Novel IL-15R alpha Fc fusion protein and use thereof

Abstract

The present application relates to IL-15R [alpha] and Fc fusion proteins, IL-15 and IL-15R [alpha] heterodimer proteins, as well as isolated nucleic acid molecules encoding any of the fusion proteins or the heterodimer proteins, vectors and host cells comprising the nucleic acid molecules, compositions, kits, and articles of manufacture comprising any of the fusion proteins or the heterodimer proteins, and methods of making and using the same.

Description

[0001] Submit Sequence Listing

[0002] This application claims priority to U.S. Provisional Application No. 63 / 370,605 filed on August 5, 2022; U.S. Provisional Application No. 63 / 370,606 filed on August 5, 2022; U.S. Provisional Application No. 63 / 370,607 filed on August 5, 2022; and U.S. Provisional Application No. 63 / 477,993 filed on December 30, 2022, the contents of each of which are incorporated herein by reference in their entirety. The contents of the electronic sequence listing (a novel IL-15R alpha Fc fusion protein and its use SEQ.xml; size: 25KB; and record date: 2022.08.05) are incorporated herein by reference in their entirety. Technical Field

[0003] The present application relates to IL-15Rα(alpha) and Fc fusion protein, IL-15 and IL-15Rα heterodimer protein, including activatable IL-15 prodrug, and preparation and use methods thereof. The present application also relates to the cleavage product of the activatable prodrug and use methods thereof. Background Art

[0004] Cytokines are potent immune agonists and are therefore considered very promising tumor therapeutic agents. For example, interleukin-15 (IL-15) is currently being studied for its anti-tumor activity and has been used in human therapy.

[0005] IL-15 is a member of the four α-helical bundle families, with a molecular weight of 14-15 kDa and 114 amino acids (Fehniger TA, Caligiuri MA. Interleukin 15: biology and relevance to human disease. Blood. 2001; 97: 14-32), produced by mononuclear phagocytes and other immune system cells. IL-15 is essential for the development and function of natural killer cells (NK), natural killer T cells (NKT) and memory CD8+ T cells.

[0006] However, the therapeutic window of cytokines is very narrow and the serum half-life is short. Therefore, the therapeutic administration of cytokines will produce undesirable systemic effects and toxicity. Since a large amount of cytokines need to be applied at the expected site of action of the cytokine (e.g., tumor) to achieve the desired cytokine level, the above problems are exacerbated. It is urgent to find a method to extend the half-life of IL-15 in vivo, promote, reduce its side effects and toxicity or enhance the biological activity of IL-15 in vivo.

[0007] Recently, studies have found that the complex formed by IL-15 and its receptor IL-15Rα can significantly enhance the biological activity of IL-15. Studies have shown that the complex formed by IL-15 and soluble IL-15Rα is significantly better than IL-15 alone in stimulating the proliferation of memory CD8+T lymphocytes and NT / NKT cells. In terms of stimulating the proliferation and survival of memory CD8+T cells, the IL-15 / IL-l5Rα complex is stronger than IL-15 alone. This mechanism may be related to cis presentation.

[0008] Although researchers have been committed to research related to IL-15 immunotherapy, including IL-15 and Fc fusion protein, IL-15Rα and IL-15 fusion protein or IL-15, IL-15Rα and Fc heterodimer protein, IL-15 has the disadvantages of short half-life in vivo and easy to cause systemic immune side effects, and there is still a need to improve the biological activity of IL-15 in vivo.

[0009] Surprisingly, the inventors found that the length of the linker between the Fc domain and IL-15Rα_sushi affects the biological activity of the IL-15 and IL-15Rα heterodimeric protein to some extent, and the present application is provided based on this.

[0010] Application Overview

[0011] The present application relates to IL-15Rα and Fc fusion proteins, IL-15 and IL-15Rα heterodimeric proteins, including activatable IL-15 prodrugs, and methods for preparing and using the same. The IL-15 and IL-15Rα heterodimeric proteins of the present application exhibit excellent activity, stability, and prolonged in vivo half-life.

[0012] In one aspect, the present application provides an isolated fusion protein, wherein the fusion protein comprises: IL-15Rα or a functional fragment thereof, which is connected to an Fc domain via a peptide linker.

[0013] In some embodiments, the fusion protein of separation provided herein, wherein the peptide linker comprises 3 to 40 amino acid residues. In some embodiments, the peptide linker comprises 3 to 25 amino acid residues. In some embodiments, the fusion protein of separation provided herein, wherein the peptide linker comprises 20 to 25 amino acid residues. In some embodiments, the peptide linker comprises 25 amino acid residues.

[0014] In some embodiments, the peptide linker is rich in G and S amino acid residues. In some embodiments, the peptide linker consists of G and S amino acid residues.

[0015] In some embodiments, the isolated fusion protein provided herein, wherein the IL-15Rα or a functional fragment thereof is linked to the N-terminus or C-terminus of the Fc domain.

[0016] In some embodiments, provided herein is an isolated fusion protein, wherein the fusion protein further comprises an IL-15 cytokine.

[0017] In some embodiments, provided herein are isolated fusion proteins wherein the IL-15 cytokine is linked to the N-terminus or the C-terminus of the Fc domain.

[0018] In some embodiments, the isolated fusion protein provided herein, wherein the IL-15 cytokine is linked to the N-terminus or C-terminus of IL-15Rα or a functional fragment thereof.

[0019] In another aspect, the present application provides an isolated heterodimeric protein comprising two polypeptide chains, wherein one polypeptide chain comprises any one of the fusion proteins described herein, and the other polypeptide chain comprises an IL-15 cytokine and / or an Fc domain.

[0020] In some embodiments, the isolated heterodimeric protein provided herein, wherein the IL-15Rα or a functional fragment thereof and the IL-15 cytokine are linked to the N-terminus or the C-terminus of the Fc domain.

[0021] In some embodiments, the isolated heterodimeric protein provided herein, wherein the Fc domain is selected from the group consisting of human IgG1 Fc domain, human IgG2 Fc domain, human IgG3 Fc domain, human IgG4 Fc domain, IgA Fc domain, IgD Fc domain, IgE Fc domain and IgM Fc domain; optionally, the Fc domain is a human IgG1 Fc domain.

[0022] In some embodiments, the isolated heterodimeric protein provided herein, wherein the Fc domain is a human IgG1 Fc domain having L234A and L235A mutations, wherein the mutation sites are numbered according to the EU numbering system.

[0023] In some embodiments, the isolated heterodimeric protein provided herein, wherein the Fc domain comprises a knobs-into-holes mutation (Fc knob and Fc hole).

[0024] In some embodiments, the isolated heterodimeric protein provided herein, wherein the Fc knob is linked to IL-15 cytokine, and the Fc hole is linked to IL-15Rα or a functional fragment thereof; or, the Fc knob is linked to IL-15Rα or a functional fragment thereof, and the Fc hole is linked to IL-15 cytokine.

[0025] In some embodiments, the isolated heterodimeric protein provided herein, wherein the Fc knob comprises a T366W mutation in the Fc domain, and the Fc hole comprises T366S, L368A and Y407V mutations in the Fc domain, and the mutation sites are numbered according to the EU numbering system.

[0026] In some embodiments, the isolated heterodimeric protein provided herein, wherein the Fc knob further comprises an S354C mutation, and the Fc hole further comprises a Y349C mutation, and the mutation sites are numbered according to the EU numbering system.

[0027] In some embodiments, the isolated fusion protein or heterodimeric protein provided herein, wherein the IL-15Rα or a functional fragment thereof is selected from the extracellular domain or sushi domain or a functional analogue of IL-15Rα.

[0028] In some embodiments, the isolated fusion protein or heterodimeric protein provided herein, wherein the IL-15Rα or a functional fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 9-11, or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 9-11.

[0029] In some embodiments, the isolated fusion protein or heterodimeric protein provided herein, wherein the IL-15 cytokine comprises the amino acid sequence shown in any one of SEQ ID NOs:7-8, or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any one of SEQ ID NOs:7-8.

[0030] In some embodiments, the isolated fusion protein or heterodimeric protein provided herein, wherein the peptide linker comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-5, or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1-5.

[0031] In some embodiments, the isolated fusion protein or heterodimeric protein provided herein, wherein the isolated fusion protein or heterodimeric protein further comprises a cleavable portion (CM) and a masking polypeptide (MP), and wherein the masking polypeptide (MP) is linked to the fusion protein or heterodimeric protein via the cleavable portion (CM).

[0032] In some embodiments, the isolated fusion protein or heterodimeric protein provided herein, wherein the masking polypeptide (MP) comprises the amino acid sequence shown in any one of SEQ ID NOs: 14-15, or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 14-15.

[0033] In some embodiments, the isolated fusion protein or heterodimeric protein provided herein, wherein the cleavable portion (CM) comprises the amino acid sequence of SEQ ID NO:6, or a variant thereof, the variant having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO:6.

[0034] In some embodiments, the isolated fusion protein or heterodimeric protein provided herein comprises two polypeptide chains, wherein one polypeptide chain comprises the amino acid sequence of SEQ ID NO:16, or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO:16; and the other chain comprises the amino acid sequence of SEQ ID NO:17, or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO:17.

[0035] In some embodiments, the isolated fusion protein or heterodimeric protein provided herein comprises two polypeptide chains, wherein one polypeptide chain comprises the amino acid sequence of SEQ ID NO:16, or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO:16; and the other chain comprises the amino acid sequence of SEQ ID NO:18, or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO:18.

[0036] In some embodiments, the isolated fusion protein or heterodimeric protein provided herein comprises two polypeptide chains, wherein one polypeptide chain comprises the amino acid sequence of SEQ ID NO:19, or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO:19; and the other chain comprises the amino acid sequence of SEQ ID NO:20, or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO:20.

[0037] In some embodiments, the isolated fusion protein or heterodimeric protein provided herein comprises two polypeptide chains, wherein one polypeptide chain comprises the amino acid sequence of SEQ ID NO:19, or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO:19; and the other chain comprises the amino acid sequence of SEQ ID NO:18, or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO:18.

[0038] In some embodiments, the isolated fusion protein or heterodimeric protein provided herein comprises two polypeptide chains, wherein one polypeptide chain comprises the amino acid sequence of SEQ ID NO:21, or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO:21; and the other chain comprises the amino acid sequence of SEQ ID NO:22, or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO:22.

[0039] In some embodiments, the isolated fusion protein or heterodimeric protein provided herein comprises two polypeptide chains, wherein one polypeptide chain comprises the amino acid sequence of SEQ ID NO:23, or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO:23; and the other chain comprises the amino acid sequence of SEQ ID NO:22, or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO:22.

[0040] In some embodiments, the isolated fusion protein or heterodimeric protein provided herein comprises two polypeptide chains, wherein one polypeptide chain comprises the amino acid sequence of SEQ ID NO:24, or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO:24; and the other chain comprises the amino acid sequence of SEQ ID NO:22, or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO:22.

[0041] On the other hand, the present application provides a method for improving the activity of any fusion protein or heterodimeric protein, wherein the fusion protein or heterodimeric protein comprises IL-15Rα or its functional fragment and Fc domain, which comprises setting the peptide linker connecting IL-15Rα or its functional fragment to Fc domain to more than 15 and less than 40 amino acid residues. In some embodiments, the peptide linker comprises 20 to 25 amino acid residues. In some embodiments, the peptide linker comprises 25 amino acid residues.

[0042] Also provided are isolated nucleic acid molecules encoding any fusion protein or heterodimer protein described herein, vectors comprising the nucleic acid molecules, host cells (e.g., CHO cells, HEK 293 cells, Hela cells or COS cells) comprising the nucleic acid or vector, compositions (e.g., pharmaceutical compositions), kits, and products comprising any fusion protein or heterodimer protein described herein. Also provided are methods for treating diseases (e.g., tumors) in individuals (e.g., humans) using any fusion protein or heterodimer protein provided herein or its pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Shown are exemplary IL-15 and IL-15Rα or functional fragments thereof, showing that in one monomer, the IL-15Rα_sushi domain is connected to the C-terminus of one Fc domain via a peptide linker, and in another monomer, IL-15 is connected to the C-terminus of another Fc domain.

[0044] Figure 2 Shown are exemplary IL-15 prodrugs with an Fc domain as a half-life extending moiety, showing that in one monomer, the IL-15Rα_sushi domain is linked to the C-terminus of one Fc domain via a non-cleavable linker, in another monomer, IL-15 is linked to the C-terminus of the other Fc domain, and a masking polypeptide (MP) is linked to IL-15 via a cleavable moiety (CM).

[0045] Figure 3 Shown is an SDS-PAGE gel electrophoresis diagram used to analyze the purity of exemplary IL-15 and IL-15Rα and heterodimeric protein SB1902-C1.

[0046] Figure 4 Shown is a graph showing the results of SEC-HPLC for analyzing the homogeneity of exemplary IL-15 and IL-15Rα and heterodimeric protein SB1902-C1.

[0047] Figure 5 Shown are exemplary IL-15 and IL-15Rα heterodimeric protein SB1902-C1 and exemplary IL-15 prodrug SB1902-C2 binding to the IL-2 / IL-15Rβγ receptor.

[0048] Figure 6 Shown are the results of exemplary IL-15 and IL-15Rα heterodimeric proteins SB1902-C1 and SB1902-C1-variant1 in a CD8+ T cell activation assay.

[0049] Figure 7Shown are the results of the exemplary IL-15 prodrugs SB1902-C2 and SB1902-C2-variant0 in a CD8+ T cell activation assay.

[0050] Figure 8 Shown are the results of exemplary IL-15 prodrugs SB1902-C9-variant 1, SB1902-C9-variant 2, and SB1902-C9-variant 3 in a CD8+ T cell activation assay.

[0051] Figure 9-10 The results are shown for the use of IgG1 subtype control antibody MOPC-21 ( Fig. 9 ) or IL-15 and IL-15Rα heterodimer protein SB1902-C1 ( Fig.10 ) Results of animals bearing WEHI-164 tumors treated at a dose of 3 mg / kg. Tumor growth in animals treated with SB1902-C1 was significantly inhibited compared to tumor growth in animals treated with a subtype control antibody. DETAILED DESCRIPTION OF THE INVENTION

[0053] Disclosed herein are IL-15Rα and Fc fusion proteins, IL-15 and IL-15Rα heterodimeric proteins, including activatable IL-15 prodrugs. The IL-15 and IL-15Rα heterodimeric proteins have long half-life and better activity. The prodrug overcomes the toxicity problem that severely limits the clinical application of IL-15 cytokines.

[0054] definition

[0055] Unless otherwise clearly indicated in the context, the present invention will be practiced using conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA technology within the technical scope of the art, and the various methods described above will be described in detail below for illustration. Such techniques are fully explained in the literature. See, for example, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., John Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.

[0056] As described herein, "treatment" or "treating" is a method of obtaining a beneficial or desired result, including clinical results. In view of the purpose of this application, the beneficial or desired result includes clinical results, but is not limited to one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the degree of the disease, stabilizing the disease (e.g., preventing or delaying the deterioration of the disease), preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, alleviating the disease (partially or completely), reducing the dosage of one or more other drugs required for the treatment of the disease, delaying the progression of the disease, improving or improving the quality of life, and / or prolonging the survival period. At the same time, "treatment" also includes the reduction of pathological results of the disease. The method of the present application takes into account any one or more aspects of these treatments. For example, if one or more symptoms associated with the disease are alleviated or eliminated, including but not limited to reducing the symptoms caused by the disease, improving the quality of life of the patient suffering from the disease, reducing the dosage of other drugs required for the treatment of the disease, and / or prolonging the survival period of the individual, the patient is considered to be successfully "treated".

[0057] The term "prevent" and similar words, such as "prevented", "preventing", etc., refer to methods of preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition. It also refers to delaying the occurrence or recurrence of a disease or condition or delaying the occurrence or recurrence of symptoms of a disease or condition. As used herein, "preventing" and similar words also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition before the disease or condition recurs.

[0058] As described herein, "delaying" the development of a disease means postponing, hindering, slowing down, slowing down, stabilizing and / or delaying the development of a disease. Depending on the history of the disease and / or the individual being treated, the time of delay may be different. A method of "delaying" the development of a disease refers to a method that reduces the probability of disease development within a given time frame and / or reduces the extent of the disease within a given time frame compared to not using the method. This comparison is usually based on clinical studies using a statistically significant number of individuals.

[0059] As described herein, the term "effective amount" refers to an amount of drug or pharmaceutical composition sufficient to treat a particular disorder, condition or disease, such as improving, alleviating, weakening and / or delaying one or more symptoms. In some embodiments, an effective amount is an amount sufficient to delay the development of a disease. In some embodiments, an effective amount is an amount sufficient to prevent or delay the occurrence or recurrence of a disease. An effective amount can be administered in one or more administrations. In the case where a disease is, for example, cancer, an effective amount can be an amount sufficient to delay the occurrence or progression of cancer (e.g., reducing tumor growth rate and / or delaying or preventing tumor angiogenesis, metastasis or cancer cells from infiltrating peripheral organs), reducing the number of epithelial cells, causing cancer regression (e.g., shrinking or eradicating a tumor), and / or preventing or delaying the occurrence or recurrence of cancer. An effective amount can be administered in one or more administrations.

[0060] As used herein, "individual" or "subject" refers to a mammal, including but not limited to humans, cows, horses, cats, dogs, rodents, or primates. In some embodiments, the individual is a human.

[0061] Antibodies are classified or typed based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by heavy chains of type α, δ, ε, γ, and μ, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain n), or IgA2 (α2 heavy chain).

[0062] As used herein, the terms "Fc", "Fc region", "fragment crystallographic region", "Fc domain" or "Fc moiety" are used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during the production or purification of the protein, or by recombinant engineering of the nucleic acid encoding the protein. Suitable native sequence Fc regions for the constructs described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

[0063] As described herein, the term IgG "subtype" or "subclass" refers to any subclass of immunoglobulins defined by the chemical and antigenic properties of the constant domains. Immunoglobulins are mainly divided into five major classes: IgA, IgD, IgE, IgG and IgM, and many of them can be further divided into subclasses (subtypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are respectively called α, γ, ε, γ and μ. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known and are described in detail in Abbas et al., Cellular and Molecular Immunology, Fourth Edition (WB Saunders, Co., 2000).

[0064] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an Fc-containing structure (e.g., an antibody or a protein containing an Fc domain, hereinafter referred to as an Fc fusion protein). The preferred FcR is a human FcR native sequence. In addition, the preferred FcR is an FcR that binds to an IgG antibody (a gamma receptor), including receptor subclasses such as FcγRI, FcγRII and FcγRIII, as well as allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences and differ primarily in the cytoplasmic domain. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (described in M. FcRs are described in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994) and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" herein encompasses other FcRs, including those identified in the future.

[0065] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transport of maternal IgG to the fetus. Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994). Methods for determining binding to FcRn are well known (see Ghetie and Ward, Immunol. Today 18:(12):592-8 (1997); Ghetie et al., Nature Biotechnology 15(7):637-40 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6 (2004); WO 2004 / 92219 (Hinton et al.)). The half-life of human FcRn high affinity binding polypeptides binding to FcRn in vivo and in serum can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides with variant Fc regions. WO2004 / 42072 (Presta) details antibody variants that enhance or attenuate binding to FcRs. See Shield et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

[0066] "Antibody effector functions" refer to those biological activities caused by the Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) in an Fc-containing structure (e.g., an antibody or Fc fusion protein), and vary with the Fc subtype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor) and B cell activation. "Reducing or minimizing" antibody effector function means a reduction of at least 50% (or 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) compared to a wild-type or unmodified Fc-containing structure (e.g., an antibody or Fc fusion protein). Determination of antibody effector function can be readily determined and measured by one of ordinary skill in the art. In preferred embodiments, the antibody effector functions of complement fixation, complement dependent cellular toxicity, and antibody dependent cellular toxicity are affected. In some embodiments, the effector function is eliminated by eliminating glycosylation by mutations in the constant domains, e.g., "no effector function mutations". In some embodiments, no effector function mutants are in C HN297A or DANA mutation (D265A+N297A) in region 2. Shields et al., J. Biol. Chem. 276 (9): 6591-6604 (2001). In addition, other mutations that result in reduced or eliminated effector function include: K322A and L234A / L235A (LALA). In addition, effector function can be reduced or eliminated by production techniques, such as expression in a host cell that does not perform glycosylation (e.g., E. coli) or in a host cell that results in an altered glycosylation pattern that is ineffective or less effective in promoting effector function (e.g., Shinkaw et al., J. Biol. Chem. 278 (5): 3466-3473 (2003)).

[0067] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a form of cytotoxicity that refers to the binding of secreted Ig to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells bearing antigens and subsequently kill the target cells using cytotoxins. Antibodies "arm" the cytotoxic cells and are required for this killing. Of the major cell types that mediate ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a target molecule, an in vitro ADCC assay can be performed, as described in U.S. Pat. Nos. 5,500,362 or 5,821,337. Effector cells suitable for such experiments include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK). Alternatively, or in addition, ADCC activity of the target molecule can also be assessed in vivo, for example, as described in an animal model disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0068] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate structural subclass) that binds to a cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed. Polypeptide variants having altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in U.S. Patent No. 6,194,551 B1 and WO 99 / 51642. The contents of these patent publications are expressly incorporated herein by reference. See also Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0069] As used herein, the terms "specifically bind," "specifically recognize," or "specific for" refer to a measurable and reproducible interaction, such as binding between a ligand and a receptor, wherein the presence of the ligand can be determined in the presence of a heterogeneous population of molecules, including biomolecules. For example, a ligand that specifically binds to a receptor binds to the target receptor with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other receptors. In some embodiments, the extent of binding of the ligand to an unrelated receptor is less than 10% of the extent of binding of the ligand to the target receptor, as determined, for example, by radioimmunoassay (RIA). In some embodiments, the equilibrium dissociation constant (Kd) of a ligand that specifically binds to a target receptor is ≤10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M or ≤10 -12 M. In some embodiments, the ligand specifically binds to a receptor that is conserved in different species. In some embodiments, specific binding may include but does not require exclusive binding. The binding specificity of the ligand can be determined experimentally using methods known in the art. Such as, but not limited to, Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE™-tests and peptide scanning.

[0070] As used herein, "amino acid" is used in its broadest sense, i.e., it includes naturally occurring amino acids, as well as non-naturally occurring amino acids, including analogs and derivatives of amino acids. The latter include molecules containing amino acid moieties. According to this broad definition, those skilled in the art will find that the amino acids described herein include, for example, natural L-amino acids that form proteins; D-amino acids; chemically modified amino acids, such as amino acid analogs and derivatives; natural amino acids that do not form proteins, such as norleucine, β-alanine, ornithine, GABA, etc.; and chemically synthesized compounds with amino acid characteristics known in the art. As used herein, the term "protein formation" refers to amino acids that can be synthesized into peptides, polypeptides, or proteins of cells through metabolic pathways.

[0071] As described herein, when referring to proteases (e.g., metalloproteases), the term "substrate" refers to any material or substance on which a protease (e.g., metalloprotease) acts. The material or substance can be, for example, an organic compound or macromolecule that occurs naturally or non-naturally, such as a polypeptide or peptidomimetic. In certain embodiments, a metalloprotease substrate interacts specifically with one or more metalloproteases and is cut by the metalloprotease. The metalloprotease utilizes appropriate conditions to cut at least one molecule of the substrate within the time frame of the experiment. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the substrate can be cut by the metalloprotease.

[0072] The term "functional analog" refers to a molecule that has the same biological specificity (eg, binds to the same ligand) and / or activity (eg, activates or inhibits a target cell) as a reference molecule.

[0073] The term "prodrug" refers to a therapeutic molecule that is inactive until activated in vivo.

[0074] The term "modulate" includes "increase," "enhance" or "stimulate," as well as "decrease" or "lower," usually by a statistically or physiologically significant amount or degree relative to a control group.

[0075] The term "variant" comprises one or more substitutions, additions, deletions and / or insertions relative to a control polypeptide or polynucleotide. As described herein, a variant of a polypeptide or polynucleotide comprises an amino acid or nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or similarity or homology with a control sequence, and substantially retains the activity of the control sequence. Also included are sequences that are different from the control sequence by adding, deleting, inserting or replacing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more amino acids or nucleotides, and substantially retain at least one activity of the control sequence. In certain embodiments, addition or deletion includes C-terminal and / or N-terminal addition and / or deletion.

[0076] The term "wild type" refers to a gene or gene product (eg, polypeptide) that is most commonly observed in a population and is thus designated as the "normal" or "wild type" form of the gene.

[0077] The term "connected" includes covalently connected or non-covalently connected, and refers to a first part, such as a first amino acid sequence or a nucleotide sequence, and a second part, such as a second amino acid sequence or a nucleic acid sequence, respectively covalently or non-covalently connected. The first part can be directly connected to the second part or adjacent to each other (hereinafter referred to as direct connection, for example, in the case of polypeptides, through a peptide bond), or, alternatively, the first part is connected to the second part using an insertion part (for example, a peptide linker) (hereinafter referred to as indirect connection), which can be expressed as the first part is connected to the second part through an insertion part. In terms of polypeptides or proteins, the term "connected" includes not only the connection at the C-terminus and / or N-terminus of the first part (or the second part), but also the connection of the entire first part (or the second part) to any position in the second part (or the first part) (for example, an amino acid residue not located at the end). On the one hand, the first part is connected to the second part through a peptide bond or a linker. In some embodiments, the first part can be connected to the second part through a phosphodiester bond or a linker. In some embodiments, the term "linker" refers to a molecule (including but not limited to unmodified or modified nucleic acids or amino acids) or a group of molecules (e.g., 2 or more, e.g., 2, 3, 4, 10, 30, 50, 100 or more) or any chemical group that connects two parts, such as two polypeptides.

[0078] As described herein, a "covalent bond" refers to a stable bond formed between two atoms by sharing one or more electrons. Examples of covalent bonds include, but are not limited to, peptide bonds and disulfide bonds. As described herein, a "peptide bond" refers to a covalent bond formed between the carboxyl group of an amino acid and the amine group of an adjacent amino acid. As described herein, a "disulfide bond" refers to a covalent bond formed between two sulfur atoms, such as two Fc fragments are bound by one or more disulfide bonds. One or more disulfide bonds between two fragments may be formed by connecting thiol groups in the two fragments. In some embodiments, one or more disulfide bonds may be formed between one or more cysteines of two Fc fragments. Oxidation of two thiol groups can form a disulfide bond. In some embodiments, the covalent bond is formed by direct connection of covalent bonds. In some embodiments, the covalent bond is directly connected by a peptide bond or a disulfide bond.

[0079] When referring to two polypeptide sequences, the term "fused" or "fusion" refers to connecting two polypeptide fragments by a backbone peptide bond. Two polypeptides can be fused directly or by a peptide linker comprising one or more amino acids. A fusion protein is a polypeptide comprising two or more regions derived from different or heterologous proteins or peptides. Fusion proteins are prepared using conventional techniques for enzyme cleavage and connection of fragments from the desired sequence. PCR techniques for synthetic oligonucleotides can be used to prepare and / or amplify the desired fragments. Overlapping synthetic oligonucleotides representing the desired sequence can also be used to prepare DNA constructs encoding fusion proteins. Fusion proteins can include multiple sequences, including leader (or signal peptide) sequences, linker sequences, leucine zipper sequences, or other sequences that form oligomers, and sequences encoding highly antigenic portions that make it possible to easily purify or rapidly detect fusion proteins. Fusion proteins can be prepared by recombinant techniques from coding sequences containing the coding sequences of the two components in the fusion protein, with or without a peptide linker between the two. In some embodiments, fusion includes chemical bonding.

[0080] The half inhibitory concentration (IC 50 ) is a measure of the effectiveness of a substance (e.g., a ligand) in inhibiting a specific biological or biochemical function. It indicates how much of a particular drug or other substance (inhibitor, e.g., a ligand) is needed to inhibit a given biological process by half. Values ​​are usually expressed as molar concentrations. IC 50 The “EC 50 "Equivalent.EC 50 It also represents the plasma concentration required to achieve 50% of the maximal effect in vivo. As used herein, "IC 50 " is used to express the effective concentration of a ligand required to neutralize 50% of the biological activity of a receptor in vitro. IC can be determined by bioassays 50 or EC 50, such as inhibition of ligand binding by FACS analysis (competitive binding assay), cell-based cytokine release assays or amplified luminescent homogeneous enzyme-linked immunosorbent assay (AlphaLISA).

[0081] "Amino acid sequence identity percentage (%)" and "homology" of a peptide or polypeptide sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a particular polypeptide or polypeptide sequence, after alignment of the sequences and introduction of gaps (if necessary) to maximize the percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. To determine the percentage of amino acid sequence identity, various alignment methods within the skill of the art can be used, for example, using BLAST, BLAST-2, ALIGN, MEGALIGN, or the like. TM Publicly available computer software such as DNASTAR software can be used. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0082] As used herein, the "C-terminus" of a polypeptide refers to the last amino acid residue of the polypeptide, which residue provides an amine group to form a peptide bond with the carboxyl group of an adjacent amino acid residue. As used herein, the "N-terminus" of a polypeptide refers to the first amino acid residue of the polypeptide, which residue provides a carboxyl group to form a peptide bond with the amine group of an adjacent amino acid residue.

[0083] As used herein, the term "moiety" refers to a portion of a molecule that has a different function within the molecule, and the function can be performed by the portion in another molecule. A moiety can be a chemical entity with a specific function, or a part of a biological molecule with a specific function.

[0084] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, may include modified amino acids, and may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as coupling with a labeling component. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including but not limited to glycine and the D or L optical isomers, as well as amino acid analogs and peptidomimetics. Standard single-letter or three-letter abbreviations are used to represent amino acids.

[0085] An "isolated" polypeptide is one that has been identified, separated, and / or recovered from components of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is free from all other components of its production environment. Contaminating components of the production environment, such as those produced by recombinant transfected cells, often interfere with the study, diagnosis, or treatment of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide will be purified to: (1) greater than 95% polypeptide by weight as determined by the Lowry method, and in some embodiments, greater than 99% polypeptide by weight; (2) to a degree sufficient to obtain at least 15 N-terminal residues or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. An isolated polypeptide includes the polypeptide in situ within recombinant cells because at least one element of the polypeptide's natural environment is absent. However, generally, an isolated polypeptide will be subjected to at least one purification step.

[0086] As described herein, the terms "polynucleotide", "nucleic acid", "nucleotide" and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure can be modified before or after polymer assembly. The nucleotide sequence can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, such as by combining with a labeling component.

[0087] An "isolated" nucleic acid molecule encoding a construct (e.g., a masked polypeptide as described herein) is one that has been identified and separated from at least one contaminating nucleic acid molecule that is typically associated with the environment in which it was produced. Preferably, the isolated nucleic acid is free of all components of the environment in which it was produced. An isolated nucleic acid molecule encoding a polypeptide as described herein is present in a form or configuration other than that in which it is found in nature. Thus, an isolated nucleic acid molecule is distinct from a nucleic acid encoding a polypeptide as described herein that is naturally present in a cell. An isolated nucleic acid includes a nucleic acid molecule contained in a cell containing the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0088] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes, for example, include promoters and, optionally, operator sequences and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.

[0089] A nucleic acid is "operably linked" when it establishes a functional relationship with another nucleic acid sequence. For example, if the DNA of a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA is operably linked to the DNA of a polypeptide; if a promoter or enhancer affects the transcription of a sequence, the promoter or enhancer is operably linked to a coding sequence; or if a ribosome binding site is in a position that facilitates translation, the ribosome binding site is operably linked to a coding sequence. In general, "operably linked" means that the DNA sequences being linked are continuous, and, for a secretory leader, they are not only continuous but also in the reading phase. However, enhancers do not have to be continuous. Linking is accomplished by ligating at suitable restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used as a matter of routine practice.

[0090] As used herein, the term "vector" refers to a nucleic acid molecule that is capable of amplifying another nucleic acid molecule to which it is attached. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are introduced into the genome of a known host cell. Certain vectors are capable of directing the expression of nucleic acids to which they are attached. Such vectors are referred to herein as "expression vectors."

[0091] As used herein, the term "transfection" or "transformation" or "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0092] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and the progeny produced therefrom, without regard to the number of passages. The progeny may not be completely identical to the parent cell in nucleic acid and may contain mutations. Screening or selecting mutant progeny having the same function or biological activity as the original transformed cell is included herein.

[0093] The term "pharmaceutical preparation" or "pharmaceutical composition" refers to a preparation that is in a form that renders the biological activity of the active ingredient effective and that contains no additional ingredients that are unacceptably toxic to a subject to which the preparation is administered. Such preparations are sterile. A "sterile" preparation is sterile or does not contain any living microorganisms or their spores.

[0094] The embodiments of the present application described herein should be understood to include "consisting of" and / or "consisting essentially of" embodiments.

[0095] Reference herein to "about" for a value or parameter includes (and describes) variations for the value or parameter itself. For example, a description involving "about X" includes a description of "X".

[0096] As used herein, reference to "not" a value or parameter generally indicates and describes "other than" a value or parameter. For example, the method cannot be used to treat disease type X, which means that the method is generally used to treat other types of disease except disease type X.

[0097] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0098] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0099] IL-15Rα or its functional fragment(s)

[0100] The IL-15Rα or a functional fragment thereof according to the present application may be any kind of IL-15Rα or a functional fragment thereof.

[0101] In some embodiments, the IL-15Rα or its functional fragment is selected from the extracellular domain or sushi domain of human IL-15Rα or a functional analog. In some embodiments, the IL-15Rα or its functional fragment is a C-terminal truncated form of the extracellular domain of IL-15Rα having IL-15Rα activity.

[0102] IL-15Rα extracellular domain: The IL-15Rα extracellular domain is generally defined as the region of the IL-15Rα sequence extending from the first amino acid at the N-terminus of the IL-15Rα sequence to the last amino acid in the tail region (or the region rich in glycosylation sites). The tail region of the IL-15Rα sequence can be defined by a technician, for example, using auxiliary software.

[0103] IL-15Rα_sushi domain: The IL-15Rα extracellular domain contains a domain called sushi domain (Wei et al. 2001, J. Immunol. 167: 277-282). The IL-15Rα_sushi domain has a beta sheet conformation.

[0104] The IL-15Rαsushi domain has most of the binding affinity for IL-15, and enhances its binding and biological effects (proliferation and prevention of apoptosis) through IL-15Rβγ heterodimers, behaving as a potent IL-15 agonist without affecting the binding and function of IL-15 (Mortier E, et al. J Biol Chem. 2006 Jan 20; 281(3): 1612-9).

[0105] It is encoded by exon 2 of IL-15Rα (Anderson DM, et al. Functional characterization of the human interleukin-15 receptor alpha chain and close linkage of IL15RA and IL2RA genes. J Biol Chem. 1995 Dec 15; 270 (50): 29862-9). It starts from the first cysteine ​​residue (C1) encoded by exon 2 and ends at the fourth cysteine ​​residue (C4) encoded by exon 2. From the IL-15Rα protein sequence (in the standard N-terminal to C-terminal direction), the sushi domain of IL-15Rα can be defined as starting from the first cysteine ​​residue (C1) after the signal peptide and ending at the fourth cysteine ​​residue (C4) after the signal peptide. Residues C1 and C4 are both included in the sushi sequence. The IL-15Rα sushi domain can also be determined by analyzing the amino acid sequence of IL-15Rα using appropriate analysis software, such as: Prosite (http: / / us.expasy.org / prosite / ), (http: / / www.ebi.ac.uk / InterProScan / ), SMART (http: / / elm.eu.org / ).

[0106] In some embodiments, the IL-15Rαsushi domain comprises the amino acid sequence of SEQ ID NO:9, or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:9.

[0107] In some embodiments, the IL-15Rαsushi domain comprises the amino acid sequence of SEQ ID NO:10, or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:10.

[0108] In some embodiments, the IL-15Rαsushi domain comprises the amino acid sequence of SEQ ID NO:11, or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:11.

[0109] IL-15 cytokine (I)

[0110] IL-15 is a member of the four α-helical bundle families, with a molecular weight of 14-15 kDa and 114 amino acids (Fehniger TA, Caligiuri MA. Interleukin 15: biology and relevance to human disease. Blood. 2001; 97: 14-32), produced by mononuclear phagocytes and other immune system cells. IL-15 is essential for the development and function of natural killer cells (NK), natural killer T cells (NKT) and memory CD8+ T cells.

[0111] IL-15 is a cytokine similar to IL-2 that was originally known as a T-cell growth factor. Both cytokines exert their cell signaling function by binding to receptors composed of a trimeric complex consisting of two shared receptor chains, the common gamma chain (γc; CD132) and the IL-2 receptor beta chain (IL-2Rβ; CD122) and an alpha chain receptor unique to each cytokine, either the IL-2 receptor alpha (IL-2Rα; CD25) or the IL-15 receptor alpha (IL-15Rα; CD215).

[0112] IL-15 shares receptor components with IL-2. The α chain of IL-2 receptor (IL-2R) is not essential, but the β chain and the co-γ chain are essential for IL-15-mediated biological activity (Giri JG, et al. IL-15, a novel T cell growth factor that shares activities and receptor components with IL-2. J Leukoc Biol. 1995 May; 57(5): 763-6.). IL-15R is composed of three subunits: IL-15Rα, IL-2 / IL-15Rβ, and the γ chain. IL-15Rα is essential for high-affinity binding, but does not transmit IL-15 signals. IL-15 mainly exerts its effects through trans-presentation (TP). During this process, APCs expressing IL-15 bind to IL-15Rα and present the ligand to the βγ receptor heterodimer on the surrounding T / NK cells (Kenesei Volkó J, et al. IL-15 Trans-Presentation Is an Autonomous, Antigen-Independent Process. J Immunol. 2021Nov15; 207(10):2489-2500).

[0113] In some embodiments, the IL-15 cytokine further comprises an IL-15 variant or a functional fragment thereof. In eukaryotic cells, IL-15 is first synthesized as a 162 amino acid precursor polypeptide, which is then processed into mature IL-15 by removing amino acid residues 1-48. The mature form of IL-15 consists of 114 amino acids (amino acid residues 49-162) and is secreted in an active mature form.

[0114] In some embodiments, the IL-15 cytokine comprises the amino acid sequence of SEQ ID NO:7 or a variant thereof having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:7.

[0115] In some embodiments, the IL-15 cytokine comprises the amino acid sequence of SEQ ID NO:8 or a variant thereof having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:8.

[0116] In some embodiments, the IL-15 cytokine comprises an IL-15 variant or a functional fragment thereof. In some embodiments, the IL-15 cytokine is a naturally occurring interleukin-15 (IL-15) protein. In some embodiments, the IL-15 cytokine is a modified variant thereof that can bind to an interleukin-15 receptor (IL-15R) or a component thereof (e.g., the IL-15Rα, IL-2 / IL-15Rβ and / or γ chain) or otherwise exhibit affinity.

[0117] In some embodiments, the IL-15 cytokine comprises an amino acid sequence obtained by modifying at least one amino acid of the amino acid sequence SEQ ID NO: 7. Each at least one amino acid modification can be any amino acid modification, such as substitution, insertion or deletion. Any combination of deletions, insertions and substitutions can be performed to obtain the final construct product, as long as the final construct product can have the desired characteristics, for example, retaining / improving ligand-receptor binding, retaining / enhancing biological activity, etc. In some embodiments, the IL-15 cytokine comprises an amino acid sequence generated by performing at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acid substitutions on the amino acid sequence SEQ ID NO: 7.

[0118] Connectors

[0119] The term "linker" refers to a molecule (including but not limited to unmodified or modified nucleic acids or amino acids) or a group of molecules (e.g., 2 or more, e.g., 2, 3, 4, 10, 30, 50, 100 or more) or any chemical group that connects two parts, such as two polypeptides.

[0120] In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker has a native sequence or a non-native sequence. For example, a sequence from only the hinge region of a heavy chain antibody can be used as a linker. See WO1996 / 34103.

[0121] In some embodiments, the peptide linker is a human IgG1, IgG2, IgG3, or IgG4 hinge. In some embodiments, the peptide linker is a mutated human IgG1, IgG2, IgG3, or IgG4 hinge.

[0122] In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers and glycine-serine polymers. In some embodiments, the non-cleavable linker is rich in amino acid residues G and S. In some embodiments, the non-cleavable linker includes a "G4S" repeat sequence. In some embodiments, the non-cleavable linker is a polypeptide chain comprising at least 3 residues. The part of this linker may be flexible, hydrophilic, and itself rarely or will not form a secondary structure (linker portion or flexible linker portion). The linker composed of at least 3 amino acids can be used to connect domains and / or regions close to each other after molecular assembly. Longer linkers can also be used. In some embodiments, the linker may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 125, 150, 175 or 200 residues. When multiple linkers are used to connect various parts of the molecule, the linkers can be the same or different (eg, the same or different length and / or amino acid sequence).

[0123] In some embodiments, the peptide linker comprises or consists of a Gly-Ser linker. As used herein, the term "Gly-Ser linker" refers to a peptide consisting of glycine and serine residues. In some embodiments, an exemplary Gly-Ser linker comprises the amino acid sequence GSG (SEQ ID NO: 1). In some embodiments, an exemplary Gly-Ser linker comprises the amino acid sequence (Gly4Ser) n , wherein n is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the preferred Gly-Ser linker is (Gly4Ser)1. In some embodiments, the preferred Gly-Ser linker is (Gly4Ser)2. In some embodiments, the preferred Gly-Ser linker is (Gly4Ser)3. In some embodiments, the preferred Gly-Ser linker is (Gly4Ser)4. In some embodiments, the preferred Gly-Ser linker is (Gly4Ser)5. In other aspects, two or more Gly-Ser linkers are connected in series in a polypeptide linker.

[0124] In some embodiments, the peptide linker comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-5 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1-5.

[0125] In some embodiments, the linker can be divided into cleavable and non-cleavable. The cleavable part and non-cleavable linker are described in detail in the IL-15 prodrug section below.

[0126] Linkage between IL-15Rα or its functional fragment and Fc

[0127] In some embodiments, the IL-15Rα or a functional fragment thereof is connected to the Fc domain via a peptide linker.

[0128] In some embodiments, the peptide linker between IL-15Rα or a functional fragment thereof and Fc comprises 3 to 40 amino acid residues.

[0129] In some embodiments, the peptide linker between IL-15Rα or a functional fragment thereof and Fc comprises 3 to 25 amino acid residues.

[0130] In some embodiments, the peptide linker between IL-15Rα or a functional fragment thereof and Fc comprises 20 to 25 amino acid residues.

[0131] In some embodiments, the peptide linker between IL-15Rα or a functional fragment thereof and Fc comprises 25 amino acid residues.

[0132] In some embodiments, the peptide linker between IL-15Rα or a functional fragment thereof and Fc is rich in G and S amino acid residues. In some preferred embodiments, the peptide linker consists of G and S amino acid residues.

[0133] In some embodiments, the peptide linker between IL-15Rα or its functional fragment and Fc is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers and glycine-serine polymers. In some embodiments, the peptide linker includes a "G4S" repeat sequence. In some embodiments, the peptide linker is a polypeptide chain comprising at least 3 residues. This portion of the linker may be flexible, hydrophilic, and rarely or not form a secondary structure (linker portion or flexible linker portion) by itself. In some embodiments, a linker consisting of at least 3 amino acids can be used to connect domains and / or regions that are close to each other after molecular assembly. Longer linkers can also be used. In some embodiments, the peptide linker may be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40 residues. When multiple linkers are used to connect the various parts of the molecule, the linkers may be the same or different (e.g., the same or different lengths and / or amino acid sequences).

[0134] In some embodiments, the peptide linker comprises or consists of a Gly-Ser linker. As used herein, the term "Gly-Ser linker" refers to a peptide consisting of glycine and serine residues. In some embodiments, an exemplary Gly-Ser linker comprises the amino acid sequence GSG (SEQ ID NO: 1). In some embodiments, an exemplary Gly-Ser linker comprises the amino acid sequence (Gly4Ser) n , wherein n is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the preferred Gly-Ser linker is (Gly4Ser)1. In some embodiments, the preferred Gly-Ser linker is (Gly4Ser)2. In some embodiments, the preferred Gly-Ser linker is (Gly4Ser)3. In some embodiments, the preferred Gly-Ser linker is (Gly4Ser)4. In some embodiments, the preferred Gly-Ser linker is (Gly4Ser)5. In other aspects, two or more Gly-Ser linkers are connected in series in a polypeptide linker.

[0135] Linkage between IL-15 and IL-15Rα or its functional fragments

[0136] In some embodiments, the IL-15 and IL-15Rα or a functional fragment thereof can be linked by a variety of methods, which are well known to those skilled in the art.

[0137] In some embodiments, the IL-15 is covalently or non-covalently linked to IL-15Rα or a functional fragment thereof.

[0138] In some embodiments, the IL-15 is linked to IL-15Rα or a functional fragment thereof via a peptide bond.

[0139] In some embodiments, the IL-15 is linked to IL-15Rα via a peptide linker.

[0140] In some embodiments, the IL-15 is non-covalently linked to IL-15Rα or a functional fragment thereof. The IL-15 cytokine and IL-15Rα or a functional fragment thereof can be non-covalently linked to form an "IL-15 / IL-15Rα complex".

[0141] Half-life extension part (C)

[0142] Preferably, the prodrug comprises an in vivo half-life extension portion (C). The term half-life extension portion refers to a portion that extends the half-life of the target component in serum. Long in vivo half-life is very important for therapeutic molecules, for example, the half-life of cytokines administered to a subject is usually very short, because mechanisms such as renal clearance and endocytic degradation will rapidly remove them from the subject's body under normal circumstances. For therapeutic molecules with shorter half-lives in nature, increasing the in vivo half-life of these molecules can obtain a more acceptable and manageable dosing regimen without sacrificing effectiveness. Therefore, in the prodrug provided herein, in order to achieve the purpose of extending the in vivo half-life, it is preferred that the half-life extension portion is connected to the biologically active portion.

[0143] As described herein, a "half-life extension moiety" increases the in vivo half-life and improves PK, for example, by changing its size (e.g., above the upper limit of kidney filtration), shape, hydrodynamic radius, charge, or absorption, biodistribution, metabolism and elimination parameters. An exemplary way to improve polypeptide PK is by expressing an element that binds to a receptor in a polypeptide chain, and the receptor can be recycled to the plasma membrane without being degraded in the lysosome, such as the FcRn receptor and transferrin receptor on endothelial cells. Three types of proteins, such as human IgG, HSA (or fragments) and transferrin, last much longer in human serum than predicted by their size, which is related to their ability to bind to receptors that are recycled in the lysosome without being degraded. These proteins or fragments thereof that retain FcRn binding ability are usually connected to other polypeptides to extend their serum half-life.

[0144] In some embodiments, the half-life extending portion (C) may also be an antibody or antigen-binding fragment that binds to a protein with a long serum half-life, such as serum albumin, transferrin, etc. Examples of such antibodies or antigen-binding fragments thereof include polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies, such as heavy chain variable domains (V H ), light chain variable domain (V L ) and camel-type nanobodies (V HH ), dAb, etc.

[0145] In some embodiments, the half-life extending moiety (C) may also function as a linker, optionally, as a non-cleavable linker (L).

[0146] In some embodiments, the half-life extension portion is an antibody Fc domain (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc) or a fragment thereof that can participate in FcRn-mediated circulation, such as any heavy chain polypeptide or a portion thereof that can participate in FcRn-mediated circulation. In some embodiments, the Fc domain is a monomer. In some embodiments, the Fc domain is a dimer comprising a first Fc domain and a second Fc domain.

[0147] Fc domain

[0148] In some embodiments, the Fc domain is from any one of IgA, IgD, IgE, IgG and IgM and their subclasses. Among all immunoglobulins, IgG has the highest serum content and the longest half-life. Unlike other immunoglobulins, IgG can be effectively recovered after binding to Fc receptors (FcRs). In some embodiments, the Fc domain is from IgG (e.g., IgG1, IgG2, IgG3 or IgG4). In some embodiments, the Fc domain is from human IgG. In some embodiments, the Fc domain comprises CH2 and CH3 domains. In some embodiments, the Fc domain further comprises all or part of the hinge region. In some embodiments, the Fc domain is from human IgG1 or human IgG4. In some embodiments, the two subunits of the Fc domain are dimerized by one or more (e.g., 1, 2, 3, 4 or more) disulfide bonds. In some embodiments, each subunit of the Fc domain comprises a full-length Fc sequence. In some embodiments, each subunit of the Fc domain comprises an N-terminal truncated Fc sequence, such as a truncated Fc domain containing fewer N-terminal cysteines to reduce mispairing of disulfide bonds during dimerization. In some embodiments, the Fc domain is truncated at the N-terminus, for example, the first 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the complete immunoglobulin Fc domain are missing.

[0149] In some embodiments, the Fc domain comprises one or more mutations, such as insertions, deletions and / or substitutions.

[0150] In some embodiments, the Fc domain comprises one or more amino acid mutations that alter effector function, the Fc domain is engineered (e.g., comprises one or more amino acid mutations) to alter its binding to FcR, in particular to alter binding to Fcγ receptors (responsible for ADCC) and / or to alter effector function, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). Preferably, such amino acid mutations do not reduce binding to FcRn receptors (responsible for half-life).

[0151] The Fc domain (e.g., human IgG1 Fc) is mutated to remove one or more effector functions, such as ADCC, ADCP or CDC, hereinafter referred to as a "non-effector" or "almost non-effector" Fc domain. For example, in some embodiments, the Fc domain is a non-effector human IgG1 Fc, and the human IgG1 Fc comprises one or more of the following mutations (e.g., in each Fc subunit): L234A, L235E, G237A, A330S, and P331S. In some embodiments, the Fc domain in the prodrug comprises L234A and L235A ("LALA") mutations. The combination of K322A, L234A, and L235A in IgG1 Fc is sufficient to completely eliminate the binding of FcγR and C1q (Hezareh et al., J Virol 75, 12161–12168, 2001). MedImmune found that a set of three mutations L234F / L235E / P331S had very similar effects (Oganesyan et al., Acta Crystallographica 64, 700–704, 2008). In some embodiments, the Fc domain comprises a glycosylation modification on N297 of the IgG1 Fc domain, which is known to be required for optimal FcR interaction. The Fc domain modification can be any suitable IgG Fc engineering mentioned by Wang et al. ("IgG Fc engineering to modulate antibody effector functions," Protein Cell. 2018 Jan; 9 (1): 63–73), the contents of which are incorporated herein by reference in their entirety.

[0152] Glycosylation variants

[0153] In some embodiments, the Fc domain is altered to increase or decrease the degree of glycosylation of the construct. Glycosylation sites can be added or deleted in the Fc domain by changing the amino acid sequence to create or remove one or more glycosylation sites.

[0154] Natural Fc-containing proteins produced by mammalian cells usually contain a branched chain of biantennary oligosaccharides, which are usually linked to the Fc region C by N-linkage. H2 domain. See Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to the GlcNAc on the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the Fc domain can be modified to produce certain improved properties.

[0155] In some embodiments, the Fc domain as described herein has a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc domain. For example, the fucose content in such an Fc domain or IL-15 prodrug or anti-TNFR2 antibody prodrug may be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. As described in WO2008 / 077546, the fucose content is determined by measuring the average fucose content within the sugar chains attached to Asn297 by MALDI-TOF mass spectrometry relative to the sum of all sugar structures attached to Asn297 (e.g., complex, hybrid and high mannose structures). Asn297 refers to the asparagine residue located at position 297 of the Fc domain (Fc region residues according to the EU numbering system); however, due to minor sequence variations in the Fc region, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have enhanced ADCC function. See US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US 2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated Fc-containing proteins include Lec13 CHO cells lacking protein fucosylation function (Ripka et al., Arch. Biochem. Biophys. 249: 533-545 (1986); US Pat Appl No US 2003 / 0157108A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and gene knockout cell lines, such as CHO cells knocked out for α-1,6-fucosyltransferase gene, FUT8 gene (see Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotech. Bioeng. 87: 615 (2004); Kanda, Y. et al., Biotech. Bioeng. 87: 616 (2004); Kanda, Y. et al., Biotech. Bioeng. 87: 617 (2004); Kanda, Y. et al., Biotech. Bioeng. 87: 618 (2004); Kanda, Y. et al., Biotech. Bioeng. 87: 619 ... al., Biotechnol. Bioeng., 94(4):680-688(2006); and WO2003 / 085107).

[0156] Effector function variants

[0157] In some embodiments, the present application contemplates an Fc domain having some but not all Fc effector functions, making it an ideal candidate for an application in which its half-life in vivo is important, but certain effector functions (such as CDC and ADCC) are non-essential or harmful. Cytotoxicity assays can be performed in vitro or in vivo to determine the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the Fc domain lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. The main cells that mediate ADCC, natural killer cells (NK), express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991) Table 2 on page 464 summarizes the expression of FcR on hematopoietic cells. US Patent No. 5,500,362 (see Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83: 7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)) details non-limiting examples of in vitro assays for evaluating ADCC activity of target molecules. Alternatively, non-radioactive detection methods (see ACTI for flow cytometry) may be used. TM Non-radioactive toxicity assay (Cell Technology, Inc. Mountain View, CA) and CytoTox Non-radioactive toxicity test (Promega, Madison, WI). Effector cells suitable for such an assay include peripheral blood mononuclear cells (PBMC) and NK cells. In addition, ADCC activity of the target molecule can also be assessed in vivo, for example, in an animal model as disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to determine that the Fc domain does not bind to C1q and therefore lacks CDC activity. See WO 2006 / 029879 and WO 2005 / 100402 for C1q and C3c binding enzyme-linked immunosorbent assays. CDC assays can be performed to assess complement activity (see Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003) and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can be performed using methods known in the art (see Petkova, SB et al., Int'l. Immunol. 18 (12): 1759-1769 (2006)).

[0158] Fc domains with reduced effector function include those with substitutions of one or more residues at positions 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Pat. No. 6,737,056). Such Fc mutants include substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutants in which residues 265 and 297 are replaced with alanine (U.S. Pat. No. 7,332,581). Certain antibody variants that enhance or reduce binding to FcRs are described in detail (see US Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001). In some embodiments, the Fc region is engineered to alter (i.e., increase or decrease) C1q binding and / or CDC, such as described in US Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4 178-4184 (2000).

[0159] In some embodiments, the Fc domain comprises one or more amino acid substitutions, which increase the half-life and / or enhance the binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-life and enhanced binding to neonatal FcRn are responsible for transporting maternal IgGs to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)), and are described in detail in US 2005 / 0014934A1 (Hinton et al.). Antibodies comprising an Fc domain with one or more substitutions thus increase the binding of the Fc region to FcRn. Such Fc variants include those with one or more Fc region residue substitutions, for example, Fc region 434 residue substitutions (US Patent No. 7,371,826).

[0160] See Duncan and Winter, Nature 322:738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc domain variants.

[0161] Cysteine ​​engineered variants

[0162] In some embodiments, it may be necessary to create a cysteine ​​engineered Fc domain, wherein one or more residues of the Fc domain are replaced by cysteine ​​residues. In some embodiments, the substituted residues appear in accessible sites on the Fc domain. By replacing these residues with cysteine, the active thiol group is therefore positioned at accessible sites of the Fc domain and can be used to couple the molecule with other moieties, such as a drug moiety or a linker-drug moiety, to create a long-acting drug or prodrug conjugate. In some embodiments, any one and multiple of the following residues may be replaced by cysteine: heavy chain A118 (according to the EU numbering system) and heavy chain Fc domain S400 (according to the EU numbering system). Cysteine ​​engineered molecules can be produced as described in US Patent No.7,521,541.

[0163] In some embodiments, the Fc domain is from IgG1 Fc. In some embodiments, the Fc domain is from human IgG1 Fc. In some embodiments, the Fc domain is from wild-type IgG1 Fc (IGHG1*05). In some embodiments, the Fc domain is a natural variant of IgG1 (e.g., IGHG1*03, comprising a double mutation of D239E and L241M relative to IGHG1*05). In some embodiments, the Fc domain does not comprise the hinge region of IgG1 Fc. In some embodiments, the Fc domain comprises a maximum of 5 amino acids truncated from the N-terminus of IgG1 Fc, for example, the first, first two, first three, first four or first five amino acids truncated from the N-terminus of IgG1 Fc. In some embodiments, the Fc domain comprises one or more no-effect mutations and / or deglycosylation mutations.

[0164] In some embodiments, the Fc domain is from IgG4 Fc. In some embodiments, the Fc domain is from human IgG4 Fc. In some embodiments, the Fc domain is a wild-type IgG4 Fc. In some embodiments, in some embodiments, the Fc domain is a natural variant of IgG4. In some embodiments, the Fc domain does not comprise the hinge region of IgG4. In some embodiments, the Fc portion comprises a maximum of 5 amino acids truncated from the N-terminus of IgG4, such as the first, first two, first three, first four or first five amino acids truncated from the N-terminus of IgG4. In some embodiments, the Fc domain comprises one or more no-effect mutations and / or deglycosylation mutations.

[0165] Strategies for forming Fc fusion protein polypeptides or bispecific antibodies are well known (see Spies et al., Mol Imm. (2015) 67(2)(A):95-106). For example, in some embodiments, the first and / or second polypeptide chains of the Fc domain each comprise one or more modifications that promote heterodimerization of the first and second Fc domains. Thus, any strategy available in the art may be used to make one or more amino acid modifications to the first Fc domain and one or more amino acid modifications to the second Fc domain, including any strategy described in Klein et al. (2012), MAbs, 4(6):653-663. Exemplary strategies and modifications are the "knob into holes" approach. In some embodiments, the first Fc domain comprising the CH3 domain is a heavy chain polypeptide or a fragment thereof. The CH3 domains of the two Fc domains can be altered by the "knob-into-holes" technique (Fc knob and Fc hole), which is described in detail in WO 1996 / 027011; Ridgway, JB et al. Protein Eng (1996) 9(7): 617-621; Merchant, AM, et al, Nat. Biotechnoi. (1998) 16(7): 677-681. See also Klein et al. (2012), MAbs, 4(6): 653-663. Using the knob-into-holes approach, the interaction surfaces of the two CH3 domains are altered to increase heterodimerization of the two parts of the CH3 structure containing the two altered. This is achieved by introducing a bulky residue into the CH3 domain of one of the Fc domains, which serves as a "knob". Then, to accommodate the bulky residues, a "hole" is formed in the other Fc region that can accommodate the knob. One of the altered CH3 domains can be the "knob" and the other can be the "hole". The introduction of disulfide bonds further stabilizes the heterodimers (Merchant, AM, et al, Nat. Biotechnoi (1998) 16(7); Atwell, S., et al, J. Mol, Biol. (1997) 270(1):26-35) and improves yield. It is well known that heterodimerization can be achieved by introducing T366W and / or S354C mutations in the heavy chain to produce the "knob" and by introducing T366S, L368A, Y407V and / or Y349C mutations in the heavy chain to form the "hole" (residues are numbered according to the Kabat EU numbering system).Carter et al. (2001), J. Immunol. Methods, 248:7-15; Kleinet al. (2012), MAbs, 4(6):653-663.

[0166] In some embodiments, the Fc domain or fragment thereof comprises T366S, L368A and Y407V mutations to form a 'hole'. In some embodiments, the Fc domain or fragment thereof comprises T366W mutation to form a 'knob'. In some embodiments, the Fc domain or fragment thereof comprises Y349C, T366S, L368A and Y407V mutations to form a 'hole'. In some embodiments, the Fc domain or fragment thereof comprises S354C and T366W mutations to form a 'knob'. In some embodiments, the first Fc domain or fragment thereof comprises a hole mutation and the second Fc domain or fragment thereof comprises a knob mutation. In some embodiments, the first Fc domain or fragment thereof comprises a knob mutation and the second Fc domain or fragment thereof comprises a hole mutation, and the numbering of residues is according to the EU numbering system.

[0167] In some embodiments, in addition to the LALA mutation, the knob-into-holes mutation is also present in the Fc domain.

[0168] In some embodiments, the first Fc domain comprises the amino acid sequence of SEQ ID NO:12 or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO:12, and the second Fc domain comprises the amino acid sequence of SEQ ID NO:13 or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO:13.

[0169] In some embodiments, the first Fc domain comprises the amino acid sequence of SEQ ID NO:13 or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO:13, and the second Fc domain comprises the amino acid sequence of SEQ ID NO:12 or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO:12.

[0170] IL-15Rα or its functional fragment and Fc fusion protein:

[0171] In one aspect, the present application provides a fusion protein comprising an Fc domain and IL-15Rα or a functional fragment (S) thereof.

[0172] In some embodiments, IL-15Rα or a functional fragment thereof and an Fc fusion protein are provided, wherein the IL-15Rα or a functional fragment thereof is connected to the Fc domain via a peptide linker. In some embodiments, the peptide linker is rich in G and S amino acid residues. In some embodiments, the peptide linker consists of G and S amino acid residues.

[0173] In some embodiments, IL-15Rα or its functional fragment and Fc fusion protein are provided, wherein the peptide linker connecting IL-15Rα or its functional fragment and Fc domain comprises 3 to 40 amino acid residues. In some embodiments, the peptide linker comprises 3 to 25 amino acid residues. In some embodiments, the peptide linker comprises 20 to 25 amino acid residues. In some embodiments, the peptide linker comprises 25 amino acid residues.

[0174] In some embodiments, a fusion protein of IL-15Rα or a functional fragment thereof and Fc is provided, wherein the IL-15Rα or a functional fragment thereof is linked to the N-terminus or the C-terminus of the Fc domain.

[0175] In some embodiments, a fusion protein of IL-15Rα or a functional fragment thereof and Fc is provided, wherein the fusion protein further comprises IL-15 cytokine (I).

[0176] In some embodiments, a fusion protein of IL-15Rα or a functional fragment thereof and Fc is provided, wherein the IL-15 cytokine is linked to the N-terminus or the C-terminus of the Fc domain.

[0177] In some embodiments, a fusion protein of IL-15Rα or a functional fragment thereof and Fc is provided, wherein the IL-15 is linked to the Fc domain via a covalent bond or a peptide linker.

[0178] In some embodiments, IL-15Rα or a functional fragment thereof and an Fc fusion protein are provided, wherein the IL-15Rα or a functional fragment thereof is linked to an Fc domain, and the IL-15 cytokine is linked to the Fc domain.

[0179] In some embodiments, an IL-15Rα or a functional fragment thereof and an Fc fusion protein is provided, wherein the IL-15Rα or a functional fragment thereof is linked to an Fc domain, and the IL-15 cytokine is linked to the IL-15Rα or a functional fragment thereof.

[0180] In some embodiments, IL-15Rα or a functional fragment thereof and an Fc fusion protein are provided, wherein the fusion protein comprises a construct of I-Fc-S from N to C-terminus or C to N-terminus, wherein "-" represents a covalent bond with or without a peptide linker, and wherein the peptide linker between S and Fc comprises 3 to 40 amino acid residues; preferably, 3 to 25 amino acid residues; more preferably, 20 to 25 amino acid residues; further more preferably, 25 amino acid residues. Preferably, the peptide linker is rich in G and S amino acid residues; more preferably, the peptide linker consists of G and S amino acid residues.

[0181] In some embodiments, IL-15Rα or a functional fragment thereof and an Fc fusion protein are provided, wherein the fusion protein comprises a construct of Fc-SI from N to C-terminus or C to N-terminus, wherein "-" represents a covalent bond with or without a peptide linker, and wherein the peptide linker between S and Fc comprises 3 to 40 amino acid residues; preferably, 3 to 25 amino acid residues; more preferably, 20 to 25 amino acid residues; further more preferably, 25 amino acid residues. Preferably, the peptide linker is rich in G and S amino acid residues; more preferably, the peptide linker consists of G and S amino acid residues.

[0182] In some embodiments, IL-15Rα or a functional fragment thereof and an Fc fusion protein are provided, wherein the IL-15Rα or a functional fragment thereof is linked to and co-expressed with an Fc domain, while the IL-15 cytokine is transfected alone and forms a non-covalent IL-15 / IL-15Rα complex.

[0183] In some embodiments, IL-15Rα or a functional fragment thereof and an Fc fusion protein are provided, wherein the IL-15Rα or a functional fragment thereof is selected from the extracellular domain or sushi domain or a functional analogue of IL-15Rα.

[0184] In some embodiments, IL-15 and IL-15Rα dimer proteins are provided, wherein the dimer protein comprises any IL-15Rα or its functional fragment described herein and an Fc fusion protein. In some embodiments, the dimer protein is monovalent. In some embodiments, the dimer protein is divalent. In some embodiments, the dimer protein is a homodimer. In some embodiments, the dimer protein is a heterodimer.

[0185] IL-15 and IL-15Rα heterodimer

[0186] In some embodiments, a heterodimeric protein of IL-15 and IL-15Rα comprising two monomers is provided, wherein the heterodimeric protein comprises IL-15Rα or a functional fragment thereof, an IL-15 cytokine and two Fc domains. In some embodiments, the peptide linker between the Fc domain and IL-15Rα or a functional fragment thereof comprises 3 to 40 amino acid residues; preferably, 3 to 25 amino acid residues; more preferably, 20 to 25 amino acid residues; further more preferably, 25 amino acid residues. In some embodiments, the monomer is a polypeptide.

[0187] In some embodiments, a heterodimeric protein of IL-15 and IL-15Rα comprising two monomers is provided, wherein the heterodimeric protein comprises IL-15Rα or a functional fragment thereof, an IL-15 cytokine and two Fc domains, wherein in one monomer, the IL-15Rα or a functional fragment thereof is connected to the first Fc domain via a peptide linker; and, in the other monomer, the IL-15 cytokine is connected to the second Fc domain. In some embodiments, the peptide linker between the Fc domain and IL-15Rα or a functional fragment thereof comprises 3 to 40 amino acid residues; preferably, 3 to 25 amino acid residues; more preferably, 20 to 25 amino acid residues; further more preferably, 25 amino acid residues. In some embodiments, the monomer is a polypeptide.

[0188] In some embodiments, a heterodimeric protein of IL-15 and IL-15Rα comprising two monomers is provided, wherