Modified interleukin 2 (IL-2) polypeptides, conjugates, and uses thereof

Abstract

The present disclosure relates to modified interleukin 2 (IL-2) polypeptides, polynucleotides, e.g., DNA, RNA, or viral vectors, encoding and configured to express the modified IL-2 polypeptides in vitro and / or in vivo, conjugates comprising the modified IL-2 polypeptides, and uses thereof.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 887,359, filed August 15, 2019, entitled "Modified Interleukin 2 (IL-2) Polypeptides, Conjugates and Uses Thereof," and U.S. Provisional Patent Application No. 63 / 025,095, filed May 14, 2020, entitled "Modified Interleukin 2 (IL-2) Polypeptides, Conjugates and Uses Thereof." The contents and disclosures of these applications are incorporated herein by reference in their entirety for all purposes.

[0003] Sequence Lists on ASCII Text

[0004] This patent or application contains a sequence list submitted in computer-readable ASCII text format (filename: 7006-2000140_SeqList_ST25.txt, record date: August 10, 2020, size: 3,014 bytes). The contents of the sequence list file are incorporated herein by reference in their entirety. Technical Field

[0005] This disclosure relates to modified interleukin-2 (IL-2) peptides, polynucleotides (e.g., DNA, RNA, or viral vectors) with or without conjugates, and their uses, wherein the conjugates comprise the modified IL-2 peptide, the polynucleotides encoding the modified IL-2 peptide and configured to express the modified IL-2 peptide in vitro and / or in vivo. Background Technology

[0006] The clinical use of interleukin-2 (IL-2) for cancer treatment is primarily limited by toxicity and short half-life in vivo [1,2]. Significantly reduced toxicity has been observed in animals lacking CD25 (IL-2 receptor α unit, IL-2Rα) [3]. Polyethylene glycolization, i.e., the covalent linking of polyethylene glycol (PEG) to the therapy, has been shown to overcome obstacles such as rapid clearance from the body, aggregation, and enzymatic degradation [4].

[0007] WO 2019 / 028419 A1 and WO 2019 / 028425 A1 disclose interleukin (IL) conjugates (e.g., IL-2 conjugates) and their use in treating one or more indications. WO 2019 / 028419 A1 and WO 2019 / 028425 also describe pharmaceutical compositions comprising one or more interleukin conjugates (e.g., IL-2 conjugates) and kits.

[0008] There is a need in the art for improved modified interleukin-2 (IL-2) peptides, with or without conjugates. This invention addresses this need, as well as other related needs in the art. Summary of the Invention

[0009] This invention relates to modified interleukin-2 (IL-2) peptides, polynucleotides (e.g., DNA, RNA, or viral vectors), conjugates comprising said modified IL-2 peptides, and their uses, wherein said polynucleotides encode said modified IL-2 peptides and are configured to express said modified IL-2 peptides in vitro and / or in vivo.

[0010] On one hand, the present invention relates to a modified interleukin-2 (IL-2) polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 and substitutions made at positions selected from the group consisting of: Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof, wherein: a) the modified IL-2 polypeptide is configured to be unconjugated or unconjugated to a water-soluble polymer, lipid, or polypeptide (e.g., protein) or peptide; b) and comprising SEQ ID NO:1 or SEQ ID NO:2 without the said substitutions. Compared to the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:2, the modified IL-2 polypeptide exhibits reduced binding to interleukin-2 receptor α (IL-2Rα); c) compared to the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, the modified IL-2 polypeptide exhibits reduced receptor signaling efficacy for IL-2Rαβγ; d) compared to the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, the modified IL-2 polypeptide exhibits an increased ratio of signaling efficacy for IL-2Rβγ to signaling efficacy for IL-2Rαβγ (i.e., an increased ratio of signaling efficacy for IL-2Rβγ to signaling efficacy for IL-2Rαβγ).and / or e) with SEQ ID NO:1 or SEQ ID that does not have the said substitution Compared to the corresponding IL-2 peptide with the amino acid sequence shown in NO:2, the modified IL-2 peptide exhibits enhanced receptor signaling efficacy for IL-2Rβγ, provided that the modified IL-2 peptide contains substitutions made with non-natural amino acids. Specifically, the modified IL-2 peptide contains substitutions at positions selected from the group consisting of: N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof, and substitutions made with natural or non-natural amino acids at positions within the IL-2Rα interaction region, the IL-2Rβ interaction region, and / or the IL-2Rγ interaction region, provided that the modified IL-2 peptide contains substitutions in the regions of amino acid residues 10-25, 80-100, and / or 100-134, compared to SEQ ID NO:2 which does not have the aforementioned substitutions. The corresponding region of the IL-2 polypeptide with the amino acid sequence shown in NO:1 or SEQ ID NO:2 has at least about 80% sequence identity, and the modified IL-2 polypeptide has at least about 50% sequence identity with the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution.

[0011] On the other hand, the present invention relates to a polynucleotide, such as DNA, RNA, or a viral vector, which encodes a modified IL-2 polypeptide and is configured to express the modified IL-2 polypeptide in vitro and / or in vivo. In some embodiments, the modified IL-2 polypeptide, with or without a conjugate as described above, may be used as a protein, a fusion protein, a protein conjugate, or as part of a nanoparticle. In some embodiments, the aforementioned polynucleotide (e.g., DNA, RNA, or a viral vector) encoding the modified IL-2 polypeptide and configured to express the modified IL-2 polypeptide in vitro and / or in vivo may be applied to cells, tissues, organs, or subjects, such as human subjects.

[0012] In another aspect, the present invention relates to modified IL-2 polypeptide conjugates comprising modified IL-2 polypeptides as described above, wherein the modified IL-2 polypeptides are conjugated with water-soluble polymers, lipids, polypeptides (e.g., proteins) or peptides.

[0013] In another aspect, the present invention relates to a pharmaceutical composition comprising an effective amount of a modified IL-2 polypeptide, a polynucleotide (e.g., DNA, RNA, or a viral vector), or a modified IL-2 polypeptide conjugate as described above, and a pharmaceutically acceptable carrier or excipient.

[0014] In another aspect, the present invention relates to a method for treating or preventing a disease or condition (e.g., a proliferative disease or condition, an autoimmune or inflammatory disease or condition, or an infectious disease or condition) in a subject of need, the method comprising administering to the subject an effective amount of modified IL-2, a polynucleotide (e.g., DNA, RNA, or a viral vector), a modified IL-2 polypeptide conjugate, or a pharmaceutical composition as described above.

[0015] In another aspect, the present invention relates to the use of an effective amount of a modified IL-2 polypeptide, a polynucleotide (e.g., DNA, RNA, or a viral vector), or a modified IL-2 polypeptide conjugate as described above in the preparation of a medicament for treating or preventing a disease or condition (e.g., a proliferative disease or condition, an autoimmune or inflammatory disease or condition, or an infectious disease or condition) in a subject.

[0016] In another aspect, the present invention relates to an amplification of CD4 + Helper cells, CD8 + A method for contacting effector initiators and memory cells, natural killer (NK) cells, or natural killer T (NKT) cell populations, the method comprising contacting the cell populations with an effective amount of a modified IL-2 peptide, polynucleotide (e.g., DNA, RNA, or a viral vector), or a modified IL-2 peptide conjugate as described above, or a pharmaceutical composition comprising said modified IL-2 peptide, polynucleotide (e.g., DNA, RNA, or a viral vector), or modified IL-2 peptide conjugate for a duration sufficient to induce the formation of a complex with IL-2Rβγ, thereby stimulating the expansion of said T cell, NK cell, and / or NKT cell populations.

[0017] In another aspect, the present invention relates to an amplification of CD4 + Helper cells, CD8 +A method for contacting a population of effector initiators and memory cells, Treg cells, natural killer (NK) cells, or natural killer T (NKT) cells, the method comprising contacting the cell population with an effective amount of a modified IL-2 peptide, a polynucleotide (e.g., DNA, RNA, or a viral vector), or a modified IL-2 peptide conjugate as described above, or a pharmaceutical composition comprising said modified IL-2 peptide, polynucleotide (e.g., DNA, RNA, or a viral vector), or a modified IL-2 peptide conjugate for a duration sufficient to induce the formation of a complex with IL-2Rβγ, thereby stimulating the expansion of said T cells, Treg cells, NK cells, and / or NKT cell populations while reducing cell death by 10% to 100%.

[0018] In another aspect, the present invention relates to an effective amount of a modified IL-2 polypeptide, polynucleotide (e.g., DNA, RNA, or viral vector), or a modified IL-2 polypeptide conjugate as described above, in the preparation of CD4 for amplifying cell populations. + Helper cells, CD8 + Use in drugs for effector initiators and memory cells, Treg cells, natural killer (NK) cells, or natural killer T (NKT) cells.

[0019] Other aspects and advantages of the invention will be apparent from the embodiments and examples provided herein.

[0020] For the sake of brevity, the disclosures of publications containing patents cited in this specification are incorporated herein by reference. Attached Figure Description

[0021] This patent or application document contains at least one color drawing. Upon request and payment of the necessary fees, the Patent Office will provide a copy of this patent or patent application publication with one or more color drawings.

[0022] Figure 1A An exemplary recombinant human IL-2 sequence with a mutation from cysteine ​​to serine at position 125 (rhIL-2) is shown [5]. The amino acid sites selected by superscript numbering as PEGylated by substitution of cysteine ​​alone and / or sites selected as those that disrupt IL-2Rα interaction and / or enhance IL-2Rβγ interaction by mutation are shown. Figure 1BThe 3D structures of IL-2 derived from the PDB structure 2b5i and the receptor IL-2Rαβγ complex are shown. See, for example, Protein Data Bank HMBerman, J. Westbrook, Z. Feng, G. Gilliland, TNBhat, H. Weissig, IN Shindyalov, PEBourne (2000), Nucleic Acids Research, 28:235-242. doi:10.1093 / nar / 28.1.235. Figure 1A The sites described in the text are shown as red spheres.

[0023] Figure 2 The expression of exemplary functional IL-2 variants with a single cysteine ​​substitution was demonstrated and determined by HEK-blue assay in cell culture supernatant diluted 1:10000.

[0024] Figure 3 shows exemplary or typical chromatographic and SDS-PAGE analyses of exemplary IL-2 mutant proteins and PEG conjugates. Figure 3A The chromatographic analysis of N29C performed using a Superdex 75Increase column is shown. Figure 3B The chromatographic analysis of the N29C-PEG30 conjugate is shown using an SP agarose FF (SP Sepharose FF) column. Figure 3C The chromatographic analysis of the N29C-PEG30 conjugate using a Superdex 75 column is shown. Figure 3D The SDS-PAGE analysis of the N29C-PEG30 fraction eluted from an SP agarose FF column and subsequently a Superdex 75 augmentation column is shown.

[0025] Figure 4 Exemplary and representative sensor diagrams are shown of an exemplary IL-2 mutant protein and a PEG conjugate that bind to IL-2Rα obtained by Octet Qke (ForteBio, San Jose, CA).

[0026] Figure 5 The study demonstrated that Y31C mutation and PEGylation do not affect the IL2Rα-binding cytokines obtained by Octet Qke (Ari Biosciences, San Jose, California).

[0027] Figure 6The study demonstrated that the Y31C mutant protein has enhanced binding to IL2Rαβγ-expressing cells (such as CTLL2 cells and CD25+ human T cells).

[0028] Figure 7 illustrates the activation of pSTAT5 by an exemplary IL-2 mutant protein and a PEG conjugate in a human T cell subset.

[0029] Figure 8 The graphs show concentration-time curves after a single injection of rhIL-2, P65C-PEG20 conjugate, and Y31C-PEG20+F42K conjugate into mice.

[0030] Figure 9 The study demonstrated that rhIL-2, the P65C-PEG20 conjugate, and the Y31C-PEG20+F42K conjugate stimulated the in vitro expansion of T cells and NK cells.

[0031] Figure 10 The study demonstrated that LAK (NK) cells expanded with rhIL-2, P65C-PEG20 conjugate, and Y31C-PEG20+F42K conjugate exhibited enhanced cytotoxicity. Detailed Implementation

[0032] A. General Technology

[0033] Unless otherwise indicated, the practice of this invention will employ conventional techniques within the scope of the art, including molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, immunology, and pharmacology. Such techniques are well explained in references such as *Molecular Cloning: A Laboratory Manual*. Molecular Cloning:A Laboratory Manual ), 2nd edition (Sambrook et al., 1989); Oligonucleotide Synthesis ( Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture ( Animal Cell Culture (RI Freshney, ed., 1987); Enzymatic Methods ( Methods in Enzymology (Academic Press, Inc.); The Contemporary Molecular Biology Program ( Current Protocols in Molecular Biology (FMAusubel et al., ed., 1987 and updated regularly); PCR: Polymerase Chain Reaction (… PCR:The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Remington: Pharmaceutical Science and Practice (Remington, The Science and Practice of Pharmacy ), 20th edition (Lippincott, Williams & Wilkins Publishing Co., 2003).

[0034] B. Definition

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents, patent applications (published or unpublished), and other publications mentioned herein are incorporated herein by reference in their entirety. Where a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth in this section shall prevail over the definition incorporated herein by reference.

[0036] As used in this article, “a / an” means “at least one” or “one or more”.

[0037] The terms “polypeptide,” “oligopeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of any length of amino acids, such as at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1,000, or more. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. These terms also cover amino acid polymers that are naturally occurring or modified by intervention; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification, such as conjugation with a labeled component. Furthermore, the definitions also include, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art.

[0038] As used herein, the term "variant" refers to a polypeptide that shares a degree of amino acid sequence identity with the parent polypeptide sequence. A variant is similar to the parent sequence, but has at least one substitution, deletion, or insertion in its amino acid sequence that makes it sequence-different from the parent polypeptide. Additionally, variants may retain the functional properties of the parent polypeptide, for example, maintaining at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the parent polypeptide's biological activity.

[0039] An "antibody" is an immunoglobulin molecule capable of specifically binding to a target (such as carbohydrates, polynucleotides, lipids, peptides, etc.) through at least one antigen recognition site located in the variable region of an immunoglobulin molecule, and can be any class of immunoglobulin, such as IgG, IgM, IgA, IgD, and IgE. IgY, which is the predominant antibody type in avian species such as chickens, is also included in this definition. As used herein, this term encompasses not only complete polyclonal or monoclonal antibodies, but also their fragments (such as Fab, Fab', F(ab')2, Fv), single chains (ScFv), their variants, naturally occurring variants, fusion proteins containing antibody portions with antigen recognition sites of desired specificity, humanized antibodies, chimeric antibodies, and any other modified conformation of an immunoglobulin molecule containing antigen recognition sites of desired specificity.

[0040] As used herein, the term "antigen" refers to a target molecule that an antibody specifically binds to through its antigen recognition site. Antigens can be monovalent or polyvalent, meaning that the antigen may have one or more epitopes recognized by one or more antibodies. Examples of various antigens that can be recognized by antibodies include peptides, oligosaccharides, glycoproteins, polynucleotides, lipids, etc.

[0041] As used herein, the term "epitope" refers to a portion of an antigen, for example, a peptide sequence of at least about 3 to 5, preferably about 5 to 10 or 15, and not exceeding about 1,000 amino acids (or any integer therewith), defined as a sequence that binds alone or as part of a larger sequence to an antibody generated in response to such a sequence. There is no critical upper limit to the length of the fragment, which may, for example, include almost the entire length of the antigen sequence, or even the entire length of a fusion protein from two or more epitopes of a target antigen. Epitopes used in this invention are not limited to peptides having a suitable sequence of a portion of their derived parent protein, but also encompass sequences identical to the natural sequence as well as modifications of the natural sequence, such as deletions, additions, and substitutions (which are essentially conserved).

[0042] As used herein, the term "specific binding" refers to the binding specificity of a specific binding pair. A characteristic of this type of binding is the recognition of a specific target by an antibody in the presence of other potential targets. Specific binding involves two distinct molecules, one of which binds specifically to a second molecule via a chemical or physical mechanism. The two molecules are related in the sense that their binding pair enables them to distinguish their binding partner from other assay components with similar properties. Members of a binding component pair are referred to as ligand and receptor (anti-ligand), specific binding pair (SBP) members, and SBP partners, etc. A molecule can also be an SBP member of a molecular aggregate; for example, an antibody generated from an immune complex against a second antibody and its corresponding antigen can be considered an SBP member of the immune complex.

