those limit values.

[0082] Unless otherwise stated, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used to practice or test the invention, some potential and preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials associated with the cited publications.

[0083] It should be noted that the singular forms "a," "an," and "the" used herein and in the appended claims include the plural meaning unless the context clearly indicates otherwise. Thus, for example, reference to "cell" includes a plurality of such cells, and reference to "the peptide" includes one or more peptides and their equivalents known to those skilled in the art, such as polypeptides, etc.

[0084] The publications discussed herein refer only to their disclosures prior to the filing date of this application. Nothing herein should be construed as an admission that the invention does not precede these publications by virtue of a prior invention. Furthermore, the publication dates provided may differ from the actual publication dates, which may require separate verification.

[0085] Unless otherwise stated, parts are by weight, molecular weight is by weight-average molecular weight, temperature is in degrees Celsius (°C), and pressure is at or near atmospheric pressure. Use standard abbreviations, including the following: bp = base pair; kb = kilobase; pl = picolitre; s or sec = second; min = minute; h or hr = hour; AA or aa = amino acid; kb = kilobase; nt = nucleotide; pg = picogram; ng = nanogram; μg = microgram; mg = milligram; g = gram; kg = kilogram; dl or dL = deciliter; μl or μL = microliter; ml or mL = milliliter; l or L = liter; μM = micromolar; mM = millimole; M = mole; kDa = kilodalton; im = intramuscular; ip = intraperitoneal; SC or SQ = subcutaneous; QD = once daily; BID = twice daily; QW = once weekly; QM = once monthly; HPLC = high performance liquid chromatography; BW = body weight; U = Unit; ns = no statistical significance; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; HSA = human serum albumin; MSA = mouse serum albumin; DMEM = Darwin's modified Ehrlich medium; EDTA = ethylenediaminetetraacetic acid.

[0086] It should be understood that amino acids are referred to by single-letter or three-letter codes in this disclosure. For the convenience of readers, the single-letter and three-letter amino acid codes are provided in Table 1 below:

[0087]

[0088] The standard methods of molecular biology are described in the scientific literature (see, for example, Sambrook and Russell (2001), Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; and Ausubel et al. (2001), Current Protocols in Molecular Biology, vols. 1–4, John Wiley and Sons, Inc., New York, NY, which describe cloning and site-directed mutagenesis in bacterial cells (vol. 1), cloning in mammalian cells and yeast (vol. 2), glycoconjugates and protein expression (vol. 3), and bioinformatics (vol. 4)). Scientific literature describes methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation and crystallization, as well as chemical analysis, chemical modification, post-translational modification, fusion protein production and protein glycosylation (see, for example, Coligan, et al. (2000), Current Protocols in Protein Science, vols. 1-2, John Wiley and Sons, Inc., NY).

[0089] Unless otherwise stated, the following terms are intended to have the following meanings. Other terms are defined throughout this specification.

[0090] definition:

[0091] activation: As used in this article, the term "activation" refers to a receptor or receptor complex to reflect the biological effects of agonist ligand binding to a receptor. An activator is a molecule that increases, activates, promotes, enhances activation, sensitizes, or upregulates, such as a gene, protein, ligand, receptor, or cell. For example, the binding of an IL2 agonist to an IL2 receptor (such as a high-affinity CD25 / CD122 / CD132 receptor complex) "activates" receptor signal transduction to produce one or more intracellular biological effects (such as STAT5 phosphorylation).

[0092] activeAs used herein, the term "activity" refers to a molecule and describes its properties in relation to a test system or biological function, such as the extent to which the molecule binds to another molecule. Examples of such biological functions include, but are not limited to, the catalytic activity of a biopharmaceutical, its ability to stimulate intracellular signal transduction, gene expression, cell proliferation, and its ability to regulate immunological activities (such as inflammatory responses). "Activity" is often expressed as the biological activity of a given reagent per unit, such as [catalytic activity] / [mg protein], [immunological activity] / [mg protein], International Units of Activity (IU), [STAT5 phosphorylation] / [mg protein], [T-cell proliferation] / [mg protein], plaque-forming units (PFU), etc.

[0093] Administration / Giving: The terms “giving” and “administering” are used interchangeably in this document and refer to the act of contacting a subject, including contacting the subject’s cells, tissues, organs or biological fluids in vitro, in vivo and / or ex vivo with a reagent (e.g., IL-2 mutant protein or a pharmaceutical preparation thereof). The administration of a drug can be achieved by any of the various methods recognized in the art, including but not limited to local, intravascular injection (including intravenous or intra-arterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, intranodal injection, percutaneous, transmucosal, iontophoretic delivery, intralymphatic injection, (Senti and Kundig (2009) Current Opinions in Allergy and Clinical Immunology 9(6):537-543), intragastric infusion, intraprostatic injection, intravesical infusion (e.g., bladder), inhaler (including nebulizer), intraocular injection, intraperitoneal injection, intralesional injection, intraovarian injection, intracerebral infusion or injection, intraventricular injection (ICVI), etc. The term "administration" includes contact between the reagent and cells, tissues, or organs, as well as contact between the reagent and liquids, wherein the liquids are in contact with cells. The term “giving” includes in vitro contact of a cell (or cell population) that can be isolated from a subject and brought into contact with a drug, whether the cell (or cell population) is given to the same subject (e.g., autologous cell transfer) or a different subject (e.g., allogeneic cell transfer).

[0094] Adverse eventsAs used herein, the term "adverse event" refers to any unintended experience associated with the use of a therapeutic or prophylactic agent in a subject. Adverse events are not necessarily caused by the administration of a therapeutic or prophylactic agent (e.g., IL2 mutant protein) and may also be caused by unrelated circumstances. Adverse events are generally classified as mild, moderate, or severe. The classification of adverse events used herein conforms to the Common Terminology Standard for Adverse Events v5.0 (CTCAE) published on November 27, 2017, by the U.S. Department of Health and Human Services, the National Institutes of Health, and the National Cancer Institute.

[0095] Affinity As used in this article, “affinity” refers to the degree to which a first molecule (e.g., a ligand) specifically binds to a second molecule (e.g., a receptor), and through K... d The binding kinetics measure is expressed as the dissociation constant (K) between the molecule and its target. off ) and the association constant between the molecule and its target (K on The ratio of ).

[0096] Agonists: As used herein, the term "agonist" refers to an agent that specifically binds to a second molecule ("target") and interacts with the target to induce or promote an increase in target activation. An agonist is an activator that modulates, enhances, sensitizes, or upregulates cellular activation, such as genes, proteins, ligands, receptors, biological pathways (including immune checkpoint pathways in cells), or cell proliferation. In some embodiments, an agonist is an agent that binds to a receptor and alters the receptor state, resulting in a biological response. This response mimics the effect of an endogenous activator of the receptor. The term "agonist" includes partial agonists, complete agonists, and superagonists. An agonist may be described as a "complete agonist" when it induces a complete response (i.e., a response associated with a naturally occurring ligand / receptor binding interaction) in the receptor under study; alternatively, an agonist may also be described as a partial agonist. In contrast to agonists, antagonists may specifically bind to a receptor but do not induce a signaling cascade normally initiated by the receptor and may alter the effect of the agonist on that receptor. An inverse agonist is a drug that produces a pharmacological response in the opposite direction to that of an agonist. A "super agonist" is an agonist capable of producing a maximum response greater than that of an endogenous agonist to the target receptor, and therefore possessing potency exceeding 100%. When evaluated at similar concentrations in comparative studies, the IL2 super agonist of this invention can have an activity greater than 110%, or greater than 120%, or greater than 130%, or greater than 140%, or greater than 150%, or greater than 160%, or greater than 170% of the activity of wild-type mature human IL2 according to WHO international standards (NIBSC code: 86 / 500).

[0097] Antagonist As used herein, the terms “antagonist” or “inhibitor” refer to a molecule that has one or more effects in contrast to an agonist. Antagonists can prevent, reduce, inhibit, or neutralize the activity of an agonist, and they can also prevent, inhibit, or reduce the constitutive activity of a target (such as a target receptor), even without an identified agonist. Inhibitors are molecules that reduce, block, prevent, delay activation, inactivate, desensitize, or downregulate, such as genes, proteins, ligands, receptors, biological pathways, or cells.

[0098] Antibody As used herein, the term “antibody” collectively refers to: (a) glycosylated and non-glycosylated immunoglobulins (including, but not limited to, mammalian immunoglobulin classes IgG1, IgG2, IgG3, and IgG4) that specifically bind to target molecules, and (b) immunoglobulin derivatives, including but not limited to IgG(1-4)ΔC H 2, F(ab')2, Fab, ScFv, V H V LAntibodies include tetraclonal antibodies, triclonal antibodies, biclonal antibodies, dsFv, F(ab')3, scFv-Fc, and (scFv)2, which compete with immunoglobulins of their origin for binding to target molecules. The term antibody is not limited to immunoglobulins, antibodies, or human antibodies derived from any particular mammalian species (including mice, humans, horses, and camels). The term antibody includes so-called “heavy chain antibodies” or “VHHs” or “nanobodies®,” typically obtained from immunizations of camelids (including camels, llamas, and alpacas) (see, for example, Hamers-Casterman, et al. (1993) Nature 363:446-448). Antibodies with a given specificity can also be derived from non-mammal sources, such as VHHs obtained from immunizations of cartilaginous fish (including, but not limited to, sharks). The term "antibody" includes antibodies that can be isolated from animals of natural origin or after immunization with an antigen, as well as engineered antibodies, including monoclonal antibodies, bispecific antibodies, trispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted, veneered, or deimmunized (e.g., T-cell epitope-removed) antibodies. The term "human antibody" includes antibodies obtained from humans, as well as antibodies obtained from transgenic mammals containing human immunoglobulin genes, such that, upon antigen stimulation, the transgenic animal produces antibodies containing the amino acid sequence characteristics of human-produced antibodies. The term includes parental antibodies and their derivatives, such as affinity-matured, veneered, CDR-grafted (including CDR-grafted VHHs), humanized, camel-derived (in the case of non-camel-derived VHHs), or binding molecules of antibody-binding domains (such as CDRs) encapsulated in non-immunoglobulin scaffolds. The term "antibody" is not limited to any particular synthetic method, including naturally occurring antibodies that can be isolated from natural sources, as well as engineered antibody molecules prepared by "recombinant" means. These engineered antibody molecules include antibodies isolated from transgenic animals carrying human immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells transformed with nucleic acid constructs that lead to antibody expression, antibodies isolated from combinatorial antibody libraries (including phage display libraries), or antibodies chemically synthesized (e.g., solid-phase protein synthesis). In one embodiment, an "antibody" is a mammalian immunoglobulin. In some embodiments, the antibody is a "full-length antibody" that includes variable and constant structural domains providing binding and effector functions. In most cases, a full-length antibody comprises two light chains and two heavy chains, each light chain including variable and constant regions. In some embodiments, the term "full-length antibody" is used to refer to a conventional IgG immunoglobulin structure, comprising two light chains and two heavy chains, each light chain including variable and constant regions providing binding and effector functions. The term antibody includes antibody-drug conjugates, which include modifications made to prolong the duration of action, such as fusion proteins or conjugated to polymers (e.g., PEGylated), as described in more detail below.

[0099] biological samples As used herein, the term "biological sample" or "sample" refers to a sample obtained from or derived from a subject. For example, a biological sample includes materials selected from the group consisting of: body fluids, blood, whole blood, plasma, serum, mucous secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), ocular fluids (such as vitreous fluid, aqueous humor), lymph, lymph node tissue, spleen tissue, bone marrow, and immunoglobulin-enriched portions of one or more of these tissues. In some embodiments, the sample is obtained from a subject who has been exposed to a treatment regimen comprising a pharmaceutical preparation containing an IL2 mutant protein, for example, through repeated exposure to the same IL2 mutant protein. In other embodiments, the sample is obtained from a subject who has not recently been exposed to an IL2 mutant protein, or from the subject prior to a planned administration of the IL2 mutant protein.

[0100] "CAR" or "Chimeric Antigen Receptor":As used herein, the terms “chimeric antigen receptor” and “CAR” are used interchangeably and refer to a chimeric polypeptide comprising multiple functional domains, the sequence of which, from the amino terminus to the carboxyl terminus, is arranged as follows: (a) an extracellular domain (ECD) containing an antigen-binding domain (ABD) and a “hinge” domain; (b) a transmembrane domain (TD); and (c) one or more cytoplasmic signal transduction domains (CSD), wherein the above domains may optionally be linked by one or more spacer subdomains. The CAR may further comprise a signal peptide sequence, which is typically removed during post-translational processing and presented on the surface of cells transformed with an expression vector containing a nucleic acid sequence encoding the CAR. The CAR can be prepared according to principles well known in the art. See, for example, Eshhaar et al., U.S. Patent No. 7,741,465 B1, granted June 22, 2010; Sadelain et al. (2013) Cancer Discovery 3(4):388-398; Campana and Imai (U.S. Patent No. 8,399,645, granted March 19, 2013); Jensen and Riddell (2015) Current Opinions in Immunology 33:9-15; Gross et al. (1989) PNAS (USA) 86(24):10024-10028; Curran et al. (2012) J Gene Med 14(6):405-15; Brogdon et al. (U.S. Patent No. 10,174,095, granted January 8, 2019); Guedan et al. (2019) Engineering and Design of Chimeric Antigen Receptors (2019) Molecular Therapy: Methods & Clinical Development Vol. 12: 145-156.

[0101] CAR-T cells As used herein, the terms “chimeric antigen receptor T-cell” and “CAR-T cell” are used interchangeably and refer to T cells that have been recombinantly modified to express a chimeric antigen receptor. Examples of commercially available CAR-T cell products include axicabtagene ciloleucel (available from Gilead Pharmaceuticals under the name Yescarta®) and tisagenlecleucel (available from Novartis under the name Kymriah®).

[0102] CD25 As used herein, the terms “CD25,” “IL2 receptor α,” “IL-2Rα,” “IL2Ra,” and “p55” are used interchangeably to refer to a 55 kDa polypeptide constitutively expressed in Treg cells and inducibly expressed on other T cells in response to activation (e.g., by CD3CD25, which is also referred to in the literature as a “low-affinity” IL-2 receptor). The nucleic acid and protein sequences of human CD25 can be found in Genbank accessions NM__000417 and NP_0004Q8, respectively. Human CD25 is expressed as a 272-amino acid preprotein containing a 21-amino acid signal sequence, which is removed post-translational to produce a 251-amino acid mature protein. Amino acids 22–240 (amino acids 1–219 of the mature protein) correspond to the extracellular domain. Amino acids 241–259 (amino acids 220–238 of the mature protein) correspond to the transmembrane domain. Amino acids 260-272 (amino acids 239-251 in the mature protein) correspond to the intracellular domain. The amino acid sequence of the mature form of hCD25 is:

[0103] ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQ

[0104] CTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYH

[0105] FVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA

[0106] SPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTW

[0107] QRRQRKSRRTI

[0108] (SEQ ID NO: 1)

[0109] CD122As used herein, the terms “CD122,” “interleukin-2 receptor β,” “IL2Rb,” “IL2Rβ,” “IL15Rβ,” and “p70-75” are used interchangeably to refer to the human CD122 transmembrane protein. Human CD122 (hCD122) is expressed as a 551-amino acid protein, with the first 26 amino acids comprising the signal sequence, which is cleaved post-translational in the mature 525-amino acid protein. Amino acids 27-240 (amino acids 1-214 of the mature protein) correspond to the extracellular domain, amino acids 241-265 (amino acids 225-239 of the mature protein) correspond to the transmembrane domain, and amino acids 266-551 (amino acids 240-525 of the mature protein) correspond to the intracellular domain. As used herein, the term CD122 includes naturally occurring variants of the CD122 protein, including S57F and D365E (according to the numbering of the mature hCD122 protein). hCD122 is cited in the UniProtKB database as entry P14784. The nucleic acid and protein sequences of human CD122 can be found in GenBank accessions NM_000878 and NP_000869, respectively. The amino acid sequence of the mature hCD122 protein is as follows:

[0110] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWAC

[0111] NLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETH

[0112] RCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEF

[0113] QVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWLGHLLVGLSGAFGFIILVYLLIN

[0114] CRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLE

[0115] VLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDP

[0116] YSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPG

[0117] GSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPR

[0118] EGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0119] (SEQ ID NO:2)

[0120] The amino acid sequence of the extracellular domain of hCD122 is:

[0121] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWAC

[0122] NLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETH

[0123] RCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEF

[0124] QVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDT

[0125] (SEQ ID NO:3)

[0126] CD132As used herein, the terms "CD132," "IL2 receptor γ," "IL2Rg," and "IL2Rγ" refer to type 1 cytokine receptors shared by the receptor complexes of IL-4, IL-7, IL-9, IL-15, and IL-21, hence the term "shared" γ chain. Human CD132 (hCD132) is expressed as a 369-amino acid preprotein, including a 22-amino acid N-terminal signal sequence. Amino acids 23-262 (amino acids 1-240 of the mature protein) correspond to the extracellular domain, and amino acids... Amino acids 263-283 (amino acids 241-262 in the mature protein) correspond to a 21-amino acid transmembrane domain, and amino acids 284-369 (amino acids 262-347 in the mature protein) correspond to an intracellular domain. hCD132 is cited in the UniProtKB database as entry P31785. The nucleic acid and protein sequences of human CD132 can be found in GenBank accessions NM_000206 and NP_000197, respectively. The amino acid sequence of the mature hCD132 protein is as follows:

[0127] LNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNCTWNSSSEPQP

[0128] TNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFVVQLQDPREPRRQA

[0129] TQMLKLQNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSV

[0130] DYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEA

[0131] VVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQ

[0132] PDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

[0133] (SEQ ID NO:4)

[0134] CDR.As used herein, the term “CDR” or “complementarity-determining region” refers to a discontinuous antigen-binding site found within the variable region of both the heavy and light chain immunoglobulin polypeptides (or, in the case of VHH, the heavy chain). CDRs have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977); Kabat et al., US Department of Health and Human Services, “Sequences of Proteins of Immunological Interest” (also referred to herein as “Kabat 1991”); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definition includes overlap or subsets of amino acid residues when compared to each other. In the context of this invention, the numbering of CDR locations is provided according to the Kabat numbering system.

[0135] Quite As used herein, the term "comparable" is used to describe the degree of difference between two measurements of an evaluable quantitative or qualitative parameter. For example, two measurements are considered "comparable" when a first measurement of an evaluable quantitative parameter (e.g., IL-2 activity level determined by a CTLL-2 proliferation or phosphorylation-STAT5 assay) and a second measurement of the same evaluable parameter do not deviate from a range that a person skilled in the art would consider not to present a statistically significant difference between the two results. In some cases, a measurement may be considered "comparable" if one measurement deviates from another measurement by less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 10%, or less than 7%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%. In a particular embodiment, a measurement is considered comparable to a reference standard if it deviates from a reference standard by less than 15%, or less than 10%, or less than 5%.

[0136] Source As used herein, the term "derived from" in the context of an amino acid sequence or polynucleotide sequence (e.g., an amino acid sequence "derived from" an IL-2 polypeptide) means that the polypeptide or nucleic acid has a sequence based on a reference polypeptide or nucleic acid (e.g., a naturally occurring IL-2 polypeptide or a nucleic acid encoding IL-2), and is not intended to be limited to a source or method for preparing a protein or nucleic acid. For example, the term "derived from" includes homologs or variants of the reference amino acid or DNA sequence.

[0137] enrichedAs used herein, the term “enriched” means that a sample has been subjected to non-natural manipulation so that the molecule of interest is present in a concentration that is (a) higher than that in the starting sample (e.g., at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold, or at least 1000-fold) or (b) higher than that in the environment in which the molecule was prepared (e.g., in recombinantly modified bacterial or mammalian cells).

[0138] extracellular domain As used herein, the term "extracellular domain" or its abbreviation "ECD" refers to the portion of a cell surface protein located outside the cell membrane (e.g., cell surface receptors). The term "ECD" can include the extracellular portion of a transmembrane protein or the extracellular portion of a cell surface protein (or membrane-associated protein).

[0139] identity For polypeptide or DNA sequences, the term "identity" as used herein refers to the subunit sequence identity between two molecules. Two molecules are identical at that position when the subunit position is occupied by the same monomeric subunit (i.e., the same amino acid residue or nucleotide). The similarity between two amino acid or two nucleotide sequences is a direct function of the amount of identical position. Typically, sequences are aligned to obtain the highest-order match. If necessary, identity can be calculated using publicly available techniques and widely used computer programs, such as the GCS package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990). Sequence identity can be measured using sequence analysis software, such as the sequence analysis software package from the Genetics Computing Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wisconsin 53705), using its default parameters.

[0140] IL-2:As used herein, the term "interleukin-2" or "IL-2" refers to a naturally occurring IL-2 polypeptide having IL-2 activity. In some embodiments, IL-2 refers to mature wild-type human IL-2. Mature wild-type human IL-2 (hIL2) is a mature polypeptide of 133 amino acids (minus the signal peptide consisting of an additional 20 N-terminal amino acids), as described in Fujita, et al., PNAS USA, 80, 7437-7441 (1983). The amino acid sequence of a naturally occurring variant of mature wild-type human IL-2 (hIL2) is:

[0141] APTSSSTKKT QLQLEHLLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA

[0142] TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE

[0143] TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT

[0144] (SEQ ID NO:5)

[0145] As used herein, the residue numbering of the hIL2 mutant protein is based on the hIL2 sequence UniProt ID P60568, excluding the same signal peptide as SEQ ID NO:5.

[0146] IL2 activity The term "IL2 activity" refers to one or more biological effects on cells in response to contact with an effective amount of the IL2 peptide. IL2 activity can be measured, for example, in cell proliferation assays using CTLL-2 mouse cytotoxic T cells, largely following the teachings of Gearing, AJH, and CB Bird (1987) in *Lymphokines and Interferons, A Practical Approach* (Clemens, MJ, et al., eds.): IRL Press. 295. The specific activity of recombinant human IL-2 (rhIL2) is approximately 2.1 x 10⁻⁶. 4IU / μg, which is calibrated against the WHO international standard for recombinant human IL2 (NIBSC code: 86 / 500). IL2 activity can be expressed as the level of STAT5 phosphorylation, which can be determined by flow cytometry methods known in the art (Bitar et al. (2019) Evaluating STAT5 Phosphorylation As A Mean to Assess T Cell Proliferation (Frontiers In Immunology, Vol. 10, Article 722, pp. 1-11).

[0147] IL-2 mutant protein As used herein, the term "IL-2 mutant protein" refers to a mutant protein derived from naturally occurring IL-2 that contains modifications to the amino acid sequence of the IL-2 molecule. IL-2 mutant proteins are characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites or other residues in the natural parental IL-2 polypeptide chain. In some embodiments, the IL-2 mutant protein of the present invention retains CD122 binding activity and, when evaluated at similar concentrations in comparative assays, is comparable to the activity of wild-type mature human IL-2 according to WHO international standards (NIBSC code: 86 / 500). Exemplary mutant proteins may contain substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

[0148] Quantity sufficient to produce change As used herein, the phrase "amount sufficient to produce a change" refers to an amount of test reagent sufficient to produce a detectable difference between a level of the indicator measured before (e.g., baseline level) and after administration of the test reagent, such as in cell-based assays assessing the biological function of a response to an amount of test reagent administered. "Amount sufficient to produce a change" can be an amount sufficient to be therapeutically effective, but it may be more or less than a therapeutically effective amount.

[0149] Needs treatment The term "in need of treatment" as used in this article refers to a physician's or other caregiver's judgment about a subject and whether the subject needs or is likely to benefit from treatment. This judgment is based on a number of factors within the physician's or caregiver's area of ​​expertise.

[0150] Preventable The term "in need of prevention" as used in this article refers to a physician's or other caregiver's judgment that the subject needs or is likely to benefit from preventive care. This judgment is based on a number of factors within the physician's or caregiver's area of ​​expertise.

[0151] Inhibitors As used herein, an inhibitor is a molecule that reduces, blocks, prevents, delays activation, inactivates, desensitizes, or downregulates, such as genes, proteins, ligands, receptors, or cells. Inhibitors can also be defined as molecules that reduce, block, or inactivate the constitutive activity of cells or organisms.

[0152] Separate :

[0153] As used herein, the term "isolated" refers to a polypeptide of interest that, if naturally occurring, exists in an environment different from that in which it may naturally occur. "Isolated" means a polypeptide included in a sample that is substantially enriched with the polypeptide of interest and / or that the polypeptide of interest has been partially or substantially purified. If the polypeptide is not naturally occurring, "isolated" means that the polypeptide has been isolated from the environment in which it was prepared by synthetic or recombinant means.

[0154] Kabat ID: As used herein, the term "Kabat numbering" is a recognized term in the field of antibody engineering, referring to a system for numbering amino acid residues (e.g., hypervariable residues) that are more variable than other amino acid residues in the heavy and light chain regions of immunoglobulins (Kabat, et al., (1971) Ann. NY Acad. Sci. 190:382-93; Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the purposes of this disclosure, the location of CDRs in the variable regions of antibodies described herein follows the Kabat numbering system or is simply referred to as "Kabat".

[0155] ligands As used herein, the term "ligand" refers to a molecule that exhibits specific binding to a receptor and causes alteration in the biological activity of the receptor, thereby altering the activity of the receptor to which it binds. In one embodiment, the term "ligand" refers to a molecule or complex thereof that can act as an agonist or antagonist of a receptor. As used herein, the term "ligand" includes both natural and synthetic ligands. "Ligand" also includes small molecules, such as peptide mimics of cytokines and peptide mimics of antibodies. A complex of a ligand and a receptor is referred to as a "ligand-receptor complex."

[0156] Modified IL-2 mutant protein:As used herein, the term "modified IL-2 mutant protein" refers to an IL-2 mutant protein that includes one or more additional further modifications (i.e., modifications beyond the core amino acid sequence of the IL-2 mutant protein), such as PEGylation, glycosylation (N- and O-linking), acylation, or polysialylation, or by coupling with other peptide carrier molecules (whether chemical or fusion proteins), including but not limited to albumin fusion peptides containing serum albumin (e.g., human serum albumin (HSA) or bovine serum albumin (BSA)) or Fc-fusion proteins, or having a targeting moiety (e.g., containing IL-2 orthogonal... The modified IL-2 mutant protein can be prepared by means of a polypeptide fusion protein (IgG) targeting IL-2 mutant protein polypeptides, such as ScFv-IL2 mutant protein polypeptide fusion protein and VHH-IL-2 mutant protein polypeptide fusion protein. Modified IL-2 mutant proteins can be prepared to enhance one or more properties, such as modulating immunogenicity; increasing water solubility, bioavailability, serum half-life and / or therapeutic half-life; and / or modulating biological activity. Certain modifications can also be used, for example, to enhance antibodies (e.g., epitope tags) used in detection assays and to facilitate protein purification. In some embodiments, the modified IL-2 mutant protein is used with SEQ SEQ ID NO:5 must have at least 95, 96, 97, 98, or 99% identity and have one of the three combinations of modifications relative to SEQ ID NO:5 listed in Table 2. Suitable algorithms for determining sequence identity percentages and sequence similarity percentages are BLAST and BLAST 2.0 algorithms, described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI) website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence. These HSPs match or satisfy a threshold score T of some positive value when compared with words of the same length in the database sequence. T is called the adjacent word score threshold (Altschul et al., ibid.). These initial adjacent word hits (words) The word hit is used as a seed to initiate the search in order to find longer HSPs containing them. The word hit is then extended in both directions along each sequence until the cumulative alignment score is increased. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix.The extension of a word match in any direction is halted if: the cumulative alignment score decreases by X from its maximum attainable value; the cumulative score becomes zero or below due to the accumulation of one or more negatively scored residues; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The default values ​​used in the BLASTN program (for nucleotide sequences) are: word length (W) 28, expected value (E) 10, M=1, N=-2, and comparison of two strands. For amino acid sequences, the default values ​​used in the BLASTP program are: word length (W) 3, expected value (E) 10, and a BLOSUM62 score matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0157] adjust As used in this article, the terms “modulation” and “modulation” refer to the ability of a test reagent to affect a reaction in a system (including biological systems or biochemical pathways), which can be positive or negative, and can be direct or indirect.

[0158] Mutant protein: As used herein, the term "mutant protein" refers to a modified form of a wild-type polypeptide, including modifications to the primary structure (i.e., amino acid sequence) of such a polypeptide. The term mutant protein can refer to the polypeptide itself, a composition comprising the polypeptide, or the nucleic acid sequence encoding it. In some embodiments, the mutant protein polypeptide includes about 1 to about 10 amino acid modifications relative to the parent polypeptide, or about 1 to about 5 amino acid modifications relative to the parent, or about 1 to about 3 amino acid modifications relative to the parent, or 1 to 2 amino acid modifications relative to the parent, or a single amino acid modification relative to the parent. The mutant protein may share at least about 99% identity with the parent polypeptide, or at least about 98% identity, or at least about 97% identity, or at least about 95% identity, or at least about 90% identity.

[0159] N-terminus As used in the context of polypeptide structure, “N-terminus” (or “amino terminus”) and “C-terminus” (or “carboxyl terminus”) refer to the polar amino and carboxyl terms of the polypeptide, respectively, while the terms “N-terminal” and “C-terminal” refer to the relative positions of the amino acid sequences of the polypeptide toward the N-terminus and C-terminus, respectively, and may include N-terminal and C-terminal residues, respectively. “Near-neighbor N-terminus” or “near-neighbor C-terminus” refers to the position of the first amino acid residue relative to the second amino acid residue, wherein the first and second amino acid residues are covalently linked to provide a continuous amino acid sequence.

[0160] Nucleic acidThe terms “nucleic acid,” “nucleic acid molecule,” and “polynucleotide” are used interchangeably in this document and refer to any polymer of nucleotides of any length, namely deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers, etc.

[0161] According to IL-2 number As used herein, the term "according to IL-2 number" refers to the identification of the position of a specific amino acid by referring to the position of a naturally occurring amino acid in the mature sequence of mature wild-type hIL-2. For example, R81 refers to the 81st amino acid, arginine, present in SEQ ID NO:5.

[0162] Operable connection The term "operably linked" in this paper refers to the relationship between nucleic acid sequences encoding different functions when combined into a single nucleic acid sequence that, upon introduction into a cell, provides the ability to enable transcription and / or translation of a specific nucleic acid sequence within the cell. For example, if it is expressed as a preprotein involved in polypeptide secretion, the signal sequence DNA is operably linked to the polypeptide DNA; if it affects the transcription of the sequence, a promoter or enhancer is operably linked to the coding sequence; or if it is localized to promote translation, a ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the linked DNA sequences are contiguous, and in the case of secretory leaders, contiguous and in the reading segment. However, some genetic elements, such as enhancers, do not require the sequences that interact with them to be contiguous.

[0163] Parental polypeptides As used herein, the terms “parental polypeptide,” “parental protein,” “precursor polypeptide,” or “precursor protein” are used interchangeably to refer to an unmodified polypeptide that is subsequently modified to produce a variant polypeptide or mutant protein. Parental polypeptides can be wild-type (natural) polypeptides.

[0164] Partial agonistsAs used herein, the term "partial agonist" refers to a molecule that specifically binds to and activates a given receptor, but only partially activates the receptor as opposed to a full agonist. Partial agonists can exhibit both agonist and antagonist effects. For example, when a full agonist and a partial agonist are present, the partial agonist acts as a competitive antagonist, competing with the full agonist for receptor binding, resulting in a net reduction in receptor activation compared to the receptor's contact with the full agonist in its absence. Clinically, partial agonists can be used to activate receptors to provide the desired submaximal response when the amount of endogenous ligands present is insufficient, or they can reduce overstimulation of the receptor when the amount of endogenous ligands is excessive. The maximum response (Emax) produced by a partial agonist is referred to as its intrinsic activity, which can be expressed as a percentage when a full agonist produces a 100% response. When evaluated at similar concentrations in comparative tests, the IL2 partial agonist of the present invention can have greater than 10%, or greater than 20%, or greater than 30%, or greater than 40%, or greater than 50%, or greater than 60%, or greater than 70% of the activity of wild-type mature human IL2 as defined by WHO international standards (NIBSC code: 86 / 500).

[0165] PEG-IL2 mutant protein:As used herein, the term "PEG-IL2 mutant protein" refers to an IL2 mutant protein covalently bound to at least one polyethylene glycol (PEG) molecule, wherein at least one PEG molecule is covalently attached to at least one amino acid residue of the IL-2 mutant protein. PEGylated peptides may also be referred to as mono-PEGylated, di-PEGylated, tri-PEGylated (etc.), to indicate a PEG-IL2 mutant protein comprising one, two, three (or more) PEG moieties attached to the IL-2 mutant protein, respectively. In some embodiments, PEG may be directly covalently attached to the IL-2 mutant protein (e.g., via a lysine side chain, a sulfhydryl group of cysteine, or an N-terminal amine) or optionally a linker may be used between the PEG and the IL-2 mutant protein. In some embodiments, the PEG-IL2 mutant protein comprises more than one PEG molecule, each attached to a different amino acid residue. In some embodiments, the PEG-IL2 mutant protein is derived from sequence ID NO:2 (naturally occurring hIL2). PEGylated forms of IL2 and methods for PEGylating IL2 peptides are well known in the art (e.g., see Katre et al., U.S. Patent 4,931,544, issued June 5, 1990; Katre et al., U.S. Patent 5,206,344, issued April 27, 1993; and Bossard et al., U.S. Patent No. 9,861,705, issued January 9, 2018). In some embodiments, IL2 mutant proteins can be modified by incorporating non-natural amino acids with non-natural amino acid side chains to facilitate site-specific PEGylation, as described in U.S. Patent Application Publication US20170369871A1, published December 28, 2017, by Ptacin et al. In other embodiments, cysteine ​​residues can be incorporated at different positions within the IL2 molecule to facilitate site-specific PEGylation via the cysteine ​​side chain, as illustrated in Greve et al.'s PCT international patent application number PCT / US2015 / 044462, published on February 18, 2016, under WO2016 / 025385.

[0166] polypeptide As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably and refer to a polymer of amino acids of any length, which may include genetically encoded or non-genetically encoded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a modified polypeptide backbone. These terms include fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences; fusion proteins having heterologous and homologous leader sequences; fusion proteins with or without N-terminal methionine residues; fusion proteins having immunotagged proteins; fusion proteins of immunologically active proteins (e.g., fragments of antigenic diphtheria or tetanus toxin), etc.

[0167] prevent The terms "prevention" and "avoidance" as used in this article refer to actions initiated before the onset of a disease, disorder, condition, or its symptoms, thereby temporarily or permanently preventing, mitigating, suppressing, or reducing the risk of the subject developing a certain disease, disorder, condition, or similar illness (e.g., determined by the absence of clinical symptoms), or delaying its onset. This generally applies when the subject is susceptible to a particular disease, disorder, or condition due to genetic, experiential, or environmental factors. In some cases, the terms "prevention" and "avoidance" are also used to refer to slowing the progression of a disease, disorder, or condition from its current state to a more harmful state.

[0168] receptor As used herein, the term "receptor" refers to a polypeptide having a domain that specifically binds a ligand, the binding of which results in a change in at least one biological property of the polypeptide. In some embodiments, the receptor is a "soluble" receptor that is not associated with the cell surface. The soluble form of hCD25 is an example of a soluble receptor that specifically binds to hIL2. In some embodiments, the receptor is a cell surface receptor comprising an extracellular domain (ECD) and a membrane-associated domain, the latter used to anchor the ECD to the cell surface. In some embodiments of cell surface receptors, the receptor is a transmembrane polypeptide comprising an intracellular domain (ICD) and an extracellular domain (ECD) connected by a transmembrane domain (TM), commonly referred to as a transmembrane domain. Ligand binding to the receptor results in a conformational change in the receptor, thereby producing a measurable biological effect. In some cases, if the receptor is a transmembrane polypeptide comprising ECD, TM, and ICD, ligand binding to the ECD results in measurable intracellular biological effects mediated by one or more domains of the ICD in response to ligand binding to the ECD. In some embodiments, the receptor is a component of a multi-component complex to facilitate intracellular signal transduction. For example, the ligand may bind a cell surface molecule that is not individually associated with any intracellular signal transduction but, upon ligand binding, promotes the formation of heteromultimers (including heterodimers (e.g., intermediate-affinity CD122 / CD132 IL2 receptors), heterotrimers (e.g., high-affinity CD25 / CD122 / CD132 hIL2 receptors), or homomultimers (e.g., homodimers, homotrimers, homotetramers) complexes, leading to activation of intracellular signal transduction cascades (e.g., the Jak / STAT pathway).

[0169] Reorganization As used herein, the term recombinant refers to polypeptides produced using recombinant DNA technology. The techniques and methods for recombinant DNA technology are well known in the art.

[0170] reaction: As used herein, the terms “response” or “reaction” refer to, for example, the response of a cell, tissue, organ, or organism, including changes in biochemical or physiological behavior, such as concentration, density, adhesion, or migration within biological compartments, gene expression rates, or differentiation states, where such changes are associated with activation, stimulation, or treatment, or with internal mechanisms such as genetic programming. In some cases, the terms “activation,” “stimulation,” etc., refer to cellular activation regulated by internal mechanisms and external or environmental factors; while the terms “inhibition,” “downregulation,” etc., refer to the opposite effect.

[0171] Selective As used herein, the term "selectivity" refers to the property of a substance to preferentially bind to and / or activate a particular cell type based on a certain characteristic of a certain cell population. In some embodiments, the present invention provides a CD25-selective mutant protein, i.e., a mutant protein that exhibits preferential activation on cells expressing CD25 and / or CD25 / CD122 receptors relative to cells expressing the CD132 receptor. Selectivity is typically assessed by activity measurements in characterization assays of ligand / receptor binding response-induced activity. In some embodiments, selective IL2 mutant proteins exhibit significantly reduced binding. In some embodiments, selectivity is measured by comparing CD25-expressing cells (e.g., YTCD25POS or YT...). CD25+ Cells showing significantly lower (preferably undetectable) CD25 levels compared to those showing activation of YTCD25NEG or YT cells (e.g., YTCD25NEG or YT cells) were compared. CD25- The activation of CD25-expressing T cells (e.g., Tregs) is performed. In some embodiments, the selectivity is measured by the activation of CD25-expressing T cells (e.g., Tregs) compared to the activation of T cells with low CD25 levels (unstimulated CD8+ or CD4+ T cells). In some embodiments, in the same assay, the IL2 mutant protein of the present invention exhibits a difference of at least 3-fold, at least 5-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 100-fold, or at least 200-fold in EC50 measured on CD25+ versus CD25- cells.

[0172] Significantly reduced binding: As used herein, the term "exhibiting significantly reduced binding" is used to indicate the binding affinity of a ligand (such as an IL2 mutant or a modified IL2 mutant) to a receptor relative to the binding affinity of the naturally occurring form of the modified ligand to the homologous receptor. An IL2 mutant exhibits significantly reduced binding if the binding of the IL2 mutant to the receptor in its natural form is less than 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 2%, or less than about 1% of the binding affinity of the naturally occurring ligand.

[0173] Specific binding: As used herein, the term "specific binding" refers to the degree of selectivity or affinity of one molecule to another. In the context of binding pairs (such as ligand / receptor, antibody / antigen, antibody / ligand, antibody / receptor binding pairs), a molecule is said to specifically bind to a second molecule when the first molecule of the binding pair does not bind to other components present in the sample in a significant amount. A molecule is said to specifically bind to a second molecule when the affinity of the first molecule to the second molecule is at least 2, 5, 10, 20, or 100 times greater than the affinity of the first molecule to other components present in the sample. In a specific embodiment where the first molecule in the binding pair is an antibody, the equilibrium dissociation constant between the antibody and the second molecule of the binding pair is greater than about 10. 6 M, or greater than approximately 10 8 M, or greater than approximately 10 10 M, or greater than approximately 10 11 M, or greater than approximately 10 10 M, or greater than approximately 10 12 M, for example, as determined by Scatchard analysis, indicates that the antibody specifically binds to the second molecule (e.g., protein, antigen, ligand, or receptor) in the binding pair (Munsen, et al. 1980 Analyt. Biochem. 107:220-239). In one embodiment where the ligand is an IL2 mutant protein and the receptor includes an orthogonal CD122 ECD, if the equilibrium dissociation constant of the IL2 mutant protein / orthogonal CD122 ECD is greater than about 10... 5 M, or greater than approximately 10 6 M, or greater than approximately 10 7 M, or greater than approximately 10 8 M, or greater than approximately 10 9 M, or greater than approximately 10 10 M, or greater than approximately 10 11M, then the IL2 mutant protein specifically binds. Specific binding can be assessed using techniques known in the art, including but not limited to competitive ELISA, radioligand binding assays (e.g., saturation binding, Scatchard plot, nonlinear curve fitting procedures, and competitive binding assays); non-radioligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), and surface plasmon resonance assays (see, e.g., Drescher et al., Methods Mol Biol 493:323-343 (2009)), using commercially available instruments from GE Healthcare Bio-Sciences, such as Biacore 8+, Biacore S200, and Biacore T200 (GE Healthcare Bio-Sciences, 100 Results Way, Marlborough, MA). 01752); liquid-phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR) and immunoprecipitation); and solid-phase ligand binding assays (e.g., multi-well plate assays, bead-based ligand binding assays, column-based ligand binding assays, and filtration assays).

[0174] object The terms “recipient,” “individual,” “object,” and “patient” are used interchangeably herein and refer to any mammalian object requiring diagnosis, treatment, or therapy, particularly a human. For therapeutic purposes, “mammal” means any animal classified as a mammal, including humans, domestic and farm animals, non-human primates, and zoo, sporting, or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In some embodiments, the mammal is a human.

[0175] Suffering from As used herein, the term "having" refers to a physician's judgment, based on existing information recognized in the field for identifying a disease, disorder, or condition (including but not limited to X-rays, CT scans, routine laboratory diagnostic tests (e.g., blood cell counts), genomic data, protein expression data, and immunohistochemistry), that a subject needs or will benefit from treatment. The term "having" is often used in conjunction with a specific disease state; for example, "having an inflammatory, infectious, or autoimmune disease, disorder, or condition" means that the subject has been diagnosed with an inflammatory, infectious, or autoimmune disease, disorder, or condition.

[0176] Basically pureAs used herein, the term "substantially pure" means that a component (e.g., a polypeptide) constitutes more than about 50% of the total composition, typically more than about 60% of the total polypeptide content. More generally, "substantially pure" means that at least 75%, at least 85%, at least 90% or more of the total composition is the component of interest. In some cases, the polypeptide will constitute more than about 90%, or more than about 95%, of the total composition.

[0177] T cells: As used herein, the term "T-cell" or "T cell" in its conventional sense refers to a lymphocyte differentiated in the thymus that possesses specific cell surface antigen receptors, including some that control the initiation or suppression of cell-mediated immunity and humoral immunity, as well as the lysis of antigen-carrying cells. In some embodiments, T cells include, but are not limited to, naïve CD8 cells. + T cells, cytotoxic CD8 + T cells, immature CD4 + T cells, helper T cells, such as T cells H 1. T H 2. T H 9. T H 11. T H 22. T FH Regulatory T cells, such as T cells R 1. Tregs, induced Tregs; memory T cells, such as central memory T cells, effector memory T cells, NKT cells, tumor-infiltrating lymphocytes (TILs) and engineered variants of these T cells, including but not limited to CAR-T cells, recombinant modified TILs and TCR engineered cells.

[0178] Therapeutic effective doseThe term "therapeutic effective dose" as used herein refers to the amount of an agent administered to a subject, alone or as part of a pharmaceutical composition or treatment regimen, in a single dose or as part of a series of doses, to produce any detectable, positive effect on any symptom, aspect, or characteristic of a disease, disorder, or condition. Therapeutic effective doses can be determined by measuring the associated physiological effects and can be adjusted in conjunction with the dosing regimen and diagnostic analysis of the subject's condition. The assessment parameters used to determine the therapeutic effective dose of an agent are determined by a physician using recognized diagnostic criteria, including but not limited to indicators such as age, weight, sex, general health status, ECOG score, observable physiological parameters, blood concentrations, blood pressure, electrocardiogram, computed tomography, X-ray, etc. Alternatively, other parameters typically assessed in clinical settings can be monitored to determine whether the subject has received a therapeutically effective dose of the agent, such as normalization of body temperature, heart rate, blood chemistry, blood pressure, cholesterol levels, or any symptoms, aspects, or characteristics of the disease, disorder, or condition; reductions in biomarkers (such as inflammatory cytokines, IFN-γ, granzymes, etc.); reductions in serum tumor markers; improvements in the Responsive Response Standard for Solid Tumors (RECIST); improvements in the Immune Related Response Standard (irRC); prolonged survival; prolonged progression-free survival; prolonged time to progression; prolonged time to treatment failure; prolonged event-free survival; prolonged time to next treatment; improved objective response rate; improved duration of response; reduced tumor burden; complete remission; partial remission; stable disease, etc. Clinicians in this field assess improvements in the subject's response to the administered agent based on these parameters. As used herein, the terms “complete response (CR),” “partial response (PR),” “stable disease (SD),” and “progressive disease (PD)” relating to target lesions, and the terms “complete response (CR),” “incomplete response / stable disease (SD),” and “progressive disease (PD)” relating to non-target lesions, should be understood as defined in the RECIST criteria. The terms “immune-related complete response (irCR),” “immune-related partial response (irPR),” “immune-related progressive disease (irPD),” and “immune-related stable disease (irSD)” as used herein are as defined according to the immune-related response criteria (irRC).As used herein, the term “immune-related response criteria (irRC)” refers to a system used to assess the response to immunotherapy, as described in Wolchok et al. (2009), Guidelines for the Evaluation of Immune Therapy Activity in Solid Tumors: Immune-Related Response Criteria, Clinical Cancer Research 15(23): 7412-7420. During the course of treatment, the therapeutically effective dose may be adjusted based on the dosing regimen and / or assessment of the patient’s condition and changes in the foregoing factors. In one embodiment, the therapeutically effective dose is the amount of reagent that, when used alone or in combination with another reagent, does not cause irreversible serious adverse events during administration to mammalian subjects.

[0179] Transmembrane domains: The term "transmembrane domain" or "TM" refers to a domain of a transmembrane polypeptide (such as CD122, CD132, or CAR) that, when associated with the cell membrane, is embedded within the cell membrane and linked by peptidyl linkage to both the extracellular domain (ECD) and intracellular domain (ICD) of the transmembrane polypeptide. The transmembrane domain can be homologous (naturally associated) or heterologous (not naturally associated) to one or both of the extracellular and / or intracellular domains. In some embodiments, the transmembrane domain is a transmembrane domain naturally associated with the ECD domain of a homologous receptor of a derived orthogonal receptor. In some embodiments, the transmembrane domain is a transmembrane domain naturally associated with the ICD domain of a homologous receptor of a derived orthogonal receptor. In some embodiments, the transmembrane domain is a transmembrane domain naturally associated with a proliferation signal transduction domain. In some embodiments, the transmembrane domain is a transmembrane domain naturally associated with a different protein. Alternatively, the transmembrane domain of a receptor can be an artificial amino acid sequence that crosses the plasma membrane. In some embodiments, when the receptor is a chimeric receptor comprising an intracellular domain derived from a first parent receptor and a second extracellular domain derived from a second different parent receptor, the transmembrane domain of the chimeric receptor is a transmembrane domain typically associated with the ICD or ECD of the parent receptor from which the chimeric receptor is derived.

[0180] treatThe terms “treatment,” “therapy,” “management,” etc., refer to an action process (e.g., administration of a mutant IL-2 protein or a pharmaceutical composition containing it) or similar procedure initiated after the diagnosis or observation of a subject’s disease, disorder, or symptom or its symptoms, in order to temporarily or permanently eliminate, reduce, suppress, alleviate, or improve at least one underlying cause of the disease, disorder, or symptom troubling the subject, or at least one symptom associated with the disease, disorder, or symptom. Treatment includes actions taken on a subject suffering from a disease, wherein such actions result in the suppression of the subject’s disease (e.g., preventing the development of the disease, disorder, or symptom or improving one or more symptoms associated therewith).

[0181] Treg cells or regulatory T cells. The terms "regulatory T cells" or "Treg cells" used in this article refer to CD4+ cells. + T cells are types of T cells that can suppress the responses of other T cells, including but not limited to effector T cells (Teff). Treg cells are characterized by the expression of CD4, the α subunit of the IL-2 receptor (CD25), and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, AnnuRev Immunol 22, 531-62 (2004). "Conventional CD4 + "T cells" refers to CD4 cells other than regulatory T cells. + T cells.

[0182] variants The terms "protein variant" or "variant protein" or "variant polypeptide" are used interchangeably herein and refer to a polypeptide that differs from a parent polypeptide due to at least one amino acid modification. The parent polypeptide can be a naturally occurring or wild-type (WT) polypeptide or a modified form of a WT polypeptide (i.e., a mutant protein).

[0183] wild type: "Wild-type," "WT," or "natural" in this article refers to an amino acid or nucleotide sequence that exists in nature, including allelic variations. Wild-type proteins, peptides, antibodies, immunoglobulins, IgG, etc., have amino acid or nucleotide sequences that have not been artificially modified.

[0184] In some embodiments, the IL2 mutant protein of the present invention is provided with modifications that modify the binding of the IL2 mutant protein to other proteins, particularly CD25, CD122, and CD132, as well as combinations of these proteins, such as CD122 / CD132 (“intermediate affinity IL2 receptor”), CD25 (“low affinity IL2 receptor”), and CD25 / CD122 / CD132 (“high affinity IL2 receptor”).

[0185] The present invention provides methods and compositions for treating and / or preventing inflammatory, infectious or autoimmune diseases, disorders or conditions, which involve administering a therapeutically effective amount of a human IL-2 mutant protein, which has a reduced binding affinity for CD132 while retaining a significant binding affinity for CD122 and / or CD25 comparable to that of wild-type human IL-2.

[0186] In some embodiments, the IL-2 mutant protein exhibits decreased binding affinity to the extracellular domain of hCD132 (e.g., <50% of wild-type hIL2, or <45% of wild-type hIL2, or <40% of wild-type IL2, or <35% of wild-type hIL2, or <25% of wild-type hIL2, or <20% of wild-type hIL2, or <15% of wild-type IL2, or <10% of wild-type IL2, or <5% of wild-type IL2), while retaining basal affinity for the extracellular domain of the wild-type human CD122 receptor (e.g., 20% of wild-type hIL2, or >30% of wild-type hIL2, or >40%, or >50% of wild-type hIL2). Affinity to wild-type hIL2, or >60% affinity to wild-type hIL2, or >65% affinity to wild-type hIL2, or >70% affinity to wild-type hIL2, or >75% affinity to wild-type hIL2, or >80% affinity to wild-type hIL2, or >85% affinity to wild-type hIL2, or >90% affinity to wild-type IL2, or >90% affinity to wild-type IL2. (Affinity to wild-type IL2, or >95% affinity to wild-type IL2, or >100% affinity to wild-type IL2, or >105% affinity to wild-type hIL2, or >110% affinity to wild-type IL2, or >115% affinity to wild-type hIL2, or >125% affinity to wild-type IL2, or >150% affinity to wild-type hIL2).

[0187] In some embodiments, the IL-2 mutant protein with reduced binding affinity to the CD132 receptor, useful in the practice of the methods of the present invention, further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mutations that increase the binding affinity to CD122. In some embodiments, the host IL-2 mutant protein useful in the practice of the methods of this disclosure comprises at least one mutation relative to wild-type IL-2 (e.g., SEQ ID NO: 5) (e.g., deletion, addition, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) and binds to CD122 with a higher affinity than wild-type IL-2. In some embodiments, the affinity of the IL-2 mutant protein for CD122 is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% greater than that of wild-type hIL-2. The binding affinity of the IL-2 mutant protein can also be expressed as an affinity for CD122 that is 1.2, 1.4, 1.5, 2, 5, 10, 15, 20, 25, 50, 100, 200, 250, or more times greater than that of wild-type hIL-2.

[0188] In some embodiments, the IL-2 mutant protein exhibits decreased binding affinity to the extracellular domain of hCD132 (e.g., <50% of wild-type hIL2, or <45% of wild-type hIL2, or <40% of wild-type hIL2, or <35% of wild-type hIL2, or <25% of wild-type hIL2, or <20% of wild-type hIL2, or <15% of wild-type hIL2, or <10% of wild-type hIL2, or <5% of wild-type hIL2), while retaining basal affinity for the hCD25 / hCD122 receptor complex (e.g., >50% of wild-type hIL2, or >60% of wild-type hIL2). Affinity to wild-type hIL2, or >65% affinity to wild-type hIL2, or >70% affinity to wild-type hIL2, or >75% affinity to wild-type hIL2, or >80% affinity to wild-type hIL2, or >85% affinity to wild-type hIL2, or >90% affinity to wild-type hIL2, or >90% affinity to wild-type hIL2, or >95% affinity to wild-type hIL2, or >100% affinity to wild-type hIL2, or >105% affinity to wild-type hIL2, or >110% affinity to wild-type hIL2, or >115% affinity to wild-type hIL2, or >125% affinity to wild-type hIL2, or >150% affinity to wild-type IL2. In some embodiments, the IL2 mutant protein of the present invention has a reduced affinity for CD132. In some embodiments, this IL2 mutant protein incorporates modifications to the primary structure of wild-type IL2, with one or more modifications incorporated at positions 18, 22, and 126 according to the wild-type hIL-2 number.

[0189] In some implementations, the IL-2 mutant protein exhibits decreased binding affinity to CD132 while retaining a basic binding affinity to hCD25 (e.g., >50% of wild-type hIL2, or >60% of wild-type hIL2, or >65% of wild-type hIL2, or >70% of wild-type hIL2, or >75% of wild-type hIL2, or >80% of wild-type hIL2, or >85% of wild-type hIL2, or >90% of wild-type hIL2, or >90% of wild-type hIL2). Affinity, or >95% affinity for wild-type hIL2, or >100% affinity for wild-type IL2, or >105% affinity for wild-type hIL2, or >110% affinity for wild-type hIL2, or >115% affinity for wild-type hIL2, or >125% affinity for wild-type hIL2, or >150% affinity for wild-type hIL2, or >200% affinity for wild-type hIL2, or >300% affinity for wild-type IL2, or >400% affinity for wild-type hIL2, or >500% affinity for wild-type IL2.

[0190] On the one hand, the present invention provides a mutant hIL-2 protein that, compared with wild-type human IL-2 (hIL-2), exhibits a significant or enhanced binding affinity for hCD25 and a reduced binding affinity for the extracellular domain of the hCD132 receptor.

[0191] In some embodiments, the IL-2 mutant protein includes one or more amino acid substitutions that reduce the binding affinity to the CD132 receptor. The substitutions are selected from amino acids 18, 22, and 126, numbered according to the mature wild-type hIL-2.

[0192] In some embodiments, the host IL-2 mutant protein, which is useful as a partial agonist in the practice of the method of the present invention, has one or more reduced functions compared with wild-type IL-2.

[0193] In some embodiments, the IL-2 mutant protein useful in the practice of the method of the present invention disrupts the association between CD122 and CD132, such that the CD122 / CD132 interaction is reduced by about 2%, about 5%, about 10%, about 15%, about 20%, about 50%, about 75%, about 90%, about 95%, or more relative to wild-type hIL-2. In some embodiments, one or more mutations that reduce the binding affinity of the IL-2 mutant protein to CD132 are amino acid substitutions. In some embodiments, the host hIL-2 mutant protein has only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions compared to wild-type IL-2 (SEQ ID NO:5).

[0194] In some embodiments, the IL2 mutant protein useful in the practice of the method of the present invention is an inhibitor of IL-2 and / or IL-15 phosphorylation in CD8+ T cells. In some embodiments, the mutant protein is an inhibitor of IL-2 and / or IL-15-induced CD8+ T cell proliferation. In some embodiments, the mutant protein is an inhibitor of IL-2-dependent, TCR-induced cell proliferation.

[0195] In some embodiments, the IL2 mutant protein useful in the practice of the methods of the present invention is an inhibitor of IL-2-dependent natural killer (NK) cell activation. IL-2 activation of NK cells can be measured by any suitable method known in the art, for example, by measuring IL-2-induced CD69 expression and / or cytotoxicity, as described herein.

[0196] In some embodiments of the invention, the IL-2 mutant protein is a partial agonist. In some embodiments, the IL-2 mutant protein useful in the practice of the method of the invention is a partial agonist with reduced ability to stimulate one or more CD122 / CD132 heterodimerization-dependent signaling pathways. In some embodiments, the host IL-2 mutant protein has a reduced ability to stimulate phosphorylation in CD122+ cells compared to wild-type hIL-2. In some embodiments, the level at which the IL-2 mutant protein stimulates STAT5 phosphorylation in IL-2RP+ cells is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or lower than the level at which wild-type IL-2 stimulates STAT5 phosphorylation in the same cells. In some embodiments, IL-2Rp+ cells are T cells. In certain embodiments, the T cells are CD8+ T cells. In some embodiments, the CD8+ T cells are freshly isolated CD8+ T cells. In other embodiments, CD8+ T cells are activated CD8+ T cells. In other embodiments, CD122+ cells are natural killer (NK) cells.

[0197] In some embodiments, the IL-2 mutant protein useful in the practice of the method of the present invention is a partial agonist with a reduced ability to stimulate signal transduction in CD122+ cells compared to wild-type hIL-2. In some embodiments, the level at which the IL-2 mutant protein stimulates pERK1 / ERK2 signal transduction in CD122+ cells is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or lower than the level at which wild-type IL-2 stimulates pERK1 / ERK2 signal transduction in the same cells. In some embodiments, the CD122+ cells are T cells. In a particular embodiment, the CD122+ T cells are CD8+ T cells. In some embodiments, the CD122+ CD8+ T cells are CD122+ CD8+ T cells isolated from the subject. In other embodiments, the CD8+ T cells are activated CD122+ CD8+ T cells. In other embodiments, CD122+ cells are natural killer (NK) cells. STAT5 and ERK1 / 2 signaling can be measured, for example, by phosphorylation of STAT5 and ERK1 / 2 using any suitable method known in the art. For example, phosphorylation of STAT5 and ERK1 / 2 can be measured using specific antibodies against the phosphorylated forms of these molecules.

[0198] In some embodiments, the mutant protein useful in the practice of the methods of the present invention is a partial agonist with a reduced ability to induce lymphocyte proliferation compared to wild-type hIL-2. In some embodiments, the lymphocytes are T cells. In a particular embodiment, the lymphocytes are primary CD8+ T cells. In other embodiments, the lymphocytes are activated CD8+ T cells. Cell proliferation can be determined using any suitable method known in the art. For example, lymphocyte proliferation can be measured using a carboxyfluorescein diacetate succinimidyul diester (CFSE) dilution assay or by [31-1]-thymidine inclusion, as described herein. In some embodiments, the level of lymphocyte proliferation induced by the IL-2 mutant protein of the present invention is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or lower than the level of lymphocyte proliferation induced by wild-type hIL-2.

[0199] In some embodiments, the IL-2 mutant protein of the present invention is a partial agonist, exhibiting a reduced ability to activate CD25 expression compared to wild-type IL-2. In some embodiments, the level at which the IL-2 mutant protein activates IL-2Ra expression in lymphocytes is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or lower than the level at which wild-type IL-2 activates CD25 expression in the same cells. In some embodiments, the lymphocytes are CD8+ T cells. In some embodiments, the CD8+ T cells are freshly isolated CD8+ T cells. + T cells. In other embodiments, CD8+ T cells are activated CD8+ T cells.

[0200] In some embodiments of the present invention, the IL-2 mutant protein is a complete agonist.

[0201] In some embodiments of the present invention, the IL-2 mutant protein is a super agonist.

[0202] In some embodiments, the methods and compositions of the present invention for treating and / or preventing inflammatory, infectious, or autoimmune diseases, disorders, or symptom-related illnesses involve administering a therapeutically effective amount of a human IL-2 mutant protein in combination with a supplement that has reduced binding affinity for CD132 but retains a significant binding affinity for CD122 and / or CD25 comparable to that of wild-type hIL-2. The supplement includes, but is not limited to, one or more of chemotherapy, immune checkpoint modulators, radiotherapy, and / or physical interventions (such as surgery).

[0203] In some embodiments, the present invention provides a human interleukin-2 (IL-2) mutant protein that provides binding properties to modifications of one or more IL2 receptors for the treatment of inflammatory, infectious, or autoimmune diseases, disorders, or conditions.

[0204] In various embodiments, the present invention provides a polypeptide comprising an amino acid sequence according to Formula 1:

[0205] (AA1) a –(AA2) b -(AA3) c -(AA4) d -(AA5) e -(AA6) f -(AA7) g -(AA8) h -(AA9) i-T10-Q11-L12-Q13-L14-E15-H16-L17-(AA18)-L19-D20-L21-(AA22)-M23-I24-L25-N26 -G27-I28-N29-N30-Y31-K32-N33-P34-(AA35)-L36-T37-(AA38)-(AA39)-L40-T41-F42- K43-F44-Y45-M46-P47-K48-K49-A50-T51-E52-L53-K54-(AA55)-L56-Q57-C58-L59-E60 -E61-E62-L63-K64-P65-L66-E67-E68-(AA69)-L70-N71-L72-A73-(AA74)-S75-K76-N77- F78-H79-(AA80-(AA81)-P82-R83-D84-(AA85)-(AA86)-S87-N88-(AA89)-N90-(AA91)-( AA92)-V93-L94-E95-L96-(AA97)-G98-S99-E100-T101-T102-F103-(AA104)-C105-E106- Y107-A108-(AA109)-E110-T111-A112-(AA113)-I114-V115-E116-F117-L118-N119-R12 0-W121-I122-T123-F124-(AA125)-(AA126)-S127-I128-I129-(AA130)-T131-L132-T133

[0206] Formula 1 (SEQ ID NO: 97)

[0207] in:

[0208] a, b, c, d, e, f, g, h, and i are each individually selected from 0 or 1;

[0209] AA1 is A (wild-type, a=1) or missing (a=0);

[0210] AA2 is P (wild type, b=1) or deleted (b=0);

[0211] AA3 is T (wild type, c=1), C, A, G, Q, E, N, D, R, K, P or deleted (c=0);

[0212] AA4 is S (wild type, d=1) or missing (d=0);

[0213] AA5 is S (wild-type, e=1) or deleted (e=0);

[0214] AA6 is S (wild type, f=1) or missing (f=0);

[0215] AA7 is T (wild type, g=1) or deleted (g=0);

[0216] AA8 is K (wild-type, h=1) or deleted (h=0);

[0217] AA9 is either K (wild type, i=1) or missing (i=0);

[0218] AA18 is L (wild type) or R, L, G, M, F, E, H, W, K, Q, S, V, I, Y, H, D or T;

[0219] AA22 is Q (wild type) or F, E, G, A, L, M, F, W, K, S, V, I, Y, H, R, N, D, T or F;

[0220] AA35 is either K (wild type) or E;

[0221] AA38 is R (wild type), W, or G;

[0222] AA39 is M (wild type), L, or V;

[0223] AA55 is either H (wild type) or Y;

[0224] AA69 is either V (wild type) or A;

[0225] AA74 is Q (wild type), P, N, H, S;

[0226] AA80 is L (wild type), F, or V;

[0227] AA81 is R (wild type), I, D, or T;

[0228] AA85 is either L (wild type) or V;

[0229] AA86 is either I (wild type) or V;

[0230] AA89 is either I (wild type) or V;

[0231] AA91 is V (wild type), R, or K;

[0232] AA92 is either I (wild type) or F;

[0233] AA97 is either K (wild type) or Q;

[0234] AA104 is either M (wild type) or A;

[0235] AA109 is a non-natural amino acid, either D (wild-type), C, or with an activated side chain;

[0236] AA113 is either T (wild type) or N;

[0237] AA125 is C (wild type), A, or S;

[0238] AA126 is Q (wild type) or H, M, K, C, D, E, G, I, R, S, or T; and

[0239] AA130 is S (wild type), T, G, or R; and

[0240] The premise is that if AA18 is R and AA22 is E, then AA126 is not H, M, K, C, D, E, G, I, R, S or T.

[0241] In some embodiments, the present invention provides an IL2 mutant protein comprising the following mutations:

[0242] AA18 is selected from the following group: L (wild type) or R, L, G, M, F, E, H, W, K, Q, S, V, I, Y, H, D or T;

[0243] AA22 is selected from the following group: Q (wild type) or F, E, G, A, L, M, F, W, K, S, V, I, Y, H, R, N, D, T, or F; and

[0244] AA126 is selected from the following groups: Q (wild type) or H, M, K, C, D, E, G, I, R, S, or T.

[0245] The premise is that if AA18 is R and AA22 is E, then AA126 is not H, M, K, C, D, E, G, I, R, S or T.

[0246] In some embodiments, the present invention provides an IL2 mutant protein comprising the following mutations:

[0247] a=0;

[0248] AA18 is selected from the following group: L (wild type) or R, L, G, M, F, E, H, W, K, Q, S, V, I, Y, H, D or T;

[0249] AA22 is selected from the following group: Q (wild type) or F, E, G, A, L, M, F, W, K, S, V, I, Y, H, R, N, D, T, or F; and

[0250] AA126 is selected from the following groups: Q (wild type) or H, M, K, C, D, E, G, I, R, S or T.

[0251] The premise is that if AA18 is R and AA22 is F or V, then AA126 is not H, M, K, C, D, E, G, I, R, S or T.

[0252] In some embodiments, the present invention provides IL-2 mutant proteins comprising amino acid substitutions at positions 18, 22, and 126 according to wild-type hIL-2 numbering, as described in Table 2 below. It should be noted that the three-letter abbreviations for specific IL2 mutant proteins reflect IL2 mutant proteins having mutations at positions 18, 22, and 126; for example, “FEH” is a shorthand term for IL2 mutant proteins containing L18F, Q22E, and Q126H substitutions. In particular, the IL2 mutant proteins of the present invention comprise amino acid substitutions at positions 18 and / or 22 and 126, as described in Table 2 below:

[0253]

[0254]

[0255]

[0256] The IL2 mutant proteins listed in Table 2 were prepared and tested essentially according to the embodiments described herein. The experimental results are shown in the attached figures. Figure 1 and 2 Provided in [the text]. For example... Figure 1 As illustrated, the IL2 mutant protein of this invention retains significant IL2 activity. For example... Figure 2 As can be seen, the IL2 mutant protein of the present invention exhibits significantly preferred activity on CD25-expressing cells compared to wild-type IL2.

[0257] IL2 mutant proteins may also contain more than one substitution, deletion, or insertion in the wild-type IL-2 amino acid sequence. The following nomenclature is used herein to indicate substitutions, deletions, or insertions. Residues are designated herein by a one-letter or three-letter amino acid code followed by the IL-2 amino acid position, for example, “Cys125” or “C125” refers to the cysteine ​​residue at position 125 of SEQ ID NO:5. Substitutions are designated herein by a one-letter amino acid code followed by the IL-2 amino acid position, followed by the one-letter amino acid code of the substitution, for example, “K35A” refers to the substitution of the lysine (K) residue at position 35 of SEQ ID NO:5 with an alanine (A) residue. Deletions are referred to as “des”, followed by the deleted amino acid residue and its position in SEQ ID NO:5. For example, the term “des-Ala1” or “desA1” refers to the deletion of alanine at position 1 of the polypeptide in SEQ ID NO:5.

[0258] Mutations that increase CD122 affinity

[0259] In some embodiments of the present invention, the IL2 mutant protein may include amino acid substitutions that enhance CD122 binding affinity. Examples of amino acid substitutions that enhance CD122 binding affinity include, but are not limited to, Q74N, Q74H, Q74S, L80F, L80V, R81D, R81T, L85V, I86V, I89V, and / or I92F, or combinations thereof. In some embodiments, amino acid substitutions that increase CD122 binding affinity include: L80F, R81D, L85V, I86V, and I92F. In some embodiments, amino acid substitutions that increase CD122 binding affinity include: N74Q, L80F, R81D, L85V, I86V, I89V, and I92F. In some embodiments, amino acid substitutions that increase CD122 binding affinity include: Q74N, L80V, R81T, L85V, I86V, and I92F. In some embodiments, amino acid substitutions that increase CD122 binding affinity include: Q74H, L80F, R81D, L85V, I86V, and I92F. In some embodiments, amino acid substitutions that increase CD122 binding affinity include: Q74S, L80F, R81D, L85V, I86V, and I92F. In some embodiments, amino acid substitutions that increase CD122 binding affinity include: Q74N, L80F, R81D, L85V, I86V, and I92F. In some embodiments, amino acid substitutions that increase CD122 binding affinity include: Q74S, R81T, L85V, and I92F.

[0260] In some embodiments, the IL2 mutant protein is affinity-matured to enhance its affinity for CD25 and / or CD122. An “affinity-matured” polypeptide is a polypeptide with one or more alterations at one or more residues, resulting in improved affinity of the orthogonal polypeptide for a homologous orthogonal receptor, or vice versa, compared to a parent polypeptide that does not possess these alterations. Affinity maturation increases the binding affinity of the IL2 mutant protein by at least about 10%, or at least about 50%, or at least about 100%, or at least about 150%, or 1-5 times compared to the “parent” polypeptide.

[0261] Mutations that increase CD25 affinity:

[0262] In some embodiments, the IL-2 mutant protein contains one or more mutations at the position of the IL-2 sequence, which either contact CD25 or alter the orientation of other positions contacting CD25, resulting in an increased affinity of the IL-2 mutant protein for CD25. In some embodiments, the IL-2 mutant protein of the present invention comprises one or more substitutions for V69A and Q74P, which have been described as increasing the binding affinity of IL2 for CD25.

[0263] Removal of Thr3 glycosylation sites

[0264] The IL2 mutant protein of the present invention may also provide, or optionally provide, the elimination of the O-glycosylation site at Thr3 to promote the production of a non-glycosylated IL2 mutant protein when expressed in mammalian cells (e.g., CHO or HEK cells). Therefore, in some embodiments, the IL2 mutant protein further comprises a modification that eliminates the O-glycosylation site at the residue 3 corresponding to human IL-2. In some embodiments, the modification that eliminates the O-glycosylation site at the residue 3 corresponding to human IL-2 is an amino acid modification. Exemplary amino acid substitutions include T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, T3S, T3C, and T3P, which remove the glycosylation site at position 3 without eliminating biological activity (see U.S. Patent No. 5,116,943; Weiger et al., (1989) Eur. J. Biochem., 180:295-300). In a particular embodiment, the modification is the substitution of T3A with an amino acid.

[0265] Minimize vascular leakage syndrome

[0266] In some embodiments of the invention, the IL2 mutant protein may contain amino acid substitutions to avoid vascular leakage syndrome, a substantial negative and dose-limiting side effect of IL2 therapy in humans without a substantial loss of efficacy. See Epstein, et al., U.S. Patent No. 7,514,073B2, issued April 7, 2009. Examples of such modifications included in the IL2 mutant protein of the present invention include one or more R38W, R38G, R39L, R39V, F42K, and H55Y.

[0267] Antioxidant M104A

[0268] The IL2 mutant protein disclosed herein may optionally further include a modification at the M104 position, in one embodiment replacing methionine 104 with an alanine residue (M104A) to provide a more antioxidant IL2 mutant protein (see Koths, et al., U.S. Patent 4,752,585, issued June 21, 1988).

[0269] Cys125

[0270] In some implementations, the cysteine ​​at position 125 is replaced with alanine or serine (C125A or C125S) to minimize potential protein misfolding during recombinant expression in bacteria and isolation from inclusion bodies, as described herein.

[0271] N-terminal deletion

[0272] When recombinantly produced in a bacterial expression system, in the absence of a leader sequence, endogenous proteases cause the deletion of the N-terminal Met-Ala1 residue to provide the "desAla1" IL2 mutant protein. In some embodiments, the present invention provides an hIL2 mutant protein, which is an hIL2 polypeptide comprising one of the following groups of amino acid modifications:

[0273] IL2 mutant proteins may include the deletion of the first two amino acids (desAla1-desPro2), and Thr3 glycosylation with cysteine ​​residues to promote selective N-terminal modification, particularly PEGylation of the thiol group of cysteine ​​(see, for example, U.S. Patent No. 5,206,344, Katre et al., issued April 27, 1993).

[0274] IL2 mutant proteins may further include compounds with zero N-terminal amino acids at positions 1-9 (compounds in which a, b, c, d, e, f, g, h, and i are all zero), or positions 1-8 (compounds in which a, b, c, d, e, f, g, and h are all zero), or positions 1-7 (compounds in which a, b, c, d, e, f, and g are all zero), or positions 1-6 (compounds in which a, b, c, d, e, and f are all zero), or positions 1-5 (compounds in which a, b, c, d, and e are all zero), or positions 1-4 (compounds in which a, b, c, and d are all zero), or positions des1-3 (compounds in which a, b, and c are all zero), or positions 1-2 (compounds in which a and b are both zero), while retaining IL2 activity.

[0275] Conservative amino acid substitution

[0276] In some embodiments, the IL2 mutant protein of the present invention may further include a more conserved amino acid substitution in the wild-type IL-2 amino acid sequence. Such conserved substitutions include those described by Dayhoff in *The Atlas of Protein Sequence and Structure 5* (1978) and by Argos in *EMBO J., 8:779-785* (1989). Generally, conserved substitutions are made according to the following table shown in Table 3.

[0277]

[0278] Substantial alterations in functional or immunological properties can be made by selecting less conserved amino acid substitutions than those shown in Table 3. For example, substitutions that more significantly affect the polypeptide backbone structure or disrupt secondary or tertiary elements can be made, including replacing amino acids with small, uncharged side chains (e.g., glycine) with large, highly charged side chains (asparagine). In particular, substitutions of IL2 residues involved in one or more interactions with CD25, CD122, and / or CD123 can be observed in the crystal structures of IL2 associated with its described receptors.

[0279] Modifications that prolong in vivo persistence

[0280] As described above, the compositions of the present invention comprise IL2 mutant proteins that have been modified to provide for extending lifespan in vivo and / or extending duration of action in a subject.

[0281] Primary sequence modification

[0282] In some embodiments, the IL2 mutant protein may contain certain amino acid substitutions that result in extended lifespan in vivo. For example, Dakshinamurthi et al. (International Journal of Bioinformatics Research (2009) 1(2):4-13) describe that one or more substitutions of V91R, K97E, and T113N in the IL2 peptide produce IL2 variants with enhanced stability and activity. In some embodiments, the IL2 mutant protein of the present invention comprises one, two, or all three V91R, K97E, and T113N modifications.

[0283] Conjugates and carrier molecules

[0284] In some embodiments, the IL-2 mutant protein is modified to provide an extended duration of action in the subject, which can be achieved by conjugation to a carrier molecule to provide the desired pharmacological properties (e.g., an extended half-life). In some embodiments, the IL-2 mutant protein can be covalently linked to the Fc domain of IgG, albumin, or other molecules to extend its half-life, for example, through PEGylation, glycosylation, fatty acid acylation, etc., as known in the art. In some embodiments, the plasma half-life of the IL-2 conjugate in human subjects is greater than 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, or 30 days.

[0285] Albumin fusion

[0286] In some embodiments, the IL2 mutant protein is expressed as a fusion protein with an albumin molecule (e.g., human serum albumin), as is known in the art, to facilitate prolonged in vivo exposure.

[0287] In embodiments of the invention, an hIL2 analog is conjugated to albumin, referred herein as an “IL2 mutant protein albumin fusion.” When the term “albumin” is used in the context of an hIL2 analog albumin fusion, it includes albumin such as human serum albumin (HSA), cynomolgus monkey serum albumin, and bovine serum albumin (BSA). In some embodiments, the HAS sequence contains a C34S or K573P amino acid substitution relative to the wild-type HAS sequence. According to the invention, albumin can be conjugated to an IL2 mutant protein at the carboxyl terminus, amino terminus, carboxyl and amino terminus, and internally (see, for example, U.S. Patent Nos. 5,876,969 and 7,056,701). In the HSA-hIL2 mutant protein peptide conjugates contemplated in this disclosure, various forms of albumin can be used, such as albumin secretion pre-sequences and their variants, fragments and variants thereof, and HSA variants. This form typically possesses one or more desired albumin activities. In other embodiments, the present invention relates to fusion proteins comprising hIL2 analog peptides directly or indirectly fused to albumin, albumin fragments, and albumin variants, wherein the fusion protein exhibits higher plasma stability than the unfused drug molecule and / or the fusion protein retains the therapeutic activity of the unfused drug molecule. In some embodiments, direct fusion is achieved via a linker, such as a peptide linker or a modified version thereof, as discussed more fully below.

[0288] Alternatively, the hIL2 analog albumin fusion comprises an IL2 mutant protein, which is a fusion protein containing an albumin-binding domain (ABD) polypeptide sequence and an IL2 mutant protein polypeptide. As described above, the fusion protein containing the albumin-binding domain (ABD) polypeptide sequence and the hILI2 analog polypeptide can, for example, be achieved through genetic manipulation, such that a nucleic acid encoding HSA or a fragment thereof is bound to a nucleic acid encoding one or more IL2 mutant protein sequences. In some embodiments, the albumin-binding peptide comprises the amino acid sequence DICLPRWGCLW (SEQ ID NO:6).

[0289] The IL2 mutant protein peptide can also be conjugated to large, slowly metabolizing macromolecules, such as proteins; polysaccharides, such as sepharose, cellulose, or cellulose beads; polyamino acids, such as polyglutamic acid or polylysine; amino acid copolymers; inactivated viral particles; flammable bacterial toxins, such as toxoids or leukocyte toxin molecules from diphtheria, tetanus, or cholera; inactivated bacteria, dendritic cells, or thyroglobulins; tetanus toxoid; diphtheria toxoid; polyamino acids, such as poly(D-lysine:D-glutamic acid); rotavirus VP6 peptide; influenza virus hemaglutinin; influenza virus nucleoprotein; keyhole limpet hemocyanin (KLH); and hepatitis B virus core protein and surface antigen. If desired, this conjugation can be used to generate antibodies against the peptides disclosed herein.

[0290] In some implementations, the IL2 mutant protein is coupled to XTEN (chemically or as a fusion protein), which provides an extended duration similar to that of polyethylene glycolation, and can be produced in E. coli as a recombinant fusion protein. XTEN polymers suitable for conjugation with the IL2 mutant protein of the present invention are provided in Podust et al. (2016), “Extension of in vivo half-life of biologically active molecules by XTEN protein polymers,” J Controlled Release 240:52-66, and Haeckel et al. (2016), “XTEN as Biological Alternative toPEGylation Allows Complete Expression of a Protease-Activatable Killin-Based Cytostatic,” PLOS ONE | DOI:10.1371 / journal.pone.0157193, June 13, 2016. XTEN polymer fusion proteins can incorporate protease-sensitive cleavage sites, such as the MMP-2 cleavage site, between the XTEN peptide and the IL2 mutant protein.

[0291] Other candidate components and molecules for conjugation include other components and molecules suitable for isolation or purification. Specific non-limiting examples include binding molecules such as biotin (a biotin-avidin specific binding pair), antibodies, receptors, ligands, lectins, or molecules containing a solid support (including, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test strips, and membranes).

[0292] PEGylation:

[0293] In some embodiments, the IL2 mutant protein is coupled to one or more water-soluble polymers. Examples of water-soluble polymers useful in the practice of this invention include polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (polymers of polyvinylpyrrolidone, ethylene glycol, and propylene glycol, poly(oxyethylated polyols), polyolefinic alcohols, polysaccharides, poly-α-hydroxy acids, polyvinyl alcohol (PVA), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof.

[0294] In some embodiments, the IL2 mutant protein is coupled to one or more polyethylene glycol molecules or "PEGylated / PEGylated". Although the method or site of PEG attachment to the IL2 mutant protein may vary, in some embodiments PEGylation does not alter or only minimally alters the activity of the IL2 mutant protein.

[0295] In some embodiments, cysteine ​​can be replaced with threonine at position 3 (3TC) to facilitate N-terminal polyethylene glycolation using specific chemical methods.

[0296] In some embodiments, selective PEGylation of the IL2 mutant protein (e.g., by incorporating non-natural amino acids with side chains to facilitate selective PEG coupling chemistry, as described in Ptacin, et al. (PCT International Application No. PCT / US2018 / 045257 filed August 3, 2018, and published as International Publication No. WO on February 7, 2019) (Disclosed in 2019 / 028419A1) can be used to generate IL2 mutant proteins with reduced affinity for one or more subunits of the IL2 receptor complex (such as CD25, CD132). For example, hIL2 mutant proteins wherein non-natural amino acids with a PEGylated specific moiety are incorporated at those sequences or residues of IL2 identified as interacting with CD25 (including amino acids 34-45, 61-72, and 105-109) typically provide IL2 mutant proteins with reduced binding to CD25. Similarly, hIL2 mutant proteins wherein non-natural amino acids with a PEGylated specific moiety are incorporated at those sequences or residues of IL2 identified as interacting with hCD132 (including amino acids 18, 22, 109, 126, or 119-133) typically provide IL2 mutant proteins with reduced binding to hCD132.

[0297] In some embodiments, the increase in half-life outweighs any decrease in biological activity. PEGs suitable for coupling to peptide sequences are generally water-soluble at room temperature and have the general formula R(O-CH2-CH2). n OR, where R is hydrogen or a protecting group, such as alkyl or alkylol, where n is an integer from 1 to 1000. When R is a protecting group, it typically has 1 to 8 carbons. PEG coupled to the polypeptide sequence can be linear or branched. This invention considers branched PEG derivatives, “star-PEG” and multi-arm PEG.

[0298] The molecular weight of PEG used in this invention is not limited to any particular range. The PEG component of the PEG-IL2 mutant protein may have a molecular weight greater than about 5 kDa, greater than about 10 kDa, greater than about 15 kDa, greater than about 20 kDa, greater than about 30 kDa, greater than about 40 kDa, or greater than about 50 kDa. In some embodiments, the molecular weight is about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, or about 10 kDa to about 30 kDa. The molecular weight of linear or branched PEG molecules ranges from about 2,000 to about 80,000 Daltons, or about 2,000 to about 70,000 Daltons, or about 5,000 to about 50,000 Daltons, or about 10,000 to about 50,000 Daltons, or about 20,000 to about 50,000 Daltons, or about 30,000 to about 50,000 Daltons, or about 20,000 to about 40,000 Daltons, or about 30,000 to about 40,000 Daltons. In one embodiment of the invention, the PEG is a 40 kD branched PEG comprising two 20 kD arms.

[0299] This disclosure also considers compositions of conjugates in which PEG has different n values, thus allowing for a variety of different PEGs at specific ratios. For example, some compositions comprise mixtures of conjugates where n = 1, 2, 3, and 4. In some compositions, the percentage of conjugate with n = 1 is 18-25%, the percentage of conjugate with n = 2 is 50-66%, the percentage of conjugate with n = 3 is 12-16%, and the percentage of conjugate with n = 4 is up to 5%. Such compositions can be produced using reaction conditions and purification methods known in the art. Chromatography can be used to distinguish the fractions of the conjugates, and then identify the fractions containing, for example, conjugates with the desired amount of PEG attached, purified from unmodified protein sequences and conjugates with other amounts of PEG attached.

[0300] PEGs suitable for coupling to peptide sequences are typically water-soluble at room temperature and have the general formula R(O-CH2-CH2). n OR, where R is a hydrogen or protecting group, such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, it typically has 1 to 8 carbons.

[0301] Two widely used first-generation activated monomethoxy PEGs (mPEGs) are succinimide carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotehnol. Appl. Biochem 15:100-114) and benzotriazole methyl PEG (BTC-PEG; see, e.g., Dolence, et al., U.S. Patent No. 5,650,234), which preferably react with lysine residues to form a carbamate link, but are also known to react with histidine and tyrosine residues. PEG-aldehyde linkers are used to target a single N-terminal site of the polypeptide via reductive amination.

[0302] Polyglycolation frequently occurs at the N-terminus of peptides, specifically at the α-amino group, the ε-amino group on the lysine residue side chain, and the imidazole group on the histidine residue side chain. Since most recombinant peptides possess a single α-group and several ε- and imidazole groups, numerous positional isomers can be generated depending on the chemical nature of the linker. General polyglycolation strategies known in the art are applicable herein.

[0303] PEG can bind to the IL2 mutant protein of the present invention via a terminal reactive group (“spacer”) that mediates the binding between a free amino or carboxyl group of one or more polypeptide sequences and polyethylene glycol. PEG having a spacer that can bind to a free amino group comprises N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating a succinate of polyethylene glycol with N-hydroxysuccinimide.

[0304] In some embodiments, the PEGylation of IL2 mutant proteins is facilitated by incorporating non-natural amino acids with unique side chains to promote site-specific PEGylation. Incorporating non-natural amino acids into peptides to provide a functional moiety for site-specific PEGylation of such peptides is known in the art. See, for example, Ptacin, et al., (PCT International Application No. PCT / US2018 / 045257, filed August 3, 2018 and published February 7, 2019, International Publication No. WO 2019 / 028419A1). In one embodiment, the IL2 mutant protein of the present invention incorporates a non-natural amino acid at position D109 of the IL2 mutant protein. In one embodiment of the present invention, the IL2 mutant protein is PEGylated at position 109 of the IL2 mutant protein, and the PEG molecule has a molecular weight of about 20 kDa, or about 30 kDa, or about 40 kDa.

[0305] The PEG coupled to the polypeptide sequence can be linear or branched. This invention considers branched PEG derivatives, "star-PEG," and multi-arm PEG. In practice, the specific embodiments of the present invention useful PEGs include 10kDa linear PEG-aldehydes (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601 USA), 10kDa linear PEG-NHS esters (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), 20kDa linear PEG-aldehydes (e.g., Sunbright® ME-200AL, NOF, 20kDa linear PEG-NHS esters (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS, Sunbright® ME-200GS, Sunbright® ME-200HS, NOF), and 20kDa 2-arm branched PEG-aldehydes. PEG-aldehyde comprising two 10kDa straight-chain PEG molecules (e.g., Sunbright® GL2-200AL3, NOF), a 20kDa 2-arm branched PEG-NHS ester, the 20kDa PEG-NHS ester comprising two 10kDa straight-chain PEG molecules (e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF), a 40kDa 2-arm branched PEG-aldehyde, the 40kDa PEG-aldehyde comprising two 20kDa straight-chain PEG molecules (e.g., Sunbright® GL2-400AL3), a 40kDa 2-arm branched PEG-NHS ester, the 40kDa PEG-NHS ester comprising two 20kDa straight-chain PEG molecules (e.g., Sunbright® GL2-400AL3, Sunbright® GL2-400GS2, NOF), and a straight-chain 30kDa PEG-aldehyde (e.g., Sunbright® ME-300AL) and linear 30kDa PEG-NHS ester.

[0306] As previously mentioned, PEG can be attached to IL2 mutant proteins directly or via adapter molecules. Suitable adapters include “flexible adapters” whose length is generally sufficient to allow some movement between the modified polypeptide sequence and the linked component and molecule. Adapter molecules are typically about 6–50 atoms long. Adapter molecules can also be, for example, arylaceyne, ethylene glycol oligomers containing 2–10 monomer units, diamines, diacids, amino acids, or combinations thereof. Suitable adapters can be readily chosen and can be of any suitable length, such as 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10–20, 20–30, 30–50, or more than 50 amino acids. Examples of flexible adapters include glycine polymers (G). n Glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible joints. Glycine and glycine-serine polymers are relatively unstructured and therefore can be used as neutral tethers between components. Other examples of flexible joints include glycine polymers (G...). n Glycine-alanine polymers, alanine-serine polymers, and glycine-serine polymers. Glycine and glycine-serine polymers are relatively unstructured and can therefore be used as neutral tethers between components. Polymers of these linker sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) can be linked together to provide flexible linkers for coupling heterologous amino acid sequences to the peptides disclosed herein.

[0307] In addition, this linker can be used to link IL2 mutant proteins to other heterologous peptide components described herein, whereby the heterologous amino acid sequence can be a signal sequence and / or fusion chaperone, such as albumin, Fc sequence, etc.

[0308] acylation

[0309] In some embodiments, the IL2 mutant protein of the present invention may be coupled to an acylated fatty acid molecule, as described in Resh (2016) Progress in Lipid Research 63: 120–131. Examples of fatty acids that can be coupled include myristic acid, palmitic acid, and palmitoleic acid. Myristic acid is typically linked to an N-terminal glycine, but lysine can also be myristicated. Palmitoylation is typically achieved by enzymatic modification of a free cysteine ​​-SH group, such as S-palmitoylation catalyzed by DHHC proteins. Palmitoylation of serine and threonine residues is typically achieved by enzymatic modification using a PORCN enzyme.

[0310] acetylation

[0311] In some embodiments, the IL-2 mutant protein is acetylated at the N-terminus by an enzymatic reaction with an N-terminal acetyltransferase, for example, acetyl-CoA. Alternatively, in addition to N-terminal acetylation, the IL-2 mutant protein is acetylated at one or more lysine residues, for example by an enzymatic reaction with a lysine acetyltransferase. See, for example, Choudhary et al. (2009) Science 325 (5942):834L2 ortho840.

[0312] Fc Fusion

[0313] In some embodiments, the IL-2 fusion protein may incorporate an Fc region derived from an IgG antibody subclass that lacks the IgG heavy chain variable region. The "Fc region" may be a naturally occurring or synthetic polypeptide homologous to the C-terminal domain of IgG produced by papain digestion of IgG. The molecular weight of the IgG Fc is approximately 50 kDa. The mutant IL-2 polypeptide may contain the entire Fc region or retain a smaller portion that extends its circulating half-life as a part of a chimeric polypeptide. Furthermore, the full-length or fragmented Fc region may be a variant of the wild-type molecule. That is, they may contain mutations that may or may not affect polypeptide function; in all cases, native activity is neither required nor desired, as further described below. In some embodiments, the IL-2 mutant protein fusion protein (such as the IL-2 partial agonists or antagonists described herein) includes an IgG1, IgG2, IgG3, or IgG4 Fc region. Exemplary Fc regions may include mutations that inhibit complement fixation and Fc receptor binding, or they may be cleavable, i.e., capable of binding complement or lysing cells via another mechanism such as antibody-dependent complement cleavage (ADCC).

[0314] In some embodiments, the IL2 mutant protein contains the functional domain of an Fc fusion chimeric polypeptide molecule. Fc fusion conjugates have been shown to increase the systemic half-life of biologics, thus reducing the need for frequent dosing. Fc binds to nascent Fc receptors (FcRn) lining the endothelial cells of blood vessels. Upon binding, the Fc fusion molecule is protected from degradation and re-release into circulation, allowing the molecule to remain in circulation for a longer period. This Fc binding is considered a mechanism by which endogenous IgG maintains its long plasma half-life. Recent Fc fusion technologies link a single copy of a biologic to the Fc region of an antibody, optimizing the pharmacokinetic and pharmacodynamic properties of the biologic compared to conventional Fc fusion conjugates. The "Fc region" used to prepare the Fc fusion can be a naturally occurring or synthetic polypeptide homologous to the C-terminal domain of IgG produced by papain digestion of IgG. The molecular weight of the IgG Fc is approximately 50 kDa. The IL2 mutant protein can provide the entire Fc region or retain a smaller portion that extends its ability to prolong the circulating half-life as a chimeric polypeptide. Furthermore, the full-length or fragmented Fc region can be a variant of the wild-type molecule. In a typical presentation, each monomer of the dimer Fc carries a heterologous polypeptide, which may be the same or different.

[0315] In some implementations, when the IL2 mutant protein is to be administered as an Fc fusion, particularly when the polypeptide chains coupled to the various subunits of the Fc dimer are different, the Fc fusion can be engineered to have a “knob-into-hole modification.” Knob-into-hole modification is described more fully in Ridgway et al. (1996), Protein Engineering 9(7):617-621, and U.S. Patent No. 5,731,168, issued March 24, 1998. The mortar modification refers to the modification of the interface between two immunoglobulin heavy chains in the CH3 domain, wherein: i) in the CH3 domain of the first heavy chain, amino acid residues are replaced with amino acid residues having larger side chains (e.g., tyrosine or tryptophan), creating a protrusion (“mortar”) from the surface; and ii) in the CH3 domain of the second heavy chain, amino acid residues are replaced with amino acid residues having smaller side chains (e.g., alanine or threonine), thereby creating a cavity (“mortar”) within the interface of the second CH3 domain, wherein the protruding side chain (“mortar”) of the first CH3 domain is received by the cavity in the second CH3 domain. In one embodiment, the “mortar modification” includes amino acid substitution T366W and optionally amino acid substitution S354C in one antibody heavy chain, and amino acid substitution T366S, L368A, Y407V and optionally Y349C in the other antibody heavy chain. Furthermore, the Fc domain can be modified by introducing cysteine ​​residues at the S354 and Y349 positions, thereby generating stable disulfide bonds between the two antibody heavy chains in the Fe region (Carter, et al. (2001) Immunol Methods 248, 7-15). The mortar and pestle design is used to promote the expression of a first polypeptide (such as an IL2 mutant protein) on a first Fc monomer with a mortar modification and the expression of a second polypeptide on a second Fc monomer with a mortar modification, thereby promoting the expression of heterodimeric polypeptide conjugates.

[0316] The Fc region can be "soluble" or "insoluble," but is usually insoluble. Insoluble Fc regions typically lack both high-affinity Fc receptor binding sites and Clq binding sites. The high-affinity Fc receptor binding site of mouse IgG Fc includes the Leu residue at position 235. Therefore, the Fc receptor binding site can be inhibited by mutation or deletion of Leu 235. For example, replacing Leu 235 with Glu inhibits the ability of the Fc region to bind to the high-affinity Fc receptor. The mouse Clq binding site can be functionally disrupted by mutation or deletion of Glu 318, Lys 320, and Lys 322 residues in IgG. For example, replacing Glu 318, Lys 320, and Lys 322 with Ala residues prevents IgG1 Fc from guiding antibody-dependent complement cleavage. In contrast, soluble IgG Fc regions possess both high-affinity Fc receptor binding sites and Clq binding sites. High-affinity Fc receptor binding sites include the Leu residue at position 235 of IgG Fc, while Clq binding sites include Glu 318, Lys 320, and Lys 322 residues of IgG 1. Soluble IgG Fc contains wild-type residues or conserved amino acid substitutions at these sites. Soluble IgG Fc can induce antibody-dependent cytotoxicity or complement-directed cytolysis (CDC) against cells. Appropriate mutations in human IgG are also known (see, for example, Morrison et al., The Immunologist 2:119-124, 1994; and Brekke et al., The Immunologist 2:125, 1994).

[0317] In some embodiments, the amino or carboxyl terminus of the IL2 mutant protein of the present invention may be fused to the Fc region of an immunoglobulin (such as human Fc) to form a fusion conjugate (or fusion molecule). Fc fusion conjugates have been shown to increase the systemic half-life of biologics, thus reducing the need for frequent dosing. Fc binds to nascent Fc receptors (FcRn) lining the endothelial cells of blood vessels. Upon binding, the Fc fusion molecule is protected from degradation and re-release into circulation, allowing the molecule to remain in circulation for a longer period. This Fc binding is considered a mechanism by which endogenous IgG maintains its long plasma half-life. Recent Fc fusion technologies link a single copy of a biologic to the Fc region of an antibody, optimizing the pharmacokinetic and pharmacodynamic properties of the biologic compared to conventional Fc fusion conjugates.

[0318] In some embodiments, the Fc domain monomer includes at least one mutation relative to the Fc region of wild-type human IgG1, IgG2, or IgG4, as described in U.S. Patent No. US10259859B2, the entire teaching of which is incorporated herein by reference. As disclosed herein, the Fc domain monomer includes:

[0319] One of the following amino acid substitutions relative to wild-type human IgG1:

[0320] T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351 N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L36 8Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T or K409I;

[0321] or

[0322] (b)(i) N297A mutation, relative to the human IgG1 Fc region;

[0323] (ii) L234A, L235A and G237A mutations, relative to the human IgG1 Fc region;

[0324] (iii) L234A, L235A, G237A and N297A mutations, relative to the human IgG1 Fc region;

[0325] (iv) N297A mutation, relative to the human IgG2 Fc region;

[0326] (v) A330S and P331S mutations, relative to the human IgG2 Fc region;

[0327] (vi) A330S, P331S and N297A mutations, relative to the human IgG2 Fc region;

[0328] (vii) S228P, E233P, F234V, L235A, and delG236 mutations, relative to the human IgG4 Fc region; or

[0329] (viii) S228P, E233P, F234V, L235A, delG236 and N297A mutations, relative to the human IgG4 Fc region.

[0330] In some implementations, the Fc domain monomer includes:

[0331] The following amino acid substitutions relative to wild-type human IgG1 are: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L35IT, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q. K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T or K409I;

[0332] as well as

[0333] (b) The Fc domain monomer further includes

[0334] (i) N297A mutation, relative to the human IgG1 Fc region;

[0335] (ii) L234A, L235A and G237A mutations, relative to the human IgG1 Fc region;

[0336] (iii) L234A, L235A, G237A and N297A mutations, relative to the human IgG1 Fc region;

[0337] (iv) N297A mutation, relative to the human IgG2 Fc region;

[0338] (v) A330S and P331S mutations, relative to the human IgG2 Fc region;

[0339] (vi) A330S, P331S and N297A mutations, relative to the human IgG2 Fc region;

[0340] (vii) S228P, E233P, F234V, L235A, and delG236 mutations, relative to the human IgG4 Fc region; or

[0341] (viii) S228P, E233P, F234V, L235A, delG236 and N297A mutations, relative to the human IgG4 Fc region.

[0342] In some embodiments, the polypeptide exhibits reduced phagocytosis in phagocytosis assays compared to polypeptides possessing the Fc region of wild-type human IgG. In some embodiments, an Fc domain monomer is linked to a second polypeptide containing a second Fc domain monomer to form an Fc domain dimer.

[0343] Chimeric peptides / fusion proteins

[0344] In some embodiments, the IL2 mutant protein may include the functional domain of a chimeric polypeptide. The IL2 mutant protein fusion protein of the present invention can be readily produced by a recombinant DNA method using techniques known in the art, by constructing a recombinant vector containing a nucleic acid sequence comprising, within a frame, a nucleic acid sequence encoding the IL2 mutant protein and a nucleic acid sequence encoding an N-terminal or C-terminal fusion partner of the IL2 mutant protein, the sequence optionally further comprising, within a frame, a nucleic acid sequence encoding an adapter or spacer polypeptide.

[0345] In some embodiments, the IL2 mutant protein is coupled (chemically, or in the case of peptide reagents (e.g., antibodies or vaccines) in fusion protein form) to other chemical or biological agents, including therapeutic compounds such as anti-inflammatory, antimicrobial, or antiviral compounds, or other agents useful in treating autoimmune diseases, including biologics (e.g., entaracept) and monoclonal antibodies. Antimicrobial agents include aminoglycosides (including gentamicin), antiviral compounds such as rifampicin, 3′-azido-3′-deoxythymidine (AZT), and acyclovir, antifungal agents such as azoles including fluconazole, macrolides such as amphotericin B and candicidin, and antiparasitic compounds such as antimonial agents, etc. Mutant IL2 proteins can be coupled to other cytokines such as CSF, GSF, GMCSF, TNF, erythropoietin, immunomodulators or cytokines such as interferon or interleukin, neuropeptides, reproductive hormones such as HGH, FSH, or LH, thyroid hormones, neurotransmitters such as acetylcholine, and hormone receptors such as estrogen receptors. Also included are nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin, salicylic acid acetate, ibuprofen, sulindac, piroxicam, and naproxen, as well as anesthetics or analgesics. Radioisotopes, such as those useful for imaging and treatment, are also included.

[0346] IL-2 mutant proteins can also be conjugated to corticosteroids (including but not limited to prednisone, budesonide, and prednisolone), Janus kinase inhibitors (including but not limited to tofacitinib (Xeljanz®), calcineurin inhibitors (including but not limited to cyclosporine and tacrolimus), mTor inhibitors (including but not limited to sirolimus and everolimus), and IMDH inhibitors (including but not limited to azathioprine, leflunomide, and mycophenolate mofetil). IL-2 mutant proteins can also be conjugated to biologics such as abatacept (Orencia®) or etanercept. (Enbrel®). IL-2 mutant proteins can also be conjugated to therapeutic antibodies, such as anti-CD25 antibodies (e.g., dacrolimus and baliximab), anti-VLA-4 antibodies (e.g., natetuzumab), anti-CD52 antibodies (e.g., alemtuzumab), anti-CD20 antibodies (e.g., rituximab, olizumab), anti-TNF antibodies (e.g., infliximab and adalimumab), anti-IL-6R antibodies (e.g., tocilizumab), anti-TNFα antibodies (e.g., adalimumab (Humira®), golimumab, and infliximab), anti-integrin-α4β7 antibodies (e.g., vedolizumab), anti-IL-17a antibodies (e.g., brodalumab or secukinumab), anti-IL-4Rα antibodies (e.g., dupilumab), and anti-RANKL antibodies. (For example), antibodies against IL-6R, anti-IL-1β (e.g., cannabinoids), anti-CD11a (e.g., efalizumab), anti-CD3 (e.g., muramonab), anti-IL5 (e.g., mepolizumab, rellizumab), anti-BLyS (e.g., belimumab); and anti-IL-12 / IL-23 (e.g., ustekinumab).

[0347] IL-2 mutant proteins can also be conjugated to, for example, HSV vaccines, Bordetella pertussis vaccines, Escherichia coli vaccines, pneumococcal vaccines (including multivalent pneumococcal vaccines such as Prevnar® 13), diphtheria toxoid, tetanus toxoid and pertussis vaccines (including combination vaccines such as Pediatrix® and Pentacel®), varicella vaccines, Haemophilus influenzae type B (HIB) vaccines, human papillomavirus (HPV) vaccines such as Garasil®, polio vaccines, leptospirosis vaccines, combination respiratory vaccines, Moraxella vaccines, and attenuated or inactivated virus products such as bovine respiratory disease vaccine (RSV), human influenza vaccines such as Fluzone® and Quadravlent Fluzone®, and feline leukemia virus vaccines. Leukemia vaccine, infectious gastroenteritis vaccine and rabies vaccine.

[0348] The IL2 mutant protein of the present invention can be chemically coupled to such other reagents using well-known chemical coupling methods. Bifunctional cross-linking agents, such as homofunctional and heterofunctional cross-linking agents known in the art, can be used for this purpose. The type of cross-linking agent used depends on the nature of the molecule to be coupled to the IL-2 mutant protein, which can be readily determined by those skilled in the art. Alternatively, the IL2 mutant protein and / or the molecule intended to be coupled thereto can be chemically derivatized so that they are coupled in separate reactions, as is also well known in the art.

[0349] Flag tag

[0350] In other embodiments, the IL2 mutant protein may be modified to include other polypeptide sequences that function as antigen tags, such as the FLAG sequence. As described herein, the FLAG sequence can be recognized by biotinylated, highly specific anti-FLAG antibodies (see, for example, Blanar et al. (1992) Science 256:1014 and LeClair et al. (1992) PNAS-USA89:8145). In some embodiments, the IL2 mutant protein polypeptide further includes a C-terminal c-myc epitope tag.

[0351] His tags

[0352] In some embodiments, the IL2 mutant protein of the present invention (including fusion proteins of such IL2 mutant proteins) is expressed as a fusion protein having one or more transition metal chelating polypeptide sequences. Incorporation of such a transition metal chelating domain facilitates purification by immobilized metal affinity chromatography (IMAC), as described in U.S. Patent No. 4,569,794, issued February 11, 1986, by Smith et al. Examples of transition metal chelating polypeptides useful in the practice of the present invention are described in U.S. Patent No. 5,320,663, issued May 10, 1995, by Smith et al. (ibid.) and Dobeli et al., the entire teachings of which are incorporated herein by reference. Specific transition metal chelating polypeptides useful in the practice of the present invention are peptides comprising 3-6 consecutive histidine residues (SEQ ID NO: 98), such as 6-histidine peptide (His)6 (SEQ ID NO: 99), often referred to in the art as “His-tag”.

[0353] Targeted portion:

[0354] In some embodiments, the IL2 mutant protein is provided as a fusion protein, which is fused to a polypeptide sequence (“targeting domain”) that promotes selective binding to cell surface molecules expressing specific cell surface molecules that bind specifically to such targeting domain. In some embodiments, the targeting domain is an antibody (particularly a single-domain antibody, scFv, or VHH) or ligand that specifically binds to a surface protein selected from the group consisting of: BLyS, CD11a, CD20, CD25, CD3, CD52, IgEIL-12 / IL-23, IL-17a, IL-1β, IL-4Rα, IL-5, IL-6R, integrin-α4β7, RANKL, TNFα, VEGF-A, and VLA-4.

[0355] Preparation of IL2 mutant protein

[0356] IL2 mutant proteins can be produced using conventional methods for constructing peptides, including recombinant or solid-phase synthesis.

[0357] Chemical synthesis:

[0358] In addition to generating mutant peptides through the expression of nucleic acid molecules that have been replaced by recombinant molecular biotechnology, the host IL-2 mutant protein can be chemically synthesized. Chemically synthesized peptides can be routinely produced by those skilled in the art. Chemical synthesis involves the direct synthesis of peptides encoding protein sequences of IL-2 mutant proteins exhibiting the aforementioned properties via chemical means. This method can incorporate natural and non-natural amino acids at positions affecting the interactions between IL2 and CD25, CD122, and CD132.

[0359] In some embodiments, the IL2 mutant protein of the present invention can be prepared by chemical synthesis. The chemical synthesis of the IL2 mutant protein can be carried out in a liquid phase or a solid phase. Solid-phase peptide synthesis (SPPS) allows for the inclusion of non-natural amino acids and / or peptide / protein backbone modifications. Various forms of SPPS that can be used to synthesize the IL2 mutant protein of the present invention are known in the art (e.g., Ganesan A. (2006) Mini Rev. Med. Chem. 6:3-10; and Camarero et al., (2005) Protein Pept Lett. 12:723-8). During chemical synthesis, the α-functional group and any reactive side chain can be protected with acid-insecure or base-insecure groups that are stable under conditions of amide bond linkage but readily cleaved without damaging the formed peptide chain.

[0360] In solid-phase synthesis, N-terminal or C-terminal amino acids can be coupled to a suitable support material. Suitable support materials are those that are inert to the kit reaction conditions of stepwise condensation and cleavage reactions during synthesis and are insoluble in the reaction medium being used. Examples of commercially available support materials include styrene / divinylbenzene copolymers modified with active groups and / or polyethylene glycol; chloromethylated styrene / divinylbenzene copolymers; hydroxymethylated or aminomethylated styrene / divinylbenzene copolymers, etc. Continuous coupling of the protected amino acids can be performed according to conventional methods in peptide synthesis, typically in an automated peptide synthesizer.

[0361] At the end of solid-phase synthesis, the peptide is cleaved from the support material, along with the side-chain protecting groups. The obtained peptide can be purified by various chromatographic methods, including but not limited to hydrophobic adsorption chromatography, ion exchange chromatography, partition chromatography, high-performance liquid chromatography, and reversed-phase HPLC.

[0362] Reorganization of production:

[0363] Alternatively, the IL2 mutant protein of the present invention can be produced using recombinant DNA technology. In typical practice of recombinant peptide production, a nucleic acid sequence encoding the desired peptide is incorporated into an expression vector suitable for use in a host cell to which expression will be performed. The nucleic acid sequence is operatively linked to one or more expression control sequences encoded by the vector and functions in the target host cell. If a secretory leader sequence (signal peptide) is incorporated into the peptide, the recombinant protein can be recovered by disrupting the host cell or from the cellular medium. The recombinant protein can be purified and concentrated for further use, including incorporation. The process for recombinant production of the IL2 peptide is known in the art and described in Fernandes and Taforo, U.S. Patent No. 4,604,377, August 5, 1986, and the IL2 mutant protein is described in Mark et al., U.S. Patent No. 4,512,584, May 21, 1985, and Gillis, U.S. Patent No. 4,401,756, August 30, 1983, the entire teachings of which are incorporated herein by reference.

[0364] Construction of the nucleic acid sequence encoding the IL2 mutant protein

[0365] In some embodiments, the IL mutant protein is produced via a recombinant method using a nucleic acid sequence encoding an IL2 mutant protein (or a fusion protein containing an IL2 mutant protein). Alternatively, the nucleic acid sequence encoding the desired IL-2 mutant protein can be chemically synthesized using an oligonucleotide synthesizer.

[0366] Nucleic acid sequences are not limited to sequences encoding polypeptides; they may also include partial or complete non-coding sequences located upstream or downstream of a coding sequence (e.g., the coding sequence for IL-2). Those skilled in molecular biology are familiar with the routine procedures used to isolate nucleic acid molecules. For example, they can be produced by treating genomic DNA with restriction endonucleases or by performing a polymerase chain reaction (PCR). If the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription.

[0367] Nucleic acid molecules (and their fusions) encoding IL2 mutant proteins can contain naturally occurring sequences or sequences different from naturally occurring sequences, but due to the degeneracy of the genetic code, they encode the same polypeptide. These nucleic acid molecules can be composed of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis) or combinations or modifications of nucleotides within these types of nucleic acids. Furthermore, nucleic acid molecules can be double-stranded or single-stranded (i.e., sense or antisense strands).

[0368] The nucleic acid sequence encoding the IL mutant protein can be obtained from a variety of commercial sources that provide customized nucleic acid sequences. The amino acid sequence variants of the IL polypeptide that produce the IL2 mutant protein of the present invention are prepared by incorporating suitable nucleotide changes into the coding sequence based on the genetic code well known in the art. Such variants represent the insertion, substitution, and / or specific deletion of the indicated residues. Any combination of insertions, substitutions, and / or specific deletions can yield the final construct, provided that the final construct possesses the desired biological activity as defined herein.

[0369] Methods for constructing DNA sequences encoding mutant IL-2 proteins and expressing these sequences in suitable transformed hosts include, but are not limited to, the use of PCR-assisted mutagenesis. Mutations consisting of deletions or additions of amino acid residues can also be created in the IL-2 polypeptide using standard recombinant techniques. If deletions or additions are made, the nucleic acid molecule encoding IL-2 is optionally digested with a suitable restriction endonuclease. The resulting fragment can be expressed directly or further manipulated, for example, by ligation to a second fragment. Ligation can be facilitated if the two ends of the nucleic acid molecule contain overlapping complementary nucleotides, but blunt-ended fragments can also be ligated. PCR-generated nucleic acids can also be used to generate a variety of mutant sequences.

[0370] The IL2 mutant protein of this invention can be produced not only through direct recombinant production but also as a fusion polypeptide with a heterologous polypeptide (e.g., a signal sequence or other polypeptides having specific cleavage sites at the N- or C-terminus of the mature IL2 mutant protein). Typically, the signal sequence can be a component of a vector or a portion of a coding sequence inserted into the vector. The selected heterologous signal sequence is preferably a sequence that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). In some embodiments, the signal sequence is a signal sequence naturally associated with the IL2 mutant protein (i.e., a human IL2 signal sequence). The inclusion of the signal sequence depends on whether it is required to secrete the IL-2 mutant protein from the prepared recombinant cells. If the selected cells are prokaryotic cells, it is generally preferred that the DNA sequence does not encode the signal sequence. If the selected cells are eukaryotic cells, it is generally preferred that a signal sequence is encoded, with the wild-type IL-2 signal sequence being the most preferred. Alternatively, a heterologous mammalian signal sequence is also suitable, such as a signal sequence of a secretory polypeptide from the same or related species, and a viral secretion leader sequence, such as the herpes simplex gD signal. When the recombinant host cell is a yeast cell, such as Saccharomyces cerevisiae, an alpha mating factor secretion signal sequence can be used to achieve the extracellular secretion of the IL2 mutant protein into the culture medium, as described in Singh, U.S. Patent No. 7,198,919 B1, issued April 3, 2007.

[0371] If the IL-2 mutant protein to be expressed is to be expressed in chimera form (e.g., a fusion protein comprising the IL-2 mutant protein and a heterologous polypeptide sequence), the chimera protein may be encoded by a hybrid nucleic acid molecule comprising a first sequence encoding all or part of the IL-2 mutant protein and a second sequence encoding all or part of the heterologous polypeptide. For example, the host IL-2 mutant protein described herein may be fused to a hexahistidine tag (SEQ ID NO: 99) to facilitate the purification of the protein expressed in bacteria, or fused to a hemagglutinin tag to facilitate the purification of the protein expressed in eukaryotic cells. The terms "first" and "second" should not be construed as limiting the element orientation of the fusion protein; the heterologous polypeptide may be linked to the N-terminus and / or C-terminus of the IL-2 mutant protein. For example, the N-terminus may be linked to a targeting portion, and the C-terminus to a purification handle of the hexahistidine tag (SEQ ID NO: 99).

[0372] The complete amino acid sequence of the polypeptide (or fusion / chimera) to be expressed can be used to construct a back-translated gene. DNA oligomers containing nucleotide sequences encoding IL-2 mutant proteins can be synthesized. For example, small oligonucleotides encoding a portion of the desired polypeptide can be synthesized and then linked together. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0373] Codon optimization:

[0374] In some implementations, the nucleic acid sequence encoding the IL2 mutant protein may be "codon-optimized" to facilitate expression in a specific host cell type. Techniques for codon optimization in various expression systems, including mammalian, yeast, and bacterial host cells, are well-known in the art, and online tools are available to provide codon-optimized sequences for expression in multiple host cell types. See, for example, Hawash, et al. (2017) 9:46-53 and Mauro and Chappell in... "feed Recombinant Protein Expression in Mammal Cells: Methods and Protocols Mammalian Cells: Methods and Protocols), Edited by David Hacker (HumanPress, New York). In addition, several web-based online software programs are available free of charge to assist in the preparation of codon-optimized nucleic acid sequences.

[0375] Construction of expression carriers:

[0376] After assembly (through synthesis, site-directed mutagenesis, or another method), the nucleic acid sequence encoding the IL-2 mutant protein is inserted into an expression vector. Multiple expression vectors can be used for various host cells, typically based on the host cell chosen for expression. Expression vectors typically contain, but are not limited to, one or more of the following: origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. Vectors include viral vectors, plasmid vectors, integration vectors, etc. Plasmids are examples of non-viral vectors.

[0377] To promote efficient expression of recombinant peptides, the nucleic acid sequence encoding the peptide sequence to be expressed is operatively linked to transcriptional and translational regulatory sequences that function in the selected expression host.

[0378] Optional markers:

[0379] Expression vectors typically contain a select gene, also known as a selectable marker. This gene encodes a protein essential for the survival or growth of transformed host cells in selective media. Host cells not transformed with a vector containing a select gene cannot survive in the medium. A typical select gene encodes a protein that will (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate, or tetracycline; (b) compensate for auxotrophic deficiencies; or (c) provide a key nutrient that is not available from a complex culture medium.

[0380] Regulatory sequence:

[0381] The expression vector for the IL2 mutant protein used in this invention comprises a regulatory sequence that is recognized by the host organism and operatively linked to the nucleic acid sequence encoding the IL2 mutant protein. The terms “regulatory sequence,” “regulatory sequence,” or “expression control sequence” are used interchangeably herein to refer to promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). See, for example, Goeddel (1990) in *Gene Expression Technology: Methods in Enzymology*, 185 (Academic Press, San Diego, California, USA). Regulatory sequences include nucleotide sequences that guide constitutive expression in many types of host cells and nucleotide sequences that guide expression only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will understand that the design of the expression vector may depend on factors such as, for example, the choice of host cells to be transformed, the desired protein expression level, etc. In the selection of the expression control sequence, those skilled in the art will understand that a variety of factors will be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the host IL-2 mutant protein, particularly regarding potential secondary structures.

[0382] Promoter:

[0383] In some implementations, the regulatory sequence is a promoter, the selection of which is based, for example, on the cell type in which expression is sought. A promoter is a non-translated sequence (typically within about 100 to 1000 bp) located upstream (5') of the start codon of a structural gene that controls the transcription and translation of a specific nucleic acid sequence operatively linked to it. Such promoters are generally classified into two categories: inducible and constitutive. Inducible promoters are promoters that respond to certain changes in culture conditions (such as the presence or absence of nutrients or changes in temperature), increasing the level of transcription from the DNA they control. A large number of promoters recognized by a variety of potential host cells are well known.

[0384] In bacteria, the T7 promoter can be used; in insect cells, the polyhedrosis protein promoter can be used; and in mammalian cells, the cytomegalovirus or metallothionein promoter can be used. Additionally, tissue-specific and cell-specific promoters are widely used in the case of higher eukaryotic cells. These promoters are named for their ability to direct the expression of nucleic acid molecules in a given in vivo tissue or cell type. Technologists are familiar with many promoters and other regulatory elements that can be used to direct the expression of nucleic acids.

[0385] Transcription of vectors in mammalian host cells can be controlled by promoters, for example, derived from viral genomes such as polyomavirus, vaccinia virus, adenovirus (e.g., human adenovirus serotype 5), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retroviruses (e.g., mouse stem cell virus), hepatitis B virus, and most ideally simian virus 40 (SV40); promoters from heterologous mammals such as actin promoters, PGK (phosphoglycerate kinase) or immunoglobulin promoters; and promoters from heat shock, provided these promoters are compatible with the host cell system. Early and late promoters of SV40 virus are readily available as SV40 restriction fragments, which also contain the SV40 virus's origin of replication.

[0386] Enhancer:

[0387] In higher eukaryotes, transcription is typically amplified by inserting enhancer sequences into vectors. Enhancers are cis-acting elements of DNA, typically 10 to 300 bp in length, that act on the promoter to amplify its transcription. The orientation and location of enhancers are relatively independent, found at the 5' and 3' of transcription units, within introns, and within the coding sequence itself. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, enhancers from eukaryotic viruses are commonly used. Examples include the SV40 enhancer post-OMI, the cytomegalovirus early promoter enhancer, the polyoma enhancer post-OMI, and the adenovirus enhancer. Enhancers can be spliced ​​into the 5' or 3' position of the coding sequence in the expression vector, but are preferably located 5' from the promoter. Expression vectors for eukaryotic host cells will also contain sequences necessary for terminating transcription and for stabilizing mRNA. These sequences are typically obtained from the 5', and occasionally 3', untranslated regions of eukaryotic or viral DNA or cDNA. The construction of suitable carriers containing one or more of the above components employed standard techniques.

[0388] In addition to sequences that promote transcription of the inserted nucleic acid molecule, vectors can contain origins of replication and other genes encoding selectable markers. For example, the neomycin resistance (neoR) gene confers resistance to G418 on cells that express it, thus allowing phenotypic selection of transfected cells. Other examples of markers or reporter genes include β-lactamases, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), dihydrofolate reductase (DHFR), hygromycin-β-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding β-galactosidase), and xanthine-guanine phosphoribosyltransferase (XGPRT). Those skilled in the art can readily determine whether a given regulatory element or selectable marker is suitable or unsuitable for a particular expression environment.

[0389] The correct assembly of expression vectors can be confirmed by nucleic acid sequencing, restriction mapping, and expression of bioactive peptides in a suitable host.

[0390] Host cell:

[0391] This invention further provides prokaryotic or eukaryotic cells containing and expressing nucleic acid molecules encoding a mutant IL-2 protein. The cells of this invention are transfected cells, i.e., cells in which nucleic acid molecules have been introduced, for example, by introducing nucleic acid molecules encoding a mutant IL-2 polypeptide using recombinant DNA technology. Progeny of such cells are also considered to be within the scope of this invention.

[0392] Host cells are typically selected based on their compatibility with the chosen expression vector, the toxicity of the product encoded by the DNA sequence of this invention, their secretion characteristics, their ability to correctly fold polypeptides, their fermentation or culture requirements, and the ease of purification of the product encoded by the DNA sequence. Suitable host cells for cloning or expressing the DNA in the vectors described herein are the aforementioned prokaryotic cells, yeast, or higher eukaryotic cells.

[0393] In some implementations, recombinant IL-2 mutant proteins or their bioactive variants can also be prepared in eukaryotic cells, such as yeast or human cells. Suitable eukaryotic host cells include insect cells (e.g., baculovirus vectors for protein expression in cultured insect cells, such as the pAc series (Smith et al. (1983) Mol. CellBiol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)); yeast cells (e.g., vectors for expression in Saccharomyces cerevisiae (including pYepSecl) (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA) and pPicZ). (Ingenie, San Diego, California)); or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)).

[0394] Examples of useful mammalian host cell lines include: mouse L cells (LM[TK-], ATCC CRL-2648); monkey kidney CV1 cell line transformed from SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (HEK293 or HEK293 cell subclones, grown in suspension culture); juvenile hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); and human lung cells (W138, ATCC CCL 1651). 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human hepatocellular carcinoma cell line (Hep G2). In mammalian cells, the control function of expression vectors is usually provided by viral regulatory elements. For example, commonly used promoters are derived from polyomaviruses, adenovirus type 2, cytomegaloviruses, and simian virus 40.

[0395] IL-2 mutant proteins can be produced in prokaryotic hosts, such as the bacteria *Escherichia coli*, or in eukaryotic hosts, such as insect cells (e.g., Sf21 cells) or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells can be obtained from many sources, including the U.S. Type Culture Collection (Massanas, Virginia). When selecting an expression system, compatibility between the components is paramount. Those skilled in the art are capable of making this decision. Furthermore, if guidance is needed in selecting an expression system, those skilled in the art can consult Ausubel et al. (*Current Protocols in Molecular Biology*, John Wiley and Sons, NY, 1993) and Pouwels et al. (*Cloning Vectors: A Laboratory Manual*, 1985, Supplement 1987).

[0396] In some implementations, the resulting IL-2 mutant protein is glycosylated or unglycosylated, depending on the host organism used to produce the mutant protein. If bacteria are chosen as the host, the produced IL-2 mutant protein will be unglycosylated. On the other hand, eukaryotic cells will glycosylate the IL-2 mutant protein, although perhaps in a different manner than native IL-2 glycosylation.

[0397] For other expression systems used in prokaryotic and eukaryotic cells, see Sambrook et al. (1989), *Molecular Cloning: A Laboratory Manual* (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY), Chapters 16 and 17. See also Goeddel (1990), *Gene Expression Technology: Methods in Enzymology*, 185 (Academic Press, San Diego, CA).

[0398] Transfection:

[0399] This expression construct can be introduced into host cells to produce the IL-2 mutant protein disclosed herein, or to produce its biologically active mutant protein. The vector DNA can be introduced into prokaryotic or eukaryotic cells using conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989), *Molecular Cloning: A Laboratory Manual* (2nd edition, Cold Spring Harbor Laboratory Press, Plainview, NY), and other standard molecular biology laboratory manuals.

[0400] To facilitate the transfection of target cells, the target cells can be directly exposed to a non-viral vector under conditions that favor uptake of the non-viral vector. Examples of conditions that promote the uptake of exogenous nucleic acids by mammalian cells are well known in the art, including but not limited to chemical methods (e.g., Lipofectamine®, Thermo-Fisher Scientific), high salt, and magnetic fields (electroporation).

[0401] Cell culture:

[0402] Cells can be cultured in conventional nutrient media modified to be suitable for inducing promoters, selecting transformants, or amplifying genes encoding desired sequences. Mammalian host cells can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI 1640 (Sigma), and DMEM (DSigma) are suitable for culturing host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine and thymidine), antibiotics, trace elements, and glucose or equivalent energy sources. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those previously used for selecting expression in host cells and are obvious to those skilled in the art.

[0403] Recombinant protein recovery:

[0404] If a secretory leader sequence is used, the recombinantly produced IL2 mutant protein peptide can be recovered from the culture medium as a secreted peptide. Alternatively, the IL2 mutant protein peptide can also be recovered from host cell lysates. Protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF), can be used in the recovery stage from cell lysates to inhibit protease degradation during purification and may include antibiotics to prevent the growth of exogenous contaminants.

[0405] purification:

[0406] Various purification steps are known in the art and are available, such as affinity chromatography. Affinity chromatography utilizes highly specific binding sites commonly found in biological macromolecules to separate them based on their ability to bind to specific ligands. Covalent bonds attach ligands to an insoluble porous support medium, making the ligands readily visible on the protein sample, thus utilizing the natural specific binding of one type of molecule to separate and purify a second type from the mixture. Antibodies are commonly used in affinity chromatography. Size selection steps can also be used, for example, gel filtration chromatography (also known as size exclusion or molecular sieve chromatography) for separating proteins based on their size. In gel filtration, a protein solution is passed through a column filled with a semi-permeable porous resin. The semi-permeable resin has a range of pore sizes that determines the size of proteins that can be separated using the column.

[0407] The IL-2 mutant protein produced by the transformed host can be purified by any suitable method. Various methods for purifying IL-2 are known. See, for example, *Current Protocols in Protein Science*, Volume 2. Edited by John E. Coligan, Ben M. Dunn, Hidde L. Ploehg, David W. Speicher, Paul T. Wingfield, Unit 6.5 (1997, Copyright John Wiley and Sons, Inc.). The IL-2 mutant protein can be isolated from inclusion bodies produced in *E. coli*, or from conditioned media of mammalian or yeast cultures producing the specific mutant protein, using cation exchange, gel filtration, and / or reversed-phase liquid chromatography.

[0408] The basic purified form of recombinant peptides can be purified from the expression system using conventional biochemical procedures and can be used as a therapeutic agent, as described herein.

[0409] The biological activity of the IL-2 mutant protein can be assessed by any suitable method known in the art, and can be assessed as a basic purified form, or as a portion of cell lysate or cell culture medium when expressed using a secretory leader sequence. Such activity assays include CTLL-2 proliferation, induction of phosphorylated-STAT5 (pSTAT5) activity in T cells, PHA-blast proliferation, and NK cell proliferation.

[0410] preparation

[0411] For therapeutic applications, the mutant protein can be administered to mammals. Administration can be intravenous, by bolus injection or by continuous infusion over a period of time. Alternative routes of administration include intramuscular, intraperitoneal, intraspinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, local, or inhalation. The IL2 mutant protein can also be suitably administered intratumorally, adjacent to, within the lesion, intranodally, or peritumorally, or via the lymphatic system to exert local and systemic therapeutic effects.

[0412] In some embodiments, the host IL-2 mutant protein (and / or nucleic acid) may be incorporated into the composition, including a pharmaceutical composition. Such compositions typically comprise a polypeptide or nucleic acid molecule and a pharmaceutically acceptable carrier. The pharmaceutical composition is prepared to be compatible with the intended route of administration and for therapeutic use, for administering the IL-2 mutant protein to a subject requiring treatment or prevention.

[0413] Parenteral preparations:

[0414] The mutant IL-2 polypeptide of this invention can be administered orally, but is more likely to be given via a parenteral route. Examples of parenteral routes include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), mucosal, and rectal administration. Parenteral formulations include solutions or suspensions for parenteral application and may include carriers and buffers. Pharmaceutical formulations suitable for parenteral administration include sterile aqueous solutions (when water-soluble) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions.

[0415] carrier The carrier includes sterile diluents such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Suitable flowability can be maintained, for example by using coating materials such as lecithin, maintaining the desired particle size in the case of dispersions, and by using surfactants such as sodium dodecyl sulfate. For intravenous administration, suitable carriers include physiological saline, antibacterial water, CremophorEL™ (BASF, Parsipney, NJ), or phosphate-buffered saline (PBS).

[0416] Buffer: The term buffer includes buffers such as acetates, citrates, or phosphates, as well as agents used to adjust pH, such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as mono- and / or di-basic sodium phosphate, hydrochloric acid, or sodium hydroxide (e.g., to a pH of approximately 7.2–7.8, such as 7.5).

[0417] dispersion Typically, the active ingredient is incorporated into a sterile carrier containing an alkaline dispersion medium and the other desired components mentioned above to prepare a dispersion. In preparing sterile powders for sterile injections, the preferred preparation method is vacuum drying and freeze-drying, yielding a powder of the active component and any other desired components from a previously sterile filtered solution.

[0418] preservativePharmaceutical preparations intended for parenteral administration should be sterile and fluid to facilitate injectability. They should be stable under manufacturing and storage conditions and resistant to contamination during storage. Microbial protection can be achieved through various antimicrobial and antifungal agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; and chelating agents, such as ethylenediaminetetraacetic acid, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. A sterile solution can be prepared by incorporating the required amount of the active compound and one or more of the above components, or combinations thereof, into a suitable solvent, followed by filtration and sterilization.

[0419] tension agent In many cases, the composition may preferably contain isotonic agents, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride.

[0420] Oral Composition Oral compositions typically include inert diluents or edible carriers. For oral therapeutic purposes, excipients may be used to incorporate the active compound and administer it in tablet, lozenge, or capsule form, such as gelatin capsules. Oral compositions may also be prepared using liquid carriers for use as mouthwashes. Pharmaceutically compatible binders and / or adjuvants may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds with similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginate, carboxymethyl starch (Primogel™), or corn starch; lubricants such as magnesium stearate or fully hydrogenated vegetable oil (Sterotes™); flow aids such as silica gel; sweeteners such as sucrose or saccharin; or flavorings such as peppermint, methyl salicylate, or orange flavoring.

[0421] Inhaled formulations In the case of administration by inhalation, the main IL-2 mutant protein or the nucleic acid encoding it is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant (e.g., carbon dioxide gas) or a spray. Such methods include those described in U.S. Patent No. 6,468,798.

[0422] Mucosa and transdermalThe host IL-2 mutant protein or nucleic acid can also be administered systemically via mucosal or transdermal methods. For mucosal or transdermal administration, a permeabilizer suitable for the permeation barrier is used in the formulation. Such permeabilizers are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives used for mucosal administration. Mucosal administration can be performed by preparing the compound for rectal delivery using nasal sprays or suppositories (e.g., using conventional suppository bases such as cocoa butter or other glycerides) or retention enemas. For transdermal administration, the active compound is formulated as an ointment, ointment, gel, or cream, as is generally known in the art, and may incorporate a penetration enhancer such as ethanol or lanolin.

[0423] Extended release and storage formulations: In some embodiments, the IL2 mutant protein is administered in formulation form to provide prolonged release of the IL2 mutant protein agent. Examples of prolonged-release formulations of injectable compositions can be achieved by including agents that delay absorption (such as aluminum monostearate and gelatin) in the composition. In one embodiment, the host IL-2 mutant protein or nucleic acid is prepared using a carrier that will protect the compound from rapid clearance by the body, such as controlled-release formulations, including implantable and microencapsulated delivery systems. Biodegradable biocompatible polymers, such as ethylene-vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Such formulations can be prepared using standard techniques. The materials are also available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes of cells infected with viral antigens targeted by monoclonal antibodies) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0424] Administration of nucleic acids encoding the mutant IL2 protein (gene therapy)In some embodiments, the compound (the host IL-2 mutant protein or nucleic acid) may also be administered via transfection or infection using methods known in the art, including but not limited to those described in McCaffrey et al. (Nature 418:6893, 2002), Xia et al. (Nature Biotechnol. 20: 1006-1010, 2002), or Putnam (Am. J. Health Syst. Pharm. 53: 151-160, 1996, erratum in Am. J. Health Syst. Pharm. 53:325, 1996). In some embodiments, the IL-2 mutant protein is administered to the recipient via a pharmaceutically acceptable formulation of a recombinant expression vector. In some embodiments, the recombinant expression vector is a viral vector. In some embodiments, the recombinant vector is a recombinant viral vector. In some embodiments, the recombinant viral vector is a recombinant adeno-associated virus (rAAV) or a recombinant adenovirus (rAd), particularly a replication-deficient adenovirus derived from human adenovirus serotypes 3 and / or 5. In some embodiments, the replication-deficient adenovirus has one or more modifications to the E1 region, which interferes with the virus's ability to initiate cell circulation and / or apoptosis pathways in human cells. The replication-deficient adenoviral vector may optionally include a deletion in the E3 domain. In some embodiments, the adenovirus is a replication-capable adenovirus. In some embodiments, the adenovirus is a replication-capable recombinant virus engineered to selectively replicate in lymphocytes.

[0425] In one embodiment, an IL2 mutant protein formulation is provided, based on the teachings of Fernandes and Taforo, U.S. Patent No. 4,604,377, issued August 5, 1986, which are incorporated herein by reference. And Yasui, et al., U.S. Patent No. 4,645,830.

[0426] Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. In one embodiment, the preparation is delivered in a pre-filled syringe for parenteral administration.

[0427] How to use

[0428] The present invention also provides a method for treating a subject suffering from a disease disorder or condition by administering a therapeutically effective amount of the IL-2 mutant protein of the present invention (or a nucleic acid encoding the IL-2 mutant protein, including a recombinant virus encoding the IL-2 mutant protein). In the treatment of such a disease, the IL-2 mutant protein of the present invention can be modified to provide advantageous properties, such as reduced vascular leakage syndrome. Disorders suitable for treatment with the IL-2 mutant protein of the present invention (including pharmaceutically acceptable formulations comprising the IL-2 mutant protein and / or a nucleic acid molecule encoding it, including a recombinant virus encoding such IL-2 mutant protein) include inflammatory or autoimmune diseases, including but not limited to viral infections (e.g., AIDS, influenza, chronic HCV, chronic hepatitis B, C, or D), Helicobacter pylori infection, HTLV, organ rejection, graft-versus-host disease, autoimmune thyroid disease, multiple sclerosis, allergies, asthma, and neurodegenerative diseases, including Alzheimer's disease and systemic lupus erythematosus (SLE). Autoinflammatory diseases, inflammatory bowel disease (IBD), Crohn's disease, diabetes, including type 1 or type 2 diabetes, inflammation, autoimmune diseases, atopic diseases, adjacent autoimmune diseases, chondritis, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, generalized juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Ritter syndrome, SEA syndrome (seronegative osteopathic arthropathy syndrome). Enthesopathy, Arthropathy Syndrome, Juvenile Dermatomyositis, Juvenile Psoriatic Arthritis, Juvenile Scleroderma, Juvenile Systemic Lupus Erythematosus, Juvenile Vasculitis, Oligoarthritis Rheumatoid Arthritis, Polyarthritis Rheumatoid Arthritis, Systemic Rheumatoid Arthritis, Ankylosing Spondylitis, Enteropathic Arthritis, Reactive Arthritis, Ritter's Syndrome, SEA Syndrome (Seronephropathy, Bone-Setting Lesions, Arthropathy Syndrome).

[0429] Other examples of proliferative and / or differentiation disorders for treatment with the IL-2 mutant protein to which this invention is applicable (including pharmaceutically acceptable formulations comprising the IL-2 mutant protein and / or nucleic acid molecules encoding it, including recombinant viruses encoding such IL-2 mutant protein) include, but are not limited to, dermatological disorders. Dermatological disorders may involve abnormal activity of one or more cells or layers of the dermis, epidermis, or hypodermis, or abnormalities at the dermal-epidermal junction. For example, dermatological disorders may involve abnormal activity of keratinocytes (such as overproliferating basal and superbasal keratinocytes), melanocytes, Langerhans cells, Merkel cells, immune cells, and other cells found in one or more epidermal layers (such as the basal layer (germinal layer), spinous layer, granular layer, stratum lucidum, or stratum corneum). In other embodiments, the disorder may involve abnormal activity of dermal cells, such as dermal endothelial cells, fibroblasts, and immune cells (such as mast cells or macrophages) found in the dermis, such as the papillary layer or reticular layer.

[0430] Examples of skin diseases include psoriasis, psoriatic arthritis, dermatitis (eczema), exfoliative dermatitis or atopic dermatitis, pityriasis rubra pilaris, pityriasis rosacea, psoriasis-like diseases, lichenoid pityriasis, lichen planus, lichen luster, ichthyosis-like skin diseases, keratosis, skin diseases, alopecia areata, pyoderma gangrenosa, vitiligo, bullous pemphigoid (e.g., ocular cicatricial bullous pemphigoid or bullous bullous pemphigoid), urticaria, keratosis, and rheumatoid arthritis, which involve Excessive proliferation and inflammation of the epithelial-associated cells lining the joint capsule; dermatitis such as seborrheic dermatitis and photodermatitis; keratosis such as seborrheic keratosis, senile keratosis, actinic keratosis, photoinduced keratosis, and follicular keratosis; acne vulgaris; keloids and prevention of keloid formation; nevi; warts, including verruca vulgaris, condyloma acuminata, or genital warts, and human papillomavirus (HPV) infection, such as genital warts; leukoplakia of the mucous membranes; lichen planus and keratitis. Skin diseases can be dermatitis, such as atopic dermatitis or allergic dermatitis, such as psoriasis.

[0431] The compositions of the present invention (including pharmaceutically acceptable formulations comprising the IL2 mutant protein and / or nucleic acid molecules encoding it, including recombinant viruses encoding such IL2 mutant protein) may also be given to patients who have (or may have) psoriasis or psoriatic disease. The term "psoriasis" is intended to have its medical meaning as a disease that primarily affects the skin and produces raised, thickened, scaly, non-scarring lesions. Lesions are typically well-defined erythematous papules covered with overlapping, shiny scales. The scales are usually silvery or slightly milky white. Nails are frequently affected, resulting in pitting, onycholysis, thickening, and discoloration. Psoriasis is sometimes associated with arthritis and can have serious consequences. Excessive keratinocyte proliferation is a key feature of epidermal hyperplasia in psoriatic arthritis, accompanied by epidermal inflammation and reduced keratinocyte differentiation. Several mechanisms have been cited to explain the keratinocyte overproliferation characteristic of psoriasis. Disordered cellular immunity is also thought to be involved in the pathogenesis of psoriasis. Examples of psoriasis include chronic quiescent psoriasis, plaque psoriasis, moderate to severe plaque psoriasis, common psoriasis, eruptive psoriasis, erythrodermic psoriasis, generalized pustular psoriasis, annular pustular psoriasis, or focal pustular psoriasis.

[0432] In some embodiments, the host IL-2 mutant protein, functioning as an IL-2 antagonist as described herein, is used to treat one or more conditions, wherein inhibition of one or more IL-2 and / or IL-15-dependent functions is useful. In some embodiments, the IL-2 mutant protein described herein is used to treat one or more diseases or conditions, wherein inhibition of CD122 / CD132 heterodimerization and downstream signal transduction is useful (e.g., GVDH or leukemia). In one embodiment, the treatment method is for treating graft-versus-host disease (GVHD). In some embodiments, the treatment includes the step of administering a therapeutically effective amount of the IL-2 mutant protein (including pharmaceutically acceptable formulations comprising the IL2 mutant protein and / or nucleic acid molecules encoding it, including recombinant viruses encoding such IL2 mutant protein) to a subject suffering from GVHD.

[0433] IL2 mutant protein in combination with other therapeutic agents for autoimmune diseases:

[0434] This invention provides the use of the IL2 mutant protein of the present invention in combination with one or more other active agents (“supplements”) for the treatment of autoimmune diseases. As used herein, the term “supplement” includes agents that can be administered or introduced individually, such as agents formulated separately for administration alone (e.g., available in a kit) and / or treatments that can be administered or introduced in combination with the IL2 mutant protein.

[0435] As used herein, the term "in combination with" when referring to administering multiple agents to a subject means administering the first agent to the subject at least one other (i.e., the second, third, fourth, fifth, etc.) agent. For the purposes of this invention, if the biological effect resulting from the administration of the first agent persists in the subject's body when the second agent is administered, resulting in an additive therapeutic effect of the first and second agents, then an agent (e.g., an IL2 mutant protein) is considered to be administered in combination with a second agent (e.g., a therapeutic autoimmune antibody, such as Humira®). For example, therapeutic antibodies are sometimes administered every two weeks via IV infusion (e.g., adalimumab for treating Crohn's disease), while the IL2 mutant protein of this disclosure can be administered more frequently, such as daily, by-the-dosage, or weekly. However, if the administration of the first agent (e.g., etanercept) provides a therapeutic effect over a longer period, and the administration of the second agent (e.g., the IL2 mutant protein) provides its therapeutic effect while the therapeutic effect of the first agent is still ongoing, then the second agent is considered to be administered in combination with the first agent, even if the first agent may have been administered at a point in time (e.g., days or weeks) distant from the time of administration of the second agent. In one embodiment, if the first and second reagents are administered simultaneously (within 30 minutes of each other), concurrently, or sequentially, then a reagent is considered to be administered in combination with the second reagent. In some embodiments, if the first and second reagents are administered within approximately 24 hours of each other, preferably within approximately 12 hours of each other, preferably within approximately 6 hours of each other, preferably within approximately 2 hours of each other, or preferably within approximately 30 minutes of each other, then the first reagent is considered to be administered "concurrently" with the second reagent. The term "in combination with" should also be understood to apply to situations where the first and second reagents are co-formulated in a single pharmaceutically acceptable formulation and that co-formulation is administered to the subject. In some embodiments, the IL2 mutant protein and one or more supplements are administered or applied sequentially, for example, after administering one reagent, one or more other reagents are administered. In other embodiments, the IL2 mutant protein and one or more supplements are administered simultaneously, for example, two or more reagents are administered at the same time or approximately at the same time; these two or more reagents may be present in two or more separate formulations or combined into a single formulation (i.e., a co-formulation). Regardless of whether these reagents are administered sequentially or simultaneously, they are considered to be administered in combination for the purposes of this invention.

[0436] In some embodiments, the supplement is an agent of one or more corticosteroids selected from the group consisting of (including but not limited to prednisone, budesonide, and prednisolone), Janus kinase inhibitors (including but not limited to tofacitinib (Xeljanz®), calcineurin inhibitors (including but not limited to cyclosporine and tacrolimus), mTor inhibitors (including but not limited to sirolimus and everolimus), IMDH inhibitors (including but not limited to azathioprine, leflunomide, and mycophenolate mofetil), or biologics such as abatacept (Orencia®) or etanercept. (Enbrel®) and therapeutic antibodies. Therapeutic antibodies that can be given as supplements in combination with the IL2 mutant protein of the present invention to treat autoimmune diseases include, but are not limited to, anti-CD25 antibodies (e.g., dacrolimus and baliximab), anti-VLA-4 antibodies (e.g., natetzumab), anti-CD52 antibodies (e.g., alemtuzumab), anti-CD20 antibodies (e.g., rituximab, olizumab), anti-TNF antibodies (e.g., infliximab and adalimumab), anti-IL-6R antibodies (e.g., tocilizumab), anti-TNFα antibodies (e.g., adalimumab (Humira®), golimumab, and infliximab), and anti-intrinsic antibodies. Anti-IL-17a antibodies (e.g., vedolizumab), anti-IL-4Rα antibodies (e.g., dupilumab), anti-IL-6R antibodies, anti-IL-1β antibodies (e.g., cannabinoids), anti-CD11a antibodies (e.g., efalizumab), anti-CD3 antibodies (e.g., muramonab), anti-IL5 antibodies (e.g., mepolizumab, retalizumab), anti-BLyS antibodies (e.g., belimumab); and anti-IL-12 / IL-23 antibodies (e.g., ustekinumab).

[0437] Many therapeutic antibodies have been approved for clinical use to combat autoimmune diseases. Table 4 provides examples of antibodies approved by the U.S. Food and Drug Administration (FDA) for the treatment of subjects with autoimmune diseases, which may be administered as supplements in combination with the IL2 mutant protein of this disclosure (and optionally additional supplements) to treat the indicated autoimmune disease.

[0438]

[0439]

[0440] In the practice of the method of the present invention, the aforementioned antibodies, which are useful as supplements, can be given alone or in the form of any antibody-drug conjugate (ADC), including antibodies, linkers, and one or more drugs (such as 1, 2, 3, 4, 5, 6, 7, or 8 drugs) or in modified forms (such as PEGylation).

[0441] In some embodiments, the supplement is a vaccine. Based on the teachings of U.S. Patent No. 5,800,819 (granted September 1, 1998) to Doyle et al., the IL2 mutant protein of the present invention can be administered to a subject in combination with a vaccine as an adjuvant to enhance the immune response to the vaccine. Examples of vaccines that can be combined with the IL-2 mutant protein of the present invention include HSV vaccines, Bordetella pertussis vaccines, Escherichia coli vaccines, pneumococcal vaccines (including multivalent pneumococcal vaccines such as Prevnar® 13), diphtheria, tetanus and pertussis vaccines (including combination vaccines such as Pediatrix® and Pentacel®), varicella vaccines, Haemophilus influenzae type B vaccines, human papillomavirus vaccines such as Garasil®, polio vaccines, leptospirosis vaccines, combination respiratory vaccines, Moraxella vaccines, and attenuated or inactivated virus products such as bovine respiratory disease vaccine (RSV), multivalent human influenza vaccines such as Fluzone® and Quadravlent Fluzone®, and feline leukemia virus vaccines. (vaccines), including infectious gastroenteritis vaccine and rabies vaccine.

[0442] dose

[0443] The dosage, toxicity, and efficacy of such main IL-2 mutant protein or nucleic acid compound can be determined in cell cultures or laboratory animals using standard pharmaceutical procedures. Data obtained from cell culture experiments and animal studies can be used to establish dosage ranges for human use. The dosage of this compound is preferably within a circulating concentration range that includes an ED50 with minimal acceptable toxicity. This dosage may vary within this range depending on the dosage form and route of administration used. For any compound used in the methods of this invention, the therapeutically effective dose can be initially estimated from cell culture experiments. Doses can be formulated in animal models to achieve circulating plasma concentration ranges, including the IC50 (i.e., the concentration of the test compound that achieves half-maximal symptom inhibition) determined in cell cultures. This type of information can be used to determine the human dosage more precisely. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0444] As defined herein, the therapeutically effective amount (i.e., effective dose) of the main IL-2 mutant protein depends on the peptide selected. For example, a single dose of about 0.001 to 0.1 mg / kg of patient body weight may be administered; in some embodiments, about 0.005, 0.01, or 0.05 mg / kg may be administered. In some embodiments, 600,000 IU / kg (IU can be determined by lymphocyte proliferation bioassay and expressed in international units (IU) as established by the World Health Organization first-generation international standard for interleukin-2 (human)) may be administered.

[0445] In some embodiments, the pharmaceutically acceptable form of the invented IL2 mutant protein is administered to the subject according to the “low-dose” treatment regimens described in U.S. Patents 9,669,071 and 10,293,028B2 to Klatzman et al., the entire teachings of which are incorporated herein by reference. Other low-dose regimens are described in Smith, KA (1993) Blood 81(6):1414-1423, and He, et al. (2016) Nature Medicine 22(9): 991-993.

[0446] According to another aspect of the invention, a method for stimulating the immune system of an animal by administering the IL-2 mutant protein of the invention is provided. This method is useful for treating disease states in which the host immune response is insufficient. In treating the subject, a therapeutically effective dose of the compound (i.e., the active ingredient) is administered. The therapeutically effective dose refers to the amount of the active ingredient that produces improvement in symptoms or prolongs the survival of the subject. The effective dose will vary depending on the characteristics of the IL-2 mutant protein to be administered, the physical characteristics of the subject, the nature of the disease or condition, etc. A single administration can range from about 50,000 IU / kg to about 1,000,000 IU / kg or more, more typically about 600,000 IU / kg. This can be repeated several times a day (e.g., 2-3 times a day) for several days (e.g., about 3-5 consecutive days), and then repeated once or more after a rest period (e.g., about 7-14 days). Thus, the effective dose can comprise a single single administration or multiple administrations over a period of time (e.g., about 20-30 single administrations over about 10-20 days, each at about 600,000 IU / kg).

[0447] This composition may be administered once daily or several times, or once weekly or several times, including every other day. Those skilled in the art will also understand that certain factors may affect the dosage and duration required for effective treatment of a subject, including, but not limited to, the severity of the disease or disorder, prior treatment, the subject's age and / or general health condition, and any other pre-existing conditions. Additionally, treatment of a subject with a therapeutically effective amount of the host IL-2 mutant protein may comprise a single treatment or a series of treatments. In one embodiment, the composition is administered every 8 hours for 5 days, followed by a rest period of 2–14 days, e.g., 9 days, followed by administration every 8 hours for another 5 days. In another embodiment, the composition is administered every other day for a period of at least 6 days, optionally at least 10 days, optionally at least 14 days, optionally at least 30 days, optionally at least 60 days. Those skilled in the art will recognize that this therapy can be used to treat chronic conditions and prevent the recurrence of symptoms of chronic diseases, such as autoimmune diseases (e.g., psoriasis, IBD, etc.).

[0448] These pharmaceutical compositions may be included in containers, packages, or dispensers along with the instructions for use.

[0449] While reagents exhibiting toxic side effects can be used, care must be taken when designing delivery systems that target the compound to sites in affected tissues to minimize potential damage to untransfected cells and thus reduce side effects. The toxicity and efficacy of IL-2 mutant proteins can be determined in cell cultures or laboratory animals using standard pharmaceutical procedures. Cell culture assays and animal studies can be used to determine the LD50. 50 (The dose that is lethal to 50% of the population) and ED 50 (The dose that has a therapeutic effect on 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as LD50. 50 / ED 50 The ratio. Preferably, IL-2 mutants exhibiting a large therapeutic index are preferred. Data obtained from cell culture assays and animal studies are suitable for establishing dosage ranges for human use. The dosage of such mutants is preferably within the cyclic concentration range, which includes ED50 with little or no toxicity. 50 The dosage can vary within this range depending on various factors, such as the dosage form used, the route of administration, and the patient's condition.

[0450] The effective therapeutic dose can be determined by determining the IC50. 50 This was initially estimated from cell culture experiments. Dosage can then be formulated in animal models to achieve the range of circulating plasma concentrations, including the IC50 determined in cell culture. 50This type of information can be used to determine human dosage more accurately. Plasma levels can be measured, for example, by HPLC. The specific formulation, route of administration, and dosage can be chosen by an individual physician based on the patient's condition.

[0451] The attending physician for patients treated with IL-2 mutant protein and optional supplements will know how and when to discontinue, interrupt, or adjust medication due to toxicity, organ dysfunction, or other reasons. Conversely, if the clinical response is inadequate (excluding toxicity), the attending physician will also be aware of the need to adjust treatment to a higher level. In the management of the disorder of interest, the dosage will vary depending on the severity of the condition being treated, the route of administration, and other factors. For example, the severity of the condition can be assessed in part using standard prognostic methods. Furthermore, the dosage and possible dosing frequency will also vary based on the individual patient's age, weight, and response.

[0452] The IL-2 mutant of the present invention can be administered to an individual as a pharmaceutical formulation, formulated to suit the route of administration and the condition to be treated. Suitable routes may include: oral, rectal, transdermal, vaginal, transmucosal, or enteral administration; parenteral administration, including intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection, etc. For transmucosal administration, a penetrant is used in the formulation suitable for the penetration barrier. Such penetrants are generally known in the art.

[0453] IL-2 mutants can be formulated with one or more pharmaceutically acceptable carriers or excipients, as is well known in the art. Techniques for formulation and administration can be found in Remington's Pharmaceutical Sciences (18th edition, Easton Mead, PA). Specific examples of IL-2 formulations are described in U.S. Patent Nos. 4,604,377 and 4,766,106. IL-2 mutants can be formulated as liquids, wherein the carrier may include buffers and / or salts, such as phosphate-buffered saline. Alternatively, IL-2 mutants can be formulated as solids, wherein the carrier or filler, such as lactose, binder such as starch, and / or lubricant such as talc or magnesium stearate, and, optionally, stabilizers.

[0454] Reagent test kit

[0455] The present invention also contemplates kits comprising a pharmaceutical composition of a mutant IL2 protein and pharmaceutical compositions thereof. Such kits are typically in a physical structural form that accommodates various components, as described below, and can be used, for example, to perform the methods described above. The kit may include the mutant IL2 protein in the form of a pharmaceutical composition suitable for administration, which may be readily available or in a form that needs to be prepared, for example, thawed, reconstituted, or diluted prior to administration. When the mutant IL2 protein is in a form that needs to be reconstituted by the user, the kit may also include a sterile container providing a reconstitution medium comprising buffers, pharmaceutically acceptable excipients, etc.

[0456] In addition to other components, the kit of the present invention may further include one or more supplements.

[0457] The kit of the present invention can be designed to properly maintain the necessary conditions for the components contained therein (e.g., refrigeration or freezing).

[0458] The kit may further include labels or packaging inserts containing identification information and instructions for use for the components. The individual components in the kit may be packaged in separate containers, and all the various containers may be in a single package. Labels or inserts may include manufacturer information such as batch number and expiration date. Labels or packaging inserts may, for example, be integrated into the physical structure containing the components, be contained separately within the physical structure, or be affixed to the components of the kit (e.g., ampoules, syringes, or vials). Labels or inserts may be provided in physical form or on a computer-readable medium. In some embodiments, the actual instructions are not present in the kit; instead, the kit provides a method for obtaining the instructions remotely, such as via an internet website, including secure access by providing a password (or a scannable code, such as an IL2 mutant protein or a barcode or QR code on the container containing the kit) that complies with government regulations (e.g., HIPAA).

[0459] Example

[0460] The following examples are provided to describe certain implementations of the invention and should not be construed as limiting.

[0461] Example 1: Generation of human IL2 expression vector pcDNA3.1 / hygro(+)-huIL2

[0462] Human IL2 DNA open reading frame (“ORF”) (Genbank NM_000586.3) was synthesized (Life Technologies GeneArt Services, Carlsbad, CA) and amplified by PCR using a Platinum SuperFi II DNA polymerase kit (commercially available, catalog number 12361050, Thermo Fisher Scientific) substantially according to the manufacturer’s protocol, using primers 5' TATAGTCAGCGCCACcCATGTACAGGATGCAACTCCTGTC 3' (SEQ ID NO: 100), which incorporates the NheI restriction site, and 5' TATAGGGCCCTATCAAGTCAGTGTTGAGATG 3' (SEQ ID NO: 101), which incorporates the ApaI restriction site. PCR fragments were visualized on 1% agarose gel (catalog number #54803, Lonza, Rockland, Maine), excised from the gel, and purified using the QIAquick PCR Purification Kit (commercially available, catalog number 28106, Qiagen, Germany), according to the manufacturer’s protocol.

[0463] Purified PCR fragments and mammalian expression vector pcDNA 3.1 / Hygro(+) (commercially available, catalog number V87020, Thermo Fisher Scientific, Carlsbad, CA) were digested with restriction enzymes NheI and ApaI (commercially available, catalog numbers R0111S and R0114L, New England Biolabs, Ipswich, MA). The expression vector was further processed using a rapid dephosphorylation kit (commercially available, catalog number M0508L, New England Biolabs) according to the manufacturer's protocol. PCR fragments were ligated into pcDNA 3.1 / Hygro(+) using a rapid DNA ligation kit (commercially available, catalog number 11635379001, Sigma Aldrich, St. Louis, Missouri), substantially according to the manufacturer’s protocol, transformed into One Shot TOP10 chemicompetent E. coli (commercially available, catalog number C404006, Life Technologies, Carlsbad, CA), inoculated onto LB agar plates containing 100 μg / ml carbenicillin (commercially available, catalog number L1010, Teknova, Hollister, CA) and grown overnight at 37°C.

[0464] The following day, single colonies were picked and used to initiate 3 ml bacterial cultures in LB broth (catalog number 10855-001, Life Sciences) containing 100 μg / ml ampicillin (commercially available, catalog number A9626, Teknova). The cultures were incubated overnight at 37°C. The next day, *E. coli* were precipitated (6,000 rpm, 10 min, benchtop centrifuge, catalog number 5424, commercially available, catalog number Eppendorf, Hopoorg, NY), and the DNA expression vector was isolated using the QIAprep Spin Kit (catalog number 27106, Qiagen). The plasmid DNA was sequence validated (MCLab, South San Francisco, CA).

[0465] Example 2. Generation of human IL2 REH expression vector pcDNA3.1 / hygro(+)-huIL2-REH

[0466] The expression vector introducing three mutations into the human IL2 ORF (L38R, Q42E, and Q146H; all numbers are based on the full-length human IL2 ORF NM_000586.3 number, i.e., the expressed hIL2, including the signal peptide instead of the 20 amino acid sequence of the mature hIL2 molecule) was assembled substantially according to the teachings of Example 1, except for the following: the initial template DNA for PCR was synthesized containing the L38R (L18R of the mature protein), Q42E (Q22E of the mature protein), and Q146H (Q126H of the mature protein) mutations.

[0467] Example 3. Generation of human IL2 REM expression vector pcDNA3.1 / hygro(+)-huIL2-REM

[0468] The expression vector introduced three mutations into the human IL2 ORF (L38R, Q42E, and Q146M; all numbering is based on the full-length human IL2 ORF NM_000586.3 numbering), assembled exactly as described in the human IL2 expression vector in pcDNA3.1 / Hygro(+), except for the following: the initial template DNA synthesized for PCR contained the L38R, Q42E, and Q146M mutations.

[0469] Example 4. Introducing mutations or reversion mutations into pcDNA3.1 / hygro(+)-huIL2 and pcDNA3.1 / hygro(+)-huIL2 REH expression vectors.

[0470] All mutations or reversion mutations (restoring mutations in pcDNA3.1 / hygro(+)-huIL2-REH to match wild-type IL2 ORF) were introduced into the pcDNA3.1 / Hygro(+)-huIL2 or pcDNA3.1 / Hygro(+)-huIL2-REH expression vectors, using the Quik Change II site-directed mutagenesis kit (sample number 200524, Agilent Technologies, Santa Clara, CA) following the manufacturer's protocol. Tables 5 and 6 list the generated mutations, the templates used to introduce the mutations, and the primer sets used to introduce the mutations. Transformation into E. coli using the Quik Change PCR reaction, as well as the isolation and sequencing of the plasmid DNA, were performed using the same protocol as for generating the pcDNA3.1 / Hygro-huIL2 expression vector.

[0471]

[0472]

[0473]

[0474]

[0475]

[0476] Example 5. Transient transfection in HEK293 cells

[0477] All expression vectors were transiently transfected into HEK293 cells (CRL-1573, ATCC, Manassas, VA). Approximately 1E6 HEK293 cells were seeded into each well of a 6-well tissue culture plate in 2 ml of DMEM (Cellular 10569044, Biotech Inc.) supplemented with 10% fetal bovine serum (SH30071.03, Fisher Scientific, Chicago, ILLU, USA) and grown overnight at 37°C and 5% CO2. The next day, cells were transfected with Lipofectamine 3000 reagent (L3000150, Biotech Inc.) according to the manufacturer's protocol, using 2.5 μg DNA, 5 μL P3000 reagent, and 7.5 μL Lipofectamine 3000 per transfection. Transfected cells were grown at 37°C and 5% CO2 for 48–72 hours and then harvested from the conditioned medium.

[0478] Example 6. Protein Expression Analysis

[0479] Protein expression was measured by ELISA using the Human IL2 V-PLEX ELISA Kit (No. K151QQD-4, Mesoscale Diagnostics, Baltimore, MD), following the manufacturer's protocol (transfection medium initially diluted 1:4, then serially diluted 1:2). Plates were read using the manufacturer's preset settings for this ELISA kit on a Meso Quickplex SQ120 (Mesoscale Diagnostics). Human IL2 standards from this kit were used to calculate approximate expression levels in the conditioned medium samples.

[0480] Example 7: Determination of IL2 activity (STAT5) on CD25- and CD25+ cells

[0481] After 2-3 days of incubation, supernatant samples containing soluble IL2 protein from 293T cells were prepared according to Example 5 above. These samples were then added to YT cells (CD25NEG) and YT cells (YTCD25POS) that had been engineered to constitutively express CD25, and incubated for approximately 20 minutes. The induction level of phosphorylated-STAT5 (pSTAT5) was measured by flow cytometry. The fold change in pSTAT5 induction levels was shown in the attached... Figure 2 The selectivity of IL2 protein for CD25 status was calculated as the level of phosphorylated STAT5 (pSTAT5) on CD25+ YT cells. YTCD25 Divide by the level of phosphorylated-STAT5 in CD25-negative YT cells (pSTAT5) YT The results of these experiments are provided in the attached figures. Figure 2 middle.

[0482] As can be seen from the data presented, the IL2 mutant protein of the present invention provides selective induction of pSTAT5 on CD25 positive cells while retaining significant IL2 activity.

[0483] Example 8. Evaluation of orthologous homolog activity in human T cell clone 3F8

[0484] To evaluate the activity of a representative group of hIL-2 mutant proteins in CD4-positive human T-cell clone 3F8 cells. CD4-positive T-cell clone 3F8 was generated by activation of healthy donor PBMCs and EBV-transformed B cell line JY in two consecutive rounds of mixed leukocyte reactions, followed by production from limiting dilution single-cell clones, as described in (Yssel and Spits (2002), Current Protocols in Immunology, 7.19.1–7.19.12). CD4-positive T-cell clone 3F8 expresses CD25 and CD122, responds to IL-2, proliferates, and produces IFNγ.

[0485] 3F8 cells were contacted with the supernatant of 293T cells transfected with hIL-2 mutant protein as follows: 200,000 cells per ml were grown in a growth medium consisting of Yssel's medium (Iscove's modified Dulbecco medium, Thermo Fisher Scientific), 0.25% w / v% human albumin (Sigma), 1% penicillin / streptomycin (Thermo Fisher Scientific), 1% ITS-X insulin, transferrin, selenium (Gibco), 30 mg / L transferrin (Roche), 2 mg / L palmitic acid (Sigma), 1% LA-OA-albumin linoleic acid, oleic acid (Sigma), and 1% human serum (Gemini) (Yssel et al. (1984) J Immunol Methods 72: 219–227), 100,000 JY cells irradiated with 50 Gy per well, and 40 Gy irradiated allogeneic PBMCs at a concentration of 100,000 cells per well. One million cells. After six days of culture and amplification with 100 pM human IL-2, the cells were washed and seeded into 96-well plates (Costar) with a black clear bottom, 50,000 cells per well, with 75 μl of growth medium. The supernatant of the transfected 293T cells was serially diluted five-fold in growth medium, and 75 μl of each dilution was added to a plate of 3F8 cells, with double replicates, and the final titration ratios were from 1:2 to 1:78125. The plates were transferred to a humidified incubator (Thermo Fisher Scientific) and incubated for three days at 37°C and 5% CO2.

[0486] Remove the plate from the incubator and harvest 40 μl of culture supernatant from a 96-well flat-bottom plate (Costa). Collect the supernatant from duplicate wells. Add 100 μl of Celltiterglo (Promega) to each well to lyse the cells, following the manufacturer's instructions. Mix the cell lysates at 300 rpm for two minutes on a track-controlled shaker (VWR Scientific) and then incubate at room temperature for 10 minutes. The luminescence of the 3F8 cell lysates was read at counts per second using an Envision 2103 multi-tag reader (Perkin Elmer).

[0487] IFNγ production in culture supernatant was measured using the MSD IFNγ V-Plex Kit (MSD K151QOD) according to the manufacturer's instructions. In short, the pre-coated mAb MSD IFNγ assay plate was washed three times with 150 μL Tris wash buffer, and the IFNγ standard was diluted in dilution buffer 2. The culture supernatant was diluted 1:1 with dilution buffer 2, and 50 μL of sample and standard were added to the IFNγ assay plate and incubated at 300 rpm for 120 minutes at room temperature on a track-controlled shaker (VWR Scientific). The plate was washed three times with Tris wash buffer, and 25 μL of 1× detection antibody from dilution buffer 3 was added to each well. The plate was incubated at 300 rpm for 60 minutes at room temperature on a track-controlled shaker (VWR Scientific). The plate was washed three times with Tris wash buffer, and 150 μL of 2x readout buffer T was added to each well. The luminescence signal was read using a Mesoscale Quickplex SQ120 instrument. The concentration of IFNγ in the supernatant was calculated using MSD software based on the standard curve.

[0488] To compare the effects of each hIL-2 mutant protein on 3F8 cell proliferation and IFNγ production, the CelltiterGlo value and IFNγ concentration of cells treated with the supernatant were compared with control cells treated with growth medium alone, wild-type IL-2 transfection, or human REK IL-2 transfection. Data from these experiments are listed in Table 7 and... Figures 3A-3D These data demonstrate the association between hIL-2 mutant protein-induced proliferation and IFNγ production activity.

[0489]

Claims

1. A polypeptide having a reduced binding affinity for the extracellular domain of hCD132 compared to wild-type hIL2, said polypeptide comprising the amino acid sequence of the following formula (SEQ ID NO: 97): (AA1) a –(AA2) b -(AA3) c -(AA4) d -(AA5) e -(AA6) f -(AA7) g -(AA8) h -(AA9) i -T10-Q11-L12-Q13-L14-E15-H16-L17-(AA18)-L19-D20-L21-(AA22)-M23-I24-L25-N26 -G27-I28-N29-N30-Y31-K32-N33-P34-(AA35)-L36-T37-(AA38)-(AA39)-L40-T41-F42-K 43-F44-Y45-M46-P47-K48-K49-A50-T51-E52-L53-K54-(AA55)-L56-Q57-C58-L59-E60-E61-E62-L63-K64-P65-L66-E67-E68-(AA69)-L70-N71-L72-A73-(AA74)-S75-K76-N77-F 78-H79-(AA80)-(AA81)-P82-R83-D84-(AA85)-(AA86)-S87-N88-(AA89)-N90-(AA91)-(AA92)-V93-L94-E95-L96-(AA97)-G98-S99-E100-T101-T102-F103-(AA104)-C105-E106- Y107-A108-(AA109)-E110-T111-A112-(AA113)-I114-V115-E116-F117-L118-N119-R12 0-W121-I122-T123-F124-(AA125)-(AA126)-S127-I128-I129-(AA130)-T131-L132-T133 in: •AA126 is H; AA18 is R and AA22 is R; And among them: •AA1 is A (wild-type, a=1) or missing (a=0); •AA2 is P (wild type, b=1) or missing (b=0); •AA3 is T (wild type, c=1), C, A, G, Q, E, N, D, R, K, P or deleted (c=0); •AA4 is S (wild type, d=1) or missing (d=0); •AA5 is S (wild-type, e=1) or deleted (e=0); •AA6 is S (wild-type, f=1) or missing (f=0); •AA7 is T (wild type, g=1) or missing (g=0); •AA8 is K (wild-type, h=1) or deleted (h=0); •AA9 is K (wild type, i=1) or missing (i=0); •AA35 is K (wild type) or E; •AA38 is R (wild type), W, or G; •AA39 is M (wild type), L, or V; •AA55 is either H (wild type) or Y; •AA69 is either V (wild type) or A; •AA74 is Q (wild type), P, N, H, S; •AA80 is L (wild type), F, or V; •AA81 is R (wild type), I, D, or T; •AA85 is either L (wild type) or V; •AA86 is either I (wild type) or V; •AA89 is either I (wild type) or V; •AA92 is either I (wild type) or F; •AA97 is either K (wild type) or Q; •AA104 is either M (wild type) or A; •AA109 is a non-natural amino acid, either D (wild type), C, or with an activated side chain; •AA113 is either T (wild type) or N; •AA125 is C (wild type), A, or S; and •AA130 is S (wild type), T, G, or R.

2. The polypeptide of claim 1, wherein the polypeptide comprises a set of mutations: L18R, Q22T, and Q126H; and wherein... •AA3 is T (wild-type, c=1) or deleted (c=0); •AA35 is K (wild type); •AA38 is R (wild type); •AA39 is M (wild type); •AA55 is H (wild type); •AA69 is type V (wild type); •AA74 is Q (wild type); •AA80 is L (wild type); •AA81 is R (wild type); •AA85 is L (wild type); •AA86 is type I (wild type); •AA89 is type I (wild type); •AA92 is type I (wild type); •AA97 is K (wild type); •AA104 is M (wild type); •AA109 is type D (wild type); •AA113 is T (wild type); •AA125 is type C (wild type); and •AA130 is S (wild type).

3. The polypeptide of any one of claims 1 or 2, wherein a = 0, and optionally wherein each of ai = 0.

4. The polypeptide according to any one of claims 1-3, wherein the polypeptide is PEGylated.

5. The polypeptide of any one of claims 1-3, wherein the polypeptide is PEGylated and the molecular weight of the PEG component of the PEGylated polypeptide is about 10 kD to about 70 kD.

6. The polypeptide of any one of claims 1-5, wherein the polypeptide is a fusion protein.

7. The polypeptide of claim 6, wherein the fusion protein comprises an Fc domain.

8. A nucleic acid encoding the polypeptide of any one of claims 1-7.

9. The nucleic acid of claim 8, wherein the nucleic acid is DNA.

10. A recombinant expression vector comprising the nucleic acid as described in claim 8 or 9.

11. The vector of claim 10, wherein the vector is a viral vector.

12. The vector of claim 10, wherein the vector is a non-viral vector.

13. A host cell transformed with the vector according to any one of claims 10-12.

14. A pharmaceutical preparation comprising a polypeptide according to any one of claims 1-7, a nucleic acid according to any one of claims 8 and 9, or a carrier according to any one of claims 10-12.

15. Use of the pharmaceutical preparation of claim 14 for treating a mammalian subject suffering from an autoimmune or inflammatory disease, disorder or symptom, or viral infection, the method comprising administering a therapeutically effective amount of the pharmaceutical preparation.

16. Use of the pharmaceutical formulation of claim 15, wherein the method further comprises administering one or more supplementary agents selected from the group consisting of: steroids, Janus kinase inhibitors, calcineurin inhibitors, mTor inhibitors, IMDH inhibitors, biologics, vaccines, and therapeutic antibodies.

17. Use of the pharmaceutical formulation of claim 16, wherein the therapeutic antibody is an antibody that binds to proteins selected from the group consisting of: BLyS, CD11a, CD20, CD25, CD3, CD52, IgEIL-12 / IL-23, IL-17a, IL-1β, IL-4Rα, IL-5, IL-6R, integrin-α4β7, RANKL, TNFα, VEGF-A, and VLA-4.

18. Use of the pharmaceutical preparation according to any one of claims 15-17, wherein the disease, disorder, or condition is selected from: viral infection, Helicobacter pylori infection, HTLV, organ rejection, graft-versus-host disease, autoimmune thyroid disease, multiple sclerosis, allergy, asthma, neurodegenerative diseases including Alzheimer's disease, systemic lupus erythematosus (SLE), autoinflammatory diseases, inflammatory bowel disease (IBD), Crohn's disease, diabetes, chondritis, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, generalized juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Ritter syndrome, SEA syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile Systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Ritter syndrome, SEA syndrome, psoriasis, psoriatic arthritis, dermatitis (eczema), exfoliative dermatitis or atopic dermatitis, pityriasis rubra pilaris, rosacea, psoriasis vulgaris, lichen planus, luster Lichen, ichthyosis-like skin diseases, keratosis, skin diseases, alopecia areata, pyoderma gangrenosa, vitiligo, bullous pemphigoid, urticaria, keratosis, rheumatoid arthritis; seborrheic dermatitis, photodermatitis; seborrheic keratosis, senile keratosis, actinic keratosis, photoinduced keratosis, follicular keratosis; acne vulgaris; keloids; moles; warts, including warts, condyloma acuminata, or genital warts, and human papillomavirus (HPV) infection.

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