Engineered IL-2 Variant and Method of Using the Same

Abstract

Engineered IL-2 variant and method of use thereof. The engineered IL-2 variant contains one or more non-natural cysteine.

Description

Technology Field

[0001] Claim of priority

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 618,713 filed January 8, 2024 and U.S. Provisional Patent Application No. 63 / 655,431 filed June 3, 2024; the entire contents of which are incorporated herein by reference.

[0003] Sequence list

[0004] The present application includes a sequence list submitted electronically in an XML file named 52246-0021WO1_SL_ST26.xml. The size of the XML file created on December 6, 2024 is 108,838 bytes. The contents of the XML file are incorporated herein by reference in their entirety.

[0005] Technology field

[0006] The present disclosure relates to a engineered IL-2 variant and a method of using the same. Background Technology

[0007] Autoimmune diseases are characterized by an excessive response of the immune system against endogenous tissues. The mechanism of the immune response involves the activation of specialized cells and the acquisition of effector functions. Regulatory T cells (Tregs), formerly also described as suppressor T cells, are a specialized subgroup of T cells. Tregs regulate the immune system's self-tolerance by suppressing the activation of the immune system. Therefore, Tregs play a crucial role in preventing the onset of autoimmune diseases, and therapies capable of enhancing Tregs may be useful in the treatment of autoimmune diseases.

[0008] Interleukin-2 (IL-2) is a member of the cytokine family, each possessing four alpha-helical bundles. IL-2 is essential for the maintenance of Foxp3+ regulatory T cells (Treg cells); in its absence, Treg cells become severely deficient, leading to the development of autoimmune diseases. IL-2 performs dual and contradictory functions: it regulates the immune response by maintaining Treg cells through the promotion of their generation, survival, and functional activity, while simultaneously stimulating conventional T cells to promote the immune response. Specific IL-2 conformations selectively target Treg cells by increasing dependence on CD25 binding while impairing CD122 binding. Although therapeutic strategies have been developed to treat autoimmune diseases by enhancing the number and function of Treg cells using IL-2, monoclonal antibodies against IL-2, or IL-2 variants, there remains an ongoing challenge in minimizing the generation of effector and memory cells, natural killer cells, and other innate lymphocyte populations.

[0009] Therefore, there is a need to develop an IL-2 variant capable of selectively activating Treg cells while minimizing the activation of conventional T cells (e.g., CD8+ T cells).

[0010] The present disclosure relates to engineered IL-2 variants, protein constructs (e.g., fusion proteins), and methods of using the same. In some embodiments, the variants and protein constructs comprise one or more non-natural cysteines. In some embodiments, the variants and protein constructs comprise non-natural disulfide bonds formed by mutating one, two, three, four, five, six, or more than six residues (e.g., a pair of residues) of IL-2 to cysteines without interfering with the entire structure or residues important for IL-2 receptor interaction. As a result, the variants or protein constructs can selectively activate Treg cells while minimizing the activation of Tconv cells (e.g., T effector cells, CD8+ cells).

[0011] In addition, the present invention provides a method for screening cytokines (e.g., IL-2) that can selectively activate Treg cells while minimizing the activation of Tconv cells.

[0012] In one embodiment, the present disclosure relates to a engineered IL-2 polypeptide, wherein the engineered IL-2 polypeptide comprises non-natural cysteine.

[0013] In some embodiments, the engineered IL-2 polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO. 2.

[0014] In some embodiments, the engineered IL-2 polypeptide can bind to the trimer IL-2 receptor complex IL-2Rαβγ.

[0015] In some embodiments, the engineered IL-2 polypeptide comprises one or more of the following:

[0016] (a) The amino acid corresponding to position 11 of sequence number 2 is cysteine ​​(C);

[0017] (b) The amino acid corresponding to position 14 of sequence number 2 is C;

[0018] (c) The amino acid corresponding to position 18 of SEQ ID NO. 2 is C;

[0019] (d) The amino acid corresponding to position 40 of sequence number 2 is C;

[0020] (e) The amino acid corresponding to position 43 of sequence number 2 is C;

[0021] (f) The amino acid corresponding to position 45 of sequence number 2 is C;

[0022] (g) The amino acid corresponding to position 46 of sequence number 2 is C;

[0023] (h) The amino acid corresponding to position 48 of sequence number 2 is C;

[0024] (i) The amino acid corresponding to position 53 of sequence number 2 is C;

[0025] (j) The amino acid corresponding to position 56 of sequence number 2 is C; and

[0026] (k) The amino acid corresponding to position 7 of sequence number 2 is C.

[0027] In some embodiments, the engineered IL-2 polypeptide comprises one or more of the following:

[0028] (a) The amino acid corresponding to position 129 of sequence number 2 is C;

[0029] (b) The amino acid corresponding to position 113 of sequence number 2 is C;

[0030] (c) The amino acid corresponding to position 111 of sequence number 2 is C;

[0031] (d) The amino acid corresponding to position 108 of sequence number 2 is C;

[0032] (e) The amino acid corresponding to position 106 of sequence number 2 is C;

[0033] (f) The amino acid corresponding to position 96 of sequence number 2 is C;

[0034] (g) The amino acid corresponding to position 97 of sequence number 2 is C;

[0035] (h) The amino acid corresponding to position 129 of SEQ ID NO. 2 is C; and

[0036] (i) The amino acid corresponding to position 132 of sequence number 2 is C.

[0037] In some embodiments, the engineered IL-2 polypeptide comprises one or more of the following:

[0038] (a) The amino acid corresponding to position 11 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 129 of SEQ ID NO. 2 is C;

[0039] (b) The amino acid corresponding to position 14 of sequence number 2 is C;

[0040] (c) The amino acid corresponding to position 18 of SEQ ID NO. 2 is C;

[0041] (d) The amino acid corresponding to position 40 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 113 of SEQ ID NO. 2 is C;

[0042] (e) The amino acid corresponding to position 43 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 111 of SEQ ID NO. 2 is C;

[0043] (f) The amino acid corresponding to position 45 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 111 of SEQ ID NO. 2 is C;

[0044] (g) The amino acid corresponding to position 46 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 108 of SEQ ID NO. 2 is C;

[0045] (h) The amino acid corresponding to position 48 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 106 of SEQ ID NO. 2 is C;

[0046] (i) The amino acid corresponding to position 53 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 96 of SEQ ID NO. 2 is C;

[0047] (j) The amino acid corresponding to position 53 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 97 of SEQ ID NO. 2 is C;

[0048] (k) The amino acid corresponding to position 56 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 97 of SEQ ID NO. 2 is C;

[0049] (l) The amino acid corresponding to position 11 of SEQ ID NO. 2 is C, the amino acid corresponding to position 18 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 129 of SEQ ID NO. 2 is C;

[0050] (m) The amino acid corresponding to position 7 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 132 of SEQ ID NO. 2 is C;

[0051] (n) The amino acid corresponding to position 7 of SEQ ID NO. 2 is C, the amino acid corresponding to position 18 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 132 of SEQ ID NO. 2 is C; and

[0052] (o) The amino acid corresponding to position 7 of sequence number 2 is C, the amino acid corresponding to position 11 of sequence number 2 is C, the amino acid corresponding to position 129 of sequence number 2 is C, and the amino acid corresponding to position 132 of sequence number 2 is C.

[0053] In some embodiments, the amino acid corresponding to position 3 of SEQ ID NO. 2 is A, and the engineered IL-2 polypeptide comprises one or more of the following:

[0054] (a) The amino acid corresponding to position 15 of sequence number 2 is D;

[0055] (b) The amino acid corresponding to position 15 of sequence number 2 is N;

[0056] (c) The amino acid corresponding to position 16 of sequence number 2 is S;

[0057] (d) The amino acid corresponding to position 16 of sequence number 2 is T;

[0058] (e) The amino acid corresponding to position 19 of sequence number 2 is I;

[0059] (f) The amino acid corresponding to position 20 of sequence number 2 is N;

[0060] (g) The amino acid corresponding to position 23 of sequence number 2 is I;

[0061] (h) The amino acid corresponding to position 23 of sequence number 2 is L;

[0062] (i) The amino acid corresponding to position 84 of sequence number 2 is N;

[0063] (j) The amino acid corresponding to position 84 of sequence number 2 is T;

[0064] (k) The amino acid corresponding to position 91 of sequence number 2 is L;

[0065] (l) The amino acid corresponding to position 92 of sequence number 2 is V;

[0066] (m) The amino acid corresponding to position 92 of sequence number 2 is M;

[0067] (n) The amino acid corresponding to position 95 of SEQ ID NO. 2 is Q; and

[0068] (o) The amino acid corresponding to position 95 of sequence number 2 is N.

[0069] In some embodiments, the engineered IL-2 polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs 3 to 17 and 35 to 51.

[0070] In some embodiments, the engineered IL-2 polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO. 3 or SEQ ID NO. 14.

[0071] In some embodiments, the engineered IL-2 polypeptide comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs 39, 40, 46, 48, 50 and 51.

[0072] In some embodiments, the engineered IL-2 polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO. 43.

[0073] In some embodiments, the engineered IL-2 polypeptide comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs 39, 40, 42, 45, and 46.

[0074] In some embodiments, the engineered IL-2 polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO. 43.

[0075] In some embodiments, the engineered IL-2 polypeptide can selectively activate Treg cells while minimizing the activation of Tconv cells.

[0076] In one embodiment, the present disclosure relates to a engineered IL-2 polypeptide, wherein the engineered IL-2 polypeptide comprises one or more non-natural disulfide bonds.

[0077] In some embodiments, the engineered IL-2 polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO. 2.

[0078] In some embodiments, the engineered IL-2 polypeptide can bind to the trimer IL-2 receptor complex IL-2Rαβγ.

[0079] In some embodiments, the engineered IL-2 polypeptide comprises one or more of the following:

[0080] (a) a non-natural disulfide bond formed between the amino acid corresponding to position 11 of SEQ ID NO. 2 and the amino acid corresponding to position 129 of SEQ ID NO. 2;

[0081] (b) a non-natural disulfide bond formed between the amino acid corresponding to position 14 of SEQ ID NO. 2 and the amino acid corresponding to position 125 of SEQ ID NO. 2;

[0082] (c) a non-natural disulfide bond formed between the amino acid corresponding to position 18 of SEQ ID NO. 2 and the amino acid corresponding to position 125 of SEQ ID NO. 2;

[0083] (d) a non-natural disulfide bond formed between the amino acid corresponding to position 40 of SEQ ID NO. 2 and the amino acid corresponding to position 113 of SEQ ID NO. 2;

[0084] (e) a non-natural disulfide bond formed between the amino acid corresponding to position 43 of SEQ ID NO. 2 and the amino acid corresponding to position 111 of SEQ ID NO. 2;

[0085] (f) a non-natural disulfide bond formed between the amino acid corresponding to position 45 of SEQ ID NO. 2 and the amino acid corresponding to position 111 of SEQ ID NO. 2;

[0086] (g) a non-natural disulfide bond formed between the amino acid corresponding to position 46 of SEQ ID NO. 2 and the amino acid corresponding to position 108 of SEQ ID NO. 2;

[0087] (h) a non-natural disulfide bond formed between the amino acid corresponding to position 48 of SEQ ID NO. 2 and the amino acid corresponding to position 106 of SEQ ID NO. 2;

[0088] (i) a non-natural disulfide bond formed between the amino acid corresponding to position 53 of SEQ ID NO. 2 and the amino acid corresponding to position 96 of SEQ ID NO. 2;

[0089] (j) a non-natural disulfide bond formed between the amino acid corresponding to position 53 of SEQ ID NO. 2 and the amino acid corresponding to position 97 of SEQ ID NO. 2;

[0090] (k) a non-natural disulfide bond formed between the amino acid corresponding to position 56 of SEQ ID NO. 2 and the amino acid corresponding to position 97 of SEQ ID NO. 2;

[0091] (l) a non-natural disulfide bond formed between the amino acid corresponding to position 11 of SEQ ID NO. 2 and the amino acid corresponding to position 129 of SEQ ID NO. 2, and a non-natural disulfide bond formed between the amino acid corresponding to position 18 of SEQ ID NO. 2 and the amino acid corresponding to position 125 of SEQ ID NO. 2;

[0092] (m) A non-natural disulfide bond formed between the amino acid corresponding to position 7 of SEQ ID NO. 2 and the amino acid corresponding to position 132 of SEQ ID NO. 2;

[0093] (n) a non-natural disulfide bond formed between the amino acid corresponding to position 7 of SEQ ID NO. 2 and the amino acid corresponding to position 132 of SEQ ID NO. 2, and a non-natural disulfide bond formed between the amino acid corresponding to position 18 of SEQ ID NO. 2 and the amino acid corresponding to position 125 of SEQ ID NO. 2; and

[0094] (o) a non-natural disulfide bond formed between the amino acid corresponding to position 7 of SEQ ID NO. 2 and the amino acid corresponding to position 132 of SEQ ID NO. 2, and a non-natural disulfide bond formed between the amino acid corresponding to position 11 of SEQ ID NO. 2 and the amino acid corresponding to position 129 of SEQ ID NO. 2.

[0095] In some embodiments, the engineered IL-2 polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs 3 to 17 and 35 to 51.

[0096] In one embodiment, the present disclosure relates to a fusion protein comprising the engineered IL-2 polypeptide described herein.

[0097] In some embodiments, the fusion protein comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs 20 to 34 and 52 to 68.

[0098] In some embodiments, the fusion protein includes an Fc region.

[0099] In some embodiments, the Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 71.

[0100] In some embodiments, the Fc region comprises (1) a hole chain having an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 93 and (2) a knob chain having an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 95.

[0101] In some embodiments, the engineered IL-2 polypeptide is linked to a hole chain or a knop chain. In some embodiments, the engineered IL-2 polypeptide is linked to a hole chain.

[0102] In some embodiments, the Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 87 or SEQ ID NO. 88.

[0103] In some embodiments, the fusion protein includes a hinge region.

[0104] In some embodiments, the hinge region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 70.

[0105] In some embodiments, the engineered IL-2 polypeptide is linked to the C-terminus of the Fc region via a linker peptide. In some embodiments, the engineered IL-2 polypeptide is linked to the N-terminus of the Fc region via a linker peptide.

[0106] In some embodiments, the linker peptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 72. In some embodiments, the linker peptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs 85, 92, and 99 to 105.

[0107] In some embodiments, the fusion protein comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs 73 to 84, 96, 97, and 98.

[0108] In one embodiment, the present disclosure optionally extends from the N-terminus to the C-terminus,

[0109] (a) Hinge area;

[0110] (b) Fc region;

[0111] (c) linker peptide, and

[0112] (d) This relates to a fusion protein containing a engineered IL-2 polypeptide.

[0113] In some embodiments, the hinge region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 70; the Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 71; and the linker peptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 72, and / or; The engineered IL-2 polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs 3 to 17 and 35 to 51.

[0114] In some embodiments, the fusion protein can increase Treg activation (e.g., phosphorylation of STAT5).

[0115] In some embodiments, the fusion protein may increase Treg-mediated immunosuppression.

[0116] In one embodiment, the present disclosure relates to a pharmaceutical composition comprising the engineered IL-2 polypeptide or the fusion protein described herein; and a pharmaceutically acceptable carrier.

[0117] In one embodiment, the present disclosure relates to a nucleic acid encoding the engineered IL-2 polypeptide described herein or the fusion protein described herein.

[0118] In one embodiment, the present disclosure relates to a vector comprising the nucleic acid described herein.

[0119] In one embodiment, the present disclosure relates to a cell comprising the nucleic acid or vector described herein.

[0120] In some embodiments, the cell is an Expi293 cell or an ExpiCHO cell.

[0121] In one embodiment, the present disclosure relates to a method for producing a engineered IL-2 polypeptide or a fusion protein comprising an engineered IL-2 polypeptide, the method comprising: culturing the cells described herein under conditions sufficient for the cells to produce the engineered IL-2 polypeptide or the fusion protein; and collecting the engineered IL-2 polypeptide or the fusion protein produced by the cells.

[0122] In one embodiment, the present disclosure relates to a method for treating a subject having an immune disease, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the engineered IL-2 polypeptide described herein or the fusion protein described herein.

[0123] In some embodiments, the subject has an autoimmune disease.

[0124] In some embodiments, the autoimmune disease is rheumatoid arthritis, Crohn's disease, diabetes mellitus (e.g., type I), graft-versus-host disease (GvHD), systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel disease (IBD), ulcerative colitis, scleroderma, allergy, asthma, or atopic dermatitis.

[0125] In some embodiments, the fusion protein described herein further comprises a His tag.

[0126] In some embodiments, the His tag comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 18.

[0127] In some embodiments, the engineered IL-2 polypeptide is linked to the C-terminus of the His tag.

[0128] In one embodiment, the present disclosure relates to a method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the engineered IL-2 polypeptide described herein or the fusion protein described herein.

[0129] In some embodiments, the subject has a solid tumor or blood cancer.

[0130] In some embodiments, the cancer is breast cancer, ovarian cancer, glioma, colon cancer, hepatocellular carcinoma, prostate cancer, renal cell carcinoma, melanoma, lung cancer, pancreatic cancer, or liver cancer.

[0131] In one embodiment, the present disclosure relates to a method for reducing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising the engineered IL-2 polypeptide described herein or a fusion protein described herein.

[0132] In one embodiment, the present disclosure relates to a method for killing tumor cells, the method comprising contacting tumor cells with an effective amount of a composition comprising the engineered IL-2 polypeptide described herein or a fusion protein described herein.

[0133] In one embodiment, the present disclosure relates to a method for increasing regulatory T cell proliferation in a subject, the method comprising administering to a subject a pharmaceutical composition comprising the engineered IL-2 polypeptide described herein or the fusion protein described herein.

[0134] In one embodiment, the present disclosure relates to a method for activating regulatory T cells in a subject, the method comprising administering to the subject a pharmaceutical composition comprising the engineered IL-2 polypeptide described herein or the fusion protein described herein.

[0135] In some embodiments, non-natural cysteine ​​and / or one or more non-natural disulfide bonds stabilize the inner core of the IL-2 polypeptide or fusion protein described herein.

[0136] In some embodiments, non-natural cysteine ​​and / or one or more non-natural disulfide bonds increase the thermal stability of the IL-2 polypeptide or the fusion protein described herein.

[0137] In some embodiments, non-natural cysteine ​​and / or one or more non-natural disulfide bonds increase the serum concentration of the IL-2 polypeptide or fusion protein. In some embodiments, non-natural cysteine ​​and / or one or more non-natural disulfide bonds improve the pharmacokinetics of the IL-2 polypeptide or fusion protein.

[0138] In some embodiments, stabilization, increased thermal stability, increased serum concentration, and / or increased pharmacokinetics cannot be achieved by surface mutations.

[0139] In some embodiments, non-natural cysteine ​​and / or one or more non-natural disulfide bonds are aggregated (T agg ) and / or initiation(T onset ) Increase the temperature by at least 5℃.

[0140] In some embodiments, non-natural cysteine ​​and / or one or more non-natural disulfide bonds increase serum concentration by at least 10-fold. In some embodiments, non-natural cysteine ​​and / or one or more non-natural disulfide bonds increase pharmacokinetics by at least 10-fold.

[0141] In one embodiment, the present disclosure relates to a method for improving the stability of IL-2, and the method

[0142] (a) Providing the 3D structure of IL-2,

[0143] (b) measuring the distance between one or more C-alpha atoms of amino acid residues within the 3D structure; and

[0144] (c) comprising selecting two amino acid residues from one or more amino acid residues, wherein the C-alpha atoms of the two selected amino acid residues are within 3 to 8 Angstroms.

[0145] In some embodiments, the method comprises expressing an IL-2 variant. In some embodiments, the IL-2 variant comprises a non-natural disulfide bond formed by mutating two selected amino acid residues to cysteine.

[0146] In some embodiments, mutating two selected amino acid residues to cysteine ​​does not substantially change the 3D structure of IL-2.

[0147] In one embodiment, the present disclosure relates to a method for screening IL-2 variants, and the method

[0148] (a) Providing the 3D structure of IL-2,

[0149] (b) measuring the distance between one or more C-alpha atoms of amino acid residues within the 3D structure; and

[0150] (c) comprising selecting two amino acid residues from one or more amino acid residues, wherein the C-alpha atoms of the two selected amino acid residues are within 3 to 8 Angstroms.