[0043] As used interchangeably herein, “polynucleotide” or “nucleic acid” refers to a nucleotide polymer of any length and includes both DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any matrix in which they can be incorporated into the polymer by DNA or RNA polymerase. Polynucleotides may contain modified nucleotides (such as methylated nucleotides) and their analogs. Modifications to the nucleotide structure can be conferred, if present, before or after polymer assembly. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified post-polymerization, such as by conjugation with labeled components. Other types of modifications include, for example, "capping," substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as those with non-electrolyzed bonds (e.g., methylphosphonates, triphosphates, phosphoramides, carbamates, etc.) and those with charged bonds (e.g., thiophosphates, dithiophosphates, etc.), those containing a dangling motif such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalating agents (e.g., acridine, psoralen, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those containing modified bonds (e.g., α-anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. Furthermore, any hydroxyl group normally present in sugars can be substituted, for example, by phosphonate groups or phosphate groups, protected by standard protecting groups, activated to prepare additional linkages with other nucleotides, or conjugated to a solid support. The 5' and 3' OH groups may be phosphorylated or substituted via an amine or organic end-capping group having 1 to 20 carbon atoms. Other hydroxyl groups may also be derived as standard protecting groups. The polynucleotide may also contain similar forms of ribose or deoxyribose known in the art, including, for example, 2'-O-methyl-2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars (e.g., arabinose, xylose, or lythose), piperanose, furanose, sedoheptulose, acyclic analogs, and baseless nucleoside analogs (e.g., methylriboside). One or more phosphodiester linkages may be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, examples where phosphate esters are replaced by P(O)S (“thioester”), P(S)S (“dithioester”), (O)NR 2 (“amide”), P(O)R, P(O)OR', CO, or CH 2 (“methylal”), wherein each R or R' is independently H or a substituted or unsubstituted alkyl group (1-20 Cs), optionally containing an ether (--O--) bond, aryl, alkenyl, cycloalkyl, cycloalkenyl, or aromatic aldehyde group. Not all links in a polynucleotide need to be identical.The above description applies to all polynucleotides mentioned in this article, including RNA and DNA.

[0044] As used herein, “oligonucleotide” generally refers to a short, typically single-stranded, synthetic polynucleotide that is typically, but not necessarily, less than about 200 nucleotides in length. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The above description of polynucleotides applies equally and entirely to oligonucleotides.

[0045] As used herein, the term "homologous" refers to a nucleic acid that differs from naturally occurring nucleic acids (e.g., "prototype" or "wild-type") through minor modifications, but maintains the basic nucleotide structure of the naturally occurring form. Such modifications include, but are not limited to, changes to one or more nucleotides, including deletions (e.g., truncated versions of the nucleic acid), insertions, and / or substitutions. Homologous substances may have enhanced, diminished, or substantially similar properties compared to naturally occurring nucleic acids. Homologous substances may be complementary to or matched with naturally occurring nucleic acids. Homologous substances can be produced using techniques known in the art for producing nucleic acids, including but not limited to recombinant DNA techniques, chemical synthesis, etc.

[0046] As used herein, "substantially complementary or substantially matched" means that the two nucleic acid sequences have at least 90% sequence identity. Preferably, the two nucleic acid sequences have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. Alternatively, "substantially complementary or substantially matched" means that the two nucleic acid sequences can hybridize under one or more stringent conditions.

[0047] Typically, the stability of hybrids varies with ion concentration and temperature. Hybridization reactions are typically performed under conditions of lower stringency, followed by washings of varying but higher stringency. Moderately stringent hybridization refers to conditions that allow nucleic acid molecules, such as probes, to bind to complementary nucleic acid molecules. The hybridized nucleic acid molecules typically possess at least 60% identity, including at least any of the following: 70%, 75%, 80%, 85%, 90%, or 95% identity. Moderately stringent conditions are equivalent to hybridization at 42°C in 50% formamide, 5x Denhardt's solution, 5x SSPE, and 0.2% SDS, followed by washing at 42°C in 0.2x SSPE and 0.2% SDS. High stringency conditions can be provided by hybridization at 42°C in 50% formamide, 5x Denhardt's solution, 5x SSPE, and 0.2% SDS, followed by washing at 65°C in 0.1x SSPE and 0.1% SDS. Low-toughness hybridization refers to conditions equivalent to hybridization at 22°C in 10% formamide, 5x Dunhardt solution, 6x SSPE, and 0.2% SDS, followed by washing at 37°C in 1x SSPE and 0.2% SDS. The Dunhardt solution contains 1% sucrose, 1% polyvinylpyrrolidone, and 1% bovine serum albumin (BSA). 20x SSPE (sodium chloride, sodium phosphate, EDTA) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025M EDTA. Other suitable medium-toughness and high-toughness hybridization buffers and conditions are well known to those skilled in the art.

[0048] As used herein, “vector (or plasmid)” refers to a discrete element used to introduce heterologous DNA into a cell for expression or replication. The selection and use of such vectors are well known to those skilled in the art. Expression vectors include vectors capable of expressing DNA operatively linked to regulatory sequences such as promoter regions, which enable the expression of such DNA fragments. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector that, upon introduction into a suitable host cell, produces the expression of clonal DNA. Suitable expression vectors are well known to those skilled in the art and include those that can replicate in eukaryotic and / or prokaryotic cells, those that remain free, and those that integrate into the host cell genome.

[0049] As used herein, a “promoter region or promoter element” refers to a segment of DNA or RNA to which transcription of DNA or RNA operatively linked is controlled. A promoter region contains sequences specific enough for RNA polymerase recognition, binding, and transcription initiation. This portion of the promoter region is called the promoter. Additionally, the promoter region contains sequences that regulate this recognition, binding, and transcription initiation activity of RNA polymerase. These sequences can be cis-acting or responsive to trans-acting factors. Depending on the nature of the regulation, a promoter can be constitutive or regulatory. Exemplary promoters intended for use in prokaryotes include bacteriophage T7 and T3 promoters, among others.

[0050] As used herein, “operatively linked” or “operationally associated” refers to the functional relationship between DNA and regulatory and effector sequences of nucleotides (such as promoters, enhancers, transcription and translation termination sites) and other signaling sequences. For example, an operational link between DNA and a promoter refers to the physical and functional relationship between DNA and a promoter such that transcription of this DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA. To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' untranslated portion of the clone to eliminate additional, potentially inappropriate alternative translation start (i.e., start) codons or other sequences that may interfere with or reduce expression at the transcriptional or translational level. Alternatively, a co-occurrence site can be inserted immediately adjacent to the 5' of the start codon and may enhance expression. See, for example, Kozak (1991), *Journal of Biochemistry* 266:19867-19870. The necessity (or requirement) of such modifications can be determined empirically.

[0051] "Treating," "treatment," or "alleviation" refers to therapeutic treatment in which the goal is to slow (reduce, if not cure) the targeted pathological condition or disorder or to prevent recurrence of the condition. A subject is considered successfully "treated" if, after receiving a therapeutic dose of a therapeutic agent or treatment, the subject exhibits an observable and / or measurable reduction or absence of one or more signs and symptoms of a particular disease. The patient may also experience a reduction in disease signs or symptoms. If the patient's condition is stable, the patient is also considered treated. In some embodiments, treatment with a therapeutic agent can effectively result in the patient being disease-free for 3 months, preferably 6 months, more preferably 1 year, or even more preferably 2 years or more after treatment. These parameters used to assess successful treatment and improvement of the disease can be readily measured using routine procedures familiar to a physician with appropriate skills in the art. In some embodiments, "treatment" means any manner in which the condition, symptom, or symptoms of the disease improve or otherwise beneficially change. Treatment also encompasses any pharmaceutical use of the compositions described herein. In some embodiments, “improvement” of symptoms of a particular condition by applying a particular pharmaceutical composition means any relief attributable to or related to the application of the composition, whether permanent or temporary, persistent or transient.

[0052] The terms “prediction” or “prognosis” are often used herein to refer to the likelihood that a patient will respond favorably or unfavorably to a drug or group of drugs, or the possible outcome of a disease. In one embodiment, prediction involves the extent of those responses or outcomes. In another embodiment, prediction involves the probability that a patient will survive or improve and / or survive or improve after treatment (e.g., treatment with a specific therapeutic agent) and during a period in which no disease recurrence occurs. The predictive method of the present invention can be used clinically to make treatment decisions by selecting the most appropriate treatment for any particular patient. The predictive method of the present invention is a valuable tool for predicting whether a patient is likely to respond favorably to a treatment regimen (e.g., a given therapeutic regimen), which includes, for example, administration of a given therapeutic agent or combination, surgical intervention, steroid therapy, etc.

[0053] As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, isotonics, and absorption delay agents compatible with pharmaceutical application. The use of such media and agents for pharmaceutically active substances is well known in the art. See, for example, Remington: Pharmaceutical Science and Practice, 20th edition (Lippincott Williams and Wilkins Publishing, 2003). The use of such media or agents in compositions is contemplated except in cases where any conventional media or agents are incompatible with the active compound.

[0054] "Pharmaceutically acceptable salt" is intended to mean a salt of the free acid or base of the compound represented herein that is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to a subject. See Berge et al., *Journal of Pharmaceutical Sciences*, 1977, 66, 1-19. Preferred pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of a subject without excessive toxicity, irritation, or anaphylactic reactions. The modified interleukin-2 (IL-2) polypeptides or conjugates thereof described herein may have sufficiently acidic groups, sufficiently basic groups, both types of functional groups, or more than one group of each type, and thus react with a variety of inorganic or organic bases and inorganic and organic acids to form pharmaceutically acceptable salts.

[0055] Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfites, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, hexanoates, heptadates, propynylates, oxalates, malonates, succinates, octanoates, sebacic acid, fumarates, maleates, and butynediol. ,4-Diosyl salt, hexyn-1,6-diosyl salt, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propylsulfonate, benzenesulfonate, xylenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate, and mandelate.

[0056] As used herein, the term "therapeuticly effective amount" or "effective amount" means an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or improving a disease or condition, proliferative disease or symptom in the subject. A therapeutically effective amount further means an amount of a therapeutic agent sufficient to cause symptom improvement (e.g., treatment, cure, prevention, or improvement of an associated medical condition or an increase in the rate of treatment, cure, prevention, or improvement of such a condition). When applied to an individual active ingredient administered alone, the therapeutically effective amount refers alone to said individual active ingredient. When applied in combination, the therapeutically effective amount refers to the combined amount of active ingredients that result in a therapeutic effect, whether administered sequentially or concurrently in combination. In some embodiments, an "effective amount of a compound for treating a specific disease" is an amount sufficient to improve or alleviate symptoms associated with the disease in some way. This amount may be administered as a single dose or according to a regimen, thereby being effective. The amount may cure the disease but is typically administered to improve the symptoms of the disease. Repeated administration may be necessary to achieve the desired improvement in symptoms.

[0057] The term "combination" refers to a fixed combination or kit for combined administration in the form of a dose unit, wherein a modified interleukin-2 (IL-2) peptide or its conjugate and a combination partner (e.g., another drug described below, also referred to as a "therapeutic agent" or "adjuvant") can be administered simultaneously or separately at time intervals, particularly where these time intervals allow the combination partner to exhibit a synergistic effect, such as a co-administration effect. As used herein, the terms "co-administration" or "combination administration," etc., are intended to cover the administration of selected combination partners to a single subject (e.g., a patient) in need and are intended to include treatment regimens in which the agents are not necessarily administered via the same route of administration or simultaneously. As used herein, the term "pharmaceutical composition" means a product consisting of a mixture or combination of more than one active ingredient and includes both fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that the active ingredients, such as a modified interleukin-2 (IL-2) peptide or its conjugate and a combination partner, are administered simultaneously to a patient in the form of a single entity or dose. The term "non-fixed combination" refers to active ingredients, such as modified interleukin-2 (IL-2) peptides or their conjugates and combination partners, administered to a patient as separate entities simultaneously, concurrently, or sequentially, without a specific time limit, wherein such administration provides a therapeutically effective level of two substances in the patient's body. The latter also applies to cocktail therapy, such as the administration of three or more active ingredients.

[0058] As used herein, a “biosample” means any sample obtained from a living or viral source or other macromolecular and biomolecular source, and includes any cell type or tissue of a subject from which nucleic acids or proteins or other macromolecules may be obtained. A biosample can be a sample obtained directly from a biological source or a processed sample. For example, amplified isolated nucleic acids constitute a biosample. Biosamples include, but are not limited to, samples of bodily fluids (e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat), tissues, and organs derived from animals and plants, as well as processed samples derived therefrom.

[0059] The term "level" (or levels) is used to refer to the presence and / or quantity of a target (e.g., a substance or organism that is part of the cause of a disease or condition) and can be determined qualitatively or quantitatively. A "qualitative" change in a target level refers to the presence or absence of a target that is undetectable or absent in samples obtained from normal controls. A "quantitative" change in the level of one or more targets, compared to healthy controls, refers to a measurable increase or decrease in the level of the target.

[0060] A “healthy control” or “normal control” is a biological sample taken from an individual who does not have a disease or condition (e.g., a proliferative disease or condition). A “negative control” is a sample for which an analysis is not designed to detect any particular analyte and thus provides a reference baseline for said analysis.

[0061] As used herein, “mammal” means any species within the mammalian species category. As used herein, the term “mammal” often refers to humans, human subjects, or human patients. “Mammal” also means any species within the non-human mammalian species category, such as experimental, companion, or economically important non-human mammals. Exemplary non-human mammals include mice, rats, rabbits, cats, dogs, pigs, cattle, sheep, goats, horses, monkeys, gorillas, and chimpanzees.

[0062] As used herein, “generated by recombination” refers to a method of generating recombinant nucleic acids that relies on well-known molecular biology methods to express polypeptides or proteins encoded by cloned nucleic acids.

[0063] As used herein, the term "subject" is not limited to a specific species or sample type. For example, the term "subject" can refer to a patient, and typically a human patient. However, the term is not limited to humans and therefore encompasses a wide range of non-human animal or mammal species.

[0064] As used herein, a "prodrug" is a substance that, after administration to the body, is metabolized or otherwise converted into a substance of biological, pharmaceutical, or therapeutic activity. To produce a prodrug, a pharmaceutically active substance is modified so that the active substance will be regenerated through metabolic processes. The prodrug can be engineered to alter the metabolic stability or transport properties of the drug, mask side effects or toxicity, improve the drug's aroma, or change other characteristics or properties of the drug. With an understanding of pharmacodynamic processes and drug metabolism in vivo, those skilled in the art can engineer prodrugs of a pharmaceutically active compound once it is known (see, for example, Nogrady (1985), *Medicinal Chemistry: A Biochemical Approach*). Medicinal Chemistry A Biochemical Approach (《》, Oxford University Press, New York, pp. 388-392).

[0065] It should be understood that the aspects and embodiments of the invention described herein include "consisting of aspects and embodiments" and / or "consisting substantially of aspects and embodiments".

[0066] Throughout this disclosure, various aspects of the invention are presented in a scope format. It should be understood that the use of a scope format is merely for convenience and clarity and should not be construed as a rigid limitation on the scope of the invention. Therefore, descriptions of scopes should be considered as explicitly disclosing all possible sub-scopes and individual numerical values ​​within said scopes. For example, descriptions of scopes such as 1 to 6 should be considered as having explicitly disclosed sub-scopes, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc.; and individual numbers within said scopes, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the scope.

[0067] Other objectives, advantages and features of the present invention will become clear from the following description taken in conjunction with the accompanying drawings.

[0068] C. Modified interleukin-2 (IL-2) polypeptide and the polynucleotides encoding and expressing it.

[0069] On one hand, the present invention relates to a modified interleukin-2 (IL-2) polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 and substitutions made at the following positions with natural or non-natural amino acids: Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93 or combinations thereof, wherein: a) the modified IL-2 polypeptide is configured to conjugate with a water-soluble polymer, lipid, or polypeptide (e.g., protein) or peptide; b) with SEQ ID NO:1 or SEQ ID NO:2 containing no said substitutions. Compared to the corresponding IL-2 polypeptide with the amino acid sequence shown in NO:2, the modified IL-2 polypeptide binds less to interleukin-2 receptor α (IL-2Rα); and / or c) compared to SEQ ID NO:1 or SEQ ID NO:2 which does not have the substitution. Compared to the corresponding IL-2 peptide with the amino acid sequence shown in NO:2, the modified IL-2 peptide exhibits reduced receptor signaling efficacy for IL-2Rαβγ, provided that the modified IL-2 peptide contains substitutions made with non-natural amino acids. Specifically, the modified IL-2 peptide contains substitutions at the following positions: N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, or combinations thereof, and substitutions made with natural or non-natural amino acids at positions within the IL-2Rα interaction region, IL-2Rβ interaction region, and / or IL-2Rγ interaction region. Furthermore, the modified IL-2 peptide contains substitutions in the regions of amino acid residues 10-25, 80-100, and / or 100-134, compared to SEQ ID NO:1 or SEQ ID NO:2, which do not contain the aforementioned substitutions. The corresponding region of the IL-2 polypeptide with the amino acid sequence shown in ID NO:2 has at least about 80% sequence identity, and the modified IL-2 polypeptide has at least about 50% sequence identity with the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution.

[0070] The amino acid sequences of SEQ ID NO:1 or SEQ ID NO:2 are shown below:

[0071] SEQ ID NO:1

[0072] ( 1 APTSSSTKKTQL 13 QLEHLL 19 LDLQMILNGI 29 OF 30 OF 31 Y 32 K 33 OF 34 P 35 KLT 38 RML 41 THE 42 F 43

[0073] KF 45 YMP 48 K49KATELKHLQCLEE 62 EL 64 K 65 PLEEVL 71 NLA 74 QS 76 KNFHL 81 RPRD 85 LI 8

[0074] 7 TWO 91 V 92 I 93 VLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL 133 THE)

[0075] SEQ ID NO:2

[0076] ( 1 MPTSSSTKKTQL 13 QLEHLL 19 LDLQMILNGI 29 OF 30 OF 31 Y 32 K 33 OF 34 P 35 KLT 38 RML 41 THE 42 F 43 KF 45 YMP 4 8 K49KATELKHLQCLEE 62 EL 64 K65 PLEEVL 71 NLA 74 QS 76 KNFHL 81 RPRD 85 LI 87 SNIN 91 V 92 I 93 VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL 133 T)

[0077] In one embodiment, the modified IL-2 polypeptide has at least about 80% sequence identity in the region of amino acid residues 10-25 with the corresponding region of the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, for example, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0078] In another embodiment, the modified IL-2 polypeptide has at least about 80% sequence identity in the region of amino acid residues 80-100 with the corresponding region of the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, for example, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0079] In yet another embodiment, the modified IL-2 polypeptide has at least about 80% sequence identity in the region of amino acid residues 100-134 with the corresponding region of the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, for example, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0080] In yet another embodiment, the modified IL-2 polypeptide has at least about 80% sequence identity in the region of amino acid residues 10-25 and 80-100 with the corresponding region of the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, for example, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0081] In yet another embodiment, the modified IL-2 polypeptide has at least about 80% sequence identity in the region of amino acid residues 10-25 and 100-134 with the corresponding region of the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, for example, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0082] In yet another embodiment, the modified IL-2 polypeptide has at least about 80% sequence identity in the region of amino acid residues 80-100 and 100-134 with the corresponding region of the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, for example, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0083] In yet another embodiment, the modified IL-2 polypeptide has at least about 80% sequence identity in the regions of amino acid residues 10-25, 80-100, and 100-134 with the corresponding regions of the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, for example, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0084] In one embodiment, the modified IL-2 polypeptide has at least about 50% sequence identity with the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, for example, at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity.

[0085] The modified IL-2 polypeptide of the present invention may contain any suitable substitutions made with natural amino acids. For example, the modified IL-2 polypeptide of the present invention may contain substitutions made at the following positions with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine: Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, or combinations thereof.

[0086] In one embodiment, a) the modified IL-2 polypeptide of the present invention comprises substitutions of natural amino acids at positions selected from the group consisting of: N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof, and is configured to be conjugated at positions selected from the group consisting of: N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K76, and combinations thereof, and is configured to be conjugated to a water-soluble polymer, lipid, protein, or peptide at positions selected from the group consisting of: N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K76, and combinations thereof, and is configured to be conjugated to a water-soluble polymer, lipid, protein, or peptide at positions selected from the group consisting of: N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K76, and combinations thereof. 43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76 and combinations thereof; and / or b) the modified IL-2 polypeptide of the present invention comprises substitutions of natural amino acids at positions selected from the group consisting of: N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76 and combinations thereof, and is configured to be conjugated to a water-soluble polymer, lipid, protein or peptide at the N-terminus and / or C-terminus of the polypeptide.

[0087] In another embodiment, a) the modified IL-2 polypeptide of the present invention comprises substitutions at positions selected from the group consisting of lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine: N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q7 4. K76 and combinations thereof; and / or b) the modified IL-2 polypeptide of the present invention comprises substitutions at positions selected from the group consisting of lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine or tyrosine: N29, N30, Y31, N33, P34, K35, R38, T41, K43, K48, K49, K64, P65, N71, Q74, K76 and combinations thereof.

[0088] In yet another embodiment, a) the modified IL-2 polypeptide of the present invention comprises a substitution of cysteine ​​at a position selected from the group consisting of: N29, N30, Y31, N33, P34, K35, R38, T41, K43, K48, K49, K64, P65, N71, Q74, K76, and combinations thereof; b) the modified IL-2 polypeptide of the present invention comprises a substitution of cysteine ​​at a position selected from the group consisting of: N29, Y31, K35, P65, N71, Q74, and combinations thereof; c) the modified IL-2 polypeptide of the present invention comprises a substitution of cysteine ​​at the position of Y31; and / or d) the modified IL-2 polypeptide of the present invention comprises a substitution of cysteine ​​at the position of P65.

[0089] In yet another embodiment, the modified IL-2 polypeptide of the present invention comprises a substitution at position Y31 with any amino acid. For example, the modified IL-2 polypeptide of the present invention may comprise a substitution at position Y31 with serine or alanine.

[0090] The modified IL-2 polypeptide of the present invention may further include substitutions of natural or non-natural amino acids at positions within the IL-2Rα interaction region, the IL-2Rβ interaction region, and / or the IL-2Rγ interaction region.

[0091] The modified IL-2 polypeptide of the present invention may further include substitutions of natural amino acids at positions within the IL-2Rα interaction region. The modified IL-2 polypeptide of the present invention may further include substitutions of natural amino acids at any suitable position within the IL-2Rα interaction region. For example, the modified IL-2 polypeptide of the present invention may further include substitutions of natural amino acids at positions selected from the group consisting of: R38, F42, Y45, E62, P65, and combinations thereof.

[0092] The modified IL-2 polypeptide of the present invention may contain any suitable substitutions of natural amino acids at positions within the IL-2Rα interaction region. For example, the modified IL-2 polypeptide of the present invention may contain substitutions of lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at positions selected from the group consisting of: R38, F42, Y45, E62, P65, and combinations thereof.

[0093] In one embodiment, a) the modified IL-2 polypeptide of the present invention comprises a substitution of cysteine ​​at the position selected from the group consisting of: R38, F42, Y45, E62, P65, and combinations thereof; b) the modified IL-2 polypeptide of the present invention comprises a substitution of alanine, lysine, or serine at the F42 position; c) the modified IL-2 polypeptide of the present invention comprises a substitution of alanine at the F42 position; d) the modified IL-2 polypeptide of the present invention comprises a substitution of serine at the F42 position; e) the modified IL-2 polypeptide of the present invention comprises a substitution of lysine at the F42 position; f) the modified IL-2 polypeptide of the present invention comprises a substitution of alanine, histidine, or serine at the Y45 position; g) the modified IL-2 polypeptide of the present invention comprises a substitution of cysteine ​​at the Y45 position. h) The modified IL-2 polypeptide of the present invention contains a histidine substitution at position Y45; i) The modified IL-2 polypeptide of the present invention contains alanine, aspartic acid, or serine substitution at position R38; j) The modified IL-2 polypeptide of the present invention contains aspartic acid substitution at position R38; k) The modified IL-2 polypeptide of the present invention contains alanine substitution at position P65; l) The modified IL-2 polypeptide of the present invention contains serine substitution at position P65; m) The modified IL-2 polypeptide of the present invention contains alanine substitution at position E62; and / or n) The modified IL-2 polypeptide of the present invention contains lysine substitution at position F42, cysteine ​​substitution at position Y31, or a combination thereof.

[0094] The modified IL-2 polypeptide of the present invention may further include substitutions of natural amino acids at positions within the IL-2Rβ interaction region. The modified IL-2 polypeptide of the present invention may further include substitutions of natural amino acids at any suitable position within the IL-2Rβ interaction region. For example, the modified IL-2 polypeptide of the present invention may further include substitutions of natural amino acids at positions selected from the group consisting of: Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof.

[0095] The modified IL-2 polypeptide of the present invention may contain any suitable substitution of natural amino acids at positions within the IL-2Rβ interaction region. For example, the modified IL-2 polypeptide of the present invention may contain substitutions of lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at positions selected from the group consisting of: Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof. In one embodiment, the modified IL-2 polypeptide of the present invention may contain substitutions of cysteine ​​at positions selected from the group consisting of: Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof.

[0096] In one embodiment, the modified IL-2 polypeptide of the present invention may further comprise: a) substitutions of natural amino acids at positions within the IL-2Rα interaction region and at positions within the IL-2Rβ interaction region; b) substitutions of natural amino acids at positions within the IL-2Rα interaction region and at positions within the IL-2Rγ interaction region; or c) substitutions of natural amino acids at positions within the IL-2Rα interaction region, substitutions of natural amino acids at positions within the IL-2Rβ interaction region, and substitutions of natural amino acids at positions within the IL-2Rγ interaction region.

[0097] The modified IL-2 polypeptide of the present invention may contain any suitable substitutions made with non-natural amino acids. For example, non-natural amino acids disclosed in WO 2019 / 028425 A1 and WO 2019 / 028419 A1 may be used. In one embodiment, the non-natural amino acid may be a lysine analog, a cysteine ​​analog, or a histidine analog, containing an aromatic side chain; containing an azide group; containing an alkyne group; or containing an aldehyde or ketone group. In another embodiment, the non-natural amino acid does not contain an aromatic side chain. In another embodiment, the non-natural amino acids include N6-azidoethoxy-L-lysine (AzK), N6-propynylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetraazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propynyloxyphenylalanine, p-propynyl-phenylalanine, 3-methyl-phenylalanine, L-dihydroxyphenylalanine, fluorinated phenylalanine, and iso-phenylalanine. Propyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selenoyl)propionic acid, 2-amino-3-(phenylselenoyl)propionic acid, or selenocysteine.

[0098] Non-natural amino acids can be incorporated into modified IL-2 peptides in any suitable manner or by any method. For example, non-natural amino acids can be incorporated into modified IL-2 peptides using orthogonal tRNA synthetase / tRNA pairs. Any suitable orthogonal tRNA can be used. For example, the orthogonal tRNA of the orthogonal synthetase / tRNA pair can contain at least one non-natural nucleoside base.

[0099] Compared to the corresponding IL-2 polypeptides comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the said substitution, the modified IL-2 polypeptides of the present invention may exhibit reduced binding to IL-2Rα or may not exhibit detectable binding to IL-2Rα. In one embodiment, the binding affinity of the modified IL-2 polypeptide of the present invention to IL-2Rα may be reduced from about 10% to about 100%, for example, reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a subrange thereof. In another embodiment, the binding affinity of the modified IL-2 polypeptide of the present invention to IL-2Rα may be reduced from about 10% to about 100%, or may be reduced from about 1x to about 100,000x or more, for example, reduced by about 1x, 10x, 100x, 1,000x, 10,000x, 100,000x or more, or a subrange thereof. In yet another embodiment, the modified IL-2 polypeptide of the present invention does not have detectable binding to IL-2Rα.

[0100] Compared to the corresponding IL-2 peptides comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the said substitution, the modified IL-2 peptides of the present invention may have reduced or no receptor signaling efficacy for IL-2Rαβγ. In one embodiment, the ratio between the signaling efficacy of the modified IL-2 peptide of the present invention for IL-2Rαβγ and the signaling efficacy of the corresponding IL-2 peptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the said substitution for IL-2Rαβγ may be from about 1 / 2 to about 1 / 100,000, for example, about 1 / 2, 1 / 5, 1 / 10, 1 / 100, 1 / 1,000, 1 / 10,000, 1 / 100,000 or more, or subranges thereof. In another embodiment, the modified IL-2 peptide of the present invention does not have detectable receptor signaling efficacy for IL-2Rαβγ.

[0101] In one embodiment, compared to the corresponding IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the said substitution, the modified IL-2 polypeptide of the present invention exhibits reduced binding to IL-2Rα and reduced receptor signaling efficacy for IL-2Rαβγ compared to the corresponding IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the said substitution. In another embodiment, the modified IL-2 polypeptide of the present invention does not have detectable binding to IL-2Rα and does not have detectable receptor signaling efficacy for IL-2Rαβγ.

[0102] Compared to the corresponding IL-2 peptides containing the amino acid sequences shown in SEQ ID NO:1 or SEQ ID NO:2 without the said substitution, the modified IL-2 peptides of the present invention can substantially retain or can have a higher binding level to interleukin-2 receptor β (IL-2Rβ) or interleukin-2 receptor γ (IL-2Rγ), and / or the receptor signaling efficacy of the modified IL-2 peptides of the present invention for IL-2Rβγ can substantially retain or can have a higher binding level compared to the corresponding IL-2 peptides containing the amino acid sequences shown in SEQ ID NO:1 or SEQ ID NO:2 without the said substitution. In one embodiment, the modified IL-2 peptides of the present invention substantially retain or have a higher binding level to IL-2Rβ or IL-2Rγ compared to the corresponding IL-2 peptides containing the amino acid sequences shown in SEQ ID NO:1 or SEQ ID NO:2 without the said substitution. In another embodiment, the modified IL-2 peptide of the present invention substantially retains or enhances its receptor signaling efficacy for IL-2Rβγ compared to the corresponding IL-2 peptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the said substitution. In yet another embodiment, the modified IL-2 peptide of the present invention substantially retains or enhances its binding level for IL-2Rβ or IL-2Rγ compared to the corresponding IL-2 peptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the said substitution, and substantially retains or enhances its receptor signaling efficacy for IL-2Rβγ compared to the corresponding IL-2 peptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the said substitution.

[0103] The modified IL-2 peptide of the present invention can contain deletions at any suitable position. In one embodiment, the modified IL-2 peptide of the present invention has an N-terminal deletion, such as an N-terminal deletion of amino acid residues 1-30 or a subrange thereof. In another embodiment, the modified IL-2 peptide of the present invention has a C-terminal deletion, such as a C-terminal deletion of amino acid residues 114-134 or a subrange thereof. In yet another embodiment, the modified IL-2 peptide of the present invention has both an N-terminal and a C-terminal deletion.

[0104] The modified IL-2 peptide of the present invention may be part of a fusion peptide (e.g., a recombinant fusion protein) comprising the modified IL-2 peptide and additional amino acid sequences. The modified IL-2 peptide of the present invention may be fused with additional amino acid sequences in any suitable manner. For example, the N-terminus or C-terminus of the modified IL-2 peptide may be fused with additional amino acid sequences. The additional amino acid sequences may comprise any suitable sequence or content. For example, the additional amino acid sequences may comprise an antibody sequence or a portion or fragment thereof. In another embodiment, the additional amino acid sequences may comprise the Fc portion of an antibody.

[0105] The modified IL-2 polypeptide of the present invention can be in any suitable form. For example, the modified IL-2 polypeptide of the present invention can be in isolated or purified form.

[0106] The modified IL-2 peptide of the present invention can be prepared using any suitable technique or process. For example, the modified IL-2 peptide of the present invention can be prepared by recombinant generation, chemical synthesis, or a combination thereof.

[0107] On the other hand, the present invention relates to a polynucleotide, such as DNA, RNA or a viral vector, which encodes the modified IL-2 polypeptide as described above and is configured to express the modified IL-2 polypeptide in vitro and / or in vivo.

[0108] The modified IL-2 peptides of the present invention can be used in any suitable form. For example, the modified IL-2 peptides, with or without conjugates as described above, can be used as proteins, fusion proteins, protein conjugates, or as part of nanoparticles. In some embodiments, polynucleotides (e.g., DNA, RNA, or viral vectors) encoding the modified IL-2 peptides and configured to express the modified IL-2 peptides in vitro and / or in vivo can be applied to cells, tissues, organs, or subjects, such as human subjects.

[0109] D. Modified interleukin-2 (IL-2) polypeptide conjugate

[0110] On the other hand, the present invention relates to modified IL-2 polypeptide conjugates comprising a modified IL-2 polypeptide as described above, wherein the modified IL-2 polypeptide is conjugated to another portion (e.g., a water-soluble polymer, lipid, polypeptide (e.g., protein) or peptide).

[0111] The modified IL-2 polypeptide can be conjugated to another portion (e.g., a water-soluble polymer, lipid, protein, or peptide) in any suitable form. For example, the modified IL-2 polypeptide can be covalently conjugated to a water-soluble polymer, lipid, protein, or peptide. In another example, the modified IL-2 polypeptide can be non-covalently conjugated to a water-soluble polymer, lipid, protein, or peptide. In yet another example, the modified IL-2 polypeptide can be conjugated to a water-soluble polymer, lipid, protein, or peptide via substituted natural or non-natural amino acids at any suitable position.

[0112] In one embodiment, the modified IL-2 polypeptide is conjugated to another portion (e.g., a water-soluble polymer, lipid, protein, or peptide) by substituted natural or non-natural amino acids selected from the group consisting of: Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof. In another embodiment, the modified IL-2 polypeptide is conjugated with another portion (e.g., a water-soluble polymer, lipid, protein, or peptide) by substituted natural amino acids selected from the group consisting of: Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof. In yet another embodiment, the modified IL-2 polypeptide is conjugated with another portion (e.g., a water-soluble polymer, lipid, protein, or peptide) by substituted lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at positions selected from the group consisting of: Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof. In yet another embodiment, the modified IL-2 polypeptide is conjugated to another portion (e.g., a water-soluble polymer, lipid, protein, or peptide) by substituted cysteine ​​residues selected from the group consisting of: Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof.

[0113] The modified IL-2 polypeptide can be conjugated with another part (e.g., a water-soluble polymer, lipid, protein, or peptide) by substituted natural or non-natural amino acids at positions selected from the group consisting of: N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof. In one embodiment, the modified IL-2 polypeptide is conjugated with another portion (e.g., a water-soluble polymer, lipid, protein, or peptide) by substituted natural amino acids selected from the group consisting of: N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof. In another embodiment, the modified IL-2 polypeptide is conjugated with another portion (e.g., a water-soluble polymer, lipid, protein, or peptide) by substituted lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at positions selected from the group consisting of: N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof. In yet another embodiment, the modified IL-2 polypeptide is conjugated with another portion (e.g., a water-soluble polymer, lipid, protein, or peptide) by substituted cysteine ​​residues selected from the group consisting of: N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof.

[0114] The modified IL-2 peptide can be conjugated to another portion (e.g., a water-soluble polymer, lipid, protein, or peptide) by a single or multiple amino acid residues of the modified IL-2 peptide. In one embodiment, the modified IL-2 peptide can be conjugated to another portion (e.g., a water-soluble polymer, lipid, protein, or peptide) by: i) the α-amino group of the N-terminal amino acid residue of the modified IL-2 peptide; ii) the ε-amino group of the lysine amino acid residue of the modified IL-2 peptide; or iii) the N-glycosylation site or O-glycosylation site of the modified IL-2 peptide.

[0115] The modified IL-2 peptide can be covalently conjugated to another part (e.g., a water-soluble polymer, lipid, protein, or peptide) via a linker. The modified IL-2 peptide can also be directly covalently conjugated to another part (e.g., a water-soluble polymer, lipid, protein, or peptide) without a linker.

[0116] The modified IL-2 peptide can be conjugated to another portion (e.g., a water-soluble polymer, lipid, protein, or peptide) by a single amino acid residue in a fusion peptide comprising the modified IL-2 peptide and an additional amino acid sequence. The single amino acid residue can be located at any suitable position. For example, the single amino acid residue can be located within the modified IL-2 peptide. In another example, the single amino acid residue can be located within the additional amino acid sequence.

[0117] The additional amino acid sequence in the modified IL-2 polypeptide conjugate of the present invention may comprise any suitable sequence or content. For example, the additional amino acid sequence in the modified IL-2 polypeptide conjugate of the present invention may comprise an antibody sequence or a portion or fragment thereof. In another example, the additional amino acid sequence in the modified IL-2 polypeptide conjugate of the present invention may comprise the Fc portion of an antibody.

[0118] The modified IL-2 peptide can be conjugated to another portion (e.g., a water-soluble polymer, lipid, protein, or peptide in a fusion peptide) in any suitable form. For example, the modified IL-2 peptide can be conjugated to another portion (e.g., a water-soluble polymer, lipid, protein, or peptide) by: i) the α-amino group of the N-terminal amino acid residue of the fusion peptide; ii) the ε-amino group of the lysine amino acid residue of the fusion peptide; or iii) the N-glycosylation site or O-glycosylation site of the fusion peptide. In another example, the fusion peptide can be covalently conjugated to a water-soluble polymer, lipid, protein, or peptide directly or via a linker.

[0119] The modified IL-2 peptide of the present invention can be conjugated with any suitable water-soluble polymer. For example, the water-soluble polymer may comprise polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(enol), poly(vinylpyrrolidone), poly(hydroxyalkyl methylacrylamide), poly(hydroxyalkyl methacrylate), poly(sugar), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof. See, for example, WO 2019 / 028425A1 and WO 2019 / 028419A1.

[0120] In the modified IL-2 polypeptide conjugates of the present invention, the water-soluble polymer may comprise PEG molecules. The PEG molecules may be linear PEG or branched PEG. The branched PEG may have any suitable configuration and / or any suitable number of PEG chains. For example, the branched PEG may have about three to about ten PEG chains emanating from a central core group. In another example, the branched PEG may be a star-shaped PEG comprising about 10 to about 100 PEG chains emanating from a central core group. In yet another example, the branched PEG may be a comb-shaped PEG comprising multiple PEG chains grafted onto the polymer backbone.

[0121] The PEG molecules in the modified IL-2 polypeptide conjugates of the present invention can have any suitable molecular weight. For example, the molecular weight of the PEG molecules can range from about 300 g / mol to about 10,000,000 g / mol, such as about 300 g / mol, 500 g / mol, 1,000 g / mol, 10,000 g / mol, 100,000 g / mol, 1,000,000 g / mol, 10,000,000 g / mol or subranges thereof. In another example, the average molecular weight of the PEG molecules can range from about 5,000 Daltons to about 1,000,000 Daltons, such as about 5,000 Daltons, 10,000 Daltons, 100,000 Daltons, 1,000,000 Daltons or subranges thereof. In yet another example, the average molecular weight of the PEG molecule can be from about 20,000 Daltons to about 30,000 Daltons, for example, about 20,000 Daltons, 21,000 Daltons, 22,000 Daltons, 23,000 Daltons, 24,000 Daltons, 25,000 Daltons, 26,000 Daltons, 27,000 Daltons, 28,000 Daltons, 29,000 Daltons, 30,000 Daltons or subranges thereof.

[0122] The PEG molecules in the modified IL-2 polypeptide conjugates of the present invention can be in any suitable form. For example, the PEG molecules can be monodisperse, homogeneous, or discrete PEG molecules.

[0123] The water-soluble polymer in the modified IL-2 polypeptide conjugate of the present invention may contain polysaccharides.

[0124] The modified IL-2 peptide in the modified IL-2 peptide conjugate of the present invention can be conjugated to any suitable lipid. For example, the lipid in the modified IL-2 peptide conjugate of the present invention can comprise fatty acids.

[0125] The modified IL-2 polypeptide in the modified IL-2 polypeptide conjugate of the present invention can be conjugated to any suitable protein. For example, the protein in the modified IL-2 polypeptide conjugate of the present invention may comprise an antibody or a binding fragment thereof. The antibody or binding fragment thereof may comprise the Fc portion of an antibody.

[0126] In the modified IL-2 polypeptide conjugates of the present invention, other portions (e.g., water-soluble polymers, lipids, proteins, or peptides) can be bound to the modified IL-2 polypeptide in any suitable manner. For example, other portions (e.g., water-soluble polymers, lipids, proteins, or peptides) can be indirectly bound to substituted natural or non-natural amino acids of the modified IL-2 polypeptide via linkers. In another example, other portions (e.g., water-soluble polymers, lipids, proteins, or peptides) can be directly bound to substituted natural or non-natural amino acids of the modified IL-2 polypeptide.

[0127] The modified IL-2 polypeptide conjugates of the present invention can have any suitable in vivo half-life. For example, the in vivo half-life of the modified IL-2 polypeptide conjugates of the present invention can be from about 5 minutes to about 10 days, for example, about 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or subranges thereof.

[0128] E. Pharmaceutical Composition

[0129] On the other hand, the present invention relates to a pharmaceutical composition comprising an effective amount of a modified IL-2 polypeptide, a polynucleotide (e.g., DNA, RNA or a viral vector) or a modified IL-2 polypeptide conjugate as described above, and a pharmaceutically acceptable carrier or excipient.

[0130] The pharmaceutical compositions of the present invention can be formulated to treat or prevent any suitable disease, condition, or symptom. For example, the pharmaceutical compositions of the present invention can be formulated to treat or prevent proliferative diseases in a subject.

[0131] In one embodiment, the pharmaceutical composition of the present invention is configured to treat or prevent a solid tumor or cancer in a subject. The solid tumor or cancer may be chondrosarcoma, Ewing's sarcoma, bone / osteosarcoma, malignant fibrous histiocytoma, osteosarcoma, rhabdomyosarcoma, cardiac cancer, astrocytoma, brainstem glioma, pilocytic astrocytoma, ependymoma, primitive neuroectodermal tumor, cerebellar astrocytoma, brain astrocytoma, glioma, medulloblastoma, neuroblastoma, oligodendroglioma, pineal astrocytoma, pituitary adenoma, visual pathway and hypothalamic glioma, etc. Breast cancer, invasive lobular carcinoma, tubular carcinoma, invasive cribriform carcinoma, medullary carcinoma, male breast cancer, phyllodes tumor, inflammatory breast cancer, adrenocortical carcinoma, islet cell carcinoma (endocrine pancreas), multiple endocrine neoplasia syndrome, parathyroid carcinoma, pheochromocytoma, thyroid cancer, Merkel cell carcinoma, uveal melanoma, retinoblastoma, anal cancer, appendix cancer, bile duct cancer, carcinoid tumor, gastrointestinal cancer, colon cancer, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor Tumors, Gastrointestinal stromal tumors (GIST), hepatocellular carcinoma, pancreatic islet cell carcinoma, rectal cancer, bladder cancer, cervical cancer, endometrial cancer, gonadal germ cell tumors, ovarian cancer, ovarian epithelial carcinoma (surface epithelial-stromal tumor), ovarian germ cell tumors, penile cancer, renal cell carcinoma, renal pelvis and ureter, transitional cell carcinoma, prostate cancer, testicular cancer, gestational trophoblastic tumor, ureter and renal pelvis, transitional cell carcinoma, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and more. Muscle tumors, esophageal cancer, head and neck cancer, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, sinus and nasal cavity cancer, pharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, basal cell carcinoma, melanoma, skin cancer (non-melanoma), bronchial adenoma / carcinoid, small cell lung cancer, mesothelioma, non-small cell lung cancer, pleural pulmonary blastoma, laryngeal cancer, thymoma and thymic carcinoma, AIDS-related cancers, Kaposi's sarcoma, epithelioid hemangioendothelioma (EHE), fibroblastic small round cell tumor or liposarcoma.

[0132] In another embodiment, the pharmaceutical composition of the present invention is configured to treat or prevent malignant hematologic malignancies in a subject. Malignant hematologic malignancies may include the following: myeloid lesions, leukemia, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, anaplastic large cell lymphoma, angioimmune T-cell lymphoma, hepatocellular T-cell lymphoma, B-cell lymphoma, reticuloendothelial proliferation, reticulocytosis, small glioma, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt lymphoma, mediastinal large B-cell lymphoma, Waldenström macroglobulinemia, marginal zone B-cell lymphoma, splenic marginal zone lymphoma. Peripheral lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, lymphomatoid granulomatosis, nodular lymphocytic major Hodgkin lymphoma, plasma cell leukemia, acute polycythemia and erythroleukemia, acute erythrocytic myelopathy, acute erythrocytic leukemia, Helmholtz-Shenner disease, acute megakaryocytic leukemia, mast cell leukemia, whole bone marrow histopathy, acute whole bone marrow histopathy with myelofibrosis, lymphosarcoma cell leukemia, acute leukemia of unspecified cell type, chronic myeloid leukemia in blast crisis, stem cell leukemia, chronic leukemia of unspecified cell type, subtype of unspecified cell type Acute leukemia, accelerated phase chronic myeloid leukemia, acute myeloid leukemia, polycythemia vera, acute promyelocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, mature acute myeloblastic leukemia, acute myeloid dendritic leukemia, adult T-cell leukemia / lymphoma, aggressive NK-cell leukemia, B-cell prolymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell leukemia, chronic myeloid leukemia, chronic myeloid monocytic leukemia, chronic neutrophilic leukemia, chronic lymphocytic leukemia, hairy leukemia Cellular leukemia, chronic idiopathic myelofibrosis, multiple myeloma, Calder's disease, myeloma, solitary myeloma, plasma cell leukemia, plasmacytoma, extramedullary, malignant plasma cell tumor NOS, plasmacytoma NOS, monoclonal gammopathy, multiple myeloma, angiocentric immunoproliferative disorders, lymphoid granulomatosis, angioimmunoblastic lymphadenopathy, T-γ lymphohistioproliferative disorders, Waldenström macroglobulinemia, alpha heavy chain disease, gamma heavy chain disease, Franklin's disease, immunoproliferative small bowel disease, thalassemia, malignant immunoproliferative disorders, unspecified or immunoproliferative disorders NOS.

[0133] In yet another embodiment, the pharmaceutical composition of the present invention is configured to treat or prevent an immunodeficiency disease or condition in a subject. Immunodeficiency diseases or conditions may include agammaglobulinemia: X-linked and autosomal recessive inheritance, ataxia-telangiectasia, chronic granulomatous diseases and other phagocytic disorders, common variable immunodeficiency, complement deficiency, DeGeorge syndrome, hemophagocytic lymphohistiocytosis (HLH), hyperIgE syndrome, hyperIgM syndrome, IgG subclass deficiency, congenital immunodeficiency, NEMO deficiency syndrome, selective IgA deficiency, selective IgM deficiency, severe combined immunodeficiency, deficiency and combined immunodeficiency, and specific antibody deficiency. Transient hypogammaglobulinemia in infancy, WHIM syndrome (warts, hypogammaglobulinemia, infection and myeloablative agranulocytosis), Wiscot-Aldrich syndrome, other antibody deficiency disorders, other primary cellular immunodeficiency, severe combined immunodeficiency (SCID), common variable immunodeficiency (CVID), human immunodeficiency virus / acquired immunodeficiency syndrome (HIV / AIDS), drug-induced immunodeficiency, graft-versus-host disease, primary immunodeficiency disease (PIDD), or lymphopenia.

[0134] The pharmaceutical compositions of the present invention may further comprise another active ingredient. This other active ingredient may be an active ingredient for treating or preventing any suitable disease, condition, or symptom. For example, the other active ingredient may be an anti-vegetative substance.

[0135] Other active ingredients may be formulated in a single pharmaceutical composition of at least one exemplary modified IL-2 peptide or modified IL-2 peptide conjugate of the present disclosure, or may comprise at least one exemplary modified IL-2 peptide or modified IL-2 peptide conjugate of the present disclosure in a single pharmaceutical composition.

[0136] The pharmaceutical compositions of the present invention can be formulated for oral, parenteral, inhalation, topical, rectal, nasal, buccal, vaginal, via implanted reservoirs, or other methods of drug administration. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0137] Sterile injectable compositions, such as sterile injectable aqueous or oily suspensions, can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Sterile injectable formulations can also be sterile injectable solutions or suspensions in non-toxic, parenteral-acceptable diluents or solvents. Acceptable mediators and solvents that may be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Suitable carriers and other pharmaceutical composition components are generally sterile.

[0138] In addition, sterile, non-volatile oils are routinely used as solvents or suspension media (e.g., for the synthesis of mono- or diglycerides). Fatty acids such as oleic acid and their glyceride derivatives can be used to prepare injectables, as can pharmaceutically acceptable oils (such as olive oil or castor oil, especially in their polyoxyethyleneized form). These oil solutions or suspensions may also contain long-chain alcohols as diluents or dispersants, or carboxymethyl cellulose or similar dispersants. For formulation purposes, various emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used.

[0139] Compositions for oral administration can be in any orally acceptable dosage form, including but not limited to tablets, capsules, emulsions, and aqueous suspensions, dispersions, and solutions. In the case of tablets for oral administration, common carriers include lactose and corn starch. Lubricants such as magnesium stearate may also be added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When oral administration is an aqueous suspension or emulsion, the active ingredient can be suspended or dissolved in an oil phase combined with an emulsifier or suspending agent. If desired, certain sweeteners, flavoring agents, or coloring agents may be added. Nasal aerosols or inhalation compositions can be prepared according to techniques well known in the field of pharmaceutical formulations and can be prepared as solutions, for example, in saline, using suitable preservatives (e.g., benzyl alcohol), absorption enhancers for improving bioavailability, and / or other solubilizers or dispersants known in the art.

[0140] Any suitable formulation of the compounds described herein can be prepared. See Remington's Pharmaceutical Sciences (2000), Hoover, JE (ed.), 20th ed., Easton, Pennsylvania, Lippinger-Williams-Wilkins, pp. 780-857. Choose a formulation suitable for the appropriate route of administration. Administration of the compound as a salt may be appropriate if the compound is sufficiently basic or acidic to form a stable, non-toxic acid salt or base salt. Examples of pharmaceutically acceptable salts are addition salts of organic acids that form physiologically acceptable anions, such as toluenesulfonates, methanesulfonates, acetates, citrates, malonates, tartrates, succinates, benzoates, ascorbic acid salts, α-ketoglutarate, and α-glycerophosphates. Suitable inorganic salts, including hydrochlorides, sulfates, nitrates, bicarbonates, and carbonates, can also be formed. Pharmaceutically acceptable salts are obtained using standard procedures well known in the art, such as by means of sufficiently basic compounds, such as amines having suitable acids to provide physiologically acceptable anions. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids have also been prepared.

[0141] If the contemplated compound or substance is to be administered as a pharmacological composition, it is conceivable that the compound or substance may be formulated with pharmaceutically acceptable excipients and / or carriers. For example, the contemplated compound or substance may be administered orally as a neutral compound or substance or as a pharmaceutically acceptable salt or intravenously in a physiological saline solution. Conventional buffers such as phosphates, bicarbonates, or citrates may be used for this purpose. Of course, those skilled in the art can modify formulations within the teachings of this specification to provide large quantities of formulations for a particular route of administration. Specifically, the contemplated compound or substance may be modified to make it more soluble in water or other media, which can be easily accomplished, for example, by minor modifications (salt formulations, esterification, etc.) well known to those skilled in the art. It is also well known to those skilled in the art that the route of administration and dosage regimen of a particular compound or substance (e.g., the modified IL-2 peptide or modified IL-2 peptide conjugate of this disclosure) can be modified to maximize the beneficial effect on the patient in order to manage the pharmacokinetics of the compounds or substances of the present invention.

[0142] The modified IL-2 peptides or modified IL-2 peptide conjugates of the present invention are soluble in organic solvents (such as chloroform, dichloromethane, ethyl acetate, ethanol, methanol, isopropanol, acetonitrile, glycerol, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, etc.). In one embodiment, the present invention provides formulations prepared by mixing the modified IL-2 peptides or modified IL-2 peptide conjugates of the present invention with a pharmaceutically acceptable carrier. On one hand, the formulations can be prepared using methods comprising: a) dissolving the compound or substance in a water-soluble organic solvent, a nonionic solvent, a water-soluble lipid, cyclodextrin, vitamins such as tocopherol, fatty acids, fatty acid esters, phospholipids, or combinations thereof to provide a solution; and b) adding physiological saline or a buffer containing 1-10% carbohydrate solution. In one example, the carbohydrate comprises dextran. The pharmaceutical compositions obtained using the methods of the present invention are stable and suitable for animal and clinical use.

[0143] Illustrative examples of water-soluble organic solvents used in the pharmaceutical compositions of the present invention include, but are not limited to, polyethylene glycol (PEG), alcohols, acetonitrile, N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, or combinations thereof. Examples of alcohols include, but are not limited to, methanol, ethanol, isopropanol, glycerol, or propylene glycol.

[0144] Illustrative examples of water-soluble nonionic surfactants used in the pharmaceutical compositions of the present invention include, but are not limited to, CREMOPHOR.RTM.EL, polyethylene glycol-modified CREMOPHOR.RTM. (polyoxyethylene glycerol triricinoleate 35), hydrogenated CREMOPHOR.RTM.RH40, hydrogenated CREMOPHOR.RTM.RH60, PEG-succinate, polysorbate 20, polysorbate 80, SOLUTOL.RTM.HS (polyethylene glycol 660 12-hydroxystearate), sorbitan monooleate, poloxamer, LABRAFIL.RTM. (ethoxylated almond oil), LABRASOL.RTM. (decanoyl-hexanoyl polyethylene glycol-8-glyceryl ester), GELUCIRE.RTM. (glyceryl ester), SOFTIGEN.RTM. (PEG 6-caprylic acid glyceryl ester), glycerol, ethylene glycol polysorbate, or combinations thereof.

[0145] Illustrative examples of water-soluble lipids used in the pharmaceutical compositions of the present invention include, but are not limited to, vegetable oils, triglycerides, plant oils, or combinations thereof. Examples of lipid oils include, but are not limited to, castor oil, polyoxyethylene castor oil, corn oil, olive oil, cottonseed oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oil, hydrogenated soybean oil, coconut triglycerides, palm seed oil and its hydrogenated forms, or combinations thereof.

[0146] Illustrative examples of fatty acids and fatty acid esters used in the pharmaceutical compositions of the present invention include, but are not limited to, oleic acid, monoglycerides, diglycerides, mono- or di-fatty acid esters of PEG, or combinations thereof.

[0147] Illustrative examples of cyclodextrins used in the pharmaceutical compositions of the present invention include, but are not limited to, α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, or sulfobutyl ether-β-cyclodextrin.

[0148] Illustrative examples of phospholipids used in the pharmaceutical compositions of the present invention include, but are not limited to, soybean phosphatidylcholine or distearate phosphatidylglycerol or their hydrogenated forms or combinations thereof.

[0149] Those skilled in the art can modify formulations within the teachings of this specification to provide a large quantity of formulations for a particular route of administration. Specifically, compounds or substances can be modified to make them more soluble in water or other media. It is also well known to those skilled in the art that the route of administration and dosage regimen of a particular compound or substance can be modified to maximize the beneficial effect on the patient in order to manage the pharmacokinetics of the compounds or substances of the present invention.

[0150] F. Methods used to treat or prevent diseases or conditions.

[0151] In another aspect, the present invention relates to a method for treating or preventing a disease or condition (e.g., a proliferative disease or condition, an autoimmune or inflammatory disease or condition, or an infectious disease or condition) in a subject in need, the method comprising administering to the subject an effective amount of a modified IL-2 peptide, a polynucleotide (e.g., DNA, RNA, or a viral vector), a modified IL-2 peptide conjugate, or a pharmaceutical composition as described above.

[0152] The method of the present invention can be used to treat or prevent diseases or conditions in any suitable subject, such as proliferative diseases or conditions. For example, the method of the present invention can be used to treat or prevent human diseases or conditions, such as proliferative diseases or conditions. In another instance, the method of the present invention can be used to treat or prevent diseases or conditions in non-human mammals, such as proliferative diseases or conditions.

[0153] In one embodiment, the method of the present invention can be used to treat a subject with proliferative disease. In another embodiment, the method of the present invention can be used to prevent a subject from developing proliferative disease.

[0154] The method of the present invention can be used to treat or prevent any suitable proliferative disease or condition in a subject. For example, the method of the present invention can be used to treat or prevent a tumor in a subject. In another example, the method of the present invention can be used to treat or prevent cancer in a subject.

[0155] In one embodiment, the method of the present invention can be used to treat or prevent a solid tumor or cancer in a subject. The method of the present invention can be used to treat or prevent any suitable solid tumor or condition in a subject. For example, a solid tumor or cancer may be chondrosarcoma, Ewing's sarcoma, bone / osteosarcoma, malignant fibrous histiocytoma, osteosarcoma, rhabdomyosarcoma, cardiac cancer, astrocytoma, brainstem glioma, pilocytic astrocytoma, ependymoma, primitive neuroectodermal tumor, cerebellar astrocytoma, brain astrocytoma, glioma, medulloblastoma, neuroblastoma, oligodendroglioma, pineal astrocytoma, pituitary adenoma, visual pathway and hypothalamic glioma. Tumors, breast cancer, invasive lobular carcinoma, tubular carcinoma, invasive cribriform carcinoma, medullary carcinoma, male breast cancer, phyllodes tumor, inflammatory breast cancer, adrenocortical carcinoma, islet cell carcinoma (endocrine pancreas), multiple endocrine neoplasia syndrome, parathyroid carcinoma, pheochromocytoma, thyroid cancer, Merkel cell carcinoma, uveal melanoma, retinoblastoma, anal cancer, appendix cancer, bile duct cancer, carcinoid tumor, gastrointestinal cancer, colon cancer, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal cancer Cancer, gastrointestinal stromal tumor (GIST), hepatocellular carcinoma, pancreatic islet cell carcinoma, rectal cancer, bladder cancer, cervical cancer, endometrial cancer, gonadal germ cell tumors, ovarian cancer, ovarian epithelial carcinoma (surface epithelial-stromal tumor), ovarian germ cell tumors, penile cancer, renal cell carcinoma, renal pelvis and ureter, transitional cell carcinoma, prostate cancer, testicular cancer, gestational trophoblastic tumor, ureter and renal pelvis, transitional cell carcinoma, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, vitrectomyces cancer, etc. Ermuk tumor, esophageal cancer, head and neck cancer, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, sinus and nasal cavity cancer, pharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, basal cell carcinoma, melanoma, skin cancer (non-melanoma), bronchial adenoma / carcinoid, small cell lung cancer, mesothelioma, non-small cell lung cancer, pleural pulmonary blastoma, laryngeal cancer, thymoma and thymic carcinoma, AIDS-related cancers, Kaposi's sarcoma, epithelioid hemangioendothelioma (EHE), fibroblastic small round cell tumor or liposarcoma.

[0156] In another embodiment, the method of the present invention can be used to treat or prevent malignant hematologic malignancies in a subject. The method of the present invention can be used to treat or prevent any suitable malignant hematologic malignancy in a subject. For example, malignant hematologic malignancies may include myeloid lesions, leukemia, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, anaplastic large cell lymphoma, angioimmune T-cell lymphoma, hepatocellular T-cell lymphoma, B-cell lymphoma, reticuloendothelial proliferation, reticulocytosis, small glioma, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt lymphoma, mediastinal large B-cell lymphoma, Waldenström macroglobulinemia, marginal zone B-cell lymphoma, marginal zone splenic lymphoma, and other hematologic malignancies. Intratubular large B-cell lymphoma, primary exudative lymphoma, lymphomatoid granulomatosis, nodular lymphocytic major Hodgkin lymphoma, plasma cell leukemia, acute polycythemia and erythroleukemia, acute erythrocytic myelopathy, acute erythrocytic leukemia, Helmholtz-Shenner disease, acute megakaryocytic leukemia, mast cell leukemia, whole bone marrow histopathy, acute whole bone marrow histopathy with myelofibrosis, lymphosarcoma cell leukemia, acute leukemia of unspecified cell type, chronic myeloid leukemia in blast crisis, stem cell leukemia, chronic leukemia of unspecified cell type, subacute leukemia of unspecified cell type. Diseases, including accelerated phase chronic myeloid leukemia, acute myeloid leukemia, polycythemia vera, acute promyelocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, mature acute myeloblastic leukemia, acute myeloid dendritic cell leukemia, adult T-cell leukemia / lymphoma, aggressive NK cell leukemia, B-cell prolymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell leukemia, chronic myeloid leukemia, chronic myeloid monocytic leukemia, chronic neutrophilic leukemia, chronic lymphocytic leukemia, and hairy cell leukemia. Leukemia, chronic idiopathic myelofibrosis, multiple myeloma, Calder's disease, myeloma, solitary myeloma, plasma cell leukemia, plasmacytoma, extramedullary, malignant plasma cell tumor NOS, plasmacytoma NOS, monoclonal gammopathy, multiple myeloma, angiocentric immunoproliferative disorders, lymphoid granulomatosis, angioimmunoblastic lymphadenopathy, T-γ lymphohistioproliferative disorders, Waldenström macroglobulinemia, alpha heavy chain disease, gamma heavy chain disease, Franklin's disease, immunoproliferative small bowel disease, thalassemia, malignant immunoproliferative disorders, unspecified or immunoproliferative disorders NOS.

[0157] In another embodiment, the method of the present invention can be used to treat or prevent an immunodeficiency disease or condition in a subject. The method of the present invention can be used to treat or prevent any suitable immunodeficiency disease or condition in a subject. For example, an immunodeficiency disease or condition can be agammaglobulinemia: X-linked and autosomal recessive inheritance, ataxia-telangiectasia, chronic granulomatous diseases and other phagocytic disorders, common variable immunodeficiency, complement deficiency, DeGeorge syndrome, hemophagocytic lymphohistiocytosis (HLH), hyperIgE syndrome, hyperIgM syndrome, IgG subclass deficiency, innate immunodeficiency, NEMO deficiency syndrome, selective IgA deficiency, selective IgM deficiency, severe combined immunodeficiency, deficiency and combined immunodeficiency, specific antibodies. Deficiency disorders, transient hypogammaglobulinemia in infancy, WHIM syndrome (warts, hypogammaglobulinemia, infection and myeloablative agranulocytosis), Wiscot-Aldrich syndrome, other antibody deficiency disorders, other primary cellular immunodeficiency, severe combined immunodeficiency (SCID), common variable immunodeficiency (CVID), human immunodeficiency virus / acquired immunodeficiency syndrome (HIV / AIDS), drug-induced immunodeficiency, graft-versus-host disease, primary immunodeficiency disease (PIDD), or lymphopenia.

[0158] In another embodiment, the method of the present invention can be used to treat or prevent autoimmune diseases or conditions. For example, the method of the present invention can be used to treat or prevent inflammation, autoimmune diseases, paraneoplastic autoimmune diseases, cartilage inflammation, fibrotic diseases and / or bone degeneration, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Ritter syndrome, SEA syndrome (serologically negative, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Ritter syndrome, SEA syndrome (serologically negative, Enthes point disease, arthritis syndrome, dermatomyositis, psoriatic arthritis, scleroderma, systemic lupus erythematosus, vasculitis, myositis, polymyositis, dermatomyositis, osteoarthritis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, scleroderma, sclerosis, primary cholecystitis, sclerosing cholangitis, Sjögren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverted psoriasis, pustular psoriasis, erythroderma Autoimmune diseases include psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, Guillain-Barré disease, type 1 diabetes, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, and transplant rejection. However, autoimmune diseases or conditions are a very active area of ​​research, and other diseases or conditions as identified in this invention can be treated. In some embodiments, an autoimmune disease or condition refers to a disease or condition in which the immune system attacks its own proteins, cells, tissues, and organs. For example, in some human autoimmune diseases or conditions, the human immune system attacks its own proteins, cells, tissues, and organs, including diseased proteins, cells, tissues, and organs. A review of several autoimmune diseases or conditions, along with their lists, can be found in *The Autoimmune Diseases* (Rose and Mackay, 6th ed., 2019, Academic Press). The method of this invention may further comprise the administration of an effective amount of a second therapeutic agent to treat or prevent proliferative conditions in the subject.For example, the method of the present invention can be used to treat or prevent proliferative diseases or conditions (e.g., tumors or cancer) in a subject and further includes administering an anti-proliferative substance to the subject.

[0159] To practice the methods of this invention, the modified IL-2 peptide, polynucleotide (e.g., DNA, RNA, or viral vector), modified IL-2 peptide conjugate, or pharmaceutical composition as described above can be administered via any suitable route. For example, the modified IL-2 peptide, polynucleotide (e.g., DNA, RNA, or viral vector), modified IL-2 peptide conjugate, or pharmaceutical composition as described above can be administered orally, parenterally, by inhalation, topically, rectally, nasally, buccally, vaginally, via an implanted reservoir, or by other methods of drug administration. As used herein, the term "parenterectomy" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0160] Sterile injectable compositions, such as sterile injectable aqueous or oily suspensions, can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Sterile injectable formulations can also be sterile injectable solutions or suspensions in non-toxic, parenteral-acceptable diluents or solvents. Acceptable mediators and solvents that may be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Suitable carriers and other pharmaceutical composition components are generally sterile.

[0161] In addition, sterile, non-volatile oils are routinely used as solvents or suspension media (e.g., for the synthesis of mono- or diglycerides). Fatty acids such as oleic acid and their glyceride derivatives can be used to prepare injectables, as can pharmaceutically acceptable oils (such as olive oil or castor oil, especially in their polyoxyethyleneized form). These oil solutions or suspensions may also contain long-chain alcohols as diluents or dispersants, or carboxymethyl cellulose or similar dispersants. For formulation purposes, various emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used.

[0162] Compositions for oral administration can be in any orally acceptable dosage form, including but not limited to tablets, capsules, emulsions, and aqueous suspensions, dispersions, and solutions. In the case of tablets for oral administration, common carriers include lactose and corn starch. Lubricants such as magnesium stearate may also be added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When oral administration is an aqueous suspension or emulsion, the active ingredient can be suspended or dissolved in an oil phase combined with an emulsifier or suspending agent. If desired, certain sweeteners, flavoring agents, or coloring agents may be added. Nasal aerosols or inhalation compositions can be prepared according to techniques well known in the field of pharmaceutical formulations and can be prepared as solutions, for example, in saline, using suitable preservatives (e.g., benzyl alcohol), absorption enhancers for improving bioavailability, and / or other solubilizers or dispersants known in the art.

[0163] In another aspect, the present invention relates to the use of an effective amount of a modified IL-2 polypeptide, polynucleotide (e.g., DNA, RNA, or viral vector) or a modified IL-2 polypeptide conjugate as described above in the preparation of a medicament for treating or preventing a disease or condition (e.g., a proliferative disease or condition) in a subject.

[0164] G. Methods for expanding various immune cells

[0165] In another aspect, the present invention relates to an amplification of CD4 + Helper cells, CD8 + A method for stimulating the expansion of effector initiators and memory cells, natural killer (NK) cells, or natural killer T (NKT) cell populations, the method comprising contacting the cell population with an effective amount of a modified IL-2 peptide, a polynucleotide (e.g., DNA, RNA, or a viral vector), a modified IL-2 peptide conjugate, or a pharmaceutical composition as described above for a duration sufficient to induce the formation of a complex with IL-2Rβγ, thereby stimulating the expansion of the T cell, NK cell, and / or NKT cell populations.

[0166] In another aspect, the present invention relates to an amplification of CD4 + Helper cells, CD8 +A method for contacting effector initiators and memory cells, Treg cells, natural killer (NK) cells, or natural killer T (NKT) cell populations, the method comprising contacting the cell population with an effective amount of a modified IL-2 polypeptide, a polynucleotide (e.g., DNA, RNA, or a viral vector), a modified IL-2 polypeptide conjugate, or a pharmaceutical composition as described above for a duration sufficient to induce the formation of a complex with IL-2Rβγ, thereby stimulating the expansion of the T cells, Treg cells, NK cells, and / or NKT cell populations while reducing cell death by 10% to 100%, for example, reducing cell death by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any subrange thereof.

[0167] In one embodiment, compared to amplifying CD3 in contact with the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the said substitution, + CD4 in cell population + T regulatory (Treg) cells, modified IL-2 peptides, polynucleotides (e.g., DNA, RNA, or viral vectors), modified IL-2 peptide conjugates, or pharmaceutical compositions as described above induce CD3... + CD4 in cell population + Treg cell expansion was less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less. In another embodiment, the modified IL-2 peptide, polynucleotide (e.g., DNA, RNA, or viral vector), modified IL-2 peptide conjugate, or pharmaceutical composition as described above did not amplify CD4 in the cell population. + Treg cells. In yet another embodiment, after incubation with a modified IL-2 peptide, a polynucleotide (e.g., DNA, RNA, or a viral vector), a modified IL-2 peptide conjugate, or a pharmaceutical composition as described above, the ratio of Teff cells to Treg cells in the cell population is about or at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, 100:1, or greater.

[0168] The method described herein can be performed in any suitable manner. In one embodiment, the method described herein is performed in vivo. In another embodiment, the method described herein is performed in vitro. In yet another embodiment, the method described herein is performed ex vivo.

[0169] In another aspect, the present invention relates to an effective amount of a modified IL-2 polypeptide, polynucleotide (e.g., DNA, RNA, or viral vector), or a modified IL-2 polypeptide conjugate as described above, in the preparation of CD4 for amplifying cell populations. + Helper cells, CD8 + Use in drugs for effector initiation and memory cells, Treg cells, natural killer (NK) cells, or natural killer T (NKT) cell populations. In one embodiment, the use of the invention is configured to amplify CD4 in a subject. + Helper cells, CD8 + Effector initiators and memory cells, Treg cells, natural killer (NK) cells, or natural killer T (NKT) cell populations.

[0170] H. Example

[0171] Example 1: Design of PEG-modified IL2 mutant protein

[0172] Selection of PEG linkage sites in IL-2 Based on the human IL-2 peptide sequence, one amino acid was selected from the "Site 1" list (Table 1) and substituted with a cysteine ​​residue, allowing the mutant protein to conjugate with a maleimide-activated PEG reagent. Compared to the native IL-2 molecule, the PEG-conjugated mutant protein was expected to have an extended half-life. These PEGylations sometimes also interfere with binding to the α unit (IL-2Rα) of the IL-2 receptor, while maintaining intact binding to the β and γ units (IL-2Rβ, IL-2Rγ) (Figure 1, Table 1). All constructs were prepared in the context of wild-type human IL-2, with C125S substitution to remove this unpaired cysteine ​​residue in IL-2 (referred to herein as rhIL-2).

[0173] Table 1. IL-2 Mutation Design

[0174]

[0175]

[0176] The ultimate design of PEGylated IL-2 mutant protein molecules is expected to reduce the affinity for IL-2Rα, bind robustly to IL-2Rβγ, and prolong the half-life in humans and other animals.

[0177] Selecting additional mutation sites that disrupt IL-2Rα interaction or enhance IL-2Rβ.In addition to the polyethylene glycol modification described in step 1, other modifications are sometimes introduced. These modifications carry at least one mutation that replaces an amino acid in the "Site 2" list (Table 1) with any other amino acid. Mutations at these sites reduce binding to IL-2Rα while maintaining substantially intact binding to IL-2Rβ and IL-2Rγ (Table 1). Modifications may also carry at least one mutation that replaces an amino acid in the "Site 3" list (Table 1) with any other amino acid to enhance binding to IL-2Rβ.

[0178] Example 2: Production and purification of IL-2 mutant protein

[0179] The cDNA encoding the IL-2 mutant protein was synthesized and cloned into the pcDNA3.1(-) vector. HEK293F cells were transiently transfected with PEI MAX (Polysciences) and cultured for 96 hours. The supernatant was collected by centrifuging the culture at 4000xg for 20 minutes.

[0180] HEK-Blue TM IL-2 reporter cells (InvivoGen, hkb-il2) were used to determine IL-2 expression levels. HEK-Blue cells were stimulated with IL-2. TM IL-2 cells trigger STAT5 activation and subsequent SEAP secretion. Quanti-Blue can be used. TM Easily monitor STAT5-induced SEAP levels. Seed 100 μl of cells at 100,000 cells / well, then add 100 μl of rhIL-2 or IL-2 mutant protein to the well. After 20 to 24 hours, collect 180 μl of supernatant and mix with 20 μl of Quantiblue in a flat plate. After incubation at 37 °C for 90 min, read the absorbance at 620 nm. Figure 2 It has been shown that using culture supernatant diluted 10,000 times, IL-2 variants exhibit different detectable expression levels.

[0181] The protein of interest was separated from the supernatant using standard protein purification techniques. In short, the protein of interest was... His-Tag purification ( His-Tag Purification (HTPR) column (Roche) was used to capture the protein, which was then purified using a Superdex 75 column (GE Healthcare). The purified protein was eluted in a buffer containing 0.1 M MES and 150 mM NaCl at pH 6.0 and stored at -80°C for further use.

[0182] Example 3: Polyglycolation of IL-2 mutant protein

[0183] The purified IL-2 mutant protein (1 mg / ml) was reduced with 5 mM TCEP (Thermo Fisher Scientific) for 15 min at room temperature and then reacted with a 50-fold molar excess of maleimide-PEG 20K (Laysan Bio) for 30 min at room temperature. The reaction was stopped by adding L-cysteine ​​(Sigma) in a 2-fold molar excess of maleimide-PEG 20K. The PEG conjugate was further purified by SP agarose FF column followed by Superdex 75 column enrichment (GE Healthcare). A representative chromatogram and SDS-PAGE analysis of the purification process are shown in Figure 3.

[0184] Example 4: Binding of IL-2 mutant protein and PEG conjugate to IL-2 receptor

[0185] The binding of purified IL-2 mutant proteins or PEG conjugates to the IL-2 receptor was determined using Octet QKe (ForteBio, San Jose, California). IL-2Rα or IL-2Rβ in human Fc fusion protein format (ACROBiosystems, Beijing) was captured on an Anti-Human IgG Fc Capture (AHC) sensor. After establishing a baseline in 1x kinetic buffer, the sensor was immersed in wells containing serially diluted rhIL-2, mutant proteins, or PEG conjugates to measure the association constant. Dissociation was detected after transferring the sensor to wells containing only buffer. Data were collected and analyzed using Octet user software. A 1:1 curve fitting model was used to analyze the kinetic constants. Table 2 shows the kinetic parameters for the binding of IL-2 variants to individual IL-2 receptor subunits. Figure 4 The diagram shows a typical sensing pattern of binding. Most PEGylated mutant proteins show reduced or eliminated binding to IL-2Rα.

[0186] Table 2. Kinetic constants of the interaction between IL-2 variants and IL-2Rα

[0187] <![CDATA[K on (M -1 S -1 )]]> <![CDATA[K off (S -1 )]]> <![CDATA[K D (μM)]]> rhIL-2 <![CDATA[6.80±0.26×10 5 ]]> <![CDATA[2.03±0.02×10 -2 ]]> 0.030±0.001 Y31C-PEG20 <![CDATA[3.83±0.18×10 5 ]]> <![CDATA[2.79±0.04×10 -2 ]]> 0.073±0.004 K35C-PEG20 ND ND ND R38C-PEG20; ND ND ND P65C-PEG20 ND ND ND T41C-PEG20; ND ND ND N30C-PEG20; <![CDATA[5.93±0.89×10 5 ]]> <![CDATA[6.97±0.35×10 -2 ]]> 0.117±0.018 N33C-PEG20 ND ND ND Y31C-PEG20+F42K ND ND ND

[0188] Note: ND = Not detected

[0189] Example 5: Surface binding of IL2 mutant protein on IL2Rαβγ-expressing cells

[0190] For this analysis, two different IL2Rαβγ-expressing cell lines were tested: 1. CTLL2 cells; 2. IL2Rα+ T cells generated from human T cells derived from PBMCs reactivated by anti-CD3 / CD28 Dynabeads (at least 90% of cells were positive for IL2Rα). CTLL2 or IL2Rα+ T cells were collected and resuspended at 2–4 million cells / ml in cold binding buffer (FBB, DPBS containing 5% FBS). Histidine-labeled IL-2 and the mutant were added to the cell suspension, mixed, and incubated at 4°C for 40 min. Cells were washed once in washing buffer (FWB, DPBS containing 1% FBS). The resuspended cell clumps in FBB were reacted with 1:100 anti-His-APC (BioLegend 362605) at room temperature for 15 min. Cells were washed with 120 μL of FWB and then resuspended for flow cytometry analysis. Both Y31C and Y31C-PEG20 showed enhanced binding to CTLL2 cells and IL2Rα-positive human T cells (see [link]). Figure 6 ).

[0191] Example 6: T cell activity of pegylated IL-2 mutant protein

[0192] Thaw frozen PBMCs in serum-free AIM-V medium (Thermo Fisher Scientific) and keep them at 37°C for 2 to 4 hours before the experiment. Add 5 × 10⁻⁶ 5Cells / well were seeded in 96-well plates. Different IL-2 mutant proteins were added to the top of the cells at 4°C to avoid STAT-5 phosphorylation at different time points. Cells were mixed with the mutant proteins and incubated at 37°C for 15 minutes. The remainder of the protocol was performed at room temperature. After centrifugation, cell clumps were stained for 15 minutes in 50 μL staining buffer (PBS + 1% FBS + 2 mM EDTA) against extracellular markers (1:300-anti-human CD4 FITC, CD8 APC, CD25 BV650, R45RA BV421, BioLegend) and a viable staining agent (1:1000-eFluor 780, Thermo Fisher Scientific). Cells were washed with 200 μL washing buffer (PBS + 1% FBS) and rotated. Cell clumps were fixed in the dark for 30 minutes with 200 μL of 1x fixation buffer (FoxP3 / transcription factor staining buffer, eBioscience). Cells were then rotated and permeabilized overnight with 100 μL of cold 100% methanol at 4°C. After this period, 100 μL of wash buffer was added, cells were rotated, and staining was performed in the dark at room temperature with 50 μL of anti-human P STAT5-PE (1:80, BioLegend). 250 μL of wash buffer was added, cells were rotated, and resuspended in 110 μL of staining buffer. CD8 was assessed by flow cytometry (NovoCyte, ACEA Biosciences). + CD45RA + CD25 low Naïve (IL-2Rβγ-expressing T cells) and CD4 + CD45RA - CD25 high (IL-2Rαβγ-expressing T cells) T cells’ indicated surface markers and STAT-5 phosphorylation.

[0193] Figure 7 illustrates the dose-response phosphorylation of STAT5 in different IL-2 mutant proteins and their corresponding pegylated proteins. Compared to rhIL-2 protein, the pegylated IL-2 mutant protein showed significantly reduced activity against cells expressing IL-2Rαβγ, while the mutant protein maintained most of its activity against IL-2Rβγ-expressing T cells (Table 3). The favorable bioselectivity of the pegylated mutant protein against IL-2Rβγ-expressing T cells compared to those expressing IL-2Rαβγ was also demonstrated by the EC50 ratios in the two different cell populations.

[0194] Table 3. T cell activity of PEGylated IL2 mutant protein

[0195]

[0196] Example 7: PK study of C57BL / 6 mice

[0197] Pharmacokinetic studies of P65C-PEG20 or Y31C-PEG20+F42K were conducted in C57BL / 6 mice. The following study uses P65C-PEG20 as an example. Blood was collected from three mice at each time point. Each mouse was administered a single IV dose of 0.56 mg / kg P65C-PEG20. Blood samples were collected at 0.033, 0.083, 0.17, 0.5, 1, 4, 24, 48, 72, and 96 hours post-administration. Blood was allowed to clot at room temperature before centrifugation at 5000 rpm for 10 minutes. Serum was collected, frozen on dry ice, and maintained at -80°C until ELISA analysis.

[0198] The ELISA was performed in two phases. For Phase 1, most samples were diluted 10 times, except for early time points from 2 to 30 minutes post-dose. Samples were diluted 100 to 1,000 times. The diluted samples were added to ELISA plates coated with rabbit anti-IL-2 antibody P600 (Thermo Fisher Scientific) and detected with biotin-conjugated monoclonal IL-2 antibody M600B (Thermo Fisher Scientific). For samples collected from 1 hour onwards, the samples were further tested using a high-sensitivity IL-2 human ELISA kit (Thermo Fisher Scientific) to detect low levels of IL-2. All tests were performed in duplicate. ELISA readings were converted to concentrations using a standard curve corresponding to the IL-2 construct and the Pade approximant model (Prism).

[0199] Phenix WinNonLin version 8.1 software (Certara USA, Inc., Princeton, NJ, USA) was used to analyze and calculate PK parameters using a non-compartmental method. Figure 8 The serum concentration-time curve of P65C-PEG20 in mice was similar to that of aldesleukin [REF-1], as reported. The terminal half-life (t) of P65C-PEG20 was... 1 / 2 ) and mean residence time (MRT) inf The concentration-time intervals (AUC) of P65C-PEG20 were 23.2 hours and 2.85 hours, respectively (Table 4), while the terminal half-life and mean residence time of rhIL-2 were 4.0 hours and 0.20 hours, respectively [REF-1]. lastAt a dose of 0.56 mg / kg, the duration is 8051 hours. * The AUC of interleukin was 1380 hours at a dose of 0.8 mg / kg, while that of adeleukin was 100 ng / mL. * nanograms per milliliter [REF-1]. Compared to interleukin, P65C-PEG20 exhibited a longer terminal half-life (5.8-fold), longer residence time (14.2-fold), and higher exposure (8.3-fold, dose-normalized). REF-1: Charych D, Khalili S, Dixit V, Kirk P, Chang T, Langowski J et al. (2017) Modeling the receptor pharmacology, pharmacokinetics, and pharmacodynamics of NKTR-214, an kinetically-controlled interleukin-2 (IL2) receptor agonist for cancer immunotherapy. PLoS ONE 12(7):e0179431.

[0200] Table 4. PK parameters of IL2 P65C-PEG20

[0201]

[0202] Example 8: In vitro expansion of T cells and NK cells

[0203] T cell proliferation: PBMCs were thawed and grown at 5 million cells / ml in AIM V, 5% FBS, 5 ng / ml OKT3, and a specified concentration of IL-2 or mutant protein. Starting on day 5, cells were divided every 3–4 days with medium and IL-2 desiccant. Starting on day 7, cells were stained and total cells and lymphocyte subtypes were counted every 2–3 days. Compared with rhIL2, in the presence of P65C-PEG20 or Y31C-PEG20+F42K, ex vivo expanded T cells significantly reduced Tregs and enhanced the CD8 T / Treg ratio. Figure 9 ).

[0204] NK cell proliferation: PBMCs were thawed and grown in 24-well plates at 5 million cells / ml in AIM V, 5% FBS, and a specified concentration of IL-2 or mutant protein, 0.5 ml / well. Cells were divided every 2–3 days with medium and IL-2 desiccant. Starting from day 7, cells were stained and total cells and lymphocyte subtypes were counted every 3–6 days. Compared with rhIL2 or rhIL5, P65C-PEG20 or Y31C-PEG20+F42K promoted better NK cell proliferation. Figure 9 ).

[0205] LAK cytotoxicity: PBMC-derived LAK cells cultured for 2–4 weeks were used as effector cells. K562 cells stained with CFSE and grown overnight were used as target cells. 30,000 K562 cells were mixed with varying amounts of LAK cells in the wells of a 96-well U-plate. K562 cell viability was measured at different time points after co-culture by annexin V 7-AAD staining, and CFSE+annexin V+ populations were counted. Compared to rhIL2, P65C-PEG20 or Y31C-PEG20+F42K-activated LAK cells showed enhanced proliferation and enhanced cytotoxicity against target K562 cells. Figure 10 ).

[0206] I. References

[0207] The references mentioned are listed below.

[0208] 1. Pachella LA, Madsen LT, Dains JE. The Toxicity and Benefit of Various Dosing Strategies for Interleukin-2 in Metastatic Melanoma and Renal Cell Carcinoma. JAdv Pract Oncol. 2015;6(3):212-221.

[0209] 2. Lotze, MT, Frana, LW, Sharrow, SO, Robb, RJ, and Rosenberg, SA (1985). In vivo administration of purified human interleukin 2. Half-life and immunologic effects of the Jurkat cell line-derived interleukin 2. The Journal of Immunology, 134(1), 157LP-166.

[0210] 3. Boyman, O., Krieg, C., Létourneau, S., and Pantaleo, G. (2009). Insight into Mechanism of IL-2-Induced Toxicity Provides Rationale for Improved Treatment Strategy using IL-2 / mAb Complexes (38.8) , The Journal of Immunology, 182(1 Supplement), 38.8 LP-38.8.

[0211] 4. Maiser, B., Dismer, F. and Hubbuch, J. (2014), Optimization of random PEGylation reactions by means of highthroughput screening. Biotechnol. Bioeng., 111:104-114. doi:10.1002 / bit.25000.

[0212] 5. Aldehyde Interleukin Drug Database (DB00041(BTD00082, BIOD00082)): DB00041(BTD82, BIOD82). DB00041(BTD00082, BIOD00082). sequence list <110> Cytim Therapeutics, Inc. Xu Xiao Huang Haining Feng Yu G. Mognol Golden D. Gime <120> Modified interleukin-2 (IL-2) peptides, conjugates, and their uses <130> 7006-2000140 <150> 62 / 887,359 <151> 2019-08-15 <150> 63 / 025,095 <151> 2020-05-14 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 133 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(133) <223> Human interleukin-2 (IL-2) polypeptide <400> 1 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 2 <211> 133 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(133) <223> Human interleukin-2 (IL-2) polypeptide <400> 2 Met Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130

Claims

1. A modified interleukin-2 (IL-2) polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, and: (1) having a substitution made at a position selected from the group consisting of natural or non-natural amino acids Y31, N29 and N30, wherein the substitution is a substitution made with cysteine; or (2) A double mutation of Y31C and F42K occurs.

2. The modified IL-2 polypeptide according to claim 1, a) Conjugated to a water-soluble polymer, lipid, protein, or peptide at a position selected from the group consisting of: Y31, N29, and N30; and / or b) Conjugating the polypeptide to a water-soluble polymer, lipid, protein, or peptide at the N-terminus and / or C-terminus.

3. The modified IL-2 polypeptide according to claim 1 or 2, a) The modified IL-2 polypeptide contains a substitution of cysteine ​​at the Y31 position; or b) The modified IL-2 polypeptide contains a substitution of cysteine ​​at the N29 position.

4. The modified IL-2 polypeptide according to claim 1, compared with the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, has reduced binding to IL-2Rα.

5. The modified IL-2 polypeptide according to claim 4, wherein its binding affinity to IL-2Rα is reduced from 10% to 100%, or from 1 to 100,000 or more.

6. The modified IL-2 polypeptide according to claim 4, which does not have detectable binding to IL-2Rα.

7. The modified IL-2 polypeptide according to claim 1, compared with the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, has reduced receptor signaling efficacy for IL-2Rαβγ.

8. The modified IL-2 polypeptide of claim 7, wherein the ratio between the signal transduction efficacy of the modified IL-2 polypeptide for IL-2Rαβγ and the signal transduction efficacy of the corresponding IL-2 polypeptide for IL-2Rαβγ containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution is from 1 / 2 to 1 / 100,000.

9. The modified IL-2 polypeptide according to claim 7, which does not have detectable receptor signaling efficacy for IL-2Rαβγ.

10. The modified IL-2 polypeptide of claim 1, compared with the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, has reduced binding to IL-2Rα, and compared with the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, the modified IL-2 polypeptide has reduced receptor signaling efficacy for IL-2Rαβγ.

11. The modified IL-2 polypeptide of claim 10, which does not have detectable binding to IL-2Rα and does not have detectable receptor signaling efficacy for IL-2Rαβγ.

12. The modified IL-2 polypeptide of claim 1, compared with the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, retains or increases the binding level of the modified IL-2 polypeptide to interleukin-2 receptor β (IL-2Rβ) or interleukin-2 receptor γ (IL-2Rγ), and / or compared with the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, retains or increases the receptor signaling efficacy of the modified IL-2 polypeptide for IL-2Rβγ.

13. The modified IL-2 polypeptide of claim 12, wherein the modified IL-2 polypeptide retains or exceeds the binding level of IL-2Rβ or IL-2Rγ compared to the corresponding IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the said substitution.

14. The modified IL-2 polypeptide of claim 12, wherein the modified IL-2 polypeptide retains or has a higher receptor signaling efficacy for IL-2Rβγ compared to the corresponding IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the said substitution.

15. The modified IL-2 polypeptide of claim 12, compared with the corresponding IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, wherein the modified IL-2 polypeptide retains or exceeds the binding level of IL-2Rβ or IL-2Rγ, and compared with the corresponding IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the substitution, wherein the modified IL-2 polypeptide retains or exceeds the receptor signaling efficacy of IL-2Rβγ.

16. The modified IL-2 polypeptide according to claim 1 or 2, compared with the corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 without the said substitution, has an increased ratio of IL-2Rβγ signaling efficacy to IL-2Rαβγ signaling efficacy (i.e., an increased ratio of IL-2Rβγ signaling efficacy to IL-2Rαβγ signaling efficacy).

17. The modified IL-2 polypeptide of claim 1, which is part of a fusion polypeptide comprising the modified IL-2 polypeptide and an additional amino acid sequence.

18. The modified IL-2 polypeptide of claim 17, wherein the N-terminus or the C-terminus of the modified IL-2 polypeptide is fused to the additional amino acid sequence.

19. The modified IL-2 polypeptide of claim 18, wherein the additional amino acid sequence comprises an antibody sequence or a portion or fragment thereof.

20. The modified IL-2 polypeptide of claim 18, wherein the additional amino acid sequence comprises the Fc portion of an antibody.

21. The modified IL-2 polypeptide according to claim 1, wherein it is in isolated form.

22. A polynucleotide encoding the modified IL-2 polypeptide according to any one of claims 1 to 21 and configured to express the modified IL-2 polypeptide in vitro and / or in vivo.

23. A modified IL-2 polypeptide conjugate comprising a modified IL-2 polypeptide conjugated to a water-soluble polymer, protein, or peptide according to any one of claims 1 to 21.

24. The modified IL-2 polypeptide conjugate according to claim 23, wherein the modified IL-2 polypeptide is covalently conjugated with a water-soluble polymer, protein, or peptide.

25. The modified IL-2 polypeptide conjugate according to claim 23, wherein the modified IL-2 polypeptide is non-covalently conjugated with a water-soluble polymer, protein, or peptide.

26. The modified IL-2 polypeptide conjugate according to any one of claims 23 to 25, wherein the modified IL-2 polypeptide is conjugated to a water-soluble polymer, protein, or peptide by means of a substituted natural or non-natural amino acid selected from the group consisting of Y31, N29, and N30, wherein the substitution is a substitution with cysteine.

27. The modified IL-2 polypeptide conjugate of claim 26, wherein the modified IL-2 polypeptide is conjugated to a water-soluble polymer, protein, or peptide via a substituted cysteine ​​residue at position Y31.

28. The modified IL-2 polypeptide conjugate according to any one of claims 23 to 25 and 27, wherein the modified IL-2 polypeptide is conjugated to a water-soluble polymer, protein or peptide via a single amino acid residue of the modified IL-2 polypeptide.

29. The modified IL-2 polypeptide conjugate of claim 28, wherein the modified IL-2 polypeptide is conjugated to a water-soluble polymer, protein, or peptide by: i) The α-amino group of the N-terminal amino acid residue of the modified IL-2 polypeptide; ii) the ε-amino group of the lysine amino acid residue of the modified IL-2 polypeptide; or iii) The N-glycosylation site or O-glycosylation site of the modified IL-2 polypeptide.

30. The modified IL-2 polypeptide conjugate according to any one of claims 23 to 25, 27 and 29, wherein the modified IL-2 polypeptide is covalently conjugated to a water-soluble polymer, protein or peptide via a linker.

31. The modified IL-2 polypeptide conjugate according to any one of claims 23 to 25, 27 and 29, wherein the modified IL-2 polypeptide is conjugated to a water-soluble polymer, protein or peptide by a single amino acid residue in a fusion polypeptide comprising the modified IL-2 polypeptide and an additional amino acid sequence.

32. The modified IL-2 polypeptide conjugate according to claim 31, wherein the single amino acid residue is located within the modified IL-2 polypeptide.

33. The modified IL-2 polypeptide conjugate according to claim 31, wherein the single amino acid residue is located within the other amino acid sequence.

34. The modified IL-2 polypeptide conjugate of claim 31, wherein the additional amino acid sequence comprises an antibody sequence or a portion or fragment thereof.

35. The modified IL-2 polypeptide conjugate of claim 34, wherein the additional amino acid sequence comprises the Fc portion of the antibody.

36. The modified IL-2 polypeptide conjugate of claim 31, wherein the modified IL-2 polypeptide is conjugated to a water-soluble polymer, protein, or peptide by: i) the α-amino group of the N-terminal amino acid residue of the fusion polypeptide; ii) the ε-amino group of the lysine amino acid residue of the fusion polypeptide; or iii) The N-glycosylation site or O-glycosylation site of the fusion polypeptide.

37. The modified IL-2 polypeptide conjugate of claim 36, wherein the fusion polypeptide is covalently conjugated to a water-soluble polymer, protein, or peptide via a linker.

38. The modified IL-2 polypeptide conjugate according to claim 23, wherein the modified IL-2 polypeptide is conjugated with a water-soluble polymer.

39. The modified IL-2 polypeptide conjugate according to claim 28, wherein the water-soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(enol), poly(vinylpyrrolidone), poly(hydroxyalkyl methylacrylamide), poly(hydroxyalkyl methacrylate), poly(sugar), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof.

40. The modified IL-2 polypeptide conjugate of claim 39, wherein the water-soluble polymer comprises a PEG molecule.

41. The modified IL-2 polypeptide conjugate according to claim 40, wherein the PEG molecule is linear PEG.

42. The modified IL-2 polypeptide conjugate according to claim 40, wherein the PEG molecule is a branched PEG.

43. The modified IL-2 polypeptide conjugate according to claim 42, wherein the branched PEG has three to ten PEG chains emanating from a central core group.

44. The modified IL-2 polypeptide conjugate according to claim 42, wherein the branched PEG is a star-shaped PEG comprising 10 to 100 PEG chains emanating from a central core group.

45. The modified IL-2 polypeptide conjugate of claim 42, wherein the branched PEG is a comb-shaped PEG comprising a plurality of PEG chains transplanted onto the polymer backbone.

46. ​​The modified IL-2 polypeptide conjugate according to any one of claims 40 to 45, wherein the molecular weight of the PEG molecule is in the range of 300 g / mol to 10,000,000 g / mol.

47. The modified IL-2 polypeptide conjugate according to any one of claims 40 to 45, wherein the average molecular weight of the PEG molecule is from 5,000 Daltons to 1,000,000 Daltons.

48. The modified IL-2 polypeptide conjugate according to claim 47, wherein the average molecular weight of the PEG molecules is from 20,000 Daltons to 30,000 Daltons.

49. The modified IL-2 polypeptide conjugate according to any one of claims 40 to 45 and 48, wherein the PEG molecule is a monodisperse, homogeneous or discrete PEG molecule.

50. The modified IL-2 polypeptide conjugate of claim 38, wherein the water-soluble polymer comprises a polysaccharide.

51. The modified IL-2 polypeptide conjugate according to claim 49, wherein the modified IL-2 polypeptide is conjugated with monodisperse or homogeneous PEG molecules.

52. The modified IL-2 polypeptide conjugate according to claim 49, wherein the modified IL-2 polypeptide is conjugated with discrete PEG molecules.

53. The modified IL-2 polypeptide conjugate of claim 23, wherein the modified IL-2 polypeptide is conjugated to a protein.

54. The modified IL-2 polypeptide conjugate of claim 53, wherein the protein comprises an antibody or a binding fragment thereof.

55. The modified IL-2 polypeptide conjugate of claim 54, wherein the antibody or its binding fragment comprises the Fc portion of the antibody.

56. The modified IL-2 polypeptide conjugate according to claim 23, wherein the water-soluble polymer, lipid, protein or peptide is indirectly linked to the substituted natural or non-natural amino acid of the modified IL-2 polypeptide via a linker.

57. The modified IL-2 polypeptide conjugate of claim 23, wherein the water-soluble polymer, lipid, protein or peptide is directly bound to the substituted natural or non-natural amino acid of the modified IL-2 polypeptide.

58. The modified IL-2 polypeptide conjugate according to claim 23, having an in vivo half-life of 5 minutes to 10 days.

59. A pharmaceutical composition comprising an effective amount of a modified IL-2 polypeptide according to any one of claims 1 to 21, a polynucleotide according to claim 22, or a modified IL-2 polypeptide conjugate according to any one of claims 23 to 58, and a pharmaceutically acceptable carrier or excipient.

60. The pharmaceutical composition according to claim 59, further comprising another active ingredient.

61. The pharmaceutical composition according to claim 59 or 60, wherein it is configured to treat or prevent proliferative disorders in a subject.

62. The pharmaceutical composition according to claim 60, wherein the other active ingredient is an anti-biomass substance.

63. Use of an effective amount of the modified IL-2 polypeptide according to any one of claims 1 to 21, the polynucleotide according to claim 22, or the modified IL-2 polypeptide conjugate according to any one of claims 23 to 58 in the preparation of a medicament for treating or preventing a disease or condition of a subject, wherein the disease or condition is selected from the group consisting of: tumors, cancer, immunodeficiency diseases or conditions, autoimmune or inflammatory diseases or conditions, and infectious diseases or conditions.

64. The use according to claim 63, wherein the subject is a human.

65. The use according to claim 63, wherein the subject is a non-human mammal.

66. The use according to claim 63, wherein the disease or symptom is a tumor.

67. The use according to claim 63, wherein the disease or condition is cancer.

68. The use according to claim 66 or 67, wherein the tumor or cancer is a solid tumor or cancer.

69. The use according to claim 68, wherein the solid tumor or cancer is selected from the group consisting of: chondrosarcoma, Ewing's sarcoma, bone / osteosarcoma, malignant fibrous histiocytoma, osteosarcoma, rhabdomyosarcoma, cardiac cancer, astrocytoma, brainstem glioma, ependymoma, primitive neuroectodermal tumor, glioma, medulloblastoma, neuroblastoma, oligodendroglioma, pituitary adenoma, breast cancer, invasive lobular carcinoma, tubular carcinoma, invasive cribriform carcinoma, medullary carcinoma, phyllodes tumor, adrenocortical carcinoma, islet cell carcinoma, multiple endocrine neoplasia syndrome, parathyroid carcinoma, pheochromocytoma, thyroid cancer, Merkel cell carcinoma, uveal melanoma, retinoblastoma, anal cancer, appendix cancer, bile duct cancer, carcinoid tumor, gastrointestinal cancer, colon cancer, extrahepatic bile duct cancer, gallbladder cancer, etc. Cystic carcinoma, gastrointestinal stromal tumor (GIST), hepatocellular carcinoma, pancreatic islet cell carcinoma, rectal cancer, bladder cancer, cervical cancer, endometrial cancer, gonadal germ cell tumors, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumors, penile cancer, renal cell carcinoma, prostate cancer, testicular cancer, gestational trophoblastic tumor, ureter and renal pelvis, transitional cell carcinoma, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms' tumor, esophageal cancer, head and neck cancer, oral cancer, sinus and nasal cavity cancer, pharyngeal cancer, salivary gland cancer, basal cell carcinoma, melanoma, bronchial adenoma / carcinoid, small cell lung cancer, mesothelioma, non-small cell lung cancer, pleural pulmonary blastoma, thymoma and thymic carcinoma, AIDS-related cancers, Kaposi's sarcoma, epithelioid angioendothelioma (EHE), fibroblastic small round cell tumor and liposarcoma.

70. The use according to claim 69, wherein the astrocytoma is selected from the group consisting of: pilocytic astrocytoma, cerebellar astrocytoma, cerebral astrocytoma, and pineal astrocytoma.

71. The use according to claim 69, wherein the glioma is a visual pathway and hypothalamic glioma.

72. The use according to claim 69, wherein the breast cancer is male breast cancer or inflammatory breast cancer.

73. The use according to claim 69, wherein the gastrointestinal cancer is gastric cancer.

74. The use according to claim 69, wherein the pharyngeal cancer is selected from the group consisting of nasopharyngeal carcinoma, oropharyngeal carcinoma, hypopharyngeal carcinoma, and laryngeal carcinoma.

75. The use according to claim 66 or 67, wherein the tumor or cancer is a malignant hematologic malignancy.

76. The use according to claim 75, wherein the malignant hematologic malignancy is selected from the group consisting of: myeloid lesions, Hodgkin lymphoma, non-Hodgkin lymphoma, anaplastic large cell lymphoma, angioimmune T-cell lymphoma, hepatosplenic T-cell lymphoma, B-cell lymphoma, reticuloendothelial proliferation, reticulocytosis, small glioma, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt lymphoma, mediastinal large B-cell lymphoma, Waldenström macroglobulin. Leukemia, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, lymphomatoid granulomatosis, nodular lymphocytic major Hodgkin lymphoma, plasma cell leukemia, acute polycythemia and erythroleukemia, acute erythrocytic myelopathy, acute erythrocytic leukemia, Helmholtz-Schönlein disease, acute megakaryocytic leukemia, mast cell leukemia, panmyelopathy, acute panmyelopathy with myelofibrosis, lymphosarcoma cell leukemia, acute leukemia of unspecified cell type, chronic myeloid leukemia in blast crisis. Stem cell leukemia, unspecified cell type chronic leukemia, unspecified cell type subacute leukemia, accelerated phase chronic myeloid leukemia, acute myeloid leukemia, polycythemia vera, acute promyelocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, mature acute myeloblastic leukemia, acute myeloid dendritic cell leukemia, adult T-cell leukemia / lymphoma, aggressive NK cell leukemia, B-cell chronic lymphocytic leukemia, B-cell leukemia, chronic myeloid leukemia, chronic myeloid monocytic leukemia Nuclear cell leukemia, chronic neutrophilic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, chronic idiopathic myelofibrosis, Calder disease, myeloma, plasma cell leukemia, plasmacytoma, extramedullary, monoclonal gammopathy, angiocentric immunoproliferative disorders, lymphoid granulomatosis, angioimmunoblastic lymphadenopathy, T-γ lymphohistioproliferative disorders, Waldenström macroglobulinemia, alpha heavy chain disease, gamma heavy chain disease, Franklin's disease, immunoproliferative small bowel disease, thalassemia, malignant immunoproliferative disorders, unspecified and immunoproliferative disorders (NOS).

77. The use according to claim 76, wherein the myeloma is multiple myeloma.

78. The use according to claim 76, wherein the plasmacytoma is a malignant plasmacytoma NOS or a plasmacytoma NOS.

79. The use according to claim 63, wherein the disease or condition is an immunodeficiency disease or condition.

80. The use according to claim 79, wherein the immunodeficiency disease or condition is selected from the group consisting of: agammaglobulinemia: X-linked and autosomal recessive inheritance, ataxia-telangiectasia, chronic granulomatous diseases and other phagocytic disorders, common variable immunodeficiency, complement deficiency, DeGeorge syndrome, hemophagocytic lymphohistiocytosis (HLH), hyperIgE syndrome, hyperIgM syndrome, IgG subclass deficiency, congenital immunodeficiency, NEMO deficiency syndrome, selective IgA deficiency, selective IgM deficiency, severe combined immunodeficiency, etc. Immunodeficiency and combined immunodeficiency, specific antibody deficiency, transient hypogammaglobulinemia in infancy, WHIM syndrome (warts, hypogammaglobulinemia, infection and myeloablative agranulocytosis), Wiscot-Aldrich syndrome, other antibody deficiency disorders, other primary cellular immunodeficiency, severe combined immunodeficiency (SCID), human immunodeficiency virus / acquired immunodeficiency syndrome (HIV / AIDS), drug-induced immunodeficiency, graft-versus-host disease, primary immunodeficiency disease (PIDD), and lymphopenia.

81. The use according to any one of claims 63-65, for the treatment or prevention of an autoimmune or inflammatory disease or condition of a subject.

82. The use according to claim 81, wherein the autoimmune or inflammatory disease or condition is selected from the group consisting of: paraneoplastic autoimmune diseases, cartilage inflammation, fibrotic diseases and / or bone degeneration, arthritis, juvenile ankylosing spondylitis, juvenile Ritter syndrome, ankylosing spondylitis, Ritter syndrome, SEA syndrome (serologically negative, enthesitis, arthropathy syndrome), dermatomyositis, scleroderma, vasculitis, polymyositis, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, scleroderma, sclerosis, sclerosing cholangitis, Sjögren's syndrome, psoriasis, dermatitis, atherosclerosis, lupus, Still's disease, myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, asthma, COPD, Guillain-Barré disease, type I diabetes, thyroiditis, Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, and transplant rejection.

83. The use according to claim 82, wherein the arthritis is selected from the group consisting of: rheumatoid arthritis, juvenile arthritis, enteropathic arthritis, reactive arthritis, psoriatic arthritis, and osteoarthritis.

84. The use according to claim 82, wherein the dermatomyositis is juvenile dermatomyositis.

85. The use according to claim 82, wherein the dermatitis is atopic dermatitis.

86. The use according to claim 82, wherein the lupus is juvenile systemic lupus erythematosus or systemic lupus erythematosus (SLE).

87. The use according to claim 82, wherein the arteritis is polyarteritis nodosa.

88. The use according to claim 82, wherein the sclerosis is multiple sclerosis (MS).

89. The use according to claim 82, wherein the scleroderma is juvenile scleroderma.

90. The use according to claim 82, wherein the vasculitis is juvenile vasculitis.

91. The use according to claim 82, wherein the psoriasis is selected from the group consisting of: plaque psoriasis, guttate psoriasis, inverted psoriasis, pustular psoriasis, and erythrodermic psoriasis.

92. The use according to claim 83, wherein the rheumatoid arthritis is selected from the group consisting of: juvenile rheumatoid arthritis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, and systemic rheumatoid arthritis.

93. The use according to claim 83, wherein the juvenile arthritis is selected from the group consisting of: juvenile enteropathic arthritis, juvenile reactive arthritis, and juvenile psoriatic arthritis.

94. The use according to claim 92, wherein the juvenile rheumatoid arthritis is selected from the group consisting of: oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, and systemic juvenile rheumatoid arthritis.

95. The use according to any one of claims 63-65, for treating or preventing an infectious disease or condition of the subject.

96. The use according to claim 95, wherein the infectious disease is selected from the group consisting of: Acinetobacter infection, actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (Acquired Immunodeficiency Syndrome), amoebiasis, anaplasmosis, angiostrongylus infection, anisakiasis, anthrax, Cryptococcus hemolyticus infection, Argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, Bacillus cereus infection, bacterial meningitis, bacterial pneumonia, bacterial vaginosis, Bacteroidetes infection, giardiasis, Bartonella infection, BK virus infection, black hair nodule disease, blastomycosis, blastomycosis, Bolivian hemorrhagic fever, botulism, Brazilian hemorrhagic fever, brucellosis, Black Death, Burkholderia infection. Infections, Brulee's ulcer, Calicut virus infection, Curvularia infection, Candidiasis, Capillary nematode infection, Calion disease, Cat scratch disease, Cellulitis, Chagas disease (American trypanosomiasis), Chancroid, Chickenpox, Chikungunya fever, Chlamydia, Chlamydia pneumoniae infection, Cholera, Chromobacterium chromobacterial infection, Chytriditis, Clostridium difficile colitis, Coccidioidomycosis, Colorado tick fever (CTF), Common cold, Coronavirus disease 2019 (COVID-19), Creutzfeldt-Jakob disease (CJD), Crimean-Congo hemorrhagic fever (CCHF), Cryptococcosis, Cryptosporidiosis, Cutaneous larval migration syndrome (CLM), Cyclosporidiosis, Cysticercosis, Cytomegalovirus infection, Dengue fever, *Zygotaenia* infection, Dinuclear amoebiasis, Diphtheria, Sparganosis Tapeworm infection, dracunculiasis, Ebola hemorrhagic fever, echinococcosis, ehrlichiosis, pinworm infection, enterococcal infection, enterovirus infection, epidemic typhus, erythema infectiosum (the fifth disease), roseola infantum, fascioliasis, fascioliasis, fatal familial insomnia (FFI), filariasis, food poisoning caused by Clostridium perfringens, free-living amoeba infection, Clostridium spp. infection, gas gangrene, geomycosis, Gerstmann-Straussler-Schenck syndrome (GSS), giardiasis, glanders, gnathostomiasis, gonorrhea, granuloma inguinale (Dunofan disease), group A streptococcal infection, group B streptococcal infection, Haemophilus influenzae infection, hand-foot-mouth disease (HFMD), Hantavirus pulmonary syndrome (HPS) The following diseases are listed: Hepatitis A, Helicobacter pylori infection, Hemolytic uremic syndrome (HUS), Hemorrhagic fever with renal syndrome (HFRS), Hendra virus infection, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex, Histoplasmosis, Hookworm infection, Human bocavirus infection, Human Ehrlich's disease, Human granulocytic anaplasmosis (HGA), Human metapneumovirus infection, Human mononucleotic erythrocyte disease, Human papillomavirus (HPV) infection, Human parainfluenza virus infection, Hymenolepis taeniasis, Epstein-Barr virus infectious mononucleosis (Mono), Influenza, Coccidioidomycosis, Kawasaki disease, Keratitis, Geraniella infection, Kuru disease, Lassa fever, Legionnaires' disease.Pontiac fever, leishmaniasis, leprosy, leptospirosis, listeriosis, Lyme disease (Lyme borborygia), lymphatic filariasis (elephantiasis), lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever (MHF), measles, Middle East Respiratory Syndrome (MERS), melioidosis (Whitmore's disease), meningitis, meningococcal infection, metaclonorchiasis, microsporidiosis, molluscum contagiosum (MC), monkeypox, mumps, murine typhus (endemic typhus), mycoplasma pneumonia, genital mycoplasma infection Mycobacterium tumefaciens, myiasis, neonatal conjunctivitis, Nipah virus infection, norovirus, variant Creutzfeldt-Jakob disease, nocardiac infection, onchocerciasis (river blindness), clonorchiasis, paracoccidioidomycosis, paragonimiasis, pastoral infection, head lice, body lice, pubic lice, pelvic inflammatory disease (PID), pertussis, plague, pneumococcal infection, Pneumocystis pneumonia, pneumonia, poliomyelitis, Prevotella infection, primary amoebic meningoencephalitis (PAM), progressive multifocal disease. Leukoencephalopathy, psittacosis, Q fever, rabies, relapsing fever, respiratory syncytial virus infection, rhinocystis infection, rhinovirus infection, rickettsia infection, rickettsial pox, Rift Valley fever (RVF), Rocky Mountain spotted fever (RMSF), rotavirus infection, rubella, salmonellosis, SARS (Severe Acute Respiratory Syndrome), scabies, scarlet fever, schistosomiasis, sepsis, Shigella infection (bacillary dysentery), herpes zoster, smallpox, sporotrichosis, staphylococcal food poisoning, staphylococcal infection, strongyloidiasis, subacute sclerosis Panencephalitis, non-sexually transmitted syphilis, syphilis and yaws, tapeworm infection, tetanus, tinea corporis, toxocariasis, toxoplasmosis, trachoma, trichinosis, trichomoniasis, whipworm infection, tuberculosis, tularemia, typhus, spotted fever, Ureaplasma urealyticum infection, valley fever, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Vibrio vulnificus infection, Vibrio parahaemolyticus enteritis, viral pneumonia, West Nile fever, trichomonas vulgaris, pseudotuberculosis, Yersinia pseudotuberculosis, yellow fever, Zispallas disease, Zika fever, and mycobacteriosis.

97. The use according to claim 96, wherein the ascariasis is Beliris ascariasis infection.

98. The use according to claim 96, wherein the tinea corporis (body tinea) is selected from the group consisting of: tinea barbae (scalp tinea), tinea capitis (scalp tinea), tinea cruris (jock itch), tinea manuum (hand tinea), tinea pedis (athlete's foot), onychomycosis (nail fungus), and tinea versicolor (pityriasis versicolor).

99. The use according to claim 95, wherein the infectious disease is South American budding fungal infection or tinea capitis.

100. The use according to any one of claims 63-99, further comprising the use of administering an effective amount of the second therapeutic agent in the preparation of a medicament for treating or preventing a disease or ailment of a subject.

101. The use according to claim 69, wherein the solid tumor or cancer is skin cancer.

102. The use according to claim 82, wherein the autoimmune or inflammatory disease or condition is primary cholecystitis.

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