WO2024002363A1

WO2024002363A1 - Il-2 polypeptides and methods of use

Info

Publication number: WO2024002363A1
Application number: PCT/CN2023/105095
Authority: WO
Inventors: Puwei YUAN; Junli Liu; Fei Zhang; Mingchen CHEN; Chengcheng GUAN; Hang Chen; Fan Liu
Original assignee: Beijing Neox Biotech Ltd
Current assignee: Beijing Neox Biotech Ltd
Priority date: 2022-07-01
Filing date: 2023-06-30
Publication date: 2024-01-04
Anticipated expiration: 2025-01-01
Also published as: TW202409069A

Abstract

Provided herein are IL-2 mutant polypeptides and compositions thereof that exhibit reduced binding affinity to IL-2Rα and/or enhanced binding affinity to IL-2βγ. Further provided herein are methods for administering said IL-2 mutant polypeptides and compositions thereof for treatment of a condition, such as cancer and immune disorders.

Description

IL-2 POLYPEPTIDES AND METHODS OF USE

CROSS-REFERENCE

This application claims the benefit of International Patent Application No. PCT/CN2022/103352, filed July 1, 2022, which is incorporated by reference herein in its entirety.

BACKGROUND

Interleukin-2 (IL-2) is a member of the IL-2 cytokine subfamily that includes IL-4, IL-7, IL-9, IL-15, IL-21, erythropoietin, and thrombopoietin. IL-2 mediates key functions of the immune system, tolerance, and immunity. IL-2 acts primarily as a T cell growth factor, which is essential for the proliferation and survival of T cells as well as the generation of effector and memory T cells. IL-2 signals through the IL-2 receptor (IL-2R) , a heterotrimeric complex consisting of three chains: alpha (CD25) , beta (CD122) , and gamma (CD132) . IL-2 The binds to the IL-2Rα subunit with low affinity (K_D ～ 10^-8 M) . However, binding to IL-2Rα alone does not lead to signal transduction. Heterodimerization of the β and γ subunits of IL-2R is required for signaling in T cells. IL-2 can signalize either via intermediate-affinity dimeric IL-2Rβγ (K_D ～10^-9 M) or high-affinity trimeric IL-2Rαβγ (K_D ～ 10^-11 M) . Dimeric IL-2R is expressed by memory CD8+ T cells and natural killer (NK) cells, whereas regulatory T cells (Tregs) and activated T cells express high levels of trimeric IL-2R.

SUMMARY

Provided herein are engineered IL-2 polypeptides that exhibit reduced binding affinity to IL-2Rα, while retaining significant binding affinity to IL-2Rβγ relative to wildtype human IL-2. Further provided herein are IL-2 polypeptides that exhibit elevated binding affinity to IL-2Rβγrelative to wildtype IL-2. The IL-2 polypeptides of the disclosure can significantly activate CD8+ T cells and/or NK cells, and meanwhile can reduce or eliminate the activation of Tregs. Moreover, The IL-2 polypeptides of the disclosure can further exhibit improved thermostability relative to wildtype IL-2. Further provided herein are pharmaceutical compositions and kits of IL-2 polypeptides herein effective for treatment of a condition, such as cancer.

One embodiment provides an isolated polypeptide comprising an IL-2 variant moiety with an amino acid residue mutation at K64 of a sequence of wildtype human IL-2.

One embodiment provides an isolated polypeptide comprising an IL-2 variant moiety with an amino acid residue mutation at P65 of a sequence of wildtype human IL-2.

One embodiment provides an isolated polypeptide comprising an IL-2 variant moiety with amino acid residue mutations at both K64 and P65 of a sequence of wildtype human IL-2.

One embodiment provides an isolated polypeptide comprising an IL-2 variant moiety having a sequence of any one of SEQ ID NOs: 3-41.

One embodiment provides a pharmaceutical composition comprising an isolated polypeptide described herein; and a pharmaceutically acceptable excipient.

One embodiment provides a method of treating a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an isolated polypeptide described herein; and a pharmaceutically acceptable excipient.

One embodiment provides a nucleic acid molecule comprising a nucleic acid sequence that encodes a polypeptide described herein. In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, the nucleic acid molecule is an RNA molecule. In some embodiments, the nucleic acid molecule is an mRNA molecule.

One embodiment provides a pharmaceutical composition comprising a nucleic acid molecule encoding a polypeptide described herein; and a pharmaceutically acceptable excipient.

One embodiment provides a method of treating a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a nucleic acid molecule encoding for a polypeptide described herein; and a pharmaceutically acceptable excipient.

One embodiment provides an expression vector comprising a nucleic acid sequence that encodes the polypeptide described herein.

One embodiment provides a host cell comprising expression vector comprising a nucleic acid sequence that encodes the polypeptide described herein.

One embodiment provides a method for preparing a polypeptide comprising an IL-2 variant moiety, the method comprising: performing recombinant expression with a nucleic acid sequence that encodes the polypeptide described herein, an expression vector described herein, or a host cell described herein.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an example recombinant expression plasmid vector of wildtype IL-2.

FIG. 2 illustrates sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis results of wildtype IL-2 and three IL-2 mutants of the disclosure.

FIG. 3 illustrates size exclusion chromatography (SEC) analysis results of wildtype IL-2 and three IL-2 mutants of the disclosure.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a, ” “and, ” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an agent” includes a plurality of such agents, and reference to “the cell” includes reference to one or more cells (or to a plurality of cells) and equivalents thereof known to those skilled in the art, and so forth. When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error) , and thus, the number or numerical range, in some instances, will vary between 1%and 15%of the stated number or numerical range. The term “comprising” (and related terms such as “comprise” or “comprises” or “having” or “including” ) is not intended to exclude that in other certain embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, described herein, “consist of” or “consist essentially of” the described features.

Definitions

As used in the specification and appended claims, unless specified to the contrary, the following terms have the meaning indicated below.

“Wildtype” or “WT” or “wt” or “native” refers to an amino acid sequence that is found in nature, including allelic variations. A wildtype protein or polypeptide has an amino acid sequence that has not been intentionally modified.

“Variants” of an amino acid sequence (e.g., of a peptide, protein, or polypeptide) refers to amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and/or amino acid substitution variants. Amino acid insertion variants are characterized by insertion of one or more amino acids in a particular amino acid sequence, e.g., a wildtype IL-2 sequence or a functional variant thereof. Amino acid addition variants include N-and/or C-terminal fusions of one or more amino acids. Amino acid deletion variants are characterized by removal of one or more amino acids from the sequence. The deletions may be in any position of the protein sequence. Amino acid deletion variants include deletion at the N-and/or C-terminus of the protein to produce N-and/or C-terminal truncation variants. Amino acid substitution variants are characterized by removal of one or more amino acids from the sequence, and insertion of one or more another amino acids in place of the original amino acids.

“IL-2 mutant” or “IL-2 mutein” or “IL-2 variant” or “IL-2 variant moiety” herein refers to a polypeptide in which one or more amino acids of the wildtype human IL-2 or a functional variant thereof are mutated, preferably substituted with different amino acids. In some embodiments, an IL-2 mutant can be prolonged, truncated, or modified (such as glycosylated, methylated, or PEGylated) . In some embodiments, an IL-2 mutant has a sequence that is at least 80%identical to the sequence of wildtype human IL-2 or SEQ ID NO: 1. In some embodiments, an IL-2 mutant provided herein comprises a signal peptide of wildtype human IL-2. In some embodiments, an IL-2 mutant provided herein does not comprise a signal peptide of wildtype human IL-2.

In some embodiments, the term “wildtype human IL-2” refers to the 133-amino acid sequence of native human IL-2 (less the signal peptide, consisting of an additional 20 amino acids at the N-terminus) , whose amino acid sequence is described in Fujita, et. al, PNAS USA, 80, 7437-7441 (1983) , with or without an additional N-terminal methionine, which is necessarily included when the protein is expressed as an intracellular fraction in Escherichia coli. In one embodiment, wildtype human IL-2 comprises the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the term “wildtype IL-2” or “wildtype human IL-2” refers to an IL-2 polypeptide containing one or more amino acid mutations that do not affect the binding of the polypeptide to the IL-2 receptor, for example, substitution of alanine or serine for cysteine at a position corresponding to residue 125 of wildtype human IL-2 (C125A/S) .

“CD25” or “α subunit of IL-2 receptor” or “IL-2Rα” refers to any natural CD25 from any vertebrate sources, including mammals such as primates (e.g., human) and rodents (e.g., mouse and rat) , a “full-length” unprocessed CD25 and any processed forms of CD25 derived from cells, and naturally-occurring CD25 variants, such as splice variants or allelic variants. In certain embodiments, CD25 is human CD25.

“CD122” or “β subunit of IL-2 receptor” or “IL-2Rβ” refers to any natural CD122 from any vertebrate sources, including mammals such as primates (e.g., human) and rodents (e.g., mouse and rat) , a “full-length” unprocessed CD122 and any processed forms of CD122 derived from cells, and naturally-occurring CD122 variants, such as splice variants or allelic variants. In certain embodiments, CD122 is human CD122.

“CD132” or “γ subunit of IL-2 receptor” or “IL-2Rγ” refers to any natural CD132 from any vertebrate sources, including mammals such as primates (e.g., human) and rodents (e.g., mouse and rat) , a “full-length” unprocessed CD132 and any processed forms of CD132 derived from cells, and naturally-occurring CD132 variants, such as splice variants or allelic variants. In certain embodiments, CD132 is human CD132.

“Natural amino acid” refers to an amino acid of the 20 naturally-occurring amino acids in protein. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate) , basic (lysine, arginine, histidine) , non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are classified as aromatic amino acids.

“Unnatural amino acid” refers to an amino acid other than the 20 naturally-occurring amino acids in protein.

“Amino acid residue mutation” refers to substitution, deletion, insertion, modification, or any combination thereof of one or more amino acid residues for obtaining a final construct, such that the final construct possesses desired characteristics, e.g., altered binding affinity or enhanced stability. In some embodiments, an amino acid residue mutation is an amino acid residue substitution. For example, in order to change the binding affinity of IL-2 polypeptides, amino acid residues can be substituted in which one amino acid residue is replaced with another amino acid residue having different structure and/or chemical property.

“Percentage identity” refers to a percentage of identical amino acid residues between two sequences being compared after an optimal alignment of sequences. An optimal alignment of sequences may be produced manually or by means of computer programs which use a sequence alignment algorithm (e.g., ClustalW, T-coffee, COBALT, BestFit, FASTA, BLASTP, BLASTN, and TFastA) . Percentage identity can be calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions compared, and multiplying the result obtained by 100 so as to obtain the percentage identity between the two sequences.

The term “subject” or “patient” encompasses mammals. Examples of mammals include, but are not limited to, any member of the Mammalia class: humans, non-human primates, such as chimpanzees, and other apes and monkey species; farm animals, such as cattle, horses, sheep, goats, swine; domestic animals, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice and guinea pigs, and the like. In one aspect, the mammal is a human.

As used herein, “treatment” or “treating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and/or a prophylactic benefit. By “therapeutic benefit” is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient is still afflicted with the underlying disorder. For prophylactic benefit, the compositions are, in some embodiments, administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease has not been made.

IL-2

IL-2 is a 15-kDa four-α-helix-bundle cytokine that contains a single disulfide bond, which is essential for its biological activity. IL-2 is a member of a cytokine family that includes IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, which all share the common cytokine receptor γ chain. IL-2 is responsible for regulating the immune activity of white blood cells (usually lymphocytes) in the immune system. IL-2 acts by binding to IL-2 receptors on the surface of immune cells and is a cytokine necessary for effector T cell expansion, survival, and function. IL-2 mediates key functions of the immune system, tolerance, and immunity. IL-2 acts primarily as a T cell growth factor, which is essential for the proliferation and survival of T cells as well as the generation of effector and memory T cells. IL-2 signals through the IL-2 receptor (IL-2R) , a heterotrimeric complex consisting of three subunits: alpha (α; CD25) , beta (β; CD122) , and gamma (γ; CD132) . The IL-2Rα subunit binds IL-2 with low affinity (K_D ～ 10^-8 M) . However, IL-2 binding to IL-2Rα alone does not lead to signal transduction. Heterodimerization of the β and γsubunits of IL-2R is essential for signaling in T cells. IL-2 can signalize either via intermediate-affinity dimeric IL-2Rβγ (K_D ～ 10^-9 M) or high-affinity trimeric IL-2Rαβγ (K_D ～ 10^-11 M) .

Dimeric IL-2R is expressed by memory CD8+ T cells and NK cells, whereas regulatory T cells and activated T cells express high levels of trimeric IL-2Rαβγ. As such, IL-2 polypeptides described herein exhibit a reduced affinity for IL-2Rαβγ relative to the affinity for IL-2Rβγ, the receptor complex that is predominant on and memory T cells and NK cells. Thus, treatment using the IL-2 polypeptides described herein can promote activation of T cells such as CD8+ T cells over Tregs in the tumor microenvironment to elicit anti-tumor efficacy.

IL-2 receptors are expressed mainly by lymphoid cells. In the absence of stimulation, IL-2Rα is mainly expressed on Treg cells and not expressed on T cells, but is potently induced on T cell activation. IL-2Rβ and γ are constitutively expressed on some resting T cells, especially NK cells, CD8+ T cells, B cells, macrophages, monocytes, and DCs. Like IL-2Rα, IL-2Rβγ can be further induced on stimulation of these cells, either by an antigen or by IL-2.

The pleiotropic effects of IL-2 can be attributed to IL-2 signaling transduction via three different signaling pathways: JAK-STAT, PI3K/Akt/mTOR, and MAPK/ERK pathways. After IL-2 binding to IL-2R, cytoplasmatic domains of the β and γ subunits heterodimerize. This heterodimerization leads to the activation of Janus kinases, JAK1 and JAK3, which subsequently phosphorylate T338 on the β-subunit. This phosphorylation leads to recruitment of STAT transcription factors, predominantly STAT5, which dimerize and migrate to the cell nucleus where the transcription factors bind to DNA.

In the thymus, where T cells mature, IL-2 can prevent autoimmune diseases by promoting the differentiation of certain immature T cells into regulatory T cells, which suppress other T cells that would otherwise be primed to attack normal healthy cells in the body. IL-2 can enhance activation-induced cell death. IL-2 can also promote the differentiation of T cells into effector T cells and into memory T cells when the initial T cell is also stimulated by an antigen, thus helping the body fight off infections. Together with other polarizing cytokines, IL-2 stimulates naive CD4+ T cell differentiation into Th1 and Th2 lymphocytes while impeding differentiation into Th17 and follicular Th lymphocytes.

IL-2 stimulates proliferation and enhances function of T cells, NK cells, and B cells. IL-2-activated B cells can generate secretory IgM rather than membrane-associated IgM. Macrophages can gain maturity and elaborate transforming growth factor-β (TGF-β) when stimulated with IL-2. These immunomodulatory effects of IL-2 provide rationale for use as an anticancer agent. However, IL-2 toxicity hinders widespread implementation. IL-2 toxicity includes, for example, anemia, thrombocytopenia, endothelial cell damage, and renal damage. Modulating IL-2 activity can be an effective treatment strategy for immunodeficiency, autoimmune disorders, infectious diseases, allergic conditions, and malignancies.

The polypeptide preferentially activates effector T cells and NK cells over regulatory T cells (e.g., CD4+CD25+ FoxP3+ T cells) .

IL-2 can activate Tregs expressing IL-2Rαβγ trimer and non-targeted cells, such as eosinophils, which can inhibit the tumor suppressor activity of T cells, causing significant side effects. By engineering the IL-2 protein to reduce or block the activation of IL-2 on Tregs without affecting or enhancing the activation of cytotoxic T lymphocytes (CTLs) , i.e., attenuating or eliminating the affinity of IL-2 to IL-2Rα without affecting or enhancing the affinity for IL-2Rβγ dimer can be a new direction for the clinical development of IL-2.

Provided herein are IL-2 polypeptides comprising an IL-2 variant moiety having reduced affinity to IL-2Rαβγ relative to the affinity to IL-2Rβγ. An IL-2 mutein herein includes one or more amino acid substitutions, e.g., at positions K35, R38, F42, K43, F44, E61, E62, K64, P65, E68, L72, Y107, and/or C125, of wildtype human IL-2.

Compared with the affinity of wildtype IL-2 protein and IL-2Rα, the affinity of IL-2 mutants of the present disclosure to IL-2Rα is significantly reduced, e.g., such that these IL-2 mutants can reduce or block IL-2 activation of Tregs. Further, IL-2 mutants of the present disclosure can also significantly activate CD8+ T cells and/or NK cells. In addition, the thermal stability of the IL-2 mutants of the present disclosure is significantly improved compared with the wildtype IL-2. The druggability of the IL-2 mutants with high thermal stability is significantly improved compared with the wildtype IL-2.

IL-2 Mutants of the Disclosure

IL-2 polypeptides provided herein exhibit reduced binding affinity to IL-2α and/or enhanced binding affinity to IL-2Rβγ relative to a wildtype human IL-2. The IL-2 polypeptides can include an IL-2 variant moiety having one or more amino acid residue mutations of a sequence of wildtype human IL-2.

In some embodiments, IL-2 polypeptides having modulated binding affinity to IL-2α are generated by making amino acid substitutions to the wildtype IL-2 protein at one or more positions on the binding interface between wildtype IL-2 and the α-subunit of the IL-2R.

In some embodiments, IL-2 polypeptides with modulated binding affinity to IL-2βγ are generated by making amino acid substitutions to the wildtype IL-2 protein at one or more positions on the binding interface between wildtype IL-2 and the β-subunit of IL-2R.

In some embodiments, IL-2 polypeptides provided herein are generated based on the sequence of wildtype human IL-2:

In some embodiments, wildtype human IL-2 comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, wildtype human IL-2 consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, an IL-2 variant moiety comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to SEQ ID NO: 1.

In some embodiments, an IL-2 polypeptide comprises one, two, three, four, five, or more amino acid residue mutations at positions selected from the group consisting of K35, R38, F42, K43, F44, E61, E62, K64, P65, E68, L72, Y107, and C125 of wildtype human IL-2.

In some embodiments, an IL-2 polypeptide comprises one, two, three, four, or more amino acid residue mutations at positions selected from the group consisting of K35, R38, F42, K43, F44, E61, E62, K64, P65, E68, L72, and Y107 of wildtype human IL-2.

In some embodiments, an IL-2 polypeptide comprises an amino acid residue mutation at K64 of a sequence of wildtype human IL-2. In some embodiments, the amino acid residue mutation at K64 is K64P or K64E.

In some embodiments, an IL-2 polypeptide comprises an amino acid residue mutation at P65 of a sequence of wildtype human IL-2. In some embodiments, the amino acid residue mutation at P65 is P65E, P65D, P65R, P65W, or P65K.

In some embodiments, an IL-2 polypeptide further comprises one or more amino acid residue mutations at positions selected from the group consisting of K35, R38, K43, E61, E62, L72, and Y107 of wildtype human IL-2.

In some embodiments, an IL-2 polypeptide further comprises one or more amino acid residue mutations at positions selected from the group consisting of K35, R38, K43, and Y107 of wildtype human IL-2.

In some embodiments, an IL-2 polypeptide further comprises one or more amino acid residue mutations at positions selected from the group consisting of F42, F44, and E62 of wildtype human IL-2.

In some embodiments, an IL-2 polypeptide further comprises an amino acid residue mutation at Y107 of a sequence of wildtype human IL-2. In some embodiments, the amino acid residue mutation at Y107 is Y107K or Y107R.

In some embodiments, an IL-2 polypeptide further comprises an amino acid residue mutation at K35 of a sequence of wildtype human IL-2. In some embodiments, the amino acid residue mutation at K35 is K35Y, K35D, or K35Q.

In some embodiments, an IL-2 polypeptide further comprises an amino acid residue mutation at R38 of a sequence of wildtype human IL-2. In some embodiments, the amino acid residue mutation at R38 is R38W or R38Q.

In some embodiments, an IL-2 polypeptide further comprises an amino acid residue mutation at K43 of a sequence of wildtype human IL-2. In some embodiments, the amino acid residue mutation at K43 is K43Q, K43R, or K43G.

In some embodiments, an IL-2 polypeptide further comprises an amino acid residue mutation at E61 of a sequence of wildtype human IL-2. In some embodiments, the amino acid residue mutation at E61 is E61T.

In some embodiments, an IL-2 polypeptide further comprises an amino acid residue mutation at E62 of a sequence of wildtype human IL-2. In some embodiments, the amino acid residue mutation at E62 is E62T, E62S, E62N, E62A, or E62R.

In some embodiments, an IL-2 polypeptide further comprises an amino acid residue mutation at L72 of a sequence of wildtype human IL-2. In some embodiments, the amino acid residue mutation at L72 is L72T.

In some embodiments, an IL-2 polypeptide further comprises one, two, three, more amino acid residue mutations selected from the group consisting of F42, K43, F44, and E62 of wildtype human IL-2.

In some embodiments, an IL-2 polypeptide further comprises an amino acid residue mutation at F42 of a sequence of wildtype human IL-2. In some embodiments, the amino acid residue mutation at F42 is F42K, F42R, F42E, or F42P.

In some embodiments, an IL-2 polypeptide further comprises an amino acid residue mutation at K43 of a sequence of wildtype human IL-2. In some embodiments, the amino acid residue mutation at K43 is K43R, K43G, or K43Q.

In some embodiments, an IL-2 polypeptide further comprises an amino acid residue mutation at F44 of a sequence of wildtype human IL-2. In some embodiments, the amino acid residue mutation at F44 is F44L, F44M, or F44N.

In some embodiments, an IL-2 polypeptide further comprises an amino acid residue mutation at E62 of a sequence of wildtype human IL-2. In some embodiments, the amino acid residue mutation at E62 is E62S, E62R, E62N, E62T, or E62A.

In some embodiments, an IL-2 polypeptide comprises one, two, or more amino acid residue mutations at positions selected from the group consisting of K35, R38, and Y107 of wildtype human IL-2. In some embodiments, the amino acid residue mutation at K35 is K35Y, K35D, or K35Q. In some embodiments, the amino acid residue mutation at K35 is K35Y or K35Q. In some embodiments, the amino acid residue mutation at Y107 is Y107K or Y107R. In some embodiments, the amino acid residue mutation at R38 is R38Q or R38W. In some embodiments, an IL-2 polypeptide comprises amino acid residue mutations selected from the group consisting of: K35Y; K35Q and Y107K; and R38Q and Y107R.

In some embodiments, an IL-2 polypeptide comprises amino acid residue mutations selected from the group consisting of:

1) K64P and P65E;

2) K64P and P65D;

3) K64P and P65R;

4) K64P and P65W;

5) K64P and P65K;

6) K64E and P65R;

7) K64P and P65D;

8) K64E and P65D; and

9) K64P and P65W.

10) K35Y, K64P, and P65K;

11) R38W, K64P, and P65K;

12) R38Q, K64P, and P65K;

13) K43Q, K64P, and P65K;

14) E61T, K64P, and P65K;

15) K64P, P65K, and L72T;

16) K35Y, K64P, and P65R;

17) K35W, K64P, and P65R;

18) R38D, K64P, and P65R;

19) K43Q, K64P, and P65R;

20) E62T, K64P, and P65R;

21) K64P, P65R, and L72T;

22) K64E and P65R; and

23) E62R, K64P, and P65D.

24) K35Q, K64P, P65K, and Y107K;

25) R38Q, K64P, P65K, and Y107R;

26) K35Q, K64P, P65R, and Y107K; and

27) R38Q, K64P, P65R, and Y107R.

28) K43R, K64E, and P65R;

29) F42K, K43R, K64E, and P65R;

30) F42R, K43R, F44L, K64P, and P65E;

31) F42R, K43R, F44M, E62S, K64E, and P65D;

32) F42R, K43R, F44M, E62R, K64P, and P65E;

33) F42R, K43G, F44L, E62N, K64P, and P65D;

34) F42R, K43R, F44L, K64P, and P65R;

35) F42R, K43R, F44L, E62R, K64P, and P65D;

36) F42R, K43G, F44L, E62N, K64P, and P65D; and

37) F42P, K43R, F44N, E62A, K64P, and P65W.

In some embodiments, an IL-2 polypeptide further comprises an amino acid residue mutation at C125 of a sequence of wildtype human IL-2. In some embodiments, the amino acid residue mutation at C125 is C125A or C125S.

In some embodiments, an IL-2 polypeptide further comprises a sequence of wildtype human IL-2 having one, two, or more amino acid residues replaced with a cysteine. In some embodiments, the amino acid residues replaced by cysteine are not at positions selected from K64 and P65 corresponding to SEQ ID NO: 1. In some embodiments, the amino acid residues replaced by cysteine are not at positions selected K35, R38, K43, Y107, K64, and P65 corresponding to SEQ ID NO: 1. In some embodiments, at least two of the two or more cysteine residues form a disulfide linkage.

In some embodiments, an IL-2 polypeptide comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 3-41. In some embodiments, an IL-2 polypeptide comprises a sequence of any one of SEQ ID NOs: 3-41.

In some embodiments, an amino acid residue described herein (e.g., of IL-2) is mutated to a natural amino acid, e.g., proline, lysine, cysteine, histidine, arginine, aspartic acid, asparagine, glutamic acid, glutamine, methionine, serine, threonine, tryptophan, or tyrosine. In some embodiments, an amino acid residue described herein (e.g., of IL-2) is mutated prior to binding to (or reacting with) a conjugating moiety.

In some embodiments, an amino acid residue described herein (e.g., of IL-2) is mutated to an unnatural amino acid. In some embodiments, a polypeptide herein comprises an unnatural amino acid, wherein the cytokine is conjugated to the protein, wherein the point of attachment is not the unnatural amino acid. In some embodiments, an amino acid residue described herein (e.g., of IL-2) is mutated prior to binding to (or reacting with) a conjugating moiety. In some embodiments, mutation to an unnatural amino acid reduces the likelihood of or minimizes a self-antigen response of the immune system.

Non-limiting examples of unnatural amino acid include p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3- (2-naphthyl) alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-boronophenylalanine, O-propargyltyrosine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, selenocysteine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, azido-lysine (AzK) , an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or a combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and/or photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a keto containing amino acid; an amino acid comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an α-hydroxy amino acid; an amino thioacid; an α, α-disubstituted amino acid; a β-amino acid; a cyclic amino acid other than proline or histidine, and an aromatic amino acid other than phenylalanine, tyrosine or tryptophan.

In some embodiments, the unnatural amino acid comprises a selective reactive group, or a reactive group for site-selective labeling of a target polypeptide. In some embodiments, the chemistry is a biorthogonal reaction (e.g., biocompatible and selective reactions) . In some cases, the chemistry is a Cu (I) -catalyzed or “copper-free” alkyne-azide triazole-forming reaction, the Staudinger ligation, inverse-electron-demand Diels-Alder (IEDDA) reaction, “photo-click” chemistry, or a metal-mediated process such as olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling. In some embodiments, the unnatural amino acid comprises a photoreactive group, which crosslinks, upon irradiation with, e.g., UV. In some embodiments, the unnatural amino acid comprises a photo-caged amino acid.

In some embodiments, the unnatural amino acid is a para-substituted, meta-substituted, or an ortho-substituted amino acid derivative.

In some embodiments, the unnatural amino acid comprises N6-azidoethoxy-L-lysine (AzK) , N6-propargylethoxy-L-lysine (PraK) , BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyllysine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF) , p-iodo-L-phenylalanine, p-methoxyphenylalanine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3- (2-naphthyl) alanine, 3-methyl-phenylalanine, O-allyl-L-tyrosine, O-methyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAc-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, L-3- (2-naphthyl) alanine, 2-amino-3- ( (2- ( (3- (benzyloxy) -3-oxopropyl) amino) ethyl) selanyl) propanoic acid, 2-amino-3- (phenylselanyl) propanoic, or selenocysteine. In some embodiments, the unnatural amino acid is 3-aminotyrosine, 3-nitrotyrosine, 3, 4-dihydroxy-phenylalanine, or 3- iodotyrosine. In some embodiments, the unnatural amino acid is phenylselenocysteine. In some embodiments, the unnatural amino acid is a benzophenone, ketone, iodide, methoxy, acetyl, benzoyl, or azide-containing phenylalanine derivative. In some embodiments, the unnatural amino acid is a benzophenone, ketone, iodide, methoxy, acetyl, benzoyl, or azide-containing lysine derivative.

In some embodiments, an IL-2 polypeptide comprises an amino acid sequence that is heterologous to wildtype human IL-2 or a functional variant thereof. In some embodiments, the amino acid sequence can be a conjugating moiety, a half-life extension moiety, an albumin, an immunoglobulin or a fragment thereof, a transferrin, or a PEG.

In some embodiments, the amino acid sequence can be linked to an N-terminus of an IL-2 variant moiety. In some embodiments, the amino acid sequence can be linked to a C-terminus of an IL-2 variant moiety.

In some embodiments, provided herein are methods for preparing a polypeptide comprising an IL-2 variant moiety by performing recombinant expression using a nucleic acid molecule, an expression vector comprising a nucleic acid molecule, and/or a host cell comprising an expression vector comprising a nucleic acid molecule. A nucleic acid molecule can encode a polypeptide and/or IL-2 variant moiety described herein. The nucleic acid molecule can be codon-optimized to encode a polypeptide and/or IL-2 variant moiety described herein. Such a nucleic acid molecule can be inserted in an expression vector, which can then be transformed or incorporated into a host cell. The expression vector can be a plasmid. The host cell comprising the expression vector can be used to express an IL-2 mutant described herein. The host cell can be E. coli or other suitable bacterial cell.

Unless otherwise stated, the preparation of the present invention can be performed using standard procedures known to one skilled in the art, for example, in Michael R. Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) ; Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986) ; Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley and Sons, Inc. ) , Current Protocols in Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons, Inc. ) , Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc. ) , Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005) , Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, edited by Jennie P. Mather and David Barnes, Academic Press, 1st edition, 1998) , Methods in Molecular biology, Vol. 180, Transgenesis Techniques edited by Alan R. Clark, 2nd edition, 2002, Humana Press, and Methods in Molecular Biology, Vol. 203, 2003, Transgenic Mouse, edited by Marten H. Hofker and Jan van Deursen, each of which is herein incorporated by reference in its entirety.

Conjugating Moieties

In some embodiments, IL-2 polypeptides provided herein include an IL-2 variant moiety and a conjugating moiety that is bound to one or more cytokines (e.g., interleukins, IFNs, or TNFs) . In some embodiments, a conjugating moiety is a molecule that modulates the interaction of a cytokine with its receptor, e.g., IL-2 with IL-2R subunits. In some embodiments, the conjugating moiety is a molecule that when bound to the cytokine, enables the cytokine conjugate to modulate an immune response. In some embodiments, the conjugating moiety is bound to the cytokine through a covalent bond.

In some embodiments, a cytokine described herein is attached to a conjugating moiety with a triazole group. In some embodiments, a cytokine described herein is attached to a conjugating moiety with a dihydropyridazine or pyridazine group. In some embodiments, the conjugating moiety comprises a water-soluble polymer. In other embodiments, the conjugating moiety comprises a protein or a binding fragment thereof. In additional embodiments, the conjugating moiety comprises a peptide. In additional embodiments, the conjugating moiety comprises a nucleic acid. In additional embodiments, the conjugating moiety comprises a small molecule.

In some embodiments, the conjugating moiety comprises a bioconjugate (e.g., a TLR agonist such as a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 agonist; or a synthetic ligand such as Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, Poly I: C, Poly A: U, AGP, MPLA, RC-529, MDF2p, CFA, or flagellin) . In some cases, a conjugating moiety increases serum half-life and/or improves stability in vivo. In some cases, the conjugating moiety reduces cytokine interaction with one or more cytokine receptor domains or subunits. In some embodiments, the conjugating moiety blocks cytokine interaction with one or more cytokine domains or subunits with its cognate receptor (s) . In some embodiments, cytokine conjugates described herein comprise multiple conjugating moieties. In some embodiments, a conjugating moiety is attached to an unnatural or natural amino acid in the cytokine peptide. In some embodiments, an IL-2 peptide comprises a conjugating moiety attached to a natural amino acid in the IL-2 cytokine peptide. In some embodiments, a cytokine conjugate is attached to an unnatural amino acid in the cytokine peptide. In some embodiments, a conjugating moiety is attached to the N-or C-terminus amino acid of the cytokine peptide. Various combinations sites are disclosed herein, for example, a first conjugating moiety is attached to an amino acid in the cytokine peptide, and a second conjugating moiety is attached to the N-or C-terminus of the cytokine peptide. In some embodiments, a single conjugating moiety is attached to multiple residues of the cytokine peptide (e.g., a staple) . In some embodiments, a conjugating moiety is attached to both the N-and C-terminus amino acids of the cytokine peptide.

Masking Moieties

In some embodiments, a conjugating moiety is a masking moiety. A masking moiety can block, occlude, inhibit, decrease, or otherwise reduce (i.e., masks) the activity or binding of the cytokine to its cognate receptor or protein. For example, an IL-2 cytokine can be activatable by a protease at a target site, such as in a tumor microenvironment, by inclusion of a proteolytically cleavable linker. In some embodiments, a proteolytically cleavable linker links the cytokine to the masking moiety. Upon proteolytic cleavage of the cleavable linker at the target site, the cytokine can become activated, thereby rendering the cytokine capable of binding to its cognate receptor or protein with increased affinity.

A masking moiety can include a cleavable peptide and/or linker. In some embodiments, the cleavable linker comprises a cleavable peptide. A cleavable peptide is a polypeptide that includes a cleavage site, such as a protease cleavage site. The cleavage site is a site recognizable for cleavage of a portion of the cleavable peptide of a cleavable linker described herein. In some embodiments, the cleavable peptide comprises more than one cleavage site. In some embodiments, the cleavage site is an amino acid sequence that is recognized and cleaved by a cleaving agent. Exemplary cleaving agents include proteins, enzymes, DNAzymes, RNAzymes, metals, acids, and bases.

In some embodiments, the protease cleavage site is a matrix metalloprotease (MMP) cleavage site, a disintegrin and metalloprotease domain-containing (ADAM) metalloprotease cleavage site, a prostate specific antigen (PSA) protease cleavage site, a urokinase-type plasminogen activator (uPA) protease cleavage site, a membrane type serine protease 1 (MT-SP1) protease cleavage site, a matriptase protease cleavage site (ST14) or a legumain protease cleavage site. In embodiments, the matrix metalloprotease (MMP) cleavage site is a MMP9 cleavage site, a MMP13 cleavage site or a MMP2 cleavage site. In embodiments, the disintegrin and metalloprotease domain-containing (ADAM) metalloprotease cleavage site is an ADAM9 metalloprotease cleavage site, an ADAM10 metalloprotease cleavage site or an ADAM17 metalloprotease cleavage site. Protease cleavage sites can be designated by a specific amino acid sequence.

Half-life Extension Domains

In some embodiments, a conjugating moiety is a half-life extension domain that increases serum half-life and/or improves stability of an IL-2 polypeptide. The half-life extension domain can be fused to the N-or C-terminal of the cytokine herein. In some embodiments, a proteolytically cleavable linker links the cytokine to the masking moiety, links the cytokine to a half-life extension domain, and/or links the masking moiety to a half-life extension domain.

In some embodiments, the half-life extension domain is an albumin polypeptide or a functional fragment thereof. Albumin is a natural carrier protein that has an extended serum half-life of approximately three weeks due to its size and its susceptibility to FcRn-mediated recycling, which reduces the likelihood of intracellular degradation. Thus, linking an IL-2 polypeptide to albumin can significantly extend the serum half-life of the IL-2 polypeptide.

In some embodiments, the masked or unmasked cytokine comprises a half-life extension domain that comprises an albumin polypeptide, or a fragment or a variant thereof. In some embodiments, the albumin polypeptide is mouse serum albumin. In some embodiments, the albumin polypeptide is human serum albumin.

In some embodiments, the masked or unmasked cytokine comprises a half-life extension domain that comprises an immunoglobulin Fc domain, or a fragment or a variant thereof. In some embodiments, the immunoglobulin Fc domain is mouse immunoglobulin Fc domain. In some embodiments, the immunoglobulin Fc domain is human immunoglobulin Fc domain. In some embodiments, the human immunoglobulin Fc domain is IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM.

In some embodiments, the albumin polypeptide or Fc domain is linked to a masking moiety. In some embodiments, a masking moiety is linked to the N-or C-terminus of the albumin polypeptide or Fc domain. In some embodiments, the albumin polypeptide or Fc domain is linked to a masking moiety via a linker. In some embodiments, a linker is linked to the amino-terminus or the carboxy-terminus of the albumin polypeptide or Fc domain. In some embodiments, an N-or C-terminal spacer domain of the linker is linked to the N-or C-terminus of the albumin polypeptide or Fc domain. In some embodiments, a cleavable peptide of the linker is linked to the N-or C-terminus of the albumin polypeptide or Fc domain. In some embodiments, the albumin polypeptide or Fc domain is linked to a cytokine or functional fragment thereof (e.g., IL-2 or a mutein thereof) . In some embodiments, a cytokine or functional fragment thereof is linked to the N-or C-terminus of the albumin polypeptide or Fc domain. In some embodiments, the albumin polypeptide or Fc domain is linked to a cytokine or functional fragment thereof via a linker. In some embodiments, a linker is linked to the amino-terminus or the carboxy-terminus of the albumin polypeptide or Fc domain.

Water-soluble Polymers

In some embodiments, a conjugating moiety is a water-soluble polymer. In some embodiments, the water-soluble polymer is a non-peptidic, non-toxic, and biocompatible. A substance can be considered biocompatible if the beneficial effects associated with use of the substance alone or with another substance (e.g., an active agent such as a cytokine moiety) in connection with living tissues (e.g., administration to a subject) outweighs any deleterious effects as evaluated by a clinician, e.g., a physician, a toxicologist, or a clinical development specialist.

In some embodiments, a water-soluble polymer is non-immunogenic. A substance can be considered non-immunogenic if the intended use of the substance in vivo does not produce an undesired immune response (e.g., the formation of antibodies) , or if an immune response is produced, that such a response is not deemed clinically significant or important as evaluated by a clinician, e.g., a physician, a toxicologist, or a clinical development specialist.

In some instances, the water-soluble polymer is characterized as having from about 2 to about 300 termini. Non-limiting examples of water-soluble polymers include poly (alkylene glycols) , such as polyethylene glycol (PEG) , poly (propylene glycol) (PPG) , copolymers of ethylene glycol and propylene glycol and the like, poly (oxyethylated polyol) , poly (olefinic alcohol) , poly (vinylpyrrolidone) , poly (hydroxyalkylmethacrylamide) , poly (hydroxyalkylmethacrylate) , poly (saccharides) , poly (a-hydroxy acid) , poly (vinyl alcohol) (PVA) , polyacrylamide (PAAm) , poly (N- (2-hydroxypropyl) methacrylamide) (PHPMA) , polydimethylacrylamide (PDAAm) , polyphosphazene, polyoxazolines (POZ) , poly (N-acryloylmorpholine) , and any combination thereof.

In some embodiments, the water-soluble polymer is not limited to a particular structure. In some cases, the water-soluble polymer is linear (e.g., an end capped, e.g., an alkoxy PEG or a bifunctional PEG) , branched or multi-armed (e.g., forked PEG or PEG attached to a polyol core) , a dendritic (or star) architecture, each with or without one or more degradable linkages. Moreover, the internal structure of the water-soluble polymer can be organized in any number of different repeat patterns, such as homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, or block tripolymer.

In some embodiments, the weight-average molecular weight of a water-soluble polymer is from about 100 Daltons (Da) to about 150,000 Da. Non-limiting examples of weight-average molecular weight ranges include from about 5,000 Da to about 100,000 Da, from about 6,000 Da to about 90,000 Da, from about 10,000 Da to about 85,000 Da, from about 10,000 Da to about 85,000 Da, from about 20,000 Da to about 85,000 Da, from about 53,000 Da to about 85,000 Da, from about 25,000 Da to about 120,000 Da, from about 29,000 Da to about 120,000 Da, from about 35,000 Da to about 120,000 Da, and from about 40,000 Da to about 120,000 Da.

Non-limiting examples of weight-average molecular weights for a water-soluble polymer include about 100 Da, about 200 Da, about 300 Da, about 400 Da, about 500 Da, about 600 Da, about 700 Da, about 750 Da, about 800 Da, about 900 Da, about 1,000 Da, about 1,500 Da, about 2,000 Da, about 2,200 Da, about 2,500 Da, about 3,000 Da, about 4,000 Da, about 4,400 Da, about 4,500 Da, about 5,000 Da, about 5,500 Da, about 6,000 Da, about 7,000 Da, about 7,500 Da, about 8,000 Da, about 9,000 Da, about 10,000 Da, about 11,000 Da, about 12,000 Da, about 13,000 Da, about 14,000 Da, about 15,000 Da, about 20,000 Da, about 22,500 Da, about 25,000 Da, about 30,000 Da, about 35,000 Da, about 40,000 Da, about 45,000 Da, about 50,000 Da, about 55,000 Da, about 60,000 Da, about 65,000 Da, about 70,000 Da, and about 75,000 Da. Branched versions of the water-soluble polymer (e.g., a branched 40,000 Da water-soluble polymer comprised of two 20,000 Da polymers) having a total molecular weight of any of the foregoing can also be used. In some embodiments, the conjugate does not have any PEG moieties attached, either directly or indirectly, with a PEG having a weight average molecular weight of less than about 6,000 Da.

PEGs can comprise a number of (OCH2CH2) monomers or (CH2CH2O) monomers. The number of repeating units can be identified by the subscript “n” in “ (OCH2CH2) n. ” Thus, the value of “n” typically can fall within one or more of the following ranges: from 2 to about 3400, from about 100 to about 2300, from about 100 to about 2270, from about 136 to about 2050, from about 225 to about 1930, from about 450 to about 1930, from about 1200 to about 1930, from about 568 to about 2727, from about 660 to about 2730, from about 795 to about 2730, from about 795 to about 2730, from about 909 to about 2730, and from about 1,200 to about 1, 900. For any given polymer in which the molecular weight is known, the number of repeating units (i.e., “n” ) can be determined by dividing the total weight-average molecular weight of the polymer by the molecular weight of the repeating monomer.

In some instances, the water-soluble polymer is an end-capped polymer, i.e., having at least one terminus capped with a relatively inert group, such as a lower C1-C6 alkoxy group, or a hydroxyl group. For example, the water-soluble polymer can be a methoxy-PEG (mPEG) , which is a linear form of PEG in which one terminus of the PEG is a methoxy (-OCH3) group, while the other terminus is a hydroxyl or other functional group that can be optionally chemically modified. Non-limiting examples of water-soluble polymers include linear or branched discrete PEG (dPEG) ; linear, branched, or forked PEGs; and Y-shaped PEG derivatives.

In some embodiments, an IL-2 polypeptide described herein is conjugated to a water-soluble polymer selected from poly (alkylene glycols) , such as polyethylene glycol (PEG) , poly (propylene glycol) (PPG) , copolymers of ethylene glycol and propylene glycol and the like, poly (oxyethylated polyol) , poly (olefinic alcohol) , poly (vinylpyrrolidone) , poly (hydroxyalkylmethacrylamide) , poly (hydroxyalkylmethacrylate) , poly (saccharides) , poly (a- hydroxy acid) , poly (vinyl alcohol) (PVA) , polyacrylamide (PAAm) , poly (N- (2-hydroxypropyl) methacrylamide) (PHPMA) , polydimethylacrylamide (PDAAm) , polyphosphazene, polyoxazolines (POZ) , poly (N-acryloylmorpholine) , and a combination thereof. For example, an IL-2 polypeptide is conjugated to PEG (e.g., PEGylated) .

In some embodiments, a water-soluble polymer comprises a polyglycerol (PG) , e.g., a HPG, a LPG, a midfunctional PG, a linear-block-hyperbranched PG, or a side-chain functional PG. In some cases, the polyglycerol is a hyperbranched PG (HPG) .

In some embodiments, a water-soluble polymer is a degradable synthetic PEG alternative. Non-limiting examples of degradable synthetic PEG alternatives include poly [oligo (ethylene glycol) methyl methacrylate] (POEGMA) ; backbone modified PEG derivatives generated by polymerization of telechelic, or di-end-functionalized PEG-based macromonomers; PEG derivatives comprising comonomers comprising degradable linkage such as poly [ (ethylene oxide) -co- (methylene ethylene oxide) ] [P (EO-co-MEO) ] , cyclic ketene acetals such as 5, 6-benzo-2-methylene-1, 3-dioxepane (BMDO) , 2-methylene-1, 3-dioxepane (MDO) , and 2-methylene-4-phenyl-1, 3-dioxolane (MPDL) copolymerized with OEGMA, or poly- (s-caprolactone) -graft-poly (ethylene oxide) (PCL-g-PEO) .

In some embodiments, a water-soluble polymer comprises a poly (zwitterions) . Non-limiting examples of poly (zwitterions) include poly (sulfobetaine methacrylate) (PSBMA) , poly (carboxybetaine methacrylate) (PCBMA) , and poly (2-methyacryloyloxyethyl phosphorylcholine) (PMPC) .

In some embodiments, a water-soluble polymer comprises a polycarbonate. Non-limiting examples of polycarbonates include pentafluorophenyl 5-methyl-2-oxo-1, 3-dioxane-5-carboxylate (MTC-OC6F5) .

In some embodiments, a water-soluble polymer comprises a polymer hybrid, such as for example, a polycarbonate/PEG polymer hybrid, a peptide/protein-polymer conjugate, or a hydroxyl-containing and/or zwitterionic derivatized polymer (e.g., a hydroxyl-containing and/or zwitterionic derivatized PEG polymer) .

In some embodiments, a water-soluble polymer comprises a polysaccharide. Non-limiting examples of polysaccharides include dextran, polysialic acid (PSA) , hyaluronic acid (HA) , amylose, heparin, heparan sulfate (HS) , dextrin, or hydroxyethyl starch (HES) .

In some embodiments, a water-soluble polymer comprises a glycan. Non-limiting examples of glycans include N-linked glycans, O-linked glycans, glycolipids, O-GlcNAc, and glycosaminoglycans.

In some embodiments, a water-soluble polymer comprises a polyoxazoline polymer. A polyoxazoline polymer is a linear synthetic polymer, and similar to PEG, comprises a low polydispersity. In some instances, a polyoxazoline polymer is a polydispersed polyoxazoline polymer, characterized with an average molecule weight. In some cases, the average molecule weight of a polyoxazoline polymer includes, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, 100,000, 200,000, 300,000, 400,000, or 500,000 Da. In some embodiments, a polyoxazoline polymer comprises poly (2-methyl 2-oxazoline) (PMOZ) , poly (2-ethyl 2-oxazoline) (PEOZ) , or poly (2-propyl 2-oxazoline) (PPOZ) . In some cases, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to a polyoxazoline polymer.

In some embodiments, a water-soluble polymer is a polyacrylic acid polymer.

In some embodiments, a water-soluble polymer comprises polyamine. Polyamine is an organic polymer comprising two or more primary amino groups. In some embodiments, a polyamine includes a branched polyamine, a linear polyamine, or cyclic polyamine. In some cases, a polyamine is a low-molecular-weight linear polyamine. Exemplary polyamines include putrescine, cadaverine, spermidine, spermine, ethylene diamine, 1, 3-diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, and piperazine.

Lipids

In some embodiments, a conjugating moiety is a lipid. The lipid can be a fatty acid, e.g., a saturated fatty acid or an unsaturated fatty acid. Such fatty acids can have from 6 to 26 carbon atoms, from 6 to 24 carbon atoms, from 6 to 22 carbon atoms, from 6 to 20 carbon atoms, from 6 to 18 carbon atoms, from 20 to 26 carbon atoms, from 12 to 26 carbon atoms, from 12 to 24 carbon atoms, from 12 to 22 carbon atoms, from 12 to 20 carbon atoms, or from 12 to 18 carbon atoms in length. In some cases, the fatty acid has 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbon atoms in length. Non-limiting examples of fatty acids include caproic acid (hexanoic acid) , enanthic acid (heptanoic acid) , caprylic acid (octanoic acid) , pelargonic acid (nonanoic acid) , capric acid (decanoic acid) , undecylic acid (undecanoic acid) , lauric acid (dodecanoic acid) , tridecylic acid (tridecanoic acid) , myristic acid (tetradecanoic acid) , pentadecylic acid (pentadecanoic acid) , palmitic acid (hexadecanoic acid) , margaric acid (heptadecanoic acid) , stearic acid (octadecanoic acid) , nonadecylic acid (nonadecanoic acid) , arachidic acid (eicosanoic acid) , heneicosylic acid (heneicosanoic acid) , behenic acid (docosanoic acid) , tricosylic acid (tricosanoic acid) , lignoceric acid (tetracosanoic acid) , pentacosylic acid (pentacosanoic acid) , and cerotic acid (hexacosanoic acid) . In some embodiments, the lipid binds to one or more serum proteins, thereby increasing serum stability and/or serum half-life.

Proteins

In some embodiments, a conjugating moiety described herein is a protein or a binding fragment thereof. Non-limiting examples of such proteins include albumin, transferrin, or transthyretin. In some embodiments, the protein or a binding fragment thereof comprises an antibody or a binding fragments thereof.

In some embodiments, the conjugating moiety is albumin or a functional fragment thereof. Albumin is a family of water-soluble globular proteins. Albumin is commonly found in blood plasma, comprising about 55-60%of all plasma proteins. Human serum albumin (HSA) is a 585 amino acid polypeptide comprising three domains: domain I (amino acid residues 1-195) , domain II (amino acid residues 196-383) , and domain III (amino acid residues 384-585) . Each domain further includes a binding site, which can interact either reversibly or irreversibly with endogenous ligands such as fatty acids, bilirubin, or hemin, or exogenous compounds, such as heterocyclic or aromatic compounds.

In some embodiments, the conjugating moiety is transferrin. Transferrin is a 679 amino acid polypeptide that is about 80 kDa in size and comprises two Fe³⁺ binding sites with one at the N-terminal domain and the other at the C-terminal domain. Human transferrin has a half-life of about 7-12 days.

In some embodiments, the conjugating moiety is transthyretin (TTR) . Transthyretin is a transport protein located in the serum and cerebrospinal fluid which transports the thyroid hormone thyroxine (T4) and retinol-binding protein bound to retinol.

In some embodiments, a conjugating moiety is an antibody or a binding fragment thereof. For example, the antibody or a binding fragment thereof can be a humanized antibody or a binding fragment thereof, murine antibody or a binding fragment thereof, chimeric antibody or a binding fragment thereof, monoclonal antibody or a binding fragment thereof, monovalent Fab', divalent Fab2, F (ab) '3 fragments, single-chain variable fragment (scFv) , bis-scFv, (scFv) 2, diabody, minibody, nanobody, triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv) , single-domain antibody (sdAb) , IgNAR, camelid antibody or a binding fragment thereof, bispecific antibody or a binding fragment thereof, or a chemically modified derivative thereof.

In some embodiments, a conjugating moiety is a Fragment crystallizable domain (Fc domain) of an antibody, e.g., of IgG, IgD, IgA, IgE, or IgM. In some embodiments, a polypeptide herein comprises a fusion to an Fc portion of IgG (e.g., IgG1, IgG2, IgG3, or IgG4) . In some cases, a polypeptide herein is fused to the Fc portion is further conjugated to one or more conjugation moieties described below. For example, the Fc domain can be fused to the N-or C-terminus of an IL-2 mutein.

In some embodiments, an IL-2 polypeptide herein exhibits a decreased binding affinity to the IL-2Rα subunit relative to a wildtype IL-2 polypeptide. In some embodiments, the decreased binding affinity is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or greater relative to the binding affinity of wildtype IL-2 polypeptide to IL-2Rα. In some embodiments, the decreased binding affinity is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, 1000-fold, or more relative to a wildtype IL-2 polypeptide.

In some embodiments, an IL-2 polypeptide herein exhibits about the same binding affinity (±10%) to the IL-2Rβγ subunit relative to a wildtype IL-2 polypeptide.

In some embodiments, an IL-2 polypeptide herein increases activation of CD8+ T cells relative to a wildtype IL-2 polypeptide. In some embodiments, the increased activation of CD8+T cells is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or greater relative to activation of CD8+ T cells by a wildtype IL-2 polypeptide. In some embodiments, the increased activation of CD8+ T cells is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, 1000-fold, or greater relative to activation of CD8+T cells by a wildtype IL-2 polypeptide.

In some embodiments, an IL-2 polypeptide herein increases STAT5 phosphorylation in human CD8+ T cells relative to a wildtype IL-2 polypeptide. In some embodiments, the increased STAT5 phosphorylation in human CD8+ T cells is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or greater relative to STAT5 phosphorylation in human CD8+ T cells by a wildtype IL-2 polypeptide. In some embodiments, the increased STAT5 phosphorylation in human CD8+ T cells is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, 1000-fold, or greater relative to STAT5 phosphorylation in human CD8+ T cells by a wildtype IL-2 polypeptide.

In some embodiments, an IL-2 polypeptide herein increases activation of NK cells relative to a wildtype IL-2 polypeptide. In some embodiments, the increased activation of NK cells is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or greater relative to activation of NK cells by a wildtype IL-2 polypeptide. In some embodiments, the increased activation of NK cells is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, 1000-fold, or greater relative to activation of NK cells by a wildtype IL-2 polypeptide.

In some embodiments, an IL-2 polypeptide herein increases STAT5 phosphorylation in human NK cells relative to a wildtype IL-2 polypeptide. In some embodiments, the increased STAT5 phosphorylation in human NK cells is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or greater relative to STAT5 phosphorylation in human NK cells by a wildtype IL-2 polypeptide. In some embodiments, the increased STAT5 phosphorylation in human NK cells is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, 1000-fold, or greater relative to STAT5 phosphorylation in human NK cells by a wildtype IL-2 polypeptide.

In some embodiments, an IL-2 polypeptide herein decreases activation of Tregs relative to a wildtype IL-2 polypeptide. In some embodiments, the decreased activation of Tregs is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or greater relative to activation of Tregs by a wildtype IL-2 polypeptide. In some embodiments, the decreased activation of Tregs is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, 1000-fold, or greater relative to activation of Tregs by a wildtype IL-2 polypeptide.

In some embodiments, an IL-2 polypeptide herein decreases STAT5 phosphorylation in human Tregs relative to a wildtype IL-2 polypeptide. In some embodiments, the decreased STAT5 phosphorylation in human Tregs is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or greater relative to STAT5 phosphorylation in human Tregs by a wildtype IL-2 polypeptide. In some embodiments, the decreased STAT5 phosphorylation in human Tregs is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, 1000-fold, or greater relative to STAT5 phosphorylation in human Tregs by a wildtype IL-2 polypeptide.

In any of the above embodiments, the wildtype IL-2 comprises an amino acid residue mutation at C125 of a sequence of wildtype human IL-2. In some embodiments, the amino acid residue mutation at C125 is C125A or C125S.

In some embodiments, an IL-2 polypeptide herein comprises an IL-2 mutein is fused to a heterologous polypeptide (i.e., a polypeptide that is not IL-2 and preferably is not a variant of IL-2, e.g., a conjugated moiety described herein. The heterologous polypeptide can increase the circulating half-life of the IL-2 polypeptide. For example, the polypeptide that increases the circulating half-life can be serum albumin, e.g., HSA.

In some embodiments, an IL-2 polypeptide provided herein includes an amino acid sequence that is heterologous to wildtype human IL-2 or a functional variant thereof. The amino acid sequence can be a half-life extension moiety that increases stability of the polypeptide by increasing in vivo half-life of the polypeptide, e.g., when administered to a subject. The half-life extension moiety can be a protein, an antibody, an albumin, an immunoglobulin or a fragment thereof, a transferrin, or a PEG. The antibody can be an anti-albumin antibody. The albumin can be a serum albumin, e.g., a mouse serum albumin or a human serum albumin. In some embodiments, the immunoglobulin fragment is the Fc domain of a human immunoglobulin, e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM.

In some embodiments, a polypeptide provided herein is an antibody fusion protein. For example, a polypeptide provided herein can be an immunoglobulin Fc fusion protein. The polypeptide can include an immunoglobulin Fc domain. In some embodiments, the immunoglobulin Fc domain is a human immunoglobulin Fc domain, e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, IgM, or a fragment thereof.

IL-2 Binding and Activity

Binding affinity of an IL-2 polypeptide provided herein to a target receptor or a subunit thereof can be assessed by measuring the dissociation constant (K_D) . In some embodiments, an IL-2 polypeptide provided herein binds to IL-2Rα with a K_D that is at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 100-fold, or at least 1000-fold, or at least 10,000-fold, or at least 100,000-fold higher than the K_D of wildtype IL-2. In some embodiments, an IL-2 polypeptide provided herein binds to IL-2Rα with a K_D of at least 10^-9 M, at least 10^-8 M, or greater. In some embodiments, an IL-2 polypeptide provided herein binds to IL-2Rα with a K_D of 10^-10 M, 10^-9 M, 10^-8 M, or greater. In some embodiments, an IL-2 polypeptide provided herein binds to IL-2Rα with a K_D of from 10^-10 M to 10^-9 M, from 10^-9 M to 10^-8 M, from 10^-8 M to 10^-7 M. In some embodiments, an IL-2 polypeptide provided herein binds to IL-2Rα with a K_D of about 3 x 10^-9 M, about 6 x 10^-9 M, about 1 x 10^-8 M, about 2 x 10^-8 M, or about 3 x 10^-8 M.

In some embodiments, an IL-2 polypeptide provided herein binds to IL-2Rβγ with a K_D that is approximately the K_D of wildtype IL-2 binding to IL-2Rβγ. In some embodiments, an IL-2 polypeptide provided herein binds to IL-2Rβγ with a K_D that is at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold lower than the K_D of wildtype IL-2. In some embodiments, an IL-2 polypeptide provided herein binds to IL-2Rβγ with a K_D of about 10^-9 M or less. In some embodiments, an IL-2 polypeptide provided herein binds to IL-2Rβγ with a K_D of 10^-10 M, or less.

IL-2 signaling activity by an IL-2 mutant provided herein can be assessed by IL-2-mediated phosphorylation of STAT5. Human peripheral blood mononuclear cells (PBMCs) can be treated with an IL-2 polypeptide provided herein and phosphorylation of STAT5 can be subsequently analyzed in NK cells and different T cell subsets by flow cytometry. STAT5 phosphorylation can be used to calculate the half-maximal effect concentration or EC₅₀. Relative activity of an IL-2 mutant can be determined by comparing EC₅₀ values of mutant to wildtype.

Methods of Use

In some embodiments, provided herein is a method of treating a cell proliferative disease or condition in a subject in need thereof by administering to the subject a therapeutically effective amount of an IL-2 polypeptide described herein. In some instances, the IL-2 polypeptide comprises an isolated and purified IL-2 mutein, wherein the IL-2 polypeptide has a decreased affinity to IL-2Rα relative to a wildtype IL-2. In some instances, the IL-2 polypeptide comprises an isolated and purified IL-2 mutein having one or more amino acid substitutions at positions K35, R38, F42, K43, F44, E61, E62, K64, P65, E68, L72, Y107, and/or C125 of wildtype human IL-2 (SEQ ID NO: 1) . In some cases, the IL-2 polypeptide preferentially interacts with the IL-2Rβ and IL-2Rγ subunits to form an IL-2/IL-2Rβγ complex. Formation of the IL-2/IL-2Rβγ complex can thereby stimulate or enhance expansion of CD4+ helper cells, CD8+ effector naive and memory T cells, NK cells, and/or NKT cells. In some cases, the expansion of Teff cells skews the Teff/Treg ratio toward the Teff population.

In some embodiments, the cell proliferative disease or condition is a neoplastic disease, such as cancer. In some cases, the cancer is a metastatic cancer. In some cases, the cancer is a relapsed or refractory cancer. In some embodiments, the cancer is a cancer. The treatment-naive cancer can be a cancer that has not been treated by a therapy.

In some embodiments, the cancer is leukemia, lymphoma, sarcoma, myeloma, glioma, glioblastoma, glioblastoma multiforme, glioma, head and neck cancer, colorectal cancer, colon cancer, prostate cancer, castration-resistant prostate cancer, pancreatic cancer, melanoma, breast cancer (e.g., triple negative, ER positive, ER negative, chemotherapy resistant, trastuzumab-resistant, HER2 positive, doxorubicin resistant, tamoxifen resistant, ductal carcinoma, lobular carcinoma, primary, metastatic) , neuroblastoma, lung cancer (e.g., non-small cell lung carcinoma, squamous cell lung carcinoma (e.g., head, neck, or esophagus) , adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, carcinoid, sarcoma) , ovarian cancer, bone cancer (e.g., osteosarcoma, chondrosarcoma, Ewing sarcoma) , bladder cancer, cervical cancer, liver cancer (e.g., hepatocellular carcinoma) , kidney cancer, skin cancer, testicular cancer, adrenal cancer, adenoid cystic carcinoma, anal cancer, brain cancer, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, oral cancer, thyroid cancer, retinoblastoma, parathyroid cancer, pituitary cancer, bile duct cancer, uterine cancer, acute myeloid leukemia, lymphoma, B cell lymphoma, multiple myeloma, mesothelioma, medulloblastoma, Hodgkin’s Disease, Non-Hodgkin’s Lymphoma, neuroblastoma, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant pancreatic insulinoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, neuroblastoma, genitourinary tract cancer, malignant hypercalcemia, adrenal cortical cancer, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary thyroid carcinoma, papillary thyroid cancer, hepatocellular carcinoma, Paget’s disease of the nipple, phyllodes tumors, lobular carcinoma, ductal carcinoma, cancer of the pancreatic stellate cells, or cancer of the hepatic stellate cells. Additional non-limiting examples of cancers include, cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head and neck, esophagus, liver, kidney, ovary, stomach, or uterus.

In some embodiments, the cancer is a solid tumor. Non-limiting examples of solid tumors include bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, and prostate cancer.

In some embodiments, the cancer is a hematologic malignancy, such as a leukemia, a lymphoma, or a myeloma. In some cases, the hematologic malignancy is a T cell malignancy. In other cases, the hematological malignancy is a B cell malignancy. Non-limiting examples of hematologic malignancies include chronic lymphocytic leukemia (CLL) , small lymphocytic lymphoma (SLL) , follicular lymphoma (FL) , diffuse large B-cell lymphoma (DLBCL) , mantle cell lymphoma (MCL) , Waldenstrom’s macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt’s lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B cell lymphoma (PMBL) , immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

In some embodiments, provided herein is a method of treating an inflammatory or autoimmune disease or condition in a subject in need thereof by administering to the subject a therapeutically effective amount of an IL-2 polypeptide described herein. In some embodiments, the inflammatory or autoimmune disease is atherosclerosis, obesity, inflammatory bowel disease (IBD) , rheumatoid arthritis, allergic encephalitis, psoriasis, atopic skin disease, osteoporosis, peritonitis, hepatitis, lupus, celiac disease, syndrome, polymyalgia rheumatica, multiple sclerosis (MS) , ankylosing spondylitis, type 1 diabetes mellitus, alopecia areata, vasculitis, and temporal arteritis, graft versus host disease (GVHD) , asthma, COPD, a paraneoplastic autoimmune disease, cartilage inflammation, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter’s Syndrome, seronegative enthesopathy and arthropathy (SEA) syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, pauciarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, enteropathic arthritis, reactive arthritis, Reiter’s syndrome, dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myolitis, polymyolitis, dermatomyolitis, polyarteritis nodossa, Wegener’s granulomatosis, arteritis, ploymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, Still’s disease, Systemic Lupus Erythematosus (SLE) , myasthenia gravis, Crohn’s disease, ulcerative colitis, celiac disease, rhinosinusitis, rhinosinusitis with polyps, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barre disease, thyroiditis (e.g., Graves’ disease) , Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, transplantation rejection, kidney damage, or hepatitis C-induced vasculitis.

Pharmaceutical Compositions

A pharmaceutical composition of the disclosure can be a combination of a compound, e.g., an IL-2 polypeptide described herein, with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the compound to an organism.

Pharmaceutical formulations for administration can include aqueous solutions of the active compounds in water-soluble form. Suspensions of the active compounds can be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension can also contain suitable stabilizers or agents which increase the solubility of a compound to allow for the preparation of highly concentrated solutions. The active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

In practicing the methods of treatment or use provided herein, therapeutically effective amounts of a compound described herein is administered in a pharmaceutical composition to a subject having a disease or condition to be treated. In some embodiments, the subject is a mammal such as a human. A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors.

Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Formulation can be modified depending upon the route of administration chosen. Pharmaceutical compositions comprising compounds described herein can be manufactured, for example, by mixing, dissolving, emulsifying, encapsulating, entrapping, or compression processes.

The pharmaceutical compositions can include at least one pharmaceutically acceptable carrier, diluent, or excipient and compounds described herein as free-base or pharmaceutically acceptable salt form. Pharmaceutical compositions can contain solubilizers, stabilizers, tonicity enhancing agents, buffers, and preservatives.

Methods for the preparation of compositions comprising the compounds described herein include formulating the compounds with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid, or liquid composition. Solid compositions include, for example, powders, tablets, dispersible granules, capsules, and cachets. Liquid compositions include, for example, solutions in which a compound is dissolved, emulsions comprising a compound, or a solution containing liposomes, micelles, or nanoparticles comprising a compound as disclosed herein. Semi-solid compositions include, for example, gels, suspensions, and creams. The compositions can be in liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to use, or as emulsions. These compositions can also contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically acceptable additives.

Non-limiting examples of dosage forms suitable for use in the disclosure include liquid, powder, gel, nanosuspension, nanoparticle, microgel, aqueous or oily suspensions, emulsion, and any combination thereof.

Non-limiting examples of pharmaceutically acceptable excipients suitable for use in the disclosure include binding agents, disintegrating agents, anti-adherents, anti-static agents, surfactants, anti-oxidants, coating agents, coloring agents, plasticizers, preservatives, suspending agents, emulsifying agents, anti-microbial agents, spheronization agents, and any combination thereof.

A composition of the disclosure can be, for example, an immediate release form or a controlled release formulation. An immediate release formulation can be formulated to allow the compounds to act rapidly following administration. Non-limiting examples of immediate release formulations include readily dissolvable formulations. A controlled release formulation can be a pharmaceutical formulation that has been adapted such that release rates and release profiles of the active agent can be matched to physiological and chronotherapeutic requirements, or has been formulated to effect release of an active agent at a programmed rate. Non-limiting examples of controlled release formulations include granules, delayed release granules, hydrogels (e.g., of synthetic or natural origin) , other gelling agents (e.g., gel-forming dietary fibers) , matrix-based formulations (e.g., formulations comprising a polymeric material having at least one active ingredient dispersed through) , granules within a matrix, polymeric mixtures, and granular masses.

In some embodiments, a controlled release formulation is a delayed release form. A delayed release form can be formulated to delay a compound’s action for an extended period of time. A delayed release form can be formulated to delay the release of an effective dose of one or more compounds, for example, for about 4, about 8, about 12, about 16, or about 24 hours.

A controlled release formulation can be a sustained release form. A sustained release form can be formulated to sustain, for example, the compound’s action over an extended period of time. A sustained release form can be formulated to provide an effective dose of any compound described herein (e.g., provide a physiologically effective blood profile) over about 4, about 8, about 12, about 16, or about 24 hours.

A compound described herein can be conveniently formulated into pharmaceutical compositions composed of one or more pharmaceutically acceptable carriers. See e.g., Remington’s Pharmaceutical Sciences, latest edition, by E.W. Martin Mack Pub. Co., Easton, PA, herein incorporated by reference in its entirety, which discloses pharmaceutically acceptable excipients and carriers, and methods of preparing pharmaceutical compositions. Such carriers can be carriers for administration of compositions to humans and non-humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Pharmaceutical compositions can also include one or more additional active ingredients such as antimicrobial agents, anti-inflammatory agents, and anesthetics.

Pharmaceutical formulations can include additional carriers, as well as thickeners, diluents, buffers, preservatives, and surface active agents in addition to the agents disclosed herein.

A pharmaceutical composition disclosed herein can be administered in a therapeutically effective amount by various forms and routes including, for example, oral, topical, parenteral, intravenous injection, intravenous infusion, subcutaneous injection, subcutaneous infusion, intramuscular injection, intramuscular infusion, intradermal injection, intradermal infusion, intraperitoneal injection, intraperitoneal infusion, intracerebral injection, intracerebral infusion, subarachnoid injection, subarachnoid infusion, intraocular injection, intraspinal injection, intrasternal injection, ophthalmic administration, endothelial administration, local administration, intranasal administration, intrapulmonary administration, rectal administration, intraarterial administration, intrathecal administration, inhalation, intralesional administration, intradermal administration, epidural administration, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa) , intracapsular administration, subcapsular administration, intracardiac administration, transtracheal administration, subcuticular administration, subarachnoid administration, subcapsular administration, intraspinal administration, or intrasternal administration.

A pharmaceutical composition can be administered in a local manner, for example, via injection of the compound directly into an organ, optionally in a depot or sustained release formulation or implant. A pharmaceutical composition can be provided in the form of a rapid release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation. A rapid release form can provide an immediate release. An extended release formulation can provide a controlled release or a sustained delayed release.

A compound herein may be administered in combination with one or more therapeutic agents, for example, a cytokine, antiviral agent, or antifungal agent. The term “therapeutic agent” encompasses any agent administered to treat a symptom or disease in an animal in need of such treatment. The compound can also be administered as a component of a vaccine, i.e., combined with essentially any preparation intended for active immunological prophylaxis.

Toxicity and therapeutic efficacy of a compound herein can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD50 (the dose lethal to 50%of a population) and the ED50 (the dose therapeutically effective in 50%of a population) . The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50/ED50) . A compound herein that exhibits large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon a variety of factors, e.g., the dosage form employed, the route of administration utilized, the condition of the subject, and the like.

A therapeutically effective dose can be estimated initially from cell culture assays by determining an IC50. A dose can then be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by HPLC. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

An attending physician for patients treated with a compound herein would know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity) . The magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient.

A compound herein can be administered to an individual alone as a pharmaceutical preparation appropriately formulated for the route of delivery and for the condition being treated. Suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, and the like. For transmucosal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation.

A compound herein can be formulated as a liquid with carriers that may include a buffer and or salt such as phosphate buffered saline. Alternatively, a compound herein may be formulated as a solid with carriers or fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.

For oral delivery, the formulated end product may be a tablet, pill, capsule, dragee, liquid, gel, syrup, slurry, suspension, and the like. Also, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol may be used. The push-fit capsules can contain the active ingredients in admixture with fillers as above while in soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.

Formulation for oral delivery can involve conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, lyophilizing processes, and the like. A compound herein also may be mixed with a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, sorbitol, and the like; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (PVP) , and the like, as well as mixtures of any two or more thereof. If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate, and the like.

If injection is desired, a compound herein can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Kits

In some embodiments, provided herein is a kit or article of manufacture comprising a polypeptide described herein, including a pharmaceutical composition thereof. The kit can include instructions for use of a polypeptide such as in the methods provided herein. Thus, in some embodiments, the kit includes instructions for the use of a polypeptide in methods for treating a disorder described herein (e.g., a cancer) in a subject in need thereof by administering to the subject a therapeutically effective amount of the polypeptide. In some embodiments, the subject is a human. In some embodiments, the disorder is a cancer. In some embodiments, the polypeptide is an IL-2 polypeptide provided herein.

The kit can further include a container. Non-limiting examples of suitable containers include bottles, vials (e.g., dual chamber vials) , syringes (such as single or dual chamber syringes) , test tubes, and intravenous (IV) bags. The container can be formed from a variety of materials such as glass or plastic. The container can hold a formulation of an IL-2 polypeptide. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a liquid formulation.

The kit may further comprise a label or a package insert, which is on or associated with the container, may indicate directions for reconstitution and/or use of the formulation. The label or package insert may further indicate that the formulation is useful or intended for intravenous, subcutaneous, or other modes of administration for treating a disorder (e.g., a cancer) in a subject. The container holding the formulation can be a single-use vial or a multi-use vial, which can allow for repeat administrations of the reconstituted formulation. The kit can further include a second container comprising a suitable diluent. The kit can further include other materials desirable from a commercial, therapeutic, and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

In some embodiments, provided herein is a kit for a single dose-administration unit. Such a kit includes a container of an aqueous formulation of a therapeutic IL-2 polypeptide, including single or multi-chambered pre-filled syringes.

In some embodiments, provided herein is an article of manufacture or kit comprising the formulations described herein for administration in an auto-injector device. An auto-injector can be described as an injection device that upon activation, will deliver its contents without additional necessary action from the patient or administrator. They are particularly suited for self-medication of therapeutic formulations when the delivery rate must be constant, and the time of delivery is greater than a few moments.

EXAMPLES

Example 1: Recombinant expression and preparation of mutant IL-2 proteins.

IL-2 mutants were generated by mutating select positions of wildtype human IL-2 (wt; SEQ ID NO: 1) . Key positions of the amino acid sequences of wildtype human IL-2 and IL-2 mutant proteins are shown in TABLE 1. For mutants, only mutated positions are shown; non-mutated positions are in blank and correspond to the residue in the wildtype protein. The corresponding full-length amino acid sequences are shown in TABLE 2. Each of the proteins of TABLE 1 is C-terminally linked with GGGGS linker and His-tag for purification, and N-terminally linked with an 18-amino acid signal peptide for expression (signal peptide is added to promote expression in mammalian cell lines and subsequently cleaved in the expression product) . The numbering scheme of the mutation positions in TABLE 1 corresponds to a mature human IL-2 protein as in SEQ ID NO: 1. A mature human IL-2 protein does not include methionine as the first amino acid residue at the N-terminus. As such, the numbering scheme starts with alanine as the first residue of the 133 amino acid sequence of SEQ ID NO: 1. Some variants described herein also include a C125A mutation, which reduces the likelihood of the formation of dimeric IL-2.

TABLE 1

TABLE 2

1) Construction of expression plasmid.

The sequence of the N-terminal 18-amino acid signal peptide is (from N-to C-terminus) :

An exemplary amino acid sequence of encoded IL-2 construct is (from N-to C-terminus) :

The bold segment represents the 18-amino acid signal peptide; the underlined segment represents the mature 133-amino acid IL-2 sequence; the italicized segment represents the G₄S linker and 6X-His-tag.

Based on the amino acid sequence of wildtype IL-2 and mutant IL-2 proteins (including the signal peptide and G₄S linker and 6X-His-tag) , the DNA sequence was synthesized and confirmed by gene sequencing.

An example DNA expression sequence of an IL-2 construct is (from N-to C-terminus) :

Segment (i) represents a NotI endonuclease cleavable site; Segment (ii) represents a Okaxaki fragment element; the Segment (iii) in bold represents the 18-amino acid signal peptide encoding sequence; the underlined Segment (iv) represents the mature IL-2 encoding sequence; the italicized Segment (v) represents the human G₄S linker and 6X-His-tag encoding sequence; Segment (vi) represents a stop codon; and Segment (vii) represents a XbaI endonuclease cleavable site.

The confirmed DNA sequence was constructed into the expression vector pcDNA3.1 (Thermo Fisher, catalog no. V79020) . The plasmid containing the IL-2 gene (FIG. 1) was transformed into E. coli DH5α cells. A large amount of both wildtype and mutant plasmids was obtained by culturing the E. coli DH5α cells for amplification and plasmid purification.

2) Expression of wildtype IL-2 and mutant IL-2 proteins in HEK293F cells.

Cells were inoculated in 300 mL of medium (Gibco^TM FreeStyle^TM 293 expression medium, catalog no. 12338018) in a 1-L shake flask at an inoculation volume of 0.5x10⁶ cells/ml. The cells were incubated for 48-54 hr in a shaker incubator at 37 ℃, 120 rpm, and 5%carbon dioxide concentration. The cells were then incubated for 24 hr in a bed incubator until the cell density reached 1x10⁶ cells/ml. Then, 300 μg of wildtype or mutant expression plasmid was added to 30 ml of medium. The mixture was vortexed for 3s to thoroughly mix. About 1.2 ml (0.5 mg/ml) of transfection reagent PEI MAX 40K (Polysciences, catalog no. 24765) was added to the transfection reagent /plasmid mixture. The mixture was held static for 20 min, and then added to the HEK293F cells. After transfection, the cells were incubated for 48-54 hr in a shaker incubator at 37 ℃, 120 rpm, and 5%carbon dioxide concentration. After incubation, the supernatant of the culture medium was isolated by centrifugation and prepared for protein purification.

3) Purification and detection of wildtype IL-2 and mutant IL-2 proteins.

The wildtype IL-2 and mutant IL-2 proteins (each containing a His-tag) were purified using Ni-NTA agarose microspheres (Thermo Fisher, catalog no. R901) . An appropriate amount of Ni-NTA agarose microspheres were added to the supernatant in the previous step, and incubated at 4 ℃ for 30 min. An appropriate volume of wash buffer (50 mM PBS, pH 7.4, 10 mM imidazole) was then added to remove non-specific proteins. Finally, an appropriate volume of elution buffer (50 mM PBS, pH 7.4, 250 mM imidazole) was added to elute the wildtype or mutant protein-bound microspheres and obtain the purified wildtype or mutant proteins.

The purified wildtype or mutant proteins were detected by SDS-PAGE protein gel. The results of SDS-PAGE protein gel detection showed that the molecular weights of wildtype IL-2 protein, m21, m24, and m52 mutant proteins were consistent with the theoretical molecular weights, confirming that the correct proteins were expressed (FIG. 2) . Size exclusion chromatography (SEC) was used to detect the purity and protein aggregation of the wildtype or mutant proteins. The SEC (at 280 nm) results showed that the peaks of wildtype IL-2 protein, m21, m24, and m52 mutant proteins were all around 20 min, indicating that these proteins were monomeric proteins and did not produce aggregated proteins (FIG. 3) .

Example 2: Recombinant expression and preparation of IL-2-Fc fusion proteins.

Mutant IL-2-Fc fusion proteins may be generated based on the amino acid sequences of wildtype human IL-2 and IL-2 mutant proteins shown in TABLE 2.

1) Construction of expression plasmid.

Based on the amino acid sequence of wildtype IL-2-Fc and mutant IL-2-Fc proteins (Fc fragment may be fused to the C-or N-terminus of the IL-2 molecule) , the DNA sequence may be synthesized and confirmed by gene sequencing. The confirmed DNA sequence may be constructed into the expression vector pcDNA3.1 (Thermo Fisher, catalog no. V79020) . The plasmid containing the IL-2 gene may be transformed into E. coli DH5α cells. A large amount of both wildtype and mutant IL-2-Fc plasmid may be obtained by culturing the E. coli DH5α cells for amplification and plasmid purification.

2) Expression of wildtype IL-2-Fc and mutant IL-2-Fc fusion proteins in HEK293F cells.

Cells may be inoculated in 300 mL of medium (Gibco^TM FreeStyle^TM 293 expression medium, catalog no. 12338018) in a 1-L shake flask at an inoculation volume of 0.5x10⁶ cells/ml. The cells may be incubated for 48-54 hr in a shaker incubator at 37 ℃, 120 rpm, and 5%carbon dioxide concentration. The cells may then be incubated for 24 hr in a bed incubator until the cell density reaches 1x10⁶ cells/ml. Then, 300 μg of wildtype or mutant IL2-Fc expression plasmid may be added to 30 ml of medium. The mixture may then be vortexed for 3s to thoroughly mix. About 1.2 ml (0.5 mg/ml) of transfection reagent PEI MAX 40K (Polysciences, catalog no. 24765) may be added to the transfection reagent /plasmid mixture. The mixture may be held static for 20 min, and then added to the HEK293F cells. After transfection, the cells may be incubated for 48-54 hrs in a shaker incubator at 37 ℃, 120 rpm, and 5%carbon dioxide concentration. After incubation, the supernatant of the culture medium may be isolated by centrifugation and prepared for protein purification.

3) Purification and detection of wildtype IL-2 and mutant IL-2-Fc fusion proteins.

The wildtype IL-2-Fc and mutant IL-2-Fc proteins may be purified using Protein A agarose microspheres. An appropriate amount of Protein A agarose microspheres may be added to the supernatant in the previous step, and incubated at 4 ℃ for 10 min. The mixture may then be subjected to low-speed centrifugation at 1000 rpm for 3 min to collect the IL-2-Fc fusion protein-bound microspheres. An appropriate volume of wash buffer (50 mM PBS, pH 7.4) may then be added to remove non-specific proteins. Finally, an appropriate volume of elution buffer (Glycine-HCl, pH 3) may be added to elute the wildtype or mutant protein-bound microspheres and obtain purified wildtype or mutant fusion proteins.

Example 3: Recombinant expression and preparation of IL-2-antibody fusion proteins.

Mutant IL-2-antibody fusion proteins may be generated based on the amino acid sequences of wildtype human IL-2 and IL-2 mutant proteins shown in TABLE 2. The IL-2 mutant-antibody fusion proteins comprise IL-2 mutant sequences and complete antibodies or antigen-binding moiety thereof (such as PD-1 antibody) . Among them, the IL-2 mutant may be linked to the N-terminus or C-terminus of the light chain or heavy chain of the antibody.

1) Construction of expression plasmid.

Based on the amino acid sequence of wildtype IL-2 and mutant IL-2 proteins and the gene sequence of antibody light chain or heavy chain, the DNA sequence may be synthesized and confirmed by gene sequencing. The confirmed DNA sequence may be constructed into the expression vector pcDNA3.1 (Thermo Fisher, catalog no. V79020) . The plasmid containing the IL-2 gene may be transformed into E. coli DH5α cells. A large amount of both wildtype and mutant-antibody plasmids may be obtained by culturing the E. coli DH5α cells for amplification and plasmid purification.

2) Expression of wildtype IL-2-Ab and mutant IL-2-Ab fusion proteins in HEK293F cells.

Cells may be inoculated in 300 mL of medium (Gibco^TM FreeStyle^TM 293 expression medium, catalog no. 12338018) in a 1-L shake flask at an inoculation volume of 0.5x10⁶ cells/ml. The cells may be incubated for 48-54 hr in a shaker incubator at 37 ℃, 120 rpm, and 5%carbon dioxide concentration. The cells may then be incubated for 24 hr in a bed incubator until the cell density reached 1x10⁶ cells/ml. Then, 300 μg of wildtype or mutant IL-2-Ab fusion expression plasmid may be added to 30 ml of medium. The mixture may be vortexed for 3 s to thoroughly mix. About 1.2 ml (0.5 mg/ml) of transfection reagent PEI MAX 40K (Polysciences, catalog no. 24765) may be added to the transfection reagent /plasmid mixture. The mixture may be held static for 20 min, and then added to the HEK293F cells. After transfection, the cells may be incubated for 48-54 hr in a shaker incubator at 37 ℃, 120 rpm, and 5%carbon dioxide concentration. After incubation, the supernatant of the culture medium may be isolated by centrifugation and prepared for protein purification.

3) Purification of wildtype IL-2-antibody and mutant IL-2-antibody fusion proteins.

The wildtype IL-2-antibody and mutant IL-2-antibody fusion proteins may be purified using Protein A agarose microspheres. An appropriate amount of Protein A agarose microspheres may be added to the supernatant in the previous step, and incubated at 4 ℃ for 10 min. The mixture may then be subjected to low-speed centrifugation at 1000 rpm for 3 min to collect the IL-2-antibody fusion protein-bound microspheres. An appropriate volume of wash buffer (50 mM PBS, pH 7.4) may then be added to remove non-specific proteins. Finally, an appropriate volume of elution buffer (Glycine-HCl, pH 3) may be added to elute the wildtype or mutant protein-bound microspheres and obtain purified wildtype or mutant fusion proteins.

Example 4: Determination of thermal stability of IL-2 mutant proteins.

For assessment of conformational stability, the melting point T_m (℃) values, which indicate the structural stability of the protein samples, were obtained by monitoring the intrinsic tryptophan and tyrosine fluorescence at the emission wavelengths of 330 nm and 350 nm.

IL-2 mutant protein stability was assessed by detecting small changes in tryptophan and tyrosine fluorescence in the mutant IL-2 protein using the label-free nano differential scanning fluorimetry (nanoDSF) assay (Prometheus, PR NT. 48) . Briefly, wt (C145A) IL-2 and IL-2 mutant proteins were prepared as described in Example 1. The wt (C145A) or mutant IL-2 protein samples were then diluted in PBS to a concentration of 0.5 mg/ml. About 10 μl of protein sample (wt (C125A) or mutant IL-2 proteins) were loaded in the detection capillaries and then placed on the sample holder. A temperature gradient of 1 ℃/min from 25-90 ℃ was then applied, and the intrinsic protein fluorescence at 330 and 350 nm was recorded for each sample. The data were analyzed using the data analysis software provided by the NT. 48 instrument.

As shown in TABLE 8, m24, m27, and m47 demonstrated high thermal stability with Tm of 74.2 ℃, 74.1 ℃, and 77.4℃ respectively, each of which was notably higher than that of wt (C125A) (T_m of 66.9 ℃) .

TABLE 8

Example 5: Determination of the affinity of mutant IL-2 protein to human IL-2Rαreceptor.

The affinity of mutant IL-2 protein to human IL-2Rα-Fc fusion protein (ACRO, Catalog: ILA-H5251) was determined by label-free biolayer interferometry (R8, BLI) as follows:

1: Mutant IL-2 proteins (see TABLE 3) were prepared and diluted with buffer (10 mM PBS + 0.02%TW-20 + 0.1%BSA, pH 7.4) to a final concentration of 5 μg/ml.

2: The IL-2Rα-Fc fusion protein was diluted with buffer (10 mM PBS + 0.02%tween-20 + 0.1%BSA, pH 7.4) from the initial concentration of 1000 nM to 15.6 nM in a 4-fold gradient to yield a total of 5 concentrations: 1000 nM, 250 nM, 62.5 nM, and 15.6 nM, respectively.

3: The NTA sensor was soaked in buffer (10 mM PBS + 0.02%tween-20 + 0.1%BSA, pH 7.4) for 10 min to equilibrate.

4: The IL-2 mutant protein was loaded onto the NTA sensor. The loading time was 90s. The mutant protein was then eluted and then equilibrated for 30 s. The mutant protein was then combined with 4 different concentrations of IL-2Rα-Fc fusion protein. The binding time was 120 s. Finally, the bound IL-2Rα-Fc fusion protein was eluted with buffer. The dissociation time was 180 s.

5: The Octet Data Acquisition Software ofR8 was used for data processing and data fitting.

The BLI affinity test results of wildtype IL-2 and mutant IL-2 to IL-2Rα are shown in TABLE 3. The test results include the dissociation constant (K_D) , the rate constant of association of the mutant to the receptor (k_on) , and the rate constant of dissociation of the mutant from the receptor (k_dis) . ND, undetectable. Since the affinity of some mutants to IL-2Rα was too low, the affinity could not be determined by fitting the data in the concentration range used in the experiment.

Results.

1. The affinity (K_D) of wildtype and IL-2Rα was 6.71E-10 M.

2. All IL-2 mutants exhibited at least 1 order of magnitude decrease in affinity for IL-2Rα compared to wildtype.

3. The affinity of some mutants (such as m17, m19, m20, and m21) to IL-2Rα was decreased by about 1-2 orders of magnitude compared with the affinity of wildtype to IL-2Rα.

4. Other mutants (such as m24, m52, etc. ) exhibited an even more significant decrease in affinity to IL-2Rα. These affinities were too low to be detectable (see ND data) .

TABLE 3

Example 6: Determination of the affinity of mutant IL-2 protein and human IL-2Rβγ.

The affinity of mutant IL-2 protein to human IL-2Rβγ-Fc fusion protein (ACRO, Catalog: ILG-H5254) was determined by label-free biolayer interferometry (R8, BLI) as follows:

1: Mutant IL-2 proteins (see TABLE 4) were prepared and diluted with buffer (10 mM PBS + 0.02%tween-20 + 0.1%BSA, pH 7.4) to a final concentration of 5 μg/ml.

2: The IL-2Rβγ-Fc fusion protein was diluted with buffer (10 mM PBS + 0.02%tween-20 + 0.1%BSA, pH 7.4) from the initial concentration of 1000 nM to 15.6 nM in a 4-fold gradient to yield a total of 5 concentrations: 1000 nM, 250 nM, 62.5 nM, and 15.6 nM, respectively.

4: The mutant IL-2 protein was loaded onto the NTA sensor. The loading time was 90s. The mutant protein was then eluted and then equilibrated for 30 s. The mutant protein was then combined with 4 different concentrations of IL-2Rβγ-Fc fusion protein. The binding time was 120 s. Finally, the bound IL-2Rβγ-Fc fusion protein was eluted with buffer. The dissociation time was 180 s.

The BLI affinity test results of wildtype IL-2 and mutant IL-2 with IL-2Rβγ-Fc fusion protein are shown in TABLE 4. ND, undetectable. Since the affinity of these mutants for IL-2Rα was too low, the affinity could not be obtained by fitting the data in the concentration range used in the experiment.

Results.

1. The affinity (K_D) of wildtype WT (C125A) to IL-2Rβγ was 5.25E-09 M.

2. The affinity of all IL-2 mutants to IL-2Rβγ had no significant change compared with the affinity of wildtype IL-2.

3. The affinity of some mutants (such as m17, m19, m20) to IL-2Rβγ remained unchanged or even higher compared with that of wildtype IL-2.

TABLE 4

Example 7: Determination of pSTAT5 phosphorylation in human peripheral blood by IL-2 mutants.

IL-2 biological activity of wildtype IL-2 or mutant IL-2 proteins was assessed by measuring the phosphorylation level of STAT5 in various cell types (including CD8+T cells, NK cells, and Tregs) in human peripheral blood mononuclear cells (PMBCs) .

Human PBMC STAT5 phosphorylation assay:

1. About 4x10⁵ of freshly isolated human PBMCs were placed in each well of a 96-well plate.

2. Wildtype IL-2 or mutant IL-2 was diluted in basal medium to 1000 nM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM, and 0.0001 nM to produce a total of eight concentrations.

3. About 200 μl of wildtype IL-2 or mutant IL-2 at each dilution was added to the PBMCs. The cells were stimulated at 37 ℃ for 20 min, and then washed twice with staining buffer (BD, Cat No. 554655) .

4. Cells were fixed with 100 μl BD Cytofix^TM fixation buffer (BD, Cat No. 554655) at 4 ℃ for 15 min. Then, the PBMCs were washed twice with staining buffer (BD, Cat No. 554655) .

5. Detection antibodies Alexa700 Mouse Anti-Human CD3 (BD, Cat No. 557943) , APC/Cyanine7 anti-human CD8a Antibody (Biolegend, Cat No. 300926) , BV421 Mouse Anti-Human CD25 (BD, Cat No. 562442) , and Brilliant Violet 510^TM anti-human CD56 (NCAM) Antibody (Biolegend Cat No. 318340) were diluted with staining buffer (BD, Cat No. 554655) .

6. About 100 μl of the diluted antibody was added to the PBMCs. The cells were then incubated in the dark at 4 ℃ for 30 min. The cells were then washed twice with staining buffer (BD, Cat No. 554655) .

7. About 200 μl of Phosflow^TM Perm Buffer III (BD, Cat No. 558050) was added to the PBMCs. The cells were lysed by incubation for 30 min on ice. The cells were then washed twice with staining buffer (BD, Cat No. 554655) .

8. Detection antibodies PerCP-Cy^TM 5.5 Mouse Anti-Human CD4 (BD, Cat No. 560650) , PE Mouse anti-Human FoxP3 (BD, Cat No. 560046) , and Alexa647 Mouse Anti-Stat5 (pY694) (BD, Cat No. 562076) were diluted with staining buffer (BD, Cat No. 554655) .

9. About 100 μl of the diluted antibody to was added to the PBMCs. The cells were then incubated in the dark at 4 ℃ for 60 min. The cells were then washed twice with staining buffer (BD, Cat No. 554655) .

10. The PBMCs were detected by flow cytometry (Beckman CytoFLEX) . CD8+ T cells are defined as CD3+CD4-CD8+ cells, NK cells are defined as CD3-CD56+ cells, and Treg cells are defined as CD3+CD4+CD25+Foxp3+ cells.

The pSTAT5 fluorescence values (MFI) of CD8+ T cells, NK cells, and Treg cells under different IL-2 concentrations were calculated and fitted by computer program or four-parameter regression calculation method. The relative biological activity was calculated by the following formula: Relative activity of test article (%) = control article (wildtype IL-2) EC₅₀/test article (mutant IL-2) EC₅₀, where EC₅₀ is the half-maximal effect concentration.

The results showed that, relative to wildtype IL-2, all IL-2 mutants (see TABLES 5-7) had varying degrees of reduced affinity for IL-2Rα (see TABLE 3) , suggesting that these IL-2 mutants are more susceptible to Treg activity. At the same time, the affinity of IL-2 mutants to IL-2Rβγ did not change significantly relative to wildtype IL-2 (see TABLE 4) , suggesting that these IL-2 mutants have a range of changes in the activity of CD8+ T and NK cells (i.e., both up and down) .

Detection results of wildtype IL-2 and IL-2 mutants activating STAT5 phosphorylation in human CD8+ T cells are shown in TABLE 5. The biological activities of m24 and m27 on CD8+ T cells increased by 470.89%and 455.32%, respectively. Detection results of wildtype IL-2 and IL-2 mutants activating STAT5 phosphorylation in human NK cells are shown in TABLE 6. The biological activities of m24 and m27 on NK cells increased by 578.08%and 612.56%, respectively. Detection results of wildtype IL-2 and IL-2 mutants activating STAT5 phosphorylation in human Treg cells are shown in TABLE 7. The IL-2 mutants all reduced or effectively eliminated activating STAT5 phosphorylation of Tregs.

TABLE 5

TABLE 6

TABLE 7

Example 8: Evaluation of the therapeutic effect of IL-2 mutants in the C57BL/6 mouse B16-F10 cutaneous melanoma model.

B16-F10 melanoma cancer cells (about 5 x 10⁵ cells) were implanted subcutaneously into the flanks of C57BL/6-IL2RB^tm2 ^(IL2RB) IL2RG^tm2 ^(IL2RG) /Bcgen mice (Beijing Biocytogen Co., Ltd, stock No.: 111850) . At around the 6^th day post-inoculation, the average tumor volume was about 100 mm³. The tumor-bearing mice were divided into 3 groups with 8 mice per group by random block method. Mice bearing B16-F10 cells were treated with either PBS, wt (C125A) , or m24 protein once daily for 10 days (10 doses) . Body weight and tumor volume for each group were measured every 3 or 4 days throughout the study. Tumor measurements (length (L) and width (W) ) were collected three times per week using digital calipers, and the tumor volume was calculated using the formula: (LxWxW) /2. Finally, tumors were collected from mice treated with wt (C125A) or m24 protein to detect tumor infiltrating lymphocytes by flow cytometry.

The results showed comparable tumor growth inhibition in response to treatment with wt(C125A) and m24. The flow cytometry analysis showed that the CD3+CD4-CD8+ /CD3+CD4+CD25+FOXP3+ Treg ratio was higher in m24 treated tumors than in wt (C125A) treated tumors.

Claims

An isolated polypeptide comprising an IL-2 variant moiety having an amino acid residue mutation at K64 of a sequence of wildtype human IL-2.
The polypeptide of claim 1, wherein the sequence of wildtype human IL-2 comprises SEQ ID NO: 1.
The polypeptide of claim 1, wherein the amino acid residue mutation at K64 is K64P or K64E.
The polypeptide of any one of claims 1-3, having an amino acid residue mutation at P65 of wildtype human IL-2.
The polypeptide of claim 4, wherein the amino acid residue mutation at P65 is P65E, P65D, P65R, P65W, or P65K.
The polypeptide of any one of claims 1-5, wherein the polypeptide comprises one, two, three, four, or more amino acid residue mutations at positions selected from the group consisting of K35, R38, F42, K43, F44, E61, E62, L72, and Y107 of wildtype human IL-2.
The polypeptide of any one of claims 1-5, wherein the polypeptide comprises one or more amino acid residue mutations at positions selected from the group consisting of K35, R38, K43, E61, E62, L72, and Y107 of wildtype human IL-2.
The polypeptide of any one of claims 1-5, wherein the polypeptide comprises one or more amino acid residue mutations at positions selected from the group consisting of K35, R38, K43, and Y107 of wildtype human IL-2.
The polypeptide of any one of claims 1-5, wherein the polypeptide comprises one or more amino acid residue mutations at positions selected from the group consisting of F42, F44, and E62 of wildtype human IL-2.
The polypeptide of claim 4 or 5, wherein the polypeptide comprises an amino acid residue mutation at Y107 of wildtype human IL-2.
The polypeptide of claim 10, wherein the amino acid residue mutation at Y107 is Y107K or Y107R.
The polypeptide of any one of claims 4-5 and 10-11, wherein the polypeptide comprises an amino acid residue mutation at K35 of wildtype human IL-2.
The polypeptide of claim 12, wherein the amino acid residue mutation at K35 is K35Y, K35D, or K35Q.
The polypeptide of any one of claims 4-5 and 10-11, wherein the polypeptide comprises an amino acid residue mutation at R38 of wildtype human IL-2.
The polypeptide of claim 14, wherein the amino acid residue mutation at R38 is R38W or R38Q.
The polypeptide of claim 4 or 5, wherein the polypeptide comprises an amino acid residue mutation at K43 of wildtype human IL-2.
The polypeptide of claim 16, wherein the amino acid residue mutation at K43 is K43Q, K43R, or K43G.
The polypeptide of claim 4 or 5, wherein the polypeptide comprises an amino acid residue mutation at E61 of wildtype human IL-2.
The polypeptide of claim 18, wherein the amino acid residue mutation at E61 is E61T.
The polypeptide of claim 4 or 5, wherein the polypeptide comprises an amino acid residue mutation at E62 of wildtype human IL-2.
The polypeptide of claim 20, wherein the amino acid residue mutation at E62 is E62T, E62S, E62N, E62A, or E62R.
The polypeptide of claim 4 or 5, wherein the polypeptide comprises an amino acid residue mutation at L72 of wildtype human IL-2.
The polypeptide of claim 22, wherein the amino acid residue mutation at L72 is L72T.
The polypeptide of any one of claims 4, 5, and 10-23, wherein the polypeptide comprises one, two, three, or more amino acid residue mutations at positions selected from the group consisting of F42, K43, F44, and E62 of wildtype human IL-2.
The polypeptide of claim 24, wherein the amino acid residue mutation at F42 is F42K, F42R, F42E, or F42P.
The polypeptide of claim 24 or 25, wherein the amino acid residue mutation at K43 is K43R, K43G, or K43Q.
The polypeptide of any one of claims 24-26, wherein the amino acid residue mutation at F44 is F44L, F44M, or F44N.
The polypeptide of any one of claims 24-27, wherein the amino acid residue mutation at E62 is E62S, E62R, E62T, E62N, or E62A.
The polypeptide of claim 1 or 2, wherein the polypeptide comprises amino acid residue mutations selected from the group consisting of:

1) K64P and P65E;

2) K64P and P65D;

3) K64P and P65R;

4) K64P and P65W;

5) K64P and P65K;

6) K64E and P65R;

7) K64P and P65D;

8) K64E and P65D; and

9) K64P and P65W.
The polypeptide of claim 1 or 2, wherein the polypeptide comprises amino acid residue mutations selected from the group consisting of:

10) K35Y, K64P, and P65K;

11) R38W, K64P, and P65K;

12) R38Q, K64P, and P65K;

13) K43Q, K64P, and P65K;

14) E61T, K64P, and P65K;

15) K64P, P65K, and L72T;

16) K35Y, K64P, and P65R;

17) R38W, K64P, and P65R;

18) R38D, K64P, and P65R;

19) K43Q, K64P, and P65R;

20) E62T, K64P, and P65R;

21) K64P, P65R, and L72T;

22) K64E and P65R; and

23) E62R, K64P, and P65D.
The polypeptide of claim 1 or 2, wherein the polypeptide comprises amino acid residue mutations selected from the group consisting of:

24) K35Q, K64P, P65K, and Y107K;

25) R38Q, K64P, P65K, and Y107R;

26) K35Q, K64P, P65R, and Y107K; and

27) R38Q, K64P, P65R, and Y107R.
The polypeptide of claim 1 or 2, wherein the polypeptide comprises amino acid residue mutations selected from the group consisting of:

28) K43R, K64E, and P65R;

29) F42K, K43R, K64E, and P65R;

30) F42R, K43R, F44L, K64P, and P65E;

31) F42R, K43R, F44M, E62S, K64E, and P65D;

32) F42R, K43R, F44M, E62R, K64P, and P65E;

33) F42R, K43G, F44L, E62N, K64P, and P65D;

34) F42R, K43R, F44L, K64P, and P65R;

35) F42R, K43R, F44L, E62R, K64P, and P65D;

36) F42R, K43G, F44L, E62N, K64P, and P65D; and

37) F42P, K43R, F44N, E62A, K64P, and P65W.
The polypeptide of any one of claims 1-32, wherein the polypeptide comprises an amino acid residue mutation at C125 of wildtype human IL-2.
The polypeptide of claim 33, wherein the amino acid residue mutation at C125 is C125A or C125S.
The polypeptide of claim 1, wherein the polypeptide comprises a sequence of any one of SEQ ID NOs: 3-41.
An isolated polypeptide comprising an IL-2 variant moiety having a sequence of any one of SEQ ID NOs: 3-41.
The polypeptide of any one of the preceding claims, wherein one or more amino acid residues in the polypeptide are further replaced by cysteine.
The polypeptide of any one of the preceding claims, wherein two or more amino acid residues in the polypeptide are further replaced by cysteine.
The polypeptide of claim 37 or 38, wherein the amino acid residues further replaced by cysteine are not at positions selected from K64 and P65 corresponding to SEQ ID NO: 1.
The polypeptide of claim 37 or 38, wherein the amino acid residues further replaced by cysteine are not at positions selected from K35, R38, K43, Y107, K64, and P65 corresponding to SEQ ID NO: 1.
The polypeptide of any one of claims 38-40, wherein at least two of the two or more cysteine residues form a disulfide linkage.
The polypeptide of any one of the preceding claims, wherein the polypeptide comprises an amino acid sequence that is heterologous to wildtype human IL-2 or a functional variant thereof.
The polypeptide of claim 42, wherein the amino acid sequence is a half-life extension moiety.
The polypeptide of claim 42 or 43, wherein the amino acid sequence is linked to the C-terminus of the IL-2 variant moiety.
The polypeptide of any one of claims 42-44, wherein the amino acid sequence is an albumin, an immunoglobulin or fragment thereof, a transferrin, or a PEG.
The polypeptide of any one of claims 42-44, wherein the amino acid sequence is a serum albumin.
The polypeptide of any one of claims 42-44, wherein the amino acid sequence is a mouse serum albumin.
The polypeptide of any one of claims 42-44, wherein the amino acid sequence is a human serum albumin.
The polypeptide of any one of claims 42-44, wherein the amino acid sequence is an immunoglobulin or fragment thereof.
The polypeptide of any one of claims 42-44, wherein the amino acid sequence is an immunoglobulin Fc domain.
The polypeptide of any one of claims 42-44, wherein the amino acid sequence is a human immunoglobulin Fc domain or fragment thereof.
The polypeptide of claim 51, wherein the human immunoglobulin Fc domain is IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM.
A nucleic acid molecule comprising a nucleic acid sequence that encodes the polypeptide of any one of claims 1-52.
The nucleic acid molecule of claim 53, wherein the nucleic acid molecule is a DNA molecule.
The nucleic acid molecule of claim 53, wherein the nucleic acid molecule is an RNA molecule.
The nucleic acid molecule of claim 53, wherein the nucleic acid molecule is an mRNA molecule.
An expression vector comprising the nucleic acid molecule of claim 53.
A host cell comprising the expression vector of claim 57.
A method for preparing a polypeptide comprising an IL-2 variant moiety, the method comprising: performing recombinant expression by using the nucleic acid molecule of claim 53, the expression vector of claim 57, or the host cell of claim 58.
A pharmaceutical composition comprising the polypeptide of any one of claims 1-52 or the nucleic acid molecule of any one of claims 53-56; and a pharmaceutically acceptable excipient.
A method of treating a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the polypeptide of any one of claims 1-52 or the nucleic acid molecule of any one of claims 53-56; and a pharmaceutically acceptable excipient.
The method of claim 61, wherein the polypeptide has a decreased binding affinity to IL-2Rαas compared to the binding affinity of a wildtype IL-2 to IL-2Rα in the subject.
The method of claim 61, wherein the polypeptide has about the same binding affinity to IL-2Rβγ as compared to the binding affinity of a wildtype IL-2 affinity to IL-2Rβγ in the subject.
The method of claim 61, wherein polypeptide increases activation of CD8+ T cells in the subject.
The method of claim 61, wherein polypeptide increases activation of natural killer (NK) cells in the subject.
The method of claim 61, wherein polypeptide decreases activation of regulatory T cells (Tregs) in the subject.
The method of claim 61, wherein the condition is a cancer or cell proliferative disease.
The method of claim 61, wherein the condition is an inflammatory or autoimmune condition.
The method of claim 61, wherein the subject is human.