[0151] In some embodiments, the method

[0152] (a) expressing an IL-2 variant containing a non-natural disulfide bond formed by mutating two selected amino acid residues to cysteine;

[0153] (b) administering an IL-2 variant to an animal with an immune disease; and

[0154] (c) Includes determining the effects of IL-2 variants on immune diseases.

[0155] In some embodiments, IL-2 is human IL-2.

[0156] In some embodiments, the method

[0157] (a) expressing an IL-2 variant containing a non-natural disulfide bond formed by mutating two selected amino acid residues to cysteine;

[0158] (b) administration of an IL-2 variant to tumor-bearing animals; and

[0159] (c) further includes determining tumor growth in tumor-bearing animals (e.g., by measuring tumor volume).

[0160] As used herein, the term “non-natural cysteine” refers to a cysteine ​​mutation in which a cysteine ​​amino acid residue substitutes a non-cysteine ​​amino acid residue of the wild-type protein or a cysteine ​​amino acid residue is inserted at a position where it does not naturally exist. In some embodiments, non-natural cysteine ​​is introduced by substitution or insertion. In some embodiments, at least one or two cysteine ​​residues are introduced by substitution. In some embodiments, the protein or polypeptide may have additional mutations.

[0161] As used herein, the term “non-natural disulfide bond” refers to a disulfide bond that is not naturally present in the wild-type protein. In some embodiments, the non-natural disulfide bond is formed by two cysteine ​​residues, at least one of which is a mutant. In some embodiments, two of which are mutants. In some embodiments, at least one or two cysteine ​​residues are introduced by insertion. In some embodiments, deletion changes the distance between two existing cysteine ​​residues, which then forms a disulfide bond that is not present in the wild-type protein.

[0162] As used herein, the term “engineered IL-2 polypeptide” refers to a polypeptide derived from wild-type IL-2 polypeptide or a portion thereof and optionally having one or more mutations (e.g., insertions, deletions, or substitutions). In some embodiments, the engineered IL-2 polypeptide comprises or consists of an amino acid sequence corresponding to amino acids 21 through 153 of human IL-2 (SEQ ID NO. 1). In some embodiments, the engineered IL-2 polypeptide has one or more mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) (e.g., amino acids are substituted by cysteine). In some embodiments, the engineered IL-2 polypeptide has 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer mutations.

[0163] As used herein, the term “protein construct” refers to a complex having one or more polypeptides. In some embodiments, the protein construct is a fusion protein, e.g., a fusion protein, comprising an Fc region and a engineered IL-2 polypeptide (e.g., the engineered IL-2 polypeptide described herein). In some embodiments, the protein construct has two or more polypeptides, and the polypeptides may associate with each other to form a dimer or a multimer (e.g., a trimer).

[0164] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human, or non-human to whom treatment according to the method of the present invention is provided. Both veterinary and non-veterinary fields are considered in this disclosure. A human patient may be an adult human or a child (e.g., a human under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Examples include non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other livestock, farm animals, and zoo animals.

[0165] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymer of any length of at least two amino acids.

[0166] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably to refer to a polymer of at least two nucleotides of any length, including but not limited to DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0167] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as universally understood by those skilled in the art to which this invention pertains. Although the methods and materials used in this invention are described herein; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, this specification, including definitions, will govern.

[0168] Other features and advantages of the present invention will become apparent from the following detailed description and drawings, and from the claims. Brief explanation of the drawing

[0169] Figure 1a shows a summary of the production and purification of proteins H8-IL-2_C01, H8-IL-2_C02, H8-IL-2_C03, H8-IL-2_C04, H8-IL-2_C05, H8-IL-2_C06, H8-IL-2_C07, H8-IL-2_C08, H8-IL-2_C09, H8-IL-2_C10, and H8-IL-2_C11 in ExpiCHO cells. Figure 1b shows a summary of the production, purification, and analysis of proteins H8-IL-2_C01, H8-IL-2_C02, H8-IL-2_C03, H8-IL-2_C04, H8-IL-2_C05, H8-IL-2_C06, H8-IL-2_C07, H8-IL-2_C08, H8-IL-2_C09, H8-IL-2_C10, and H8-IL-2_C11 in Expi293 cells. Figure 1c shows a summary of the production, purification, and analysis of proteins H8-IL-2_C21, H8-IL-2_C22, H8-IL-2_C23, and H8-IL-2_C24 in Expi293 cells. Figures 2a and 2b illustrate IL-2-pSTAT5 reporter signaling activity induced by H8-IL-2_WT and its variants H8-IL-2_C01, H8-IL-2_C03, and H8-IL-2_C05 in HEK-Blue™ IL-2 cells (Figure 2a) and HEK-Blue™ CD122 / CD132 cells (Figure 2b), respectively. FIGS. 2c and 2d illustrate IL-2-pSTAT5 reporter signaling activity induced by H8-IL-2_WT and its variants H8-IL-2_C21, H8-IL-2_C22, H8-IL-2_C23, and H8-IL-2_C24 in HEK-Blue™ IL-2 cells (Fig. 2c) and HEK-Blue™ CD122 / CD132 cells (Fig. 2d), respectively. Figure 3 illustrates the summary of the production, purification, and analysis of H8-IL-2_C01_T3A-derived variants in Expi293 cells. Figures 4a and 4b illustrate IL-2-pSTAT5 reporter signaling activity induced by H8-IL-2_WT_T3A, H8-IL-2_C01_T3A, H8-IL-2_C01#1, and H8-IL-2_C01#2 in HEK-Blue™ IL-2 cells (Figure 4a) and HEK-Blue™ CD122 / CD132 cells (Figure 4b), respectively. FIGS. 4c and 4d illustrate IL-2-pSTAT5 reporter signaling activity induced by H8-IL-2_C01_T3A, H8-IL-2_C01#3, H8-IL-2_C01#4, H8-IL-2_C01#5, H8-IL-2_C01#6, and H8-IL-2_C01#7 in HEK-Blue™ IL-2 cells (Fig. 4c) and HEK-Blue™ CD122 / CD132 cells (Fig. 4d), respectively. Figures 4e and 4f illustrate IL-2-pSTAT5 reporter signaling activity induced by H8-IL-2_C01_T3A, H8-IL-2_C01#8, H8-IL-2_C01#9, H8-IL-2_C01#10, H8-IL-2_C01#11, and H8-IL-2_C01#12 in HEK-Blue™ IL-2 cells (Figure 4e) and HEK-Blue™ CD122 / CD132 cells (Figure 4f), respectively. Figures 4g and 4h illustrate IL-2-pSTAT5 reporter signaling activity induced by H8-IL-2_C01_T3A, H8-IL-2_C01#13, H8-IL-2_C01#14, and H8-IL-2_C01#15 in HEK-Blue™ IL-2 cells (Figure 4g) and HEK-Blue™ CD122 / CD132 cells (Figure 4h), respectively. Figure 5 illustrates a summary of IL-2-pSTAT5 reporter signaling activity induced by H8-IL-2_C01_T3A-derived variants in HEK-Blue™ IL-2 cells and HEK-Blue™ CD122 / CD132 cells, respectively. Figures 6a and 6b illustrate STAT5 phosphorylation induced by H8-IL-2_WT_T3A, H8-IL-2_C01_T3A, H8-IL-2_C01#1, H8-IL-2_C01#6, H8-IL-2_C01#7, and H8-IL-2_C01#9 in Treg cells (Figure 6a) and Tconv cells (Figure 6b), respectively. Figures 6c and 6d illustrate STAT5 phosphorylation induced by H8-IL-2_WT_T3A, H8-IL-2_C01_T3A, H8-IL-2_C01#3, H8-IL-2_C01#4, H8-IL-2_C01#10, H8-IL-2_C01#12, H8-IL-2_C01#14, and H8-IL-2_C01#15 in Treg cells (Figure 6c) and Tconv cells (Figure 6d), respectively. Figures 6e and 6f illustrate STAT5 phosphorylation induced by H8-IL-2_WT_T3A, H8-IL-2_C01_T3A, H8-IL-2_C01#2, H8-IL-2_C01#5, H8-IL-2_C01#8, H8-IL-2_C01#11, and H8-IL-2_C01#13 in Treg cells (Figure 6e) and Tconv cells (Figure 6f), respectively. Figure 7 illustrates a summary of IL-2-pSTAT5 reporter signaling and STAT5 phosphorylation induced by H8-IL-2_C01_T3A and H8-IL-2_C01_T3A-derived variants. Figure 8 shows a summary of the production, purification, and analysis of G1LALAFc-IL-2_WT_T3A, G1LALAFc-IL-2_C01_T3A, and G1LALAFc-IL-2_C01_T3A-derived variants in Expi293 cells. Figures 9a and 9b illustrate IL-2-pSTAT5 reporter signaling activity induced by G1LALAFc-IL-2_WT_T3A, G1LALAFc-IL-2_C01_T3A, G1LALAFc-IL-2_C01#1, G1LALAFc-IL-2_C01#3, and G1LALAFc-IL-2_C01#4 in HEK-Blue™ IL-2 cells (Figure 9a) and HEK-Blue™ CD122 / CD132 cells (Figure 9b), respectively. Figures 9c and 9d illustrate IL-2-pSTAT5 reporter signaling activity induced by G1LALAFc-IL-2_WT_T3A, G1LALAFc-IL-2_C01_T3A, G1LALAFc-IL-2_C01#6, G1LALAFc-IL-2_C01#7, G1LALAFc-IL-2_C01#9, and G1LALAFc-IL-2_C01#10 in HEK-Blue™ IL-2 cells (Figure 9c) and HEK-Blue™ CD122 / CD132 cells (Figure 9d), respectively. Figures 9e and 9f illustrate IL-2-pSTAT5 reporter signaling activity induced by G1LALAFc-IL-2_WT_T3A, G1LALAFc-IL-2_C01_T3A, G1LALAFc-IL-2_C01#12, G1LALAFc-IL-2_C01#14, and G1LALAFc-IL-2_C01#15 in HEK-Blue™ IL-2 cells (Figure 9E) and HEK-Blue™ CD122 / CD132 cells (Figure 9F), respectively. Figure 10 illustrates a summary of IL-2-pSTAT5 reporter signaling induced by IL-2_C01_T3A (of H8 and G1LALAFc formats) and variants thereof. Figures 11a and 11b illustrate STAT5 phosphorylation induced by G1LALAFc-IL-2_WT_T3A, G1LALAFc-IL-2_C01_T3A, G1LALAFc-IL-2_C01#1, G1LALAFc-IL-2_C01#6, G1LALAFc-IL-2_C01#7, and G1LALAFc-IL-2_C01#9 in Treg cells (Figure 11a) and Tconv cells (Figure 11b), respectively. Figures 11c and 11d illustrate STAT5 phosphorylation induced by G1LALAFc-IL-2_WT_T3A, G1LALAFc-IL-2_C01_T3A, G1LALAFc-IL-2_C01#3, G1LALAFc-IL-2_C01#4, G1LALAFc-IL-2_C01#10, G1LALAFc-IL-2_C01#12, G1LALAFc-IL-2_C01#14, and G1LALAFc-IL-2_C01#15 in Treg cells (Figure 11c) and Tconv cells (Figure 11d), respectively. FIGS. 12a to 12c illustrate the stress stability of engineered IL-2 variants and their protein constructs determined by SEC-HPLC. Figure 13 illustrates the thermal stability of the engineered IL-2 variant and its protein construct determined by dynamic light scattering (DLS) analysis. Figure 14 shows the thermal stability of engineered IL-2 variants and their protein constructs determined by differential scanning fluorescence (DSF) analysis. Figures 15a and 15b illustrate the treatment plan and administration schedule (Figure 15a) and results (Figure 15b) of the autoimmune efficacy of engineered IL-2 variants and their protein constructs determined by the NPG extension model. Figure 16a is a table showing the treatment plan and administration schedule of engineered IL-2 variants and their protein constructs in a C57BL / 6 mouse model. Figure 16b shows the average body weight change of C57BL / 6 mice treated with engineered IL-2 variants and their protein constructs. FIGS. 16c to 16l illustrate the pharmacokinetics of engineered IL-2 variants and their protein constructs determined by ELISA. Figures 17a and 17b illustrate liver damage of engineered IL-2 variants and their protein constructs as determined by AST (Figure 17a) and ALT (Figure 17b). FIGS. 18a to 18d show the percentages of different T cell populations induced by engineered IL-2 variants and their protein constructs determined by flow cytometry. Figure 19 lists the related amino acid sequences. Specific details for implementing the invention

[0170] Human interleukin 2 (IL-2) is an interleukin, a type of cytokine signaling molecule of the immune system. It is a protein of 15.5 to 16 kDa that regulates the activity of white blood cells (leukocytes, lymphocytes) responsible for immunity. IL-2 is part of the body's natural response to microbial infection and is involved in distinguishing between foreign substances ("non-self") and self substances. Human IL-2 (NCBI reference sequence: NP_000577.2; UniProt ID: Q0GK43; Sequence No. 1) is a 153-amino acid protein containing a signal peptide (corresponding to amino acids 1 to 20 of Sequence No. 1) and a soluble chain (corresponding to amino acids 21 to 153 of Sequence No. 1) from the N-terminus to the C-terminus.

[0171] IL-2 belongs to the cytokine family, each of which possesses four alpha helix bundles. The family also includes IL-4, IL-7, IL-9, IL-15, and IL-21. IL-2 transmits signals through the IL-2 receptor, a complex comprising three chains: the interleukin-2 receptor alpha chain (or IL-2RA, CD25), the interleukin-2 receptor beta subunit (or IL-2RB, CD122), and the interleukin-2 receptor gamma subunit (or IL-2RG, common gamma chain, CD132). The gamma chain is shared by all family members. IL-2 plays an essential role in the core functions of the immune system, tolerance, and immunity, primarily through its direct effects on T cells. For example, in the thymus where T cells mature, it prevents autoimmune diseases by promoting the differentiation of specific immature T cells into regulatory T cells, which in turn suppress other T cells otherwise primed to attack normal, healthy cells in the body. IL-2 also helps the body fight infections by promoting the differentiation of early T cells into effector T cells and memory T cells when stimulated by antigens. Interestingly, IL-2 can stimulate an immune response by regulating effector T cells (primarily expressing IL-2RB and IL-2RG) and suppress an immune response by regulating regulatory T cells (primarily expressing IL-2RA, IL-2RB, and IL-2RG).

[0172] Detailed descriptions of IL-2 and its functions can be found in, for example, the literature [Abbas, Abul K., et al. "Revisiting IL-2: Biology and therapeutic prospects." Science immunology 3.25 (2018): eaat1482]; the literature [Spolski, Rosanne, Peng Li, and Warren J. Leonard. "Biology and regulation of IL-2: from molecular mechanisms to human therapy." Nature Reviews Immunology 18.10 (2018): 648-659]; and the literature [Arenas-Ramirez, Natalia, Janine Woytschak, and Onur Boyman. "Interleukin-2: biology, design and application." Trends in immunology 36.12 (2015): 763-777], each of which is cited in its entirety.

[0173] The trimeric IL-2 receptor complex IL-2Rαβγ contains three subunits: α (CD25), β (CD122), and γ (γ-common). The dimeric IL-2Rβγ contains two subunits: β (CD122) and γ (γ-common). The trimeric IL-2 receptor complex IL-2Rαβγ is typically expressed at high levels by Treg cells, whereas the dimeric IL-2Rβγ is expressed mostly in activated CD8+ T cells and NK cells. Low-dose IL-2 therapy preferentially activates Treg cells due to the high constitutive expression of IL-2Rα.

[0174] The present disclosure provides a engineered IL-2 variant having at least one non-natural cysteine. In some embodiments, one or two amino acid residues of wild-type human IL-2 (e.g., SEQ ID NO. 2) are selectively mutated to cysteine. In some embodiments, the mutation does not substantially alter the overall structure of IL-2, such as the relative positions of the four alpha helices of IL-2. In some embodiments, the engineered IL-2 variant described herein may bind to an IL-2 receptor and induce downstream signaling (e.g., STAT5 phosphorylation). In some embodiments, the engineered IL-2 variant may selectively activate Treg cells while minimizing the activation of effector T cells. In some embodiments, the engineered IL-2 variant comprises or is composed of any engineered IL-2 polypeptide described herein.

[0175] In some embodiments, the engineered IL-2 variant further comprises a fragment determinative region (Fc region). In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the effector function of the Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the Fc region is phagocytosis. In some embodiments, the engineered IL-2 variant as described herein has an Fc region without effector function. In some embodiments, Fc is human IgG1 Fc. In some embodiments, Fc does not have a functional Fc region. For example, the Fc region has an LALA mutation (the L234A and L235A mutations in EU numbering) or an LALA-PG mutation (the L234A, L235A, and P329G mutations in EU numbering). In some embodiments, the LALA mutation may reduce binding to IgG Fc receptors FcγRI, FcγRII, and FcγRIII, as well as complement component C1q. In some embodiments, the engineered IL-2 variant comprises G1LALAFc (e.g., SEQ ID NO. 71), which is an IgG1 Fc containing the LALA mutation.

[0176] In some embodiments, the Fc region may have knobs-into-holes (KIH) mutations, which can promote heteromerization between two peptide chains and avoid mispairing between them. This involves manipulating the CH3 domain to generate a "knob" or a "hole" to promote heteromerization formation. The KIH technique is described, for example, in the literature [Xu, Yiren, et al. "Production of bispecific antibodies in 'knobs-into-holes' using a cell-free expression system." MAbs. Vol. 7. No. 1. Taylor & Francis, 2015], which is incorporated by reference in its entirety. In some embodiments, the Fc region comprises a hole chain comprising G1LA-HoleFc (e.g., SEQ ID NO. 93) and a knob chain comprising G1LA-KnobFc (e.g., SEQ ID NO. 95). In some embodiments, a hole chain or a knob chain may be connected to the IL-2 variant described herein to form a heterodimer structure. In some embodiments, the IL-2 variant described herein is connected to a hole chain. In some embodiments, the IL-2 variant described herein is connected to a knob chain. An example of such a heterodimer structure is G1LA-KIHFc-IL-2_WT, which comprises a hole chain comprising G1LA-HoleFc-IL-2_WT (e.g., SEQ ID NO. 97) and a knob chain comprising G1LA-KnobFc (e.g., SEQ ID NO. 95). Another example of such a heterodimer structure is G1LA-KIHFc-IL-2_C01#6, which comprises a hole chain comprising G1LA-HoleFc-IL-2_C01#6 (e.g., SEQ ID NO. 98) and a knob chain comprising G1LA-KnobFc (e.g., SEQ ID NO. 95).

[0177] In some embodiments, the Fc region may be additionally connected to a hinge region (e.g., SEQ ID NO. 70). In some embodiments, the Fc region comprises a hole chain consisting of G1LA-HoleFc (e.g., SEQ ID NO. 93) and a hinge region (e.g., SEQ ID NO. 70). In some embodiments, the Fc region comprises an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 94. In some embodiments, the Fc region comprises a knob chain comprising G1LA-KnobFc (e.g., SEQ ID NO. 95) and a hinge region (e.g., SEQ ID NO. 70). In some embodiments, the Fc region comprises an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 96.

[0178] In some embodiments, a hole chain or a knob chain may be connected to the IL-2 variant described herein to form a heterodimeric structure. In some embodiments, the IL-2 variant described herein is connected to a hole chain. In some embodiments, the IL-2 variant described herein is connected to a knob chain. An example of such a heterodimeric structure is G1LA-KIHFc-IL-2_WT, which comprises a hole chain comprising G1LA-HoleFc-IL-2_WT (e.g., SEQ ID NO. 97) and a knob chain comprising G1 hinge + G1LA-KnobFc (e.g., SEQ ID NO. 96). Another example of such a heterodimeric structure is G1LA-KIHFc-IL-2_C01#6, which comprises a hole chain comprising G1LA-HoleFc-IL-2_C01#6 (e.g., SEQ ID NO. 98) and a knob chain comprising G1 hinge + G1LA-KnobFc (e.g., SEQ ID NO. 96). In some embodiments, the engineered IL-2 variant further comprises a His tag (e.g., SEQ ID NO. 18).

[0179] The present disclosure also provides a method for screening cytokine (e.g., IL-2) variants capable of selectively activating Treg cells while minimizing the activation of Tconv cells.

[0180] engineered IL-2 variant

[0181] IL-2 is a typical quaternary α-helical cytokine that is strictly regulated at the mRNA level by signals from T cell receptors and CD28. IL-2 transmits signals by binding to a receptor complex composed of three distinct subunits designated as IL-2Rα (CD25), IL-2Rβ (CD122), and a common γ chain (γc; CD132). Crystallization of the quaternary complex, in which IL-2 is bound to the outer domains of the three receptor chains, indicated that the sites on IL-2 interacting with the three IL-2R chains do not overlap, and the α chain itself does not come into contact with β or γc, thus forming a coherent chain. The high affinity of the IL-2 / IL-2Rα / β trimer complex clearly indicates that this complex is more stable than IL-2 bound to the α chain alone or the β chain alone. During the engagement of IL-2R, the IL-2 / α / β trimer directs the γ chain into the quaternary complex, inducing intracellular signal transduction. Since only certain residues of IL-2 interact with both the β and γc chains, the binding of IL-2 can induce stereochemical changes in the β chain, thereby further promoting the recruitment of the γc chain. Furthermore, it was found that while the α chain does not participate in signal transduction, both the β and γc chains are essential for signal transduction. In the absence of IL-2Rα expression, IL-2Rβ and γc can form intermediate affinity receptors that are fully functional for signal transduction. High affinity receptors are the most physiologically relevant forms of IL-2R, as previously mentioned, because CD25-deficient mice (which express only intermediate affinity IL-2R) are phenotypically indistinguishable from IL-2-deficient mice. Therefore, the structure of IL-2 and its receptor is flexible and can naturally exist in different stereochemical forms that appear to favor either the high-affinity trimer IL-2R or the medium-affinity dimer IL-2R, respectively, and consequently activate different immune cells.

[0182] Based on the 3D structure of human IL-2, the distance between one or more amino acid residues (e.g., adjacent residues) within the 3D structure can be determined. In some embodiments, two amino acid residues whose distance is within 3.0 to 8.0 angstroms (Å) may be selected. In some embodiments, two selected amino acid residues are each 3 to 4 Å, 3 to 5 Å, 3 to 6 Å, 3 to 7 Å, 3 to 8 Å, 4 to 5 Å, 4 to 6 Å, 4 to 7 Å, 4 to 8 Å, 5 to 6 Å, 5 to 7 Å, 5 to 8 Å, 6 to 7 Å, 6 to 8 Å, 7.5 to 8 Å, 3.5 to 4.5 Å, 3.5 to 5.5 Å, 3.5 to 6.5 Å, 3.5 to 7.5 Å, 3.5 to 8.5 Å, 4.5 to 5.5 Å, 4.5 to 6.5 Å, 4.5 to 6.5 Å, 4.5 to 7.5 Å, 4.5 to 7.5 Å, 4.5 The distance may be up to 8.5 Å, 5.5 to 6.5 Å, 5.5 to 7.5 Å, 5.5 to 8 Å, 6.5 to 7.5 Å, 6.5 to 8 Å, or 7.5 to 8 Å. In some embodiments, both selected amino acid residues are not cysteine, and the two selected amino acid residues may be mutated to cysteine ​​without interfering with the overall structure of IL-2 (e.g., the core structure formed by a 4-helical bundle). Without being bound by theory, the newly introduced cysteine ​​may form a non-natural disulfide bond to wild-type IL-2, which is expected to stabilize IL-2 and / or improve the functional efficacy of IL-2. In some embodiments, only one of the two selected amino acid residues is cysteine, and the other selected amino acid residue (non-cysteine ​​amino acid residue) can be mutated to cysteine ​​without interfering with the overall structure of IL-2 (e.g., the core structure formed by a 4-helical bundle).Without being bound by theory, the newly introduced cysteine ​​can form non-natural disulfide bonds to wild-type IL-2, which is expected to stabilize IL-2 and / or improve its functional efficacy.

[0183] In one embodiment, the engineered IL-2 variant (e.g., engineered IL-2 polypeptide) comprises a first cysteine ​​mutation and a second cysteine ​​mutation. In some embodiments, the first cysteine ​​mutation occurs at an amino acid residue corresponding to positions 7, 11, 40, 43, 45, 46, 48, 53, or 56 of SEQ ID NO. 2. In some embodiments, the second cysteine ​​mutation occurs at an amino acid residue corresponding to positions 129, 113, 111, 108, 106, 96, 97, or 132 of SEQ ID NO. 2. In some embodiments, the distance between two selected amino acid residues is 10 Å, 9 Å, 8 Å, 7.9 Å, 7.8 Å, 7.7 Å, 7.6 Å, 7.5 Å, 7.4 Å, 7.3 Å, 7.2 Å, 7.1 Å, 7 Å, 6.9 Å. Å, 6.8 Å, 6.7 Å, 6.6 Å, 6.5 Å, 6.4 Å, 6.3 Å, 6.2 Å, 6.1 Å, 6 Å, 5.9 Å, 5.8 Å, 5.7 Å, 5.6 Å, 5.5 Å, 5.4 Å, 5.3 Å, 5.2 Å, 5.1 Å, 5 Å, 4.9 Å, 4.8 Å, 4.7 Å, 4.6 Å, 4.5 Å, 4.4 Å, 4.3 Å, 4.2 Å, 4.1 Å, 4 Å, 3.9 Å, 3.8 Å, 3.7 Å, 3.6 Å, 3.5 Å, 3.4 Å, 3.3 Å, 3.2 Å, 3.1 Å, 3 Å, 2.9 Å, 2.8 Å, 2.7 Å, 2.6 Å, 2.5 Å, 2.4 Å, 2.3 Å, 2.2 Å, 2.1 Å, or within 2 Å. In some embodiments, when the distance between two selected amino acid residues is within the above range, the first cysteine ​​mutation may be paired with any of the second cysteine ​​mutations described herein. In some embodiments, one or more non-natural cysteines can further stabilize the overall structure of IL-2, for example, and the relative positions and angles of the 4-helical bundles of IL-2 are maintained.

[0184] In one embodiment, the engineered IL-2 variant (e.g., engineered IL-2 polypeptide) comprises a third cysteine ​​mutation, wherein the introduced third cysteine ​​may form a disulfide bond with the introduced cysteine ​​or naturally occurring cysteine. In some embodiments, the third cysteine ​​mutation occurs at amino acid residues corresponding to positions 11, 18, 129, 7, or 132 of SEQ ID NO. 2.

[0185] In one embodiment, the engineered IL-2 variant (e.g., engineered IL-2 polypeptide) comprises a fourth cysteine ​​mutation, wherein the introduced fourth cysteine ​​may form a disulfide bond with the introduced cysteine ​​or naturally occurring cysteine. In some embodiments, the fourth cysteine ​​mutation occurs at the amino acid residue corresponding to position 11, position 129, position 7, or position 132 of SEQ ID NO. 2.

[0186] In one embodiment, the engineered IL-2 variant (e.g., engineered IL-2 polypeptide) comprises one cysteine ​​mutation, wherein the introduced cysteine ​​can form a disulfide bond with a naturally occurring cysteine. In some embodiments, the cysteine ​​mutation occurs at an amino acid residue corresponding to position 14 or 18 of SEQ ID NO. 2.

[0187] In some embodiments, the present invention provides a engineered IL-2 polypeptide (e.g., IL-2_C01) comprising a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11) and a second cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (I129). In some embodiments, the two cysteine ​​residues form a disulfide bond. The sequence of IL-2_C01 is presented in SEQ ID NO. 3.

[0188] In some embodiments, a engineered IL-2 polypeptide (e.g., IL-2_C02) comprising a cysteine ​​residue at a position corresponding to position 14 of SEQ ID NO. 2 (e.g., L14) is provided herein. In some embodiments, the cysteine ​​residue forms a disulfide bond with cysteine ​​at a position corresponding to position 125 of SEQ ID NO. 2. The sequence of IL-2_C02 is presented in SEQ ID NO. 4.

[0189] In some embodiments, a engineered IL-2 polypeptide (e.g., IL-2_C03) comprising a cysteine ​​residue at a position corresponding to position 18 of SEQ ID NO. 2 (e.g., L18) is provided herein. In some embodiments, the cysteine ​​residue forms a disulfide bond with cysteine ​​at a position corresponding to position 125 of SEQ ID NO. 2. The sequence of IL-2_C03 is presented in SEQ ID NO. 5.

[0190] In some embodiments, the present invention provides a engineered IL-2 polypeptide (e.g., IL-2_C04) comprising a first cysteine ​​residue at a position corresponding to position 40 of SEQ ID NO. 2 (e.g., L40) and a second cysteine ​​residue at a position corresponding to position 113 of SEQ ID NO. 2 (e.g., T113). In some embodiments, the two cysteine ​​residues form a disulfide bond. The sequence of IL-2_C04 is presented in SEQ ID NO. 6.

[0191] In some embodiments, the present invention provides a engineered IL-2 polypeptide (e.g., IL-2_C05) comprising a first cysteine ​​residue at a position corresponding to position 43 of SEQ ID NO. 2 (e.g., K43) and a second cysteine ​​residue at a position corresponding to position 111 of SEQ ID NO. 2 (e.g., T111). In some embodiments, the two cysteine ​​residues form a disulfide bond. The sequence of IL-2_C05 is presented in SEQ ID NO. 7.

[0192] In some embodiments, the present invention provides a engineered IL-2 polypeptide (e.g., IL-2_C06) comprising a first cysteine ​​residue at a position corresponding to position 45 of SEQ ID NO. 2 (e.g., Y45) and a second cysteine ​​residue at a position corresponding to position 111 of SEQ ID NO. 2 (e.g., T111). In some embodiments, the two cysteine ​​residues form a disulfide bond. The sequence of IL-2_C06 is presented in SEQ ID NO. 8.

[0193] In some embodiments, the present invention provides a engineered IL-2 polypeptide (e.g., IL-2_C07) comprising a first cysteine ​​residue at a position corresponding to position 46 of SEQ ID NO. 2 (e.g., M46) and a second cysteine ​​residue at a position corresponding to position 108 of SEQ ID NO. 2 (e.g., A108). In some embodiments, the two cysteine ​​residues form a disulfide bond. The sequence of IL-2_C07 is presented in SEQ ID NO. 9.

[0194] In some embodiments, the present invention provides a engineered IL-2 polypeptide (e.g., IL-2_C08) comprising a first cysteine ​​residue at a position corresponding to position 48 of SEQ ID NO. 2 (e.g., K48) and a second cysteine ​​residue at a position corresponding to position 106 of SEQ ID NO. 2 (e.g., E106). In some embodiments, the two cysteine ​​residues form a disulfide bond. The sequence of IL-2_C08 is presented in SEQ ID NO. 10.

[0195] In some embodiments, the present invention provides a engineered IL-2 polypeptide (e.g., IL-2_C09) comprising a first cysteine ​​residue at a position corresponding to position 53 of SEQ ID NO. 2 (e.g., L53) and a second cysteine ​​residue at a position corresponding to position 96 of SEQ ID NO. 2 (e.g., L96). In some embodiments, the two cysteine ​​residues form a disulfide bond. The sequence of IL-2_C09 is presented in SEQ ID NO. 11.

[0196] In some embodiments, the present invention provides a engineered IL-2 polypeptide (e.g., IL-2_C10) comprising a first cysteine ​​residue at a position corresponding to position 53 of SEQ ID NO. 2 (e.g., L53) and a second cysteine ​​residue at a position corresponding to position 97 of SEQ ID NO. 2 (e.g., K97). In some embodiments, the two cysteine ​​residues form a disulfide bond. The sequence of IL-2_C10 is presented in SEQ ID NO. 12.

[0197] In some embodiments, the present invention provides a engineered IL-2 polypeptide (e.g., IL-2_C11) comprising a first cysteine ​​residue at a position corresponding to position 56 of SEQ ID NO. 2 (e.g., L56) and a second cysteine ​​residue at a position corresponding to position 97 of SEQ ID NO. 2 (e.g., K97). In some embodiments, the two cysteine ​​residues form a disulfide bond. The sequence of IL-2_C11 is presented in SEQ ID NO. 13.

[0198] In some embodiments, the present invention provides a engineered IL-2 polypeptide (e.g., IL-2_C21) comprising a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), a second cysteine ​​residue at a position corresponding to position 18 of SEQ ID NO. 2 (e.g., L18), and a third cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (e.g., I129). The sequence of IL-2_C21 is presented in SEQ ID NO. 14. In some embodiments, the second cysteine ​​residue forms a disulfide bond with cysteine ​​at a position corresponding to position 125 of SEQ ID NO. 2. In some embodiments, the first cysteine ​​residue and the third cysteine ​​residue form a disulfide bond.

[0199] In some embodiments, the present invention provides a engineered IL-2 polypeptide (e.g., IL-2_C22) comprising a first cysteine ​​residue at a position corresponding to position 7 of SEQ ID NO. 2 (e.g., T7) and a second cysteine ​​residue at a position corresponding to position 132 of SEQ ID NO. 2 (e.g., L132). The sequence of IL-2_C22 is presented in SEQ ID NO. 15. In some embodiments, the two cysteine ​​residues form a disulfide bond.

[0200] In some embodiments, the present invention provides a engineered IL-2 polypeptide (e.g., IL-2_C23) comprising a first cysteine ​​residue at a position corresponding to position 7 of SEQ ID NO. 2 (e.g., T7), a second cysteine ​​residue at a position corresponding to position 18 of SEQ ID NO. 2 (e.g., L18), and a third cysteine ​​residue at a position corresponding to position 132 of SEQ ID NO. 2 (e.g., L132). The sequence of IL-2_C23 is presented in SEQ ID NO. 16. In some embodiments, the second cysteine ​​residue forms a disulfide bond with cysteine ​​at a position corresponding to position 125 of SEQ ID NO. 2. In some embodiments, the first cysteine ​​residue and the third cysteine ​​residue form a disulfide bond.

[0201] In some embodiments, the present invention provides a engineered IL-2 polypeptide (e.g., IL-2_C24) comprising a first cysteine ​​residue at a position corresponding to position 7 of SEQ ID NO. 2 (e.g., T7), a second cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), a third cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (e.g., I129), and a fourth cysteine ​​residue at a position corresponding to position 132 of SEQ ID NO. 2 (e.g., L132). The sequence of IL-2_C24 is presented in SEQ ID NO. 17. In some embodiments, the first cysteine ​​residue and the fourth cysteine ​​residue form a disulfide bond. In some embodiments, the second cysteine ​​residue and the third cysteine ​​residue form a disulfide bond.

[0202] In some embodiments, the present invention provides a engineered IL-2 polypeptide comprising one or more of the following: (a) the amino acid corresponding to Q11 of SEQ NO. 2 is C, and the amino acid corresponding to I129 of SEQ NO. 2 is C; (b) the amino acid corresponding to L14 of SEQ NO. 2 is C; (c) the amino acid corresponding to L18 of SEQ NO. 2 is C; (d) the amino acid corresponding to L40 of SEQ NO. 2 is C, and the amino acid corresponding to T113 of SEQ NO. 2 is C; (e) the amino acid corresponding to K43 of SEQ NO. 2 is C, and the amino acid corresponding to T111 of SEQ NO. 2 is C; (f) the amino acid corresponding to Y45 of SEQ NO. 2 is C, and the amino acid corresponding to T111 of SEQ NO. 2 is C; (g) the amino acid corresponding to M46 of SEQ NO. 2 is C, and the amino acid corresponding to A108 of SEQ NO. 2 is C; (h) The amino acid corresponding to K48 of SEQ ID NO. 2 is C, and the amino acid corresponding to E106 of SEQ ID NO. 2 is C; (i) The amino acid corresponding to L53 of SEQ ID NO. 2 is C, and the amino acid corresponding to L96 of SEQ ID NO. 2 is C; (j) The amino acid corresponding to L53 of SEQ ID NO. 2 is C, and the amino acid corresponding to K97 of SEQ ID NO. 2 is C; (k) The amino acid corresponding to L56 of SEQ ID NO. 2 is C, and the amino acid corresponding to K97 of SEQ ID NO. 2 is C;

[0203] In some embodiments, the engineered IL-2 polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs 3, 4, 5, 6, 7, 8, 9, 16, or 17. In some embodiments, the engineered IL-2 polypeptide described herein comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 2, and the amino acid sequence comprises one or more of the mutations described herein. In some embodiments, the engineered IL-2 polypeptide described herein comprises at least one, at least two, at least three, or at least four of the cysteine ​​mutations listed in Table 1.

[0204] In some embodiments, the engineered IL-2 polypeptide may have at least or about 1 (e.g., at least or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40) amino acid insertions, deletions, or substitutions when compared to any one of SEQ ID NOs 3 to 17.

[0205] In some embodiments, the engineered IL-2 polypeptide described herein may also contain an alanine mutation at a position corresponding to position 3 of SEQ ID NO. 2 (e.g., T3). In some embodiments, the engineered IL-2 polypeptide described herein may also contain a T3A mutation.

[0206] In some embodiments, the engineered IL-2 polypeptide described herein may also contain a mutation at a position corresponding to position 15 of SEQ ID NO. 2 (e.g., E15). In some embodiments, the engineered IL-2 polypeptide described herein may also contain an E15D or E15N mutation.

[0207] In some embodiments, the engineered IL-2 polypeptide described herein may also contain a mutation at a position corresponding to position 16 of SEQ ID NO. 2 (e.g., H16). In some embodiments, the engineered IL-2 polypeptide described herein may also contain an H16S or H16T mutation.

[0208] In some embodiments, the engineered IL-2 polypeptide described herein may also contain a mutation at a position corresponding to position 19 of SEQ ID NO. 2 (e.g., L19). In some embodiments, the engineered IL-2 polypeptide described herein may also contain an L19I mutation.

[0209] In some embodiments, the engineered IL-2 polypeptide described herein may also contain a mutation at a position corresponding to position 20 of SEQ ID NO. 2 (e.g., D20). In some embodiments, the engineered IL-2 polypeptide described herein may also contain a D20N mutation.

[0210] In some embodiments, the engineered IL-2 polypeptide described herein may also contain a mutation at a position corresponding to position 23 of SEQ ID NO. 2 (e.g., M23). In some embodiments, the engineered IL-2 polypeptide described herein may also contain an M23I or M23L mutation.

[0211] In some embodiments, the engineered IL-2 polypeptide described herein may also contain a mutation at a position corresponding to position 84 of SEQ ID NO. 2 (e.g., D84). In some embodiments, the engineered IL-2 polypeptide described herein may also contain a D84N or D84T mutation.

[0212] In some embodiments, the engineered IL-2 polypeptide described herein may also contain a mutation at a position corresponding to position 91 of SEQ ID NO. 2 (e.g., V91). In some embodiments, the engineered IL-2 polypeptide described herein may also contain a V91L mutation.

[0213] In some embodiments, the engineered IL-2 polypeptide described herein may also contain a mutation at a position corresponding to position 92 of SEQ ID NO. 2 (e.g., I92). In some embodiments, the engineered IL-2 polypeptide described herein may also contain an I92V or I92M mutation.

[0214] In some embodiments, the engineered IL-2 polypeptide described herein may also contain a mutation at a position corresponding to position 95 of SEQ ID NO. 2 (e.g., E95). In some embodiments, the engineered IL-2 polypeptide described herein may also contain an E95Q or E95N mutation.

[0215] In some embodiments, the present invention provides a engineered IL-2 polypeptide IL-2_C01_T3A comprising an alanine mutation at a position corresponding to position 3 of SEQ ID NO. 2 (e.g., T3), a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), and a second cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (e.g., I129). The sequence of IL-2_C01_T3A is presented in SEQ ID NO. 36.

[0216] In some embodiments, the present invention provides an engineered IL-2 polypeptide IL-2_C01#1 comprising an alanine mutation at a position corresponding to position 3 of SEQ ID NO. 2 (e.g., T3), an E15D mutation for SEQ ID NO. 2, a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), and a second cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (I129). The sequence of IL-2_C01#1 is presented in SEQ ID NO. 37.

[0217] In some embodiments, the present invention provides an engineered IL-2 polypeptide IL-2_C01#2 comprising an alanine mutation at a position corresponding to position 3 of SEQ ID NO. 2 (e.g., T3), an E15N mutation for SEQ ID NO. 2, a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), and a second cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (I129). The sequence of IL-2_C01#2 is presented in SEQ ID NO. 38.

[0218] In some embodiments, the present invention provides an engineered IL-2 polypeptide IL-2_C01#3 comprising an alanine mutation at a position corresponding to position 3 of SEQ ID NO. 2 (e.g., T3), an H16S mutation for SEQ ID NO. 2, a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), and a second cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (I129). The sequence of IL-2_C01#3 is presented in SEQ ID NO. 39.

[0219] In some embodiments, the present invention provides an engineered IL-2 polypeptide IL-2_C01#4 comprising an alanine mutation at a position corresponding to position 3 of SEQ ID NO. 2 (e.g., T3), an H16T mutation for SEQ ID NO. 2, a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), and a second cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (I129). The sequence of IL-2_C01#4 is presented in SEQ ID NO. 40.

[0220] In some embodiments, the present invention provides an engineered IL-2 polypeptide IL-2_C01#5 comprising an alanine mutation at a position corresponding to position 3 of SEQ ID NO. 2 (e.g., T3), an L19I mutation for SEQ ID NO. 2, a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), and a second cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (I129). The sequence of IL-2_C01#5 is presented in SEQ ID NO. 41.

[0221] In some embodiments, the present invention provides an engineered IL-2 polypeptide IL-2_C01#6 comprising an alanine mutation at a position corresponding to position 3 of SEQ ID NO. 2 (e.g., T3), a D20N mutation for SEQ ID NO. 2, a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), and a second cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (I129). The sequence of IL-2_C01#6 is presented in SEQ ID NO. 42.

[0222] In some embodiments, the present invention provides an engineered IL-2 polypeptide IL-2_C01#7 comprising an alanine mutation at a position corresponding to position 3 of SEQ ID NO. 2 (e.g., T3), an M23I mutation for SEQ ID NO. 2, a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), and a second cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (I129). The sequence of IL-2_C01#7 is presented in SEQ ID NO. 43.

[0223] In some embodiments, the present invention provides an engineered IL-2 polypeptide IL-2_C01#8 comprising an alanine mutation at a position corresponding to position 3 of SEQ ID NO. 2 (e.g., T3), an M23L mutation for SEQ ID NO. 2, a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), and a second cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (I129). The sequence of IL-2_C01#8 is presented in SEQ ID NO. 44.

[0224] In some embodiments, the present invention provides an engineered IL-2 polypeptide IL-2_C01#9 comprising an alanine mutation at a position corresponding to position 3 of SEQ ID NO. 2 (e.g., T3), a D84N mutation for SEQ ID NO. 2, a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), and a second cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (I129). The sequence of IL-2_C01#9 is presented in SEQ ID NO. 45.

[0225] In some embodiments, the present invention provides an engineered IL-2 polypeptide IL-2_C01#10 comprising an alanine mutation at a position corresponding to position 3 of SEQ ID NO. 2 (e.g., T3), a D84T mutation for SEQ ID NO. 2, a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), and a second cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (I129). The sequence of IL-2_C01#10 is presented in SEQ ID NO. 46.

[0226] In some embodiments, the present invention provides an engineered IL-2 polypeptide IL-2_C01#11 comprising an alanine mutation at a position corresponding to position 3 of SEQ ID NO. 2 (e.g., T3), a V91L mutation for SEQ ID NO. 2, a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), and a second cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (I129). The sequence of IL-2_C01#11 is presented in SEQ ID NO. 47.

[0227] In some embodiments, the present invention provides a engineered IL-2 polypeptide IL-2_C01#12 comprising an alanine mutation at a position corresponding to position 3 of SEQ ID NO. 2 (e.g., T3), an I92V mutation for SEQ ID NO. 2, a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), and a second cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (I129). The sequence of IL-2_C01#12 is presented in SEQ ID NO. 48.

[0228] In some embodiments, the present invention provides an engineered IL-2 polypeptide IL-2_C01#13 comprising an alanine mutation at a position corresponding to position 3 of SEQ ID NO. 2 (e.g., T3), an I92M mutation for SEQ ID NO. 2, a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), and a second cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (I129). The sequence of IL-2_C01#13 is presented in SEQ ID NO. 49.

[0229] In some embodiments, the present invention provides an engineered IL-2 polypeptide IL-2_C01#14 comprising an alanine mutation at a position corresponding to position 3 of SEQ ID NO. 2 (e.g., T3), an E95Q mutation for SEQ ID NO. 2, a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), and a second cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (I129). The sequence of IL-2_C01#14 is presented in SEQ ID NO. 50.

[0230] In some embodiments, the present invention provides an engineered IL-2 polypeptide IL-2_C01#15 comprising an alanine mutation at a position corresponding to position 3 of SEQ ID NO. 2 (e.g., T3), an E95N mutation for SEQ ID NO. 2, a first cysteine ​​residue at a position corresponding to position 11 of SEQ ID NO. 2 (e.g., Q11), and a second cysteine ​​residue at a position corresponding to position 129 of SEQ ID NO. 2 (I129). The sequence of IL-2_C01#15 is presented in SEQ ID NO. 51.

[0231] In some embodiments, the engineered IL-2 polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs 35 to 51. In some embodiments, the engineered IL-2 polypeptide described herein comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 35, and the amino acid sequence comprises one or more of the mutations described herein. In some embodiments, the engineered IL-2 polypeptide described herein comprises at least one, at least two, at least three, or at least four of the mutations listed in Table 2.

[0232] In some embodiments, the engineered IL-2 polypeptide may have at least or about 1 (e.g., at least or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40) amino acid insertions, deletions, or substitutions when compared to any one of SEQ ID NOs 35 to 51.

[0233] IL-2 protein construct

[0234] The present disclosure provides a protein construct comprising the engineered IL-2 variant described herein (e.g., any of the engineered IL-2 polypeptides described herein). In some embodiments, the protein construct described herein has an N-terminal His-tag. In some embodiments, the protein construct described herein has a C-terminal His-tag.

[0235] In some embodiments, the protein construct described herein comprises, from the N-terminus to the C-terminus, (a) a His-tag; and (b) a engineered IL-2 polypeptide (e.g., any engineered IL-2 polypeptide described herein). In some embodiments, the His-tag comprises at least 6, at least 7, or at least 8 consecutive His residues. In some embodiments, the His-tag comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of SEQ ID NO. 18. In some embodiments, the engineered IL-2 polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs 3, 4, 5, 6, 7, 8, 9, 16, or 17. In some embodiments, the engineered IL-2 polypeptide described herein comprises at least one, at least two, at least three, or at least four of the cysteine ​​mutations listed in Table 1.

[0236] In some embodiments, the engineered IL-2 protein construct may comprise any engineered IL-2 variant as described herein. In some embodiments, the protein construct described herein comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs 20 to 34 and 52 to 68.

[0237] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, and non-identical sequences may be ignored for comparison purposes). Then, the amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is filled with the same amino acid residue or nucleotide as the corresponding position in the second sequence, the two molecules are identical at that position. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that must be introduced for optimal alignment of the two sequences and the length of each gap. For example, the comparison of sequences and the determination of the percentage of identity between the two sequences can be achieved using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0238] Engineered IL-2 variants (e.g., any of the engineered IL-2 polypeptides described herein) and protein constructs may further comprise an Fc region of an antibody. Such antibodies may be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class, or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE1, IgE2). In some embodiments, the Fc region is derived from human IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the Fc region is an IgG1 Fc region (e.g., a human IgG1 Fc region).

[0239] In some embodiments, the Fc region is connected to an antibody hinge region (e.g., IgG, IgE hinge region). Additionally, the Fc region may be modified to provide a desired effector function or serum half-life.

[0240] In some embodiments, the protein constructs described herein include an Fc region. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the effector function of the Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the Fc region is phagocytosis. In some embodiments, the effector function of the Fc region is ADCC and phagocytosis. In some embodiments, the protein constructs described herein have an Fc region without effector function. In some embodiments, Fc is human IgG1 Fc. In some embodiments, Fc does not have a functional Fc region. For example, the Fc region has an LALA mutation (EU numbering L234A and L235A mutations) or an LALA-PG mutation (EU numbering L234A, L235A, and P329G mutations).

[0241] In some embodiments, the Fc region comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 72. In some embodiments, the Fc region comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 87. In some embodiments, the Fc region comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 88.

[0242] In some embodiments, the engineered IL-2 variant (e.g., any engineered IL-2 variant described herein) is linked to the N-terminus or C-terminus of the Fc region (e.g., SEQ ID NO. 71). In some embodiments, the engineered IL-2 variant is linked to the N-terminus of the Fc region. In some embodiments, the engineered IL-2 variant is linked to the C-terminus of the Fc region. In some embodiments, the Fc region is linked to a hinge region (e.g., SEQ ID NO. 70). In some embodiments, the engineered IL-2 variant is linked to the Fc region via a linker peptide (e.g., SEQ ID NO. 72). In some embodiments, the linker peptide is a flexible linker. Details regarding the flexible linker are, for example, in the literature [Chen, X., et al. "Fusion protein linkers: property, design and functionality." [Advanced Drug Delivery Reviews 65.10 (2013): 1357-1369], the entirety of which is incorporated herein by reference. In some embodiments, the linker peptide comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 72. In some embodiments, the linker peptide comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 92. Various linker sequences may be used, including but not limited to glycine serine (GS) linkers, such as (GS)n, (SG)n, (GGS)n, (GSG)n, (GSGGS)n, and (GGGS)n—wherein n represents an integer of at least 1. In some embodiments, n is 5 to 8. In some embodiments, n is 3 to 5.An exemplary linker sequence may include an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs 85, 92, and 99 to 105.

[0243] In some embodiments, the present invention provides a protein construct comprising a human IgG1 hinge region and an Fc region from the N-terminus to the C-terminus (e.g., SEQ ID NO. 69), a linker peptide (e.g., any of the linker peptides described herein), and a engineered IL-2 polypeptide (e.g., any of the engineered IL-2 polypeptides described herein). In some embodiments, the protein construct described herein comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs. 73 to 84.

[0244] Some other modifications to the Fc region may be made. For example, cysteine ​​residue(s) are introduced into the Fc region to allow the formation of interchain disulfide bonds in this region. The resulting homomer fusion protein may have any increased half-life in vitro and / or in vivo. In some embodiments, IgG4 has the S228P mutation (indicated by EU number). The S228P mutation prevents IgG4 Fab-arm exchange in vivo and in vitro.

[0245] In some embodiments, an Fc region having a fucose-free carbohydrate structure attached (directly or indirectly) to the Fc region is provided. For example, the amount of fucose in such Fc region compositions may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain of Asn297 relative to the sum of all sugar structures (e.g., complexes, hybrids, and high mannose structures) attached to Asn297, as measured by mass spectrometry, for example, as described in International Publication WO 2008 / 077546. Asn297 refers to an asparagine residue located at position approximately 297 of the Fc region (EU numbering of Fc region residues; or position 314 in Kabat numbering); Asn297 may be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the Fc region sequence. These fucosylated variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region may be further configured to replace asparagine at position 297 with alanine (N297A).

[0246] In some embodiments, the present disclosure relates to a protein construct comprising the engineered IL-2 polypeptide described herein. In some embodiments, the protein construct comprises two or more engineered IL-2 polypeptides. In some embodiments, at least two of the engineered IL-2 polypeptides are identical. In some embodiments, at least two of the engineered IL-2 polypeptides are different. In some embodiments, the protein construct further comprises an Fc region. In some embodiments, the Fc region is an IgG4 Fc region. In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the Fc region is an IgG1 Fc region (e.g., having an LALA mutation or an LALA-PG mutation). In some embodiments, the engineered IL-2 polypeptide is linked to the C-terminus of the Fc region. In some embodiments, the engineered IL-2 polypeptide is linked to the C-terminus of the Fc region through a linker peptide (e.g., any of the linker peptides described herein). In some embodiments, the linker peptide comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 72. In some embodiments, the linker peptide comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 92. In some embodiments, the linker peptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs 85, 92, and 99 to 105.

[0247] Characterization of engineered IL-2 variants or protein constructs

[0248] In some embodiments, a engineered IL-2 variant (e.g., any of the engineered IL-2 polypeptides described herein) or a protein construct thereof described herein may bind to the trimer IL-2 receptor complex IL-2Rαβγ. In some embodiments, the engineered IL-2 variant or a protein construct thereof described herein may induce strong downstream signaling (e.g., STAT5 phosphorylation) in Treg cells (e.g., by binding to the trimer IL-2 receptor complex IL-2Rαβγ). In some embodiments, the engineered IL-2 variant or a protein construct thereof described herein does not bind to or binds minimally to the dimeric IL-2 receptor complex IL-2Rβγ. In some embodiments, the engineered IL-2 variants or protein constructs thereof described herein do not induce or minimally induce downstream signaling (e.g., STAT5 phosphorylation) in Tconv cells (e.g., by binding to the dimeric IL-2 receptor complex IL-2Rβγ).

[0249] In some embodiments, the introduction of a mutation (e.g., any of the cysteine ​​mutations described herein) may result in a change in protein structure with an RMSD (mean squared deviation of atomic positions) value of less than 10 Å, less than 9 Å, less than 8 Å, less than 7 Å, less than 6 Å, less than 5 Å, less than 4 Å, less than 3 Å, less than 2 Å, or less than 1 Å. In some embodiments, the RMSD value is calculated by structurally aligning the wild-type protein and the protein variant. In some embodiments, C 알파 Only atoms are used to determine stereostructural changes.

[0250] In some embodiments, the engineered IL-2 variants described herein (e.g., any of the engineered IL-2 polypeptides described herein) or protein constructs thereof (e.g., any of the G1LALAFc-fusion IL-2 variants described herein) may induce STAT5 signaling (e.g., STAT5 phosphorylation) in immune cells (e.g., Treg cells). In some embodiments, the engineered IL-2 variants or protein constructs thereof may induce STAT5 phosphorylation with an effect of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, or less than about 140% compared to the effect of wild-type IL-2 or protein constructs thereof. In some embodiments, engineered IL-2 variants or protein constructs thereof can induce STAT5 phosphorylation with an effect greater than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, or about 140% compared to the effect of wild-type IL-2 or protein constructs thereof.

[0251] In some embodiments, the engineered IL-2 variant or its protein construct may preferentially induce STAT5 phosphorylation in Treg cells compared to Tconv cells. In some embodiments, in Treg cells, the engineered IL-2 variant or its protein construct may induce STAT5 phosphorylation with an effect greater than about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, or about 140% compared to the effect of wild-type IL-2 or its protein construct. In some embodiments, in Tconv cells, engineered IL-2 variants or protein constructs thereof can induce STAT5 phosphorylation with an effect less than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% compared to the effect of wild-type IL-2 or protein constructs thereof.

[0252] In some embodiments, the engineered IL-2 variant or protein construct thereof can induce proliferation of immune cells (e.g., Treg cells) with an effect of about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, or about 140% compared to the effect of wild-type IL-2 or protein construct thereof.

[0253] In some embodiments, the effect of inducing STAT5 signaling (e.g., STAT5 phosphorylation) may be determined using an IL-2 reporter assay (e.g., as described in Example 3). In some embodiments, the effect of inducing STAT5 signaling (e.g., STAT5 phosphorylation) may be determined using HEK-Blue™ IL-2 cells (cells expressing CD25, CD122, and CD132) and HEK-Blue™ CD122 / CD132 cells (cells overexpressing CD122 and CD132). In some embodiments, the engineered IL-2 variant or its protein construct may preferentially induce STAT5 phosphorylation in HEK-Blue™ IL-2 cells (cells overexpressing CD25, CD122, and CD132) compared to HEK-Blue™ CD122 / CD132 cells (cells overexpressing CD122 and CD132). In some embodiments, in HEK-Blue™ IL-2 cells, the engineered IL-2 variant or its protein construct may induce STAT5 phosphorylation with an effect greater than about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, or about 140% compared to the effect of wild-type IL-2 or its protein construct. In some embodiments, in HEK-Blue™ CD122 / CD132 cells, engineered IL-2 variants or protein constructs thereof can induce STAT5 phosphorylation with an effect less than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% compared to the effect of wild-type IL-2 or protein constructs thereof.

[0254] In some embodiments, the engineered IL-2 variant or protein construct thereof described herein can induce proliferation of Treg cells (e.g., HEK-Blue™ IL-2 cells) with an effect greater than about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, or about 140% compared to the effect of wild-type IL-2 or protein construct thereof. In some embodiments, the engineered IL-2 variant or protein construct thereof described herein can induce activation of Treg cells (e.g., HEK-Blue™ IL-2 cells) with an effect greater than about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, or about 140% compared to the effect of wild-type IL-2 or protein construct thereof.

[0255] In some embodiments, the engineered IL-2 variant or protein construct thereof described herein may inhibit the immune response, activation, or number of immune cells (e.g., T cells or NK cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

[0256] In some embodiments, engineered IL-2 variants or protein constructs thereof may bind to the trimer IL-2 receptor complex IL-2Rαβγ. In some embodiments, the dissociation rate (k) off ) is 0.1 s -1 Less than, 0.01 s -1 Less than, 0.001 s -1 Less than, 0.0001 s -1 Less than, or 0.00001 s -1 It is less than. In some embodiments, the dissociation rate (k off ) is 0.01 s -1 Excess, 0.001 s -1 Excess, 0.0001 s -1 Excess, 0.00001 s-1 Exceeding, or 0.000001 s -1 It is in excess. In some embodiments, the kinetic assembly velocity (k on ) is 1 × 10 2 Exceeding / Ms, 1 × 10 3 Exceeding / Ms, 1 × 10 4 Exceeding / Ms, 1 × 10 5 Exceeding / Ms or 1 × 10 6 It is greater than / Ms. In some embodiments, the kinetic assembly velocity (k on ) is 1 × 10 5 Less than / Ms, 1 × 10 6 Less than / Ms, or 1 × 10 7 It is less than / Ms. Affinity can be inferred from the ratio of kinetic velocity constants (KD=k off / k on ). In some embodiments, KD is 1 × 10 -6 Less than M, 1 × 10 -7 Less than M, 1 × 10 -8 Less than M, 1 × 10 -9 Less than M, or 1 × 10⁻⁶ -10 It is less than M. In some embodiments, KD is 300 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or less than 10 pM. In some embodiments, KD is 1 × 10 -7 Exceeding M, 1 × 10 -8 Exceeding M, 1 × 10 -9 Exceeding M, 1 × 10 -10 Exceeding M, 1 × 10 -11 Exceeding M, or 1 × 10 -12It exceeds M.

[0257] In some embodiments, engineered IL-2 variants or protein constructs thereof may bind to the dimeric IL-2 receptor complex IL-2Rβγ. In some embodiments, the dissociation rate (k off ) is 0.1 s -1 Less than, 0.01 s -1 Less than, 0.001 s -1 Less than, 0.0001 s -1 Less than, or 0.00001 s -1 It is less than. In some embodiments, the dissociation rate (k off ) is 0.01 s -1 Excess, 0.001 s -1 Excess, 0.0001 s -1 Excess, 0.00001 s -1 Exceeding, or 0.000001 s -1 It is in excess. In some embodiments, the kinetic assembly velocity (k on ) is 1 × 10 2 Exceeding / Ms, 1 × 10 3 Exceeding / Ms, 1 × 10 4 Exceeding / Ms, 1 × 10 5 Exceeding / Ms or 1 × 10 6 It is greater than / Ms. In some embodiments, the kinetic assembly velocity (k on ) is 1 × 10 5 Less than / Ms, 1 × 10 6 Less than / Ms, or 1 × 10 7 It is less than / Ms. Affinity can be inferred from the ratio of kinetic velocity constants (KD=k off / k on ). In some embodiments, KD is 1 × 10 -6 Less than M, 1 × 10 -7 Less than M, 1 × 10 -8 Less than M, 1 × 10 -9 Less than M, or 1 × 10⁻⁶ -10It is less than M. In some embodiments, KD is 300 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or less than 10 pM. In some embodiments, KD is 1 × 10 -7 Exceeding M, 1 × 10 -8 Exceeding M, 1 × 10 -9 Exceeding M, 1 × 10 -10 Exceeding M, 1 × 10 -11 Exceeding M, or 1 × 10 -12 It exceeds M.

[0258] In some embodiments, the engineered IL-2 variant or protein construct thereof may preferentially bind to the trimer IL-2 receptor complex IL-2Rαβγ compared to the dimeric IL-2 receptor complex IL-2Rβγ. In some embodiments, the engineered IL-2 variant or protein construct thereof may bind to the trimer IL-2 receptor complex IL-2Rαβγ with a first KD, and the engineered IL-2 variant or protein construct thereof may bind to the dimeric IL-2 receptor complex IL-2Rβγ with a second KD. In some embodiments, the second KD may be at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, or at least 100 times the first KD. In some embodiments, the first KD may be less than 100%, less than 50%, less than 20%, less than 10%, less than 5%, less than 2%, or less than 1% of the second KD. In some embodiments, the first KD may be greater than 100%, greater than 50%, greater than 20%, greater than 10%, greater than 5%, greater than 2%, or greater than 1% of the second KD.

[0259] In some embodiments, the engineered IL-2 variant or its protein construct may bind to the trimer IL-2 receptor complex IL-2Rαβγ with a stronger binding affinity than wild-type IL-2. In some embodiments, the binding affinity between the engineered IL-2 variant or its protein construct and the trimer IL-2 receptor complex IL-2Rαβγ is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 400%, about 500%, or about 1000% higher than the binding affinity between wild-type IL-2 and the trimer IL-2 receptor complex IL-2Rαβγ.

[0260] In some embodiments, the engineered IL-2 variant or its protein construct may bind to the dimeric IL-2 receptor complex IL-2Rβγ with a lower binding affinity than wild-type IL-2. In some embodiments, the binding affinity between the engineered IL-2 variant or its protein construct and the dimeric IL-2 receptor complex IL-2Rβγ is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 400%, about 500%, or about 1000% lower than the binding affinity between wild-type IL-2 and the trimeric IL-2 receptor complex IL-2Rαβγ.

[0261] Conventional techniques for measuring affinity include, for example, ELISA, radioimmunoassay (RIA), and surface plasmon resonance (SPR). In some embodiments, affinity is determined by a cell-based assay.

[0262] In some embodiments, the engineered IL-2 protein constructs described herein are expressed and can be purified by methods commonly used in the art, such as affinity chromatography. In some cases, the protein constructs can be purified by size exclusion chromatography (SEC) combined with HPLC. In some embodiments, the percentage of the main peak in the SEC-HPLC analysis is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, the percentage of the high molecular weight peak (HMW%) and / or the percentage of the low molecular weight peak (LMW%) is less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0263] In some embodiments, the thermal stability of the engineered IL-2 variant or protein construct thereof described herein may be determined. The engineered IL-2 variant and protein construct thereof described herein may have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C. In some embodiments, Tm is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C. In some embodiments, the aggregation (T) of the engineered IL-2 variant and its protein constructs described herein agg ) and initiation(T onset ) Temperature can be measured by applying a temperature increase (e.g., 25 to 85°C) based on DLS / SLS. DLS / SLS is a widely known technique for determining sample interactions, particle size, and aggregation of molecules dispersed or dissolved in solution. Protein unfolding initiation (T onset ) and aggregation (T agg The temperature undergoing ) is considered a key predictor of stability. In some embodiments, T of the engineered IL-2 variant or protein construct thereof described herein agg is at least 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65°C. In some embodiments, T of the engineered IL-2 variant or protein construct thereof described herein onset is at least 59, 60, 61, 62, 63, 64, 65, or 66℃.

[0264] In some embodiments, cysteine ​​mutations (e.g., any of the cysteine ​​mutations described herein or combinations thereof) agglutinate (T agg ) and / or initiation(T onset The temperature can be increased by at least 0.5°C, at least 1°C, at least 1.5°C, at least 2°C, at least 2.5°C, at least 3°C, at least 3.5°C, at least 4°C, at least 4.5°C, at least 5°C, at least 5.5°C, at least 6°C, at least 6.5°C, at least 7°C, at least 7.5°C, at least 8°C, at least 8.5°C, or at least 9°C.

[0265] In some embodiments, the half-life of the engineered IL-2 variant or protein construct thereof described herein may be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 100-fold compared to wild-type IL-2 or protein construct thereof. In some embodiments, the half-life is determined by measuring the in vivo concentration of the molecule over time after administration to a subject.

[0266] In some embodiments, after in vivo administration, the engineered IL-2 variant or protein construct thereof described herein may have a higher serum concentration (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1x, at least 2x, at least 3x, at least 4x, at least 5x, or at least 100x) after 3, 4, 5x, or 50x, or at least 100x.

[0267] In some embodiments, after in vivo administration, the engineered IL-2 variant or protein construct thereof described herein may reduce the subject's GvHD by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, or at least 100 times.

[0268] In some embodiments, the engineered IL-2 variant or protein construct thereof described herein does not cause significant toxicity after in vivo administration.

[0269] Method for preparing engineered IL-2 variants and protein constructs

[0270] The engineered IL-2 variants or protein constructs thereof described herein may be produced by introducing appropriate nucleotide changes into DNA encoding the IL-2 peptide or a portion thereof, or by peptide synthesis. Such variants include, for example, deletion, insertion, or substitution of residues within the amino acid sequence.

[0271] Screening may be performed. The engineered IL-2 variants or protein constructs thereof described herein may be expressed and purified using methods known in the art. Their expression and purification profiles may be compared by various analytical methods (e.g., SDS-PAGE, and HPLC-SEC). Additionally, the efficacy of the engineered IL-2 variants or protein constructs thereof described herein on T cell responses may be determined by STAT-5 phosphorylation and / or IL-2 reporter assays (e.g., those described in Example 3). Based on the experimental results, the engineered IL-2 variants or protein constructs thereof described herein having good expression / purification profiles and desirable T cell response efficacy may be selected to further evaluate immunosuppressive efficacy and in vivo toxicity (e.g., in a mouse model).

[0272] Engineered IL-2 variants may be derived from any animal species, including mammals. Non-limiting examples of IL-2 variants include those derived from humans, primates, e.g., monkeys and apes, cattle, pigs, horses, sheep, camelid species, e.g., camels and llamas, chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits).

[0273] The present disclosure also provides a recombinant vector (e.g., an expression vector) comprising an isolated polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein), a host cell into which the recombinant vector is introduced (i.e., a host cell such that the host cell comprises a polynucleotide and / or a vector comprising a polynucleotide), and the production of a recombinant polypeptide or a fragment thereof by recombinant technology.

[0274] As used herein, a “vector” is any construct capable of delivering one or more polynucleotides of interest to a host cell. An “expression vector” can deliver and express one or more polynucleotides of interest as a polypeptide encoding them in a host cell into which the expression vector has been introduced. Thus, in the expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and / or poly-A tail at or near the integration site of the polynucleotide of interest within the vector or within the host cell’s genome, or flanked thereto, thereby allowing the polynucleotide of interest to be translated in the host cell into which the expression vector has been introduced.

[0275] Vectors may be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., using recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which may be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cation condensers.

[0276] In some embodiments, the polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein) are introduced using a viral expression system (e.g., vaccinia or other smallpox viruses, retroviruses, or adenoviruses), which may include the use of non-pathogenic (defective) replication-eligible viruses or replication-defective viruses. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. The DNA may also be "naked." The uptake of naked DNA may be enhanced by coating the DNA onto biodegradable beads that efficiently transport the DNA into the cell.

[0277] For expression, DNA inserts comprising polypeptide-coding polynucleotides disclosed herein may be operably linked to suitable promoters (e.g., heterogeneous promoters), such as the phage lambda PL promoter, E. coli lac, trp, and tac promoters, SV40 early and late promoters, and retrovirus LTR promoters. Other suitable promoters are known to those skilled in the art. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the promoter is a human promoter, such as the uHS or HS promoter. Human promoters can enhance the expression of human-derived proteins. For details regarding such human promoters, see, for example, the literature [Antoniou, M., et al. "Transgenes encompassing dual-promoter CpG islands from the human TBP and HNRPA2B1 loci are resistant to heterochromatin-mediated silencing." [Genomics 82.3 (2003): 269-279]; and can be found in the literature [Zhang, F., et al. "A ubiquitous chromatin opening element (UCOE) confers resistance to DNA methylation-mediated silencing of lentiviral vectors." Molecular Therapy 18.9 (2010): 1640-1649]; each of these is incorporated herein by reference in its entirety. The expression construct may further comprise a transcription initiation site, a termination site, and a ribosome binding site for translation in the transcription region. The coding portion of the mature transcript expressed by the construct may include a translation initiation codon (UAA, UGA, or UAG) appropriately located at the beginning of the polypeptide to be translated and a termination codon at the end.

[0278] As mentioned, the expression vector may include at least one selection marker. These markers include genes for dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and genes for tetracycline or ampicillin resistance for culture in E. coli and other bacteria. Representative examples of suitable hosts are bacterial cells, e.g., E. coli, Streptomyces, and Salmonella typhimurium ( Salmonella typhimurium ) cells; fungal cells, e.g., yeast cells; insect cells, e.g., drosophila( Drosophila ) S2 and Spodoptera( Spodoptera ) Sf9 cells; animal cells, e.g., CHO, COS, Bowes melanoma, HEK 293 cells; and plant cells, but not limited thereto. Suitable culture media and conditions for the host cells described herein are known in the art.

[0279] Non-restrictive vectors for use in bacteria include pQE70, pQE60, and pQE-9 available from Qiagen; pBS, Phagescript, Bluescript, pNH8A, pNH16a, pNH18A, and pNH46A available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 available from Pharmacia. Non-restrictive eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

[0280] Suitable non-restrictive bacterial promoters for use include the E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoters, lambda PR and PL promoters, and trp promoters. Suitable eukaryotic promoters include the CMV outpost promoter, HSV thymidine kinase promoter, early and late SV40 promoters, retrovirus LTR promoters, e.g., Rous sarcoma virus (RSV) promoter, and metallothionein promoters, e.g., mouse metallothionein-I promoter.

[0281] Yeast Saccharomyces cerevisiae ( Saccharomyces cerevisiae In ), a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.

[0282] Introduction of the construct into host cells may be achieved via calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. These methods are described in various standard laboratory manuals, such as [Davis et al., Basic Methods In Molecular Biology (1986)], the entirety of which is incorporated herein by reference.

[0283] In some embodiments, the host cell is a human cell suitable for protein expression, e.g., HEK 293 cell or CHO cell (e.g., CHO-S cell). In some embodiments, the host cell is Expi293™ cell and ExpiCHO™ cell. The Expi293 expression system is designed to deliver up to six times more protein in just one week compared to other transient 293 expression systems, which may take more than two weeks. This is partly due to the fact that Expi293F cells have been modified to achieve higher pg / cell / day productivity than standard HEK 293 cells, and that Expifectamine™ 293 transfection reagents and enhancers enable high-efficiency transfection and high expression levels in high-density HEK 293 cultures. Additionally, the Expi293 expression system requires fewer plastic containers, which means reduced waste and larger incubator space.

[0284] Transcription of DNA encoding the polypeptide of the present disclosure can be increased by higher eukaryotes by inserting an enhancer sequence into a vector. The enhancer is a cis-acting DNA element, typically 10 to 300 bp in length, that serves to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include an SV40 enhancer located at base pairs 100 to 270 on the late side of the replication origin, a cytomegalovirus early promoter enhancer, a polyoma enhancer on the late side of the replication origin, and an adenovirus enhancer.

[0285] To secrete the translated protein into the endoplasmic reticulum lumen, the pericytoplasmic space, or the extracellular environment, an appropriate secretion signal can be introduced into the expressed polypeptide. This signal may be endogenous to the polypeptide or may be a heterologous signal.

[0286] Polypeptides (e.g., engineered IL-2 variants) may be expressed in modified forms, such as fusion proteins (e.g., G1LALAFc fusion proteins) or those with attached histidine tags, and may contain additional heterogeneous functional regions as well as secretory signals. For example, additional amino acids, particularly charged amino acid regions, can be added to the N-terminus of the polypeptide to improve stability and persistence during purification in host cells or during subsequent handling and storage. Additionally, peptide moietyes can be added to the polypeptide to facilitate purification. These regions may be removed prior to the final preparation of the polypeptide. In particular, adding peptides to polypeptide moiety to induce secretion or excretion, improve stability, or facilitate purification is a widely known and common technique in the art.

[0287] Treatment methods

[0288] The engineered IL-2 variants and protein constructs thereof of the present disclosure can be used for various therapeutic purposes.

[0289] In one embodiment, the present disclosure provides a method for treating an immune disease in a subject. In some embodiments, the treatment may stop, slow, delay, or inhibit the progression of the immune disease. In some embodiments, the treatment may reduce the number, severity, and / or duration of one or more symptoms of the immune disease in the subject. In some embodiments, the immune disease is an autoimmune disease.

[0290] In one embodiment, the present disclosure provides a method for treating, preventing, or reducing the risk of developing disorders associated with abnormal or unwanted immune responses, such as autoimmune disorders, by administering a therapeutically effective amount of the engineered IL-2 variant and protein construct disclosed herein to a subject in need thereof. These autoimmune disorders include, but are not limited to, rheumatoid arthritis, Crohn's disease, systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel disease (IBD), ulcerative colitis, or scleroderma. In some embodiments, autoimmune disorders include allergies, asthma, and / or atopic dermatitis. Accordingly, engineered IL-2 variants and protein constructs disclosed herein may be used to suppress immune responses. In some embodiments, the immune disorders described herein are allergies, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes mellitus, pain, or neurological disorders.

[0291] In one embodiment, the present disclosure provides a method for treating, preventing, or reducing the risk of developing disorders associated with abnormal or unwanted immune responses, such as autoimmune disorders, by administering a therapeutically effective amount of the engineered IL-2 variant and protein construct disclosed herein to a subject in need thereof. These autoimmune disorders include alopecia areata, lupus, ankylosing spondylitis, Meniere's disease, antiphospholipid syndrome, mixed connective tissue disease, autoimmune Addison's disease, multiple sclerosis, autoimmune hemolytic anemia, myasthenia gravis, autoimmune hepatitis, pemphigus vulgaris, Behcet's disease, pernicious anemia, bullous pemphigus, nodular polyarthritis, cardiomyopathy, polychondritis, intra-abdominal dermatitis, polymyositis, chronic fatigue syndrome (CFIDS), polymyalgia rheumatica, chronic inflammatory demyelinating polymyositis and dermatomyositis, chronic inflammatory polyneuropathy, primary agammaglobulinemia, Churg-Strauss syndrome, primary biliary cirrhosis, cicatricial pemphigus, psoriasis, CREST syndrome, Raynaud's phenomenon, cold agglutinin disease, Reiter's syndrome, Crohn's disease, rheumatic fever, and discoid Includes, but not limited to, lupus, rheumatoid arthritis, cryoglobulinemia, sarcoidosis, fibromyalgia, scleroderma, Graves' disease, Sjögren's syndrome, Guillain-Barré syndrome, spasticity syndrome, Hashimoto's thyroiditis, Takayasu's arteritis, idiopathic pulmonary fibrosis, temporal arteritis / giant cell arteritis, idiopathic thrombocytopenia purpura (ITP), ulcerative colitis, IgA nephropathy, uveitis, diabetes mellitus (e.g., type 1), vasculitis, lichen planus, and leukoplakia.The engineered IL-2 variants and protein constructs disclosed herein may also be administered to subjects to treat, prevent, or reduce the risk of, disorders resulting from abnormal or unwanted immune responses associated with cell, tissue, or organ transplants, such as kidney, liver, and heart transplants, such as graft-versus-host disease (GvHD), or to prevent allogeneic tissue rejection. In some embodiments, the subject has a skin disease, liver disease (e.g., cirrhosis), hidradenitis suppurativa, or experimental autoimmune encephalomyelitis. In some embodiments, the subject has kidney disease, lupus, Sjögren's syndrome, ulcerative colitis, psoriasis, hidradenitis suppurativa, immune thrombocytopenia (ITP), or other inflammatory arthritis. In some embodiments, the subject has multiple sclerosis or myasthenia gravis. In some embodiments, the subject has Crohn's disease, ulcerative colitis, or diabetes mellitus (e.g., type I). In some embodiments, the subject has an autoimmune thyroid disease, Graves' disease, multiple sclerosis, psoriasis, inflammatory bowel disease (e.g., Crohn's disease (CD) and ulcerative colitis), rheumatoid arthritis, Sjögren's syndrome, autoimmune nephritis, or systemic lupus erythematosus. In some embodiments, the method comprises administering an effective amount of a composition comprising the engineered IL-2 variant and protein construct disclosed herein to the subject.

[0292] In one embodiment, the present disclosure provides a method for treating cancer in a subject, a method for reducing the rate of increase of tumor volume in the subject over time, a method for reducing the risk of metastasis, or a method for reducing the risk of additional metastasis in the subject. In some embodiments, the treatment may stop, slow, delay, or inhibit the progression of the cancer. In some embodiments, the treatment may reduce the number, severity, and / or duration of one or more symptoms of cancer in the subject.

[0293] In one embodiment, the present disclosure features a method comprising administering a therapeutically effective amount of the engineered IL-2 variant and protein construct disclosed herein to a subject who has or is identified or diagnosed with cancer (e.g., cancer, breast cancer (e.g., triple-negative breast cancer), carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematological malignancy). In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is head and neck squamous cell carcinoma (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal carcinoma. In some embodiments, the cancer is melanoma, pancreatic carcinoma, mesothelioma, hematological malignancies, particularly non-Hodgkin lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumors. In some embodiments, the cancer is breast cancer, ovarian cancer, glioma, colon cancer, hepatocellular carcinoma, prostate cancer, renal cell carcinoma, melanoma, lung cancer, pancreatic cancer, or liver cancer.

[0294] In some embodiments, the compositions and methods disclosed herein may be used to treat patients at risk of cancer. Cancer patients may be identified using various methods known in the art.

[0295] In one embodiment, the present disclosure features a method comprising administering a therapeutically effective amount of the engineered IL-2 variant and protein construct disclosed herein to a subject in need thereof, e.g., having an immune disease or cancer, or having or identified or diagnosed with cancer, e.g., breast cancer (e.g., triple-negative breast cancer), carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematological malignancy. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is head and neck squamous cell carcinoma (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal carcinoma. In some embodiments, the subject has Hodgkin lymphoma. In some embodiments, the subject has triple-negative breast cancer (TNBC), gastric cancer, urothelial carcinoma, Merkel cell carcinoma, or head and neck cancer.

[0296] In some embodiments, the cancer described herein is esophageal adenocarcinoma, lung cancer, hepatocellular carcinoma, cholangiocarcinoma, gallbladder cancer, gastric cancer, pancreatic cancer, colorectal cancer, endometrial carcinoma, ovarian cancer, bladder cancer, prostate cancer, or T-cell lymphoma. In some embodiments, the cancer is kidney cancer.

[0297] In some embodiments, the compositions and methods disclosed herein may be used to treat patients at risk of cancer. Cancer patients may be identified using various methods known in the art.

[0298] As used herein, "effective dose" means an amount or dosage sufficient to produce a beneficial or desirable result, including stopping, slowing, delaying, or inhibiting the progression of a disease, e.g., cancer. Since the effective dose depends on the age and weight, severity of symptoms, and route of administration of the subject receiving, for example, engineered IL-2 variants and protein constructs, vectors containing polynucleotides encoding engineered IL-2 variants and protein constructs, and / or compositions thereof, administration may be determined on individual criteria.

[0299] The effective dose may be administered in one or more doses. For example, the effective dose of the engineered IL-2 variant and / or protein construct is an amount sufficient to mitigate, stop, stabilize, reverse, inhibit, delay, and / or slow cancer progression in a patient, or an amount sufficient to mitigate, stop, stabilize, reverse, delay, and / or slow the proliferation of cells (e.g., biopsy cells, cancer cells described herein, or any of cell lines (e.g., cancer cell lines)) in vitro. As is understood in the art, the effective dose may vary depending on other factors, such as the type (and / or dose) of the engineered IL-2 variant and protein construct used, as well as the patient's medical history.

[0300] The effective amount and administration schedule of the engineered IL-2 variants or protein constructs thereof, polynucleotides and / or compositions encoding engineered IL-2 variants or protein constructs disclosed herein may be determined empirically, and such determination is within the knowledge of those skilled in the art. Those skilled in the art will understand that the dosage to be administered depends, for example, on the mammal to be administered the engineered IL-2 variants or protein constructs thereof, polynucleotides and / or compositions disclosed herein, the route of administration, the specific type of polynucleotide and / or composition used, and other drugs administered to the said mammal.

[0301] A typical daily dose of an effective amount of the engineered IL-2 variant or its protein construct is 0.1 mg / kg to 200 mg / kg (mg per kg of patient body weight). In some embodiments, the dose may be 150 mg / kg, 140 mg / kg, 130 mg / kg, 120 mg / kg, 110 mg / kg, 100 mg / kg, 90 mg / kg, 80 mg / kg, 70 mg / kg, 60 mg / kg, 50 mg / kg, 40 mg / kg, 30 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or less than 0.1 mg / kg. In some embodiments, the dosage may be 150 mg / kg, 140 mg / kg, 130 mg / kg, 120 mg / kg, 110 mg / kg, 100 mg / kg, 90 mg / kg, 80 mg / kg, 70 mg / kg, 60 mg / kg, 50 mg / kg, 40 mg / kg, 30 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or greater than 0.1 mg / kg. In some embodiments, the dosage is about 150 mg / kg, 140 mg / kg, 130 mg / kg, 120 mg / kg, 110 mg / kg, 100 mg / kg, 90 mg / kg, 80 mg / kg, 70 mg / kg, 60 mg / kg, 50 mg / kg, 40 mg / kg, 30 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, or 1 mg / kg.In some embodiments, the dosage is about 1 to 150 mg / kg, about 1 to 100 mg / kg, about 1 to 80 mg / kg, about 1 to 50 mg / kg, about 1 to 30 mg / kg, about 1 to 20 mg / kg, about 1 to 10 mg / kg, about 1 to 5 mg / kg, about 5 to 150 mg / kg, about 5 to 100 mg / kg, about 5 to 80 mg / kg, about 5 to 50 mg / kg, about 5 to 30 mg / kg, about 5 to 20 mg / kg, about 5 to 10 mg / kg, about 10 to 150 mg / kg, about 10 to 100 mg / kg, about 10 to 80 mg / kg, about 10 to 50 mg / kg, about 10 to 30 mg / kg, about 10 to 20 It is mg / kg, about 20 to 150 mg / kg, about 20 to 100 mg / kg, about 20 to 80 mg / kg, about 20 to 50 mg / kg, about 20 to 30 mg / kg, about 30 to 150 mg / kg, about 30 to 100 mg / kg, about 30 to 80 mg / kg, about 30 to 50 mg / kg, about 50 to 150 mg / kg, about 50 to 100 mg / kg, about 50 to 80 mg / kg, about 80 to 150 mg / kg, about 80 to 100 mg / kg, or about 100 to 150 mg / kg.

[0302] In any of the methods described herein, the engineered IL-2 variant or protein construct thereof may be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day).

[0303] Pharmaceutical composition and route of administration

[0304] The present invention also provides pharmaceutical compositions containing the engineered IL-2 variants or protein constructs thereof described herein. Such pharmaceutical compositions may be formulated in any manner known in the art.

[0305] The pharmaceutical composition is formulated to be suitable for the intended route of administration (e.g., intravenous, arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The composition may comprise a sterile diluent (e.g., sterile water or saline), a fixative oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, an antibacterial or antifungal agent, e.g., benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc., an antioxidant, e.g., ascorbic acid or sodium bisulfite, a chelating agent, e.g., ethylenediaminetetraacetic acid, a buffer, e.g., acetate, citrate, or phosphate, and an isotonic agent, e.g., sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol) or salts (e.g., sodium chloride), or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. Formulations of such compositions can be formulated and sealed in ampoules, single-use syringes, or multi-use vials. If necessary (e.g., for injectable formulations), suitable fluidity can be maintained using a coating agent, such as lecithin, or a surfactant. Absorption of the agent can be extended by adding an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems that may comprise biodegradable and biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid).

[0306] Compositions containing the engineered IL-2 variants or protein constructs thereof described herein may be formulated for parenteral administration (e.g., intravenously, intra-arterially, intramuscularly, intradermally, subcutaneously, or intraperitoneally) in the form of dosing units (i.e., physically separated units containing a predetermined amount of active compound for ease of administration and dosage uniformity).

[0307] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured in accordance with Good Manufacturing Practice (GMP). Pharmaceutical compositions may be provided in unit dosage forms (i.e., single-dose doses). Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the selected route of administration. For injectable use, engineered IL-2 variants or protein constructs thereof may be formulated in an aqueous solution, preferably in a physiologically suitable buffer to reduce discomfort at the injection site. This solution may contain formulation agents such as suspenders, stabilizers, and / or dispersants. Alternatively, engineered IL-2 variants or protein constructs thereof may exist in a lyophilized form for composition with a suitable vehicle, such as sterile non-pyrogenous water, prior to use.

[0308] The toxicity and therapeutic efficacy of the composition may be determined according to standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). For example, the LD50 (lethal dose for 50% of the population) and ED50 (dose that produces a therapeutic effect in 50% of the population) can be determined, and the therapeutic index is the ratio of LD50 to ED50. An agent with a high therapeutic index is preferred. If the agent exhibits undesirable side effects, care must be taken to minimize potential damage (i.e., to reduce unwanted side effects). Toxicity and therapeutic efficacy may be determined by other standard pharmaceutical procedures.

[0309] Exemplary doses comprise amounts in milligrams or micrograms of any of the engineered IL-2 variants or protein constructs thereof described herein per kilogram of the subject's body weight (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; about 1 μg / kg to about 50 μg / kg; about 1 mg / kg to about 10 mg / kg; or about 1 mg / kg to about 5 mg / kg). While these doses cover a wide range, those skilled in the art will understand that the efficacy of the therapeutic agent may vary and that the effective dose may be determined by methods known in the art. Generally, a relatively low dose is administered initially, and (for therapeutic purposes) the attending medical professional, veterinarian, or researcher (in the development phase) may subsequently increase the dose gradually until an appropriate response is observed. Additionally, it should be understood that the specific dose level for any particular subject depends on various factors, including the activity of the specific compound used, the subject's age, body weight, overall health status, sex, and diet, time of administration, route of administration, elimination rate, and the half-life of the engineered IL-2 variant or its protein construct in vivo.

[0310] These pharmaceutical compositions may be stored in a container, packaging, or dispenser along with administration instructions. The present disclosure also provides a method for preparing engineered IL-2 variants or protein constructs thereof for use in various applications as described herein.

[0311] Examples

[0312] The present invention is further described in the following examples, which do not limit the scope of the invention as described in the claims.

[0313] Example 1. Design of IL-2 Variant

[0314] Different IL-2 variant proteins (or IL-2 variants) were designed by mutating one or more amino acid residues in the soluble chain of human IL-2 (IL-2_WT; SEQ ID NO. 2) into cysteine ​​residues. It was thought that the introduced cysteine ​​could create artificial disulfide bonds to finely regulate the function of IL-2 in immune responses, for example, to separate the immunostimulatory and immunosuppressive functions of IL-2.

[0315] Specifically, selected amino acid residue(s) in the soluble chain of human IL-2 (IL-2_WT; SEQ ID NO. 2) were mutated into cysteine ​​residues to obtain IL-2 variants as shown in the table below.

[0316] [Table 1]

[0317]

[0318] The amino acid sequences of IL-2_WT (SEQ No. 2) and its variants IL-2_C01 (SEQ No. 3), IL-2_C02 (SEQ No. 4), IL-2_C03 (SEQ No. 5), IL-2_C04 (SEQ No. 6), IL-2_C05 (SEQ No. 7), IL-2_C06 (SEQ No. 8), IL-2_C07 (SEQ No. 9), IL-2_C08 (SEQ No. 10), IL-2_C09 (SEQ No. 11), IL-2_C10 (SEQ No. 12), IL-2_C11 (SEQ No. 13), IL-2_C21 (SEQ No. 14), IL-2_C22 (SEQ No. 15), IL-2_C23 (SEQ No. 16), and IL-2_C24 (SEQ No. 17) are shown in FIG. 12. Enumerate.

[0319] To facilitate protein purification and identification, H8-IL-2_WT (SEQ No. 19) was prepared by fusing IL-2_WT and its variants with a His tag (SEQ No. 18) at the N-terminus. The amino acid sequences of the IL-2 variants with the corresponding His tag are listed in FIG. 12.

[0320] Example 2. Generation and Purification of H8-IL-2 Variant

[0321] IL-2 variants fused with the His tag were expressed in both Expi293™ and ExpiCHO™ cells. After expression, proteins were purified by affinity chromatography using a HisTrap™ excel column (Cytiva, Cat. No.: 17371206) pre-charged with Ni Sepharose and by size exclusion chromatography (SEC) using a Superdex™ 75 Increase column (Cytiva, Cat. No.: 29148721). Additionally, the purified proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC-SEC).

[0322] As illustrated in FIGS. 1a through 1c, among 15 His-tagged IL-2 variants, only 7 variants, including H8-IL-2_C01 (SEQ No. 20), H8-IL-2_C03 (SEQ No. 22), H8-IL-2_C05 (SEQ No. 24), H8-IL-2_C21 (SEQ No. 31), H8-IL-2_C22 (SEQ No. 32), H8-IL-2_C23 (SEQ No. 33), and H8-IL-2_C24 (SEQ No. 34), were successfully generated and purified in ExpiCHO™ cells (Fig. 1a) and / or Expi293 cells (Figs. 1b and 1c). The production titers of other His-tagged IL-2 variants were too low to be determined. Therefore, H8-IL-2_C01, H8-IL-2_C03, H8-IL-2_C05, H8-IL-2_C21, H8-IL-2_C22, H8-IL-2_C23, H8-IL-2_C24, and H8-IL-2_WT were subsequently tested in vitro.

[0323] Example 3. Detection of IL-2 reporter signaling in HEK-Blue™ IL-2 cells and HEK-Blue™ CD122 / CD132 cells

[0324] An IL-2 reporter assay was performed to test the IL-2-phospho-STAT5 (pSTAT5) signaling activity induced by the designed IL-2 molecule described in Example 1. 2.8 × 10 5HEK-Blue™ IL-2 cells (InvivoGen, Cat. No.: hkb-il2) and HEK-Blue™ CD122 / CD132 cells (InvivoGen, Cat. No.: hkb-il2bg) were suspended in preheated Dulbecco's Modified Eagle's Medium (DMEM) (CORNING, Cat. No.: 10-013-CV) supplemented with 10% (v / v) heat-treated inactivated Gibco™ fetal bovine serum (FBS) (Fisher Scientific, Cat. No.: A31606-01) and 100-fold penicillin-streptomycin solution (CORNING, Cat. No.: 30-002-CI), respectively. 180 μL of HEK-Blue™ IL-2 cells or HEK-Blue™ CD122 / CD132 cells were incubated with 20 μL of serially diluted designed IL-2 variants or corresponding control proteins in a 96-well TC-treated microplate (CORNING, Cat. No.: 3599) at 37°C for 22 hours. After incubation, 20 μL of cell culture supernatant from the induced HEK-Blue™ IL-2 cells or induced HEK-Blue™ CD122 / CD132 cells was mixed with 180 μL of QUANTI-Blue™ solution (InvivoGen, Cat. No.: rep-qbs2) resuspended in a flat-bottom 96-well plate (Paul Bottger, Cat. No.: 05-031-0100) and incubated at 37°C for 2 to 3 hours. The activity of secretory alkaline phosphatase (SEAP) was evaluated by measuring absorbance at 630 nm using a Thermo Scientific™ Varioskan™ LUK spectrophotometer (Thermo Fisher Scientific, Type 3020).

[0325] As shown in Figures 2a and 2c, the IL-2-pSTAT5 reporter signaling activity induced by the tested H8-IL-2 variants H8-IL-2_C01, C03, C05, C21, C22, C23, and C24 was similar to the activity induced by H8-IL-2_WT in HEK-Blue™ IL-2 cells (cells overexpressing CD25, CD122, and CD132). However, in HEK-Blue™ CD122 / CD132 cells (cells overexpressing CD122 and CD132), the H8-IL-2 variants induced different IL-2-pSTAT5 reporter signaling activities as shown in Figures 2b and 2d. Figure 2b showed that H8-IL-2_C01 induced the weakest activity compared to H8-IL-2_WT, H8-IL-2_C03, and H8-IL-2_C05. In HEK-Blue™ CD122 / CD132 cells, the order of reporter activity from highest to lowest was H8-IL-2_WT, C05 >> H8-IL-2_C03 > H8-IL-2_C01. In a separate assay shown in Figure 2d, in HEK-Blue™ CD122 / CD132 cells, the order of reporter activity from highest to lowest was H8-IL-2_WT, C22 >> H8-IL-2_C23, C24 > H8-IL-2_C21. These results suggest that H8-IL-2 variants, particularly H8-IL-2_C01 and H8-IL-2_C21, have a greater ability to induce IL-2 signaling activity in cells overexpressing CD25, CD122, and CD132 (e.g., Treg cells) compared to cells overexpressing only CD122 and CD132 (e.g., Teff cells). Therefore, IL-2_C01 and its variants may have potential for autoimmune treatment.

[0326] Example 4. Design of H8-IL-2_C01_T3A-derived mutant

[0327] The amino acid sequences of IL-2_C01_T3A-derived variants, consisting of IL-2_WT_T3A (SEQ No. 35), IL-2_C01_T3A (SEQ No. 36), and IL-2_C01#1 to #15 (SEQ Nos. 37 to 51), are listed in FIG. 12. The mutations of IL-2_C01_T3A and its variants are summarized in Table 2 below. Each IL-2_C01_T3A-derived variant possesses additional mutations with T3A substitution, Q11C substitution, and I129C substitution in the IL-2_WT backbone. To facilitate protein purification and identification, IL-2_WT_3TA, IL-2_C01_T3A, and IL-2_C01_T3A-derived variants were fused with a His tag to produce H8-IL-2_WT_T3A (SEQ No. 52), H8-IL-2_C01_T3A (SEQ No. 53), and H8-IL-2_C01#1 to #15 (SEQ Nos. 54 to 68) H8-IL-2_C01_T3A-derived variants.

[0328] [Table 2]

[0329]

[0330] Example 5. Production and purification of H8-IL-2_C01_T3A-derived variant

[0331] IL-2_C01_T3A fused with His tag and its variants were expressed in Expi293 and ExpiCHO cells according to the same procedure as described in Example 2.

[0332] As shown in Figure 3, H8-IL-2_C01_T3A and all 15 designed H8-IL-2_C01_T3A-derived variants were successfully produced and purified in Expi293 cells. These newly designed H8-IL-2_C01_T3A-derived variants were used in the following in vitro tests.

[0333] Example 6. Detection of IL-2 reporter signaling in HEK-Blue™ IL-2 cells and HEK-Blue™ CD122 / CD132 cells

[0334] The IL-2 reporter assay was performed according to the same procedure as described in Example 3. The degree of IL-2-pSTAT5 signaling activity was determined using the following formula: [(Optical density (OD) value at 630 nm of each test molecule at 600 pM (OD630) - OD630 of the cell-alone control) / (OD630 of H8-IL-2_C01_T3A at 600 pM - OD630 of the cell-alone control)] × 100%.

[0335] As shown in Figure 5, IL-2-pSTAT5 signaling activity induced by H8-IL-2_CO1_T3A at 600 pM was used as a 100% control. The higher this value, the stronger the IL-2-pSTAT5 signaling activity. Five "+" symbols indicate 81 to 100% of IL-2-pSTAT5 signaling activity, four "+" symbols indicate 61 to 80%, three "+" symbols indicate 41 to 60%, two "+" symbols indicate 21 to 40%, and one "+" symbol indicates 11 to 20%. A "-" symbol indicates 0 to 10% activity.

[0336] As shown in Figs. 4a through 4h and Fig. 5, the designed H8-IL-2_C01_T3A-derived variants can be classified into five groups based on their IL-2-pSTAT5 reporter signaling induction activity. Group 1, consisting of H8-IL-2_C01#1 (Figs. 4a and 4b) and H8-IL-2_C01#6 (Figs. 4c and 4d), was found to have a reduced ability to induce IL-2-pSTAT5 reporter signaling activity in HEK-Blue™ IL-2 cells, whereas this effect was not observed in HEK-Blue™ CD122 / CD132 cells. Group 2, consisting of H8-IL-2_C01#7 (Figs. 4c and 4d) and H8-IL-2_C01#9 (Figs. 4e and 4f), showed moderate ability to induce IL-2-pSTAT5 reporter signaling activity in HEK-Blue™ IL-2 cells, but no such effect was observed in HEK-Blue™ CD122 / CD132 cells. Group 3, including H8-IL-2_C01#2 (Figs. 4a and 4b), showed moderate ability to induce IL-2-pSTAT5 reporter signaling activity in HEK-Blue™ IL-2 cells, but a weak effect was observed in HEK-Blue™ CD122 / CD132 cells. Group 4, consisting of H8-IL-2_C01#3 (Figs. 4c and 4d), H8-IL-2_C01#4 (Figs. 4c and 4d), H8-IL-2_C01#10 (Figs. 4e and 4f), H8-IL-2_C01#12 (Figs. 4e and 4f), H8-IL-2_C01#14 (Figs. 4g and 4h), and H8-IL-2_C01#15 (Figs. 4g and 4h), showed potent induction of IL-2-pSTAT5 reporter signaling activity in HEK-Blue™ IL-2 cells, but this effect was absent in HEK-Blue™ CD122 / CD132 cells.Group 5, consisting of H8-IL-2_C01#5 (Figs. 4c and 4d), H8-IL-2_C01#8 (Figs. 4e and 4f), H8-IL-2_C01#11 (Figs. 4e and 4f), and H8-IL-2_C01#13 (Figs. 4g and 4h), showed not only potent induction of IL-2-pSTAT5 reporter signaling activity in HEK-Blue™ IL-2 cells but also relatively potent induction of IL-2-pSTAT5 reporter signaling activity in HEK-Blue™ CD122 / CD132 cells.

[0337] Example 7. Detection of STAT5 phosphorylation in Treg and Tconv cells

[0338] The STAT5 phosphorylation activity of Treg and Tconv cells induced by the designed IL-2 molecule was measured as follows. Treg and Tconv cells were EasySep™ Human CD4 + CD127 low CD25 +Treg cells were isolated from human peripheral blood mononuclear cells (PBMCs) using the Regulatory T Cell Isolation Kit (STEMCELL™ Technologies, Cat. No.: 18063). Treg cells were incubated with Dynabeads™ Human T-Activator CD3 / CD28 beads (Thermo Fisher Scientific, Cat. No.: 11131D) in a Treg culture medium consisting of X-VIVO™15 medium (Lonza, Cat. No.: 04-418Q), 5% (v / v) human AB serum (Sigma-Aldrich, Cat. No.: H5667), 1 nM rapamycin (sirolimus) (Selleck Chemicals, Cat. No.: S1039), and Corning®100× penicillin-streptomycin solution (CORNING, Cat. No.: 30-002-CI) at 37°C for 6 days. Tconv cells were cultured in Tconv culture medium supplemented with 50 IU / mL of recombinant human IL-2 (BioLegend, Cat. No.: 589106). The Tconv culture medium was Gibco ® RPMI-1640 medium (Fisher Scientific, Cat. No.: 22400-089), 10% (v / v) heat-treated inactivated Gibco ® FBS (Fisher Scientific, Cat. No.: A31606-01) and 100x Corning ® The cells were treated with penicillin-streptomycin solution (CORNING, Cat. No.: 30-002-CI) and cultured at 37°C for 6 days. After overnight resting, Treg and Tconv cells were placed in assay medium consisting of RPMI-1640, 10% (v / v) heat-treated inactivated FBS, and 100-fold penicillin-streptomycin solution, in a quantity of 2 × 10⁶ cells. 5The cells were resuspended at a density of cells / mL. 100 μL of Treg or Tconv cells were incubated in serially diluted designed H8-IL-2_C01_T3A-derived variants and corresponding control proteins in 96-well round-bottom TC-treated microplates (SPL Life Sciences, Cat. No.: 34096) at 37°C for 30 minutes. After incubation, cells were harvested and fixed in 4% paraformaldehyde (PFA) solution (Sigma-Aldrich, Cat. No.: P6148) at 37°C for 15 minutes. Then, cells were washed with FACS buffer (PBS supplemented with 4% FBS) and permeated with BD Phosflow™ Perm Buffer III (BD Biosciences, Cat. No.: 558050) at 4°C for 30 minutes. To measure STAT5 phosphorylation levels, after washing cells with FACS buffer, Phospho-Stat5(Tyr694)(C71E5) Rabbit mAb(Alexa Fluor ® The samples were stained with 647 conjugate, Cell Signaling Technology, Cat. No.: 9365), BD Horizon™ BV650 mouse anti-human CD25 antibody (BD Biosciences, Cat. No.: 563719), CD4 monoclonal antibody (RPA-T4), PE-Cyanine5, eBioscience™ (Invitrogen, Cat. No.: 15-0049-42) at 4°C for 30 minutes. Flow cytometry was performed using a CytoFLEX flow cytometer (Beckman Coulter Inc.).

[0339] As shown in Figures 6a to 6f, the designed H8-IL-2_C01_T3A-derived variants can be classified into five groups based on the level of STAT5 phosphorylation. Group 1, consisting of H8-IL-2_C01#6, exhibited weak STAT5 phosphorylation-inducing activity in Treg cells and did not exhibit STAT5 phosphorylation-inducing activity in Tconv cells. Group 2, consisting of H8-IL-2_C01#1, H8-IL-2_C01#3, H8-IL-2_C01#4, H8-IL-2_C01#9, H8-IL-2_C01#10, and H8-IL-2_C01#12, exhibited weak STAT5 phosphorylation-inducing activity in both Treg cells and Tconv cells. Group 3, consisting of H8-IL-2_C01#7, had a relatively strong ability to induce STAT5 phosphorylation in Treg cells, but the effect was weak in Tconv cells. Group 4, consisting of H8-IL-2_C01#2, H8-IL-2_C01#5, H8-IL-2_C01#8, H8-IL-2_C01#11, H8-IL-2_C01#13, H8-IL-2_C01#14, and H8-IL-2_C01#15, had stronger STAT5 phosphorylation-inducing activity in Treg cells and also showed a relatively strong effect in Tconv cells.

[0340] Figure 7 summarizes the IL-2 reporter signaling results induced by the H8-IL-2_C01_T3A-derived variant in HEK-Blue™ IL-2 cells and HEK-Blue™ CD122 / CD132 cells, and the STAT5 phosphorylation results induced by the H8-IL-2_C01_T3A-derived variant in Treg cells and Tconv cells. The IL-2 reporter signaling results induced by the H8-IL-2_C01_T3A-derived variant are described in detail in Figure 5 and Example 6. In the pSTAT5 detection results of Figure 7, a higher value indicates more potent STAT5 phosphorylation, and indicates a stronger STAT5 phosphorylation-inducing effect of the designed H8-IL-2_C01_T3A-derived variant. Five "+" symbols indicate the strongest STAT5 phosphorylation induced by the H8-IL-2_C01_T3A-derived variant, four "+" symbols indicate stronger induction, three indicate moderate induction, two indicate weaker induction, and one indicate weakest induction. A "-" symbol indicates no STAT5 phosphorylation.

[0341] Based on the IL-2-pSTAT5 reporter activity and pSTAT5 results shown in Figure 7, the designed H8-IL-2_C01_T3A-derived variants could be classified into several groups with different characteristics. H8-IL-2_C01#2, H8-IL-2_C01#5, H8-IL-2_C01#8, H8-IL-2_C01#11, and H8-IL-2_C01#13 exhibited relatively strong IL-2-pSTAT5 signaling-inducing activity in HEK-Blue™ CD122 / CD132 cells and showed stronger effects in Tconv cells compared to other variants. Therefore, these five molecules were excluded from further studies. The remaining 10 H8-IL-2_C01_T3A-derived variant clones were selected for G1LALAFc-IL-2 synthesis and in vitro testing.

[0342] Example 8. Design of G1LALAFc-IL-2_C01_T3A-derived variant

[0343] G1LALAFc-IL-2_WT_T3A (SEQ No. 73), G1LALAFc-IL-2_C01_T3A (SEQ No. 74), and G1LALAFc-IL-2_C01#1, G1LALAFc-IL-2_C01#3, G1LALAFc-IL-2_C01#4, G1LALAFc-IL-2_C01#6, G1LALAFc-IL-2_C01#7, G1LALAFc-IL-2_C01#9, G1LALAFc-IL-2_C01#10, G1LALAFc-IL-2_C01#12, G1LALAFc-IL-2_C01#14, and G1LALAFc-IL-2_C01#15 (SEQ Nos. 75 to 84) The amino acid sequences of variants derived from G1LALAFc-IL-2_C01_T3A- are listed in FIG. 12. Each variant derived from G1LALAFc-IL-2_C01_T3A- comprises a hinge region (Sequence No. 70), a G1LALAFc region (Sequence No. 71), and a linker region (Sequence No. 72).

[0344] Example 9. Production and purification of G1LALAFc-IL-2_C01_T3A-derived variant

[0345] G1LALAFc, an IL-2_C01_T3A-derived variant fused with the Fc domain, was expressed in Expi293 cells. The expressed proteins were purified by affinity chromatography using a column packed with MabSelect PrismA™ Protein A chromatography resin (Cytiva, Cat. No.: 17549803) and size exclusion chromatography (SEC) using a Superdex® 200 Increase 10 / 300 GL column (Cytiva, Cat. No.: 28990944). The purified proteins were also analyzed by SDS-PAGE and HPLC-SEC.

[0346] As shown in Fig. 8, ten variants of G1LALAFc-IL-2_WT_T3A, G1LALAFc-IL-2_C01_T3A, and G1LALAFc-IL-2_C01_T3A were successfully produced and purified. These proteins were tested in vitro as follows.

[0347] Example 10. Detection of IL-2 reporter signaling in HEK-Blue™ IL-2 cells and HEK-Blue™ CD122 / CD132 cells

[0348] IL-2 reporter assays were performed according to the same procedure as described in Examples 3 and 6. The degree of IL-2-pSTAT5 signaling activity was determined using the following formula: [(Optical density (OD) value at 630 nm of each test molecule at 600 pM (OD630) - OD630 of the cell-alone control) / (OD630 of H8-IL-2_C01_T3A or G1LALAFc-IL-2_C01_T3A at 600 pM - OD630 of the cell-alone control)] × 100%.

[0349] As shown in Fig. 10, IL-2-pSTAT5 signaling activity induced by H8-IL-2_C01_T3A or G1LALAFc-IL-2_C01_T3A at 600 pM was used as a 100% control. The higher this value, the stronger the IL-2-pSTAT5 signaling activity. Five "+" symbols represent 81 to 100% of IL-2-pSTAT5 signaling activity, four "+" symbols represent 61 to 80%, three "+" symbols represent 41 to 60%, two "+" symbols represent 21 to 40%, and one "+" symbol represents 11 to 20%. A "-" symbol represents 0% to 10% of IL-2-pSTAT5 signaling activity.

[0350] As shown in Figures 9a through 9f, the designed G1LALAFc-IL-2_C01_T3A-derived variants can be classified into five groups based on IL-2-pSTAT5 reporter signaling activity. Group 1, consisting of G1LALAFc-IL-2_C01#1, exhibited moderate IL-2-pSTAT5 reporter signaling-inducing activity in both HEK-Blue™ IL-2 cells and HEK-Blue™ CD122 / CD132 cells. Group 2, consisting of G1LALAFc-IL-2_C01#3, #4, #6, #9, and #10, exhibited stronger IL-2-pSTAT5 reporter signaling-inducing activity in HEK-Blue™ IL-2 cells but no such activity in HEK-Blue™ CD122 / CD132 cells. Group 3, consisting of G1LALAFc-IL-2_C01#7 and #14, showed stronger IL-2-pSTAT5 reporter signaling-inducing activity in HEK-Blue™ IL-2 cells but moderate activity in HEK-Blue™ CD122 / CD132 cells. Group 4, consisting of G1LALAFc-IL-2_C01#15, showed stronger IL-2-pSTAT5 reporter signaling-inducing activity in HEK-Blue™ IL-2 cells and relatively stronger activity in HEK-Blue™ CD122 / CD132 cells.

[0351] As shown in Figure 10, the G1LALAFc type-IL-2 variant exhibited a stronger effect than the H8 type-IL-2 variant in inducing IL-2-pSTAT5 reporter signaling activity. However, the relative ranking of IL-2-pSTAT5 reporter signaling-inducing activity was indicated as "+" or "-", which was similar for both IL-2 types.

[0352] Example 11. Detection of STAT5 phosphorylation in Treg and Tconv cells

[0353] The STAT5 phosphorylation activity of Treg and Tconv cells induced by the designed IL-2 molecule was measured as described in Example 7. As shown in FIGS. 11a to 11d, based on STAT5 phosphorylation levels, G1LALAFc-IL-2_WT_T3A, G1LALAFc-IL-2_C01_T3A, and G1LALAFc-IL-2_C01#1, G1LALAFc-IL-2_C01#3, G1LALAFc-IL-2_C01#4, G1LALAFc-IL-2_C01#7, G1LALAFc-IL-2_C01#9, G1LALAFc-IL-2_C01#10, G1LALAFc-IL-2_C01#12, G1LALAFc-IL-2_C01#14, and G1LALAFc-IL-2_C01#15 G1LALAFc-IL-2_C01#15-derived variants are Treg Although STAT5 phosphorylation could be induced in both Treg cells and Tconv cells, the levels of activity varied. Only G1LALAFc-IL-2_C01#6 was able to induce STAT5 phosphorylation in Treg cells, but not in Tconv cells. Based on these preliminary experimental results, IL-2_C01#6 showed potential in the treatment of autoimmune diseases.

[0354] Example 12. Evaluation of stress stability of G1LALAFc-IL-2_C01#6

[0355] The stress stability of G1LALAFc-IL-2_WT, G1LALAFc-IL-2_C01#6, and MK-6194 analog (SEQ No. 91) was evaluated at 40°C for 1, 2, and 4 weeks. MK-6194 analog is an engineered IL-2 variant fused to a protein backbone designed to selectively activate and proliferate regulatory T cells (Treg) for potential treatment of ulcerative colitis and other autoimmune diseases. MK-6194 analog is a protein formed by fusing human IL-2 variants with L118I, N88D, V69A, Q74P, and C125S mutations to the N-terminus of human IgG1 Fc domains with L234A, L235A, and G237A mutations via a G4Sx4 linker (SEQ No. 92).

[0356] Specifically, each sample was placed in a new Eppendorf flask and immersed in 150 μL of protein solution, and incubated for 1, 2, and 4 weeks in a Constant Climate Chamber HPP110eco (Memmert GmbH) under conditions of 40°C ± 2°C / 75% ± 5% RH. At each time point, the Eppendorf flask containing 150 μL of protein solution was removed from the Constant Climate Chamber and stored in a 4°C refrigerator until the end of the experiment. After the experiment, all samples were filtered through a 0.2 μm filter prior to analysis and then analyzed using an ACQUITY UPLC H-Class (Waters Corporation).

[0357] The results are shown in Figures 12a to 12c. According to the experimental results, the SEC-HPLC profiles of G1LALAFc-IL-2_WT (Figure 12a) and G1LALAFc-IL-2_C01#6 (Figure 12b) were similar after incubation at 40°C for 4 weeks. However, the HMW (%) of the MK-6194 analog (Figure 12c) increased by more than 40%. These results demonstrated that G1LALAFc-IL-2_C01#6 exhibited better stress stability than the MK-6194 analog.

[0358] Example 13. Determination of the thermal stability of G1LALAFc-IL-2_C01#6 by DLS

[0359] The thermal stability of G1LALAFc-IL-2_WT, G1LALAFc-IL-2_C01#6, and MK-6194 analogs was measured by dynamic light scattering (DLS) analysis. Specifically, the samples were centrifuged at 12,000 rpm for 10 minutes at 8°C. 26 μL of each supernatant and 10 μL of silicone oil (Alfa Aesar, Cat. No.: A12728) were added to the wells of a 384LV plate (AURORA, Cat. No.: P8806-38403-10). The plates were centrifuged at 1,500 rpm for 10 minutes at 25°C to remove air bubbles and sealed with UltraClear Real Time PCR Pressured Sealing Film (SMBIO, Cat. No.: P4F51-C). The plates were analyzed using a DynaPro Plate Reader III (WYATT Technology) with continuous temperature ramp measurement through dynamic light scattering. The raw data were analyzed and fitted using DYNAMICS 7.10.1.21 to T onset The value of was determined.

[0360] The results are shown in Fig. 13. According to the results, the onset temperature (T) of G1LALAFc-IL-2_C01#6 onset ) was approximately 62℃; and T of G1LALAFc-IL-2_WT onset It was 53℃, and the T of the MK-6194 analogue onset It was 45℃. Compared to G1LALAFc-IL-2_WT and MK-6194 analogs, G1LALAFc-IL-2_C01#6 was T onset This was found to have increased significantly. These results demonstrated that G1LALAFc-IL-2_C01#6 exhibited better stability than G1LALAFc-IL-2_WT and MK-6194 analogs.

[0361] Example 14. Determination of the thermal stability of G1LALAFc-IL-2_C01#6 by DSF

[0362] The thermal stability of G1LALAFc-IL-2_WT, G1LALAFc-IL-2_C01#6, and MK-6194 analogs was determined using differential scanning fluorescence (DSF). Specifically, the selected proteins were resuspended in DI water at appropriate concentrations, and SYPRO ®A 50-fold stock solution was prepared by diluting 2.5 μL of a 5,000-fold concentrate of Orange Protein Gel Stain (Sigma-Aldrich, Cat. No.: S5692) in 250 μL of DI water. 10 μL of the protein solution was dispensed into each well of a Hard-Shell PCR plate (Bio-Rad, Cat. No.: HSP9601). 2.5 μL of the 50-fold SYPRO stock solution and 12.5 μL of buffer solution were added to achieve a final 5-fold SYPRO concentration in each well. This assay was performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.), and the protocol was set to a temperature analysis range increasing by 0.3°C every 10 seconds from 20°C to 95°C. Fluorescence was recorded at the end of each increase. The melting temperature (Tm) was determined by analyzing the raw fluorescence data and performing a nonlinear fitting of the thermal denaturation data.

[0363] The results are shown in Fig. 14. According to the results, the melting temperature (Tm) of G1LALAFc-IL-2_C01#6 was approximately 65°C; the Tm of G1LALAFc-IL-2_WT was 56°C; and the Tm of the MK-6194 analog was approximately 45°C. Compared to G1LALAFc-IL-2_WT and the MK-6194 analog, G1LALAFc-IL-2_C01#6 showed a significantly increased melting temperature. These results demonstrated that G1LALAFc-IL-2_C01#6 exhibited better stability than G1LALAFc-IL-2_WT and the MK-6194 analog.

[0364] Example 15. Evaluation of the autoimmune efficacy of G1LALAFc-IL-2_C01#6

[0365] The autoimmune efficacy of G1LALAFc-IL-2_C01#6 was evaluated in an NPG extension model. The treatment plan and administration schedule are shown in Figure 15a. Specifically, 8-week-old NPG mice were irradiated to eliminate endogenous immune cells, and 5 × 10 6 Human peripheral blood mononuclear cells (PBMCs) were injected intravenously (iv). Treatment group mice were injected intraperitoneally (ip) with 3 μg of G1LALAFc-IL-2_WT (G2), 1 μg of G1LALAFc-IL-2_C01#6 (G3), or 1 μg of MK-6194 analog (G4) per mouse. Control group mice (G1) were injected with 100 μL of vehicle (isotonic saline) per mouse. Treatment began on day 7 after irradiation and human peripheral blood mononuclear cell (PBMC) transplantation, and injections were administered once a week for a total of 3 weeks. To monitor for graft-versus-host disease (GvHD), mice were individually scored on a 0 to 2 point scale for five clinical indicators (body weight loss, posture, activity, coat, and skin) and on a 0 to 1 point scale for one additional clinical indicator (diarrhea). The maximum total GvHD score is 11 points. If the total GvHD score exceeded 5 points, or if the GvHD score for a single clinical indicator (body weight loss, posture, activity, coat, skin) was 2 points, the mouse was sacrificed.

[0366] The results are shown in Fig. 15b. As shown in the results, G1LALAFc-IL-2_C01#6 significantly delayed the onset of GvHD symptoms when compared to G1LALAFc-IL-2_WT and MK-6194 analogs. These results indicated that G1LALAFc-IL-2_C01#6 has excellent efficacy against autoimmune-related diseases such as GvHD.

[0367] Example 16. Evaluation of PK, drug-related hepatotoxicity, and T cell population induction ability of G1LALAFc-IL-2_C01#6

[0368] Effect on weight

[0369] The T cell population induction ability of G1LALAFc-IL-2_C01#6 was evaluated in a C57BL / 6 model. The treatment plan and administration schedule are shown in Figure 16a. Specifically, 6 to 8-week-old C57BL / 6 mice were injected intraperitoneally (ip) with (1) 3 mg / kg of G1LALAFc-IL-2_C01#6 (G2), G1LALAFc-IL-2_WT (G4), G1LA-KIHFc-IL-2_C01#6 (G6), or G1LA-KIHFc-IL-2_WT (G8), or (2) 0.3 mg / kg of G1LALAFc-IL-2_C01#6 (G3), G1LALAFc-IL-2_WT (G5), G1LA-KIHFc-IL-2_C01#6 (G7), or G1LA-KIHFc-IL-2_WT (G9). 10 mL / kg of vehicle (isotonic saline) was injected into control mice (G1).

[0370] G1LA-KIHFc-IL-2_WT comprises a hole chain comprising G1LA-HoleFc-IL-2_WT (e.g., SEQ ID NO. 97) and a knob chain comprising G1 hinge + G1LA-KnobFc (e.g., SEQ ID NO. 96). G1LA-KIHFc-IL-2_C01#6 comprises a hole chain comprising G1LA-HoleFc-IL-2_C01#6 (e.g., SEQ ID NO. 98) and a knob chain comprising G1 hinge + G1LA-KnobFc (e.g., SEQ ID NO. 96).

[0371] The results are shown in Figure 16b. According to the in vivo results, C57BL / 6 mice experienced a decrease in body weight upon administration of high doses (3 mg / kg) of G1LALAFc-IL-2_WT and G1LA-KIHFc-IL-2_WT (however, it recovered by the end of the experiment). On the other hand, G1LALAFc-IL-2_C01#6 and G1LA-KIHFc-IL-2_C01#6 did not affect the body weight of C57BL / 6 mice.

[0372] pharmacokinetics

[0373] Pharmacokinetics (PK) and drug-associated hepatotoxicity were determined. Blood was collected from the mandibular (facial) vein into a MiniCollect® TUBE (1 mL K3E K3EDTA with a purple cap) (GreinerBio-One, Cat. No.: 450531). Serum was collected by centrifugation at 3,000 rpm for 10 minutes. The supernatant (serum) was transferred to a new Eppendorf tube and stored at -20°C. For pharmacokinetic (PK) analysis, IL-2 serum concentrations were measured using an ELISA MAX™ Deluxe Set Human IL-2 (BioLegend®, Cat. No.: 431816). For drug-associated hepatotoxicity analysis, AST (aspartate aminotransferase) and ALT (alanine aminotransferase) analysis of the serum samples was performed at the National Laboratory Animal Center (NLAC).

[0374] PK results are shown in Figs. 16c to 16l. Serum concentration analysis indicated that (1) the G1LA-KIHFc-IL-2 form had better PK results than the G1LALAFc-IL-2 form, and (2) IL-2_C01#6 had superior PK results compared to IL-2_WT (see Figs. 16c and 16d). Additionally, this data indicated that while IL-2_WT at a high dose (3 mg / kg) decreased significantly on day 3, IL-2_C01#6 was more stable and remained detectable on day 7 (see Figs. 16e to 16f for the G1LALAFc-IL-2 form and Figs. 16i to 16j for the G1LA-KIHFc-IL-2 form). IL-2_WT at the low dose (0.3 mg / kg) also decreased significantly on day 3, but IL-2_C01#6 was more stable and indicated to still be detectable on day 7 (see Figs. 16g to 16h for the G1LALAFc-IL-2 form and Figs. 16k to 16l for the G1LA-KIHFc-IL-2 form). The dashed line indicates the limit of detection (LoD) value of the ELISA kit, and serum concentrations of test samples below the LoD were excluded. The limit of detection (LoD) of the ELISA kit was 7.8 pg / mL.

[0375] liver toxicity

[0376] Drug-related hepatotoxicity was determined using ASL and ALT analyses. The results are shown in Figures 17a and 17b. Normal AST levels in mice are 50 to 100 U / L, and normal ALT levels are 25 to 60 U / L. The AST levels (Figure 17a) and ALT levels (Figure 17b) detected after IL-2_C01#6 treatment were both within the normal range. Therefore, IL-2_C01#6 treatment was found not to cause liver damage.

[0377] T cell population induction ability

[0378] The T cell population induction ability of G1LALAFc-IL-2_C01#6 was evaluated in the C57BL / 6 model. Splenocytes were isolated from the spleen and treated with 1X RBC lysis buffer to remove red blood cells. Cells were stained using the following reagents: Zombie Aqua™ Fixable Viability Kit (BioLegend®, Cat. No.: 423102), PE anti-mouse CD45 antibody (BioLegend®, Cat. No.: 103106), FITC anti-mouse CD3 antibody (BioLegend®, Cat. No.: 100204), PE / Dazzle™ 594 anti-mouse CD4 antibody (BioLegend®, Cat. No.: 100456), Brilliant Violet 605™ anti-mouse CD8a antibody (BioLegend®, Cat. No.: 100744), Brilliant Violet 650™ anti-mouse CD25 antibody (BioLegend®, Cat. No.: 102038), PE / Cyanine7 anti-mouse CD49b (pan-NK cells) Antibodies (BioLegend®, Cat. No.: 108922), Alexa Fluor® 700 anti-mouse NK-1.1 antibody (BioLegend®, Cat. No.: 108730), Alexa Fluor® 647 anti-mouse FOXP3 antibody (BioLegend®, Cat. No.: 126408), and eBioscience™ Foxp3 / transcription factor staining buffer set (Thermo Fisher Scientific, Cat: 00-5523-00). Flow cytometry was performed using CytoFLEX (Beckman Coulter Inc.).

[0379] The results are shown in Figures 18a to 18d. According to the analysis of the mouse spleen T cell population, CD3 + The percentage of T cells (Fig. 18a) was not significantly affected on days 3 and 7 after IL-2 treatment. CD8 +The percentages of different T cell populations, including T cells (Fig. 18b), Treg cells (Fig. 18c), and NK cells (Fig. 18d), returned to baseline levels similar to the vehicle group on day 7 after treatment. On day 3 after treatment, a high dose (3 mg / kg) of IL-2_C01#6 significantly induced a Treg population in the spleen (Fig. 18c), and a high dose (3 mg / kg) of IL-2_WT induced CD8 in the spleen + It induced T cell (Fig. 18b) and NK cell (Fig. 18d) populations. These results indicate that IL-2_C01#6 can induce Treg populations, but CD8 + It indicated that T cell or NK cell populations were not induced.

[0380] Other embodiments

[0381] Although the present invention has been described together with its detailed description, the foregoing description is intended to illustrate the scope of the invention and not to limit it, and the scope of the invention is defined by the scope of the appended claims. Other embodiments, advantages, and variations are within the scope of the following claims.

Claims

Claim 1 Engineered IL-2 polypeptide containing non-natural cysteine. Claim 2 In claim 1, a engineered IL-2 polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO.

2. Claim 3 A engineered IL-2 polypeptide capable of binding to the trimer IL-2 receptor complex IL-2Rαβγ in claim 1 or 2. Claim 4 In any one of claims 1 to 3, a engineered IL-2 polypeptide comprising one or more of the following: (a) the amino acid corresponding to position 11 of SEQ ID NO 2 is cysteine ​​(C); (b) the amino acid corresponding to position 14 of SEQ ID NO 2 is C; (c) the amino acid corresponding to position 18 of SEQ ID NO 2 is C; (d) the amino acid corresponding to position 40 of SEQ ID NO 2 is C; (e) the amino acid corresponding to position 43 of SEQ ID NO 2 is C; (f) the amino acid corresponding to position 45 of SEQ ID NO 2 is C; (g) the amino acid corresponding to position 46 of SEQ ID NO 2 is C; (h) the amino acid corresponding to position 48 of SEQ ID NO 2 is C; (i) the amino acid corresponding to position 53 of SEQ ID NO 2 is C; (j) the amino acid corresponding to position 56 of SEQ ID NO 2 is C; and (k) the amino acid corresponding to position 7 of SEQ ID NO 2 is C. Claim 5 In any one of claims 1 to 4, a engineered IL-2 polypeptide comprising one or more of the following: (a) the amino acid corresponding to position 129 of SEQ ID NO. 2 is C; (b) the amino acid corresponding to position 113 of SEQ ID NO. 2 is C; (c) the amino acid corresponding to position 111 of SEQ ID NO. 2 is C; (d) the amino acid corresponding to position 108 of SEQ ID NO. 2 is C; (e) the amino acid corresponding to position 106 of SEQ ID NO. 2 is C; (f) the amino acid corresponding to position 96 of SEQ ID NO. 2 is C; (g) the amino acid corresponding to position 97 of SEQ ID NO. 2 is C; (h) the amino acid corresponding to position 129 of SEQ ID NO. 2 is C; and (i) the amino acid corresponding to position 132 of SEQ ID NO. 2 is C. Claim 6 In any one of claims 1 to 5, the engineered IL-2 polypeptide comprising one or more of the following: (a) the amino acid corresponding to position 11 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 129 of SEQ ID NO. 2 is C; (b) the amino acid corresponding to position 14 of SEQ ID NO. 2 is C; (c) the amino acid corresponding to position 18 of SEQ ID NO. 2 is C; (d) the amino acid corresponding to position 40 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 113 of SEQ ID NO. 2 is C; (e) the amino acid corresponding to position 43 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 111 of SEQ ID NO. 2 is C; (f) the amino acid corresponding to position 45 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 111 of SEQ ID NO. 2 is C; (g) the amino acid corresponding to position 46 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 108 of SEQ ID NO. 2 is (h) The amino acid corresponding to position 48 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 106 of SEQ ID NO. 2 is C; (i) The amino acid corresponding to position 53 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 96 of SEQ ID NO. 2 is C; (j) The amino acid corresponding to position 53 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 97 of SEQ ID NO. 2 is C; (k) The amino acid corresponding to position 56 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 97 of SEQ ID NO. 2 is C; (l) The amino acid corresponding to position 11 of SEQ ID NO. 2 is C, the amino acid corresponding to position 18 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 129 of SEQ ID NO. 2 is C; (m) The amino acid corresponding to position 7 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 132 of SEQ ID NO. 2 is C; (n) The amino acid corresponding to position 7 of SEQ ID NO. 2 is It is C, the amino acid corresponding to position 18 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 132 of SEQ ID NO. 2 is C;and (o) the amino acid corresponding to position 7 of SEQ ID NO. 2 is C, the amino acid corresponding to position 11 of SEQ ID NO. 2 is C, the amino acid corresponding to position 129 of SEQ ID NO. 2 is C, and the amino acid corresponding to position 132 of SEQ ID NO. 2 is C.; Claim 7 In any one of claims 1 to 6, the amino acid corresponding to position 3 of SEQ ID NO. 2 is A, and the engineered IL-2 polypeptide comprises one or more of the following: (a) the amino acid corresponding to position 15 of SEQ ID NO. 2 is D; (b) the amino acid corresponding to position 15 of SEQ ID NO. 2 is N; (c) the amino acid corresponding to position 16 of SEQ ID NO. 2 is S; (d) the amino acid corresponding to position 16 of SEQ ID NO. 2 is T; (e) the amino acid corresponding to position 19 of SEQ ID NO. 2 is I; (f) the amino acid corresponding to position 20 of SEQ ID NO. 2 is N; (g) the amino acid corresponding to position 23 of SEQ ID NO. 2 is I; (h) the amino acid corresponding to position 23 of SEQ ID NO. 2 is L; (i) the amino acid corresponding to position 84 of SEQ ID NO. 2 is N; (j) the amino acid corresponding to position 84 of SEQ ID NO. 2 is T; (k) The amino acid corresponding to position 91 of SEQ ID NO 2 is L; (l) the amino acid corresponding to position 92 of SEQ ID NO 2 is V; (m) the amino acid corresponding to position 92 of SEQ ID NO 2 is M; (n) the amino acid corresponding to position 95 of SEQ ID NO 2 is Q; and (o) the amino acid corresponding to position 95 of SEQ ID NO 2 is N. Claim 8 A engineered IL-2 polypeptide comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs 3 to 17 and 35 to 51 in any one of claims 1 to 7. Claim 9 In claim 8, a engineered IL-2 polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO. 3 or SEQ ID NO.

14. Claim 10 In claim 8, a engineered IL-2 polypeptide comprising an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs 39, 40, 46, 48, 50, and 51. Claim 11 In claim 8, a engineered IL-2 polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO.

43. Claim 12 In claim 8, a engineered IL-2 polypeptide comprising an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs 39, 40, 42, 45, and 46. Claim 13 In claim 8, a engineered IL-2 polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO.

43. Claim 14 A engineered IL-2 polypeptide capable of selectively activating Treg cells while minimizing the activation of Tconv cells, in any one of claims 1 to 13. Claim 15 Engineered IL-2 polypeptide containing one or more non-natural disulfide bonds. Claim 16 In claim 15, a engineered IL-2 polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO.

2. Claim 17 In claim 15 or 16, a engineered IL-2 polypeptide capable of binding to the trimer IL-2 receptor complex IL-2Rαβγ. Claim 18 In any one of claims 15 to 17, the engineered IL-2 polypeptide comprising one or more of the following: (a) a non-natural disulfide bond formed between the amino acid corresponding to position 11 of SEQ ID NO. 2 and the amino acid corresponding to position 129 of SEQ ID NO. 2; (b) a non-natural disulfide bond formed between the amino acid corresponding to position 14 of SEQ ID NO. 2 and the amino acid corresponding to position 125 of SEQ ID NO. 2; (c) a non-natural disulfide bond formed between the amino acid corresponding to position 18 of SEQ ID NO. 2 and the amino acid corresponding to position 125 of SEQ ID NO. 2; (d) a non-natural disulfide bond formed between the amino acid corresponding to position 40 of SEQ ID NO. 2 and the amino acid corresponding to position 113 of SEQ ID NO. 2; (e) a non-natural disulfide bond formed between the amino acid corresponding to position 43 of SEQ ID NO. 2 and the amino acid corresponding to position 111 of SEQ ID NO. 2; (f) an amino acid corresponding to position 45 of SEQ ID NO. 2 and (g) a non-natural disulfide bond formed between the amino acid corresponding to position 111 of SEQ ID NO. 2 and the amino acid corresponding to position 108 of SEQ ID NO. 2; (h) a non-natural disulfide bond formed between the amino acid corresponding to position 48 of SEQ ID NO. 2 and the amino acid corresponding to position 106 of SEQ ID NO. 2; (i) a non-natural disulfide bond formed between the amino acid corresponding to position 53 of SEQ ID NO. 2 and the amino acid corresponding to position 96 of SEQ ID NO. 2; (j) a non-natural disulfide bond formed between the amino acid corresponding to position 53 of SEQ ID NO. 2 and the amino acid corresponding to position 97 of SEQ ID NO. 2; (k) a non-natural disulfide bond formed between the amino acid corresponding to position 56 of SEQ ID NO. 2 and the amino acid corresponding to position 97 of SEQ ID NO. 2;(l) a non-natural disulfide bond formed between the amino acid corresponding to position 11 of SEQ ID NO. 2 and the amino acid corresponding to position 129 of SEQ ID NO. 2, and a non-natural disulfide bond formed between the amino acid corresponding to position 18 of SEQ ID NO. 2 and the amino acid corresponding to position 125 of SEQ ID NO. 2; (m) a non-natural disulfide bond formed between the amino acid corresponding to position 7 of SEQ ID NO. 2 and the amino acid corresponding to position 132 of SEQ ID NO. 2; (n) a non-natural disulfide bond formed between the amino acid corresponding to position 7 of SEQ ID NO. 2 and the amino acid corresponding to position 132 of SEQ ID NO. 2, and a non-natural disulfide bond formed between the amino acid corresponding to position 18 of SEQ ID NO. 2 and the amino acid corresponding to position 125 of SEQ ID NO. 2; and (o) a non-natural disulfide bond formed between the amino acid corresponding to position 7 of SEQ ID NO. 2 and the amino acid corresponding to position 132 of SEQ ID NO. 2, and a non-natural disulfide bond formed between the amino acid corresponding to position 11 of SEQ ID NO. 2 and the amino acid corresponding to position 129 of SEQ ID NO. 2.; Claim 19 A engineered IL-2 polypeptide comprising at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to any one of SEQ ID NOs 3 to 17 and 35 to 51 in any one of claims 15 to 18. Claim 20 A fusion protein comprising the engineered IL-2 polypeptide of any one of claims 1 to 19. Claim 21 A fusion protein according to claim 20 comprising at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to any one of SEQ ID NOs 20 to 34 and 52 to 68. Claim 22 A fusion protein comprising an Fc region further comprising the Fc region in claim 20 or 21. Claim 23 A fusion protein according to claim 22, wherein the Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO.

71. Claim 24 A fusion protein according to claim 22, wherein the Fc region comprises (1) a whole chain having an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 93 and (2) a Knob chain having an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO.

95. Claim 25 In paragraph 24, the engineered IL-2 polypeptide is a fusion protein linked to a whole chain or a knob chain. Claim 26 In paragraph 25, the engineered IL-2 polypeptide is a fusion protein linked to a whole chain. Claim 27 A fusion protein comprising a hinge region further comprising any one of claims 20 to 26. Claim 28 In claim 27, the hinge region comprises a fusion protein having an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO.

70. Claim 29 In any one of claims 20 to 24, the engineered IL-2 polypeptide is a fusion protein connected to the C-terminus or N-terminus of the Fc region via a linker peptide. Claim 30 In claim 29, the linker peptide is a fusion protein comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO.

72. Claim 31 A fusion protein comprising at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to any one of SEQ ID NOs 73 to 84 and 96 to 98 in any one of claims 20 to 30. Claim 32 A fusion protein comprising, optionally from the N-terminus to the C-terminus, (a) a hinge region; (b) an Fc region; (c) a linker peptide; and (d) a engineered IL-2 polypeptide. Claim 33 In claim 32, the hinge region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 70; the Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 71; and the linker peptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 72, and / or; A fusion protein comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs 3 to 17 and 35 to 51. Claim 34 A fusion protein capable of increasing Treg activation (e.g., phosphorylation of STAT5) in any one of claims 20 to 33. Claim 35 A fusion protein capable of increasing Treg-mediated immunosuppression in any one of claims 20 to 34. Claim 36 A pharmaceutical composition comprising: a engineered IL-2 polypeptide of any one of claims 1 to 19 or a fusion protein of any one of claims 20 to 35; and a pharmaceutically acceptable carrier. Claim 37 A nucleic acid encoding the engineered IL-2 polypeptide of any one of claims 1 to 19 or the fusion protein of any one of claims 20 to 35. Claim 38 A vector containing the nucleic acid of Article 37. Claim 39 A cell containing the nucleic acid of claim 37 or the vector of claim 38. Claim 40 In paragraph 39, a cell that is an Expi293 cell or an ExpiCHO cell. Claim 41 A method for producing a engineered IL-2 polypeptide or a fusion protein comprising an engineered IL-2 polypeptide, comprising: (a) culturing the cells of claim 39 or 40 under conditions sufficient for the cells to produce the engineered IL-2 polypeptide or fusion protein; and (b) collecting the engineered IL-2 polypeptide or fusion protein produced by the cells. Claim 42 A method for treating a subject having an immune disease, comprising administering to the subject a therapeutically effective amount of a composition comprising the engineered IL-2 polypeptide of any one of claims 1 to 19 or the fusion protein of any one of claims 20 to 35. Claim 43 In paragraph 42, a method for treating a subject having an autoimmune disease. Claim 44 A method for treating a subject having an immune disease, wherein the autoimmune disease is rheumatoid arthritis, Crohn's disease, diabetes mellitus (e.g., type I), graft-versus-host disease (GvHD), systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel disease (IBD), ulcerative colitis, scleroderma, allergy, asthma, or atopic dermatitis. Claim 45 A method for increasing regulatory T cell proliferation in a subject, comprising administering to the subject a pharmaceutical composition comprising the engineered IL-2 polypeptide of any one of claims 1 to 19 or the fusion protein of any one of claims 20 to 35. Claim 46 A method for increasing regulatory T cell proliferation, comprising administering to the subject a pharmaceutical composition comprising the engineered IL-2 polypeptide of any one of claims 1 to 19 or the fusion protein of any one of claims 20 to 35. Claim 47 A method for treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of a composition comprising the engineered IL-2 polypeptide of any one of claims 1 to 19 or the fusion protein of any one of claims 20 to 35. Claim 48 In paragraph 47, a method for treating a cancerous subject having a solid tumor or blood cancer. Claim 49 In paragraph 47, a method of treating a subject having cancer, wherein the cancer is breast cancer, ovarian cancer, glioma, colon cancer, hepatocellular carcinoma, prostate cancer, renal cell carcinoma, melanoma, lung cancer, pancreatic cancer, or liver cancer. Claim 50 A method for reducing the rate of tumor growth, comprising contacting tumor cells with an effective amount of a composition comprising the engineered IL-2 polypeptide of any one of claims 1 to 19 or the fusion protein of any one of claims 20 to 35. Claim 51 A method for killing tumor cells comprising contacting the tumor cells with an effective amount of a composition comprising the engineered IL-2 polypeptide of any one of claims 1 to 19 or the fusion protein of any one of claims 20 to 35. Claim 52 A engineered IL-2 polypeptide of any one of claims 1 to 19 or a fusion protein of any one of claims 20 to 35, wherein a non-natural cysteine ​​and / or one or more non-natural disulfide bonds stabilize the internal core of the IL-2 polypeptide or fusion protein. Claim 53 A engineered IL-2 polypeptide of any one of claims 1 to 19 or a fusion protein of any one of claims 20 to 35, wherein a non-natural cysteine ​​and / or one or more non-natural disulfide bonds increase the thermal stability of the IL-2 polypeptide or fusion protein. Claim 54 A engineered IL-2 polypeptide of any one of claims 1 to 19 or a fusion protein of any one of claims 20 to 35, wherein a non-natural cysteine ​​and / or one or more non-natural disulfide bonds improve the pharmacokinetics of the IL-2 polypeptide or fusion protein. Claim 55 A engineered IL-2 polypeptide or fusion protein, wherein in any one of paragraphs 52 to 54, stabilization, increased thermal stability, and / or increased pharmacokinetics cannot be achieved by surface mutations. Claim 56 In paragraph 53, non-natural cysteine ​​and / or one or more non-natural disulfide bonds are aggregated (T agg ) and / or initiation(T onset ) A engineered IL-2 polypeptide or fusion protein that increases the temperature by at least 5°C. Claim 57 In paragraph 54, the non-natural cysteine ​​and / or one or more non-natural disulfide bonds are engineered IL-2 polypeptides or fusion proteins that increase pharmacokinetics by at least 10-fold. Claim 58 As a method for improving the stability of IL-2, (a) providing a 3D structure of IL-2, (b) one or more C amino acid residues within the 3D structure 알파 Measuring the distance between atoms; and (c) selecting two amino acid residues from one or more amino acid residues, comprising C of the two selected amino acid residues 알파 A method for improving the stability of IL-2, wherein the atom exists within 3 to 8 Angstroms. Claim 59 A method for improving the stability of IL-2 according to claim 58, further comprising expressing an IL-2 variant, wherein the IL-2 variant comprises non-natural disulfide bonds formed by mutating two selected amino acid residues to cysteine. Claim 60 In claim 59, a method for improving the stability of IL-2, wherein mutating two selected amino acid residues to cysteine ​​does not substantially change the 3D structure of IL-2. Claim 61 A method for screening IL-2 variants, comprising: (a) providing a 3D structure of IL-2; and (b) one or more C amino acid residues within the 3D structure. 알파 Measuring the distance between atoms; and (c) selecting two amino acid residues from one or more amino acid residues, comprising C of the two selected amino acid residues 알파 A method for screening IL-2 variants in which atoms exist within 3 to 8 angstroms. Claim 62 A method for screening an IL-2 variant according to claim 61, further comprising: (a) expressing an IL-2 variant comprising a non-natural disulfide bond formed by mutating two selected amino acid residues to cysteine; (b) administering the IL-2 variant to an animal having an immune disease; and (c) determining the effect of the IL-2 variant on the immune disease. Claim 63 A method for screening an IL-2 variant according to claim 61, further comprising: (a) expressing an IL-2 variant comprising a non-natural disulfide bond formed by mutating two selected amino acid residues to cysteine; (b) administering the IL-2 variant to a tumor-bearing animal; and (c) determining tumor growth in the tumor-bearing animal (e.g., by measuring tumor volume). Claim 64 A method for screening IL-2 variants, wherein IL-2 is human IL-2, in any one of paragraphs 58 to 63.

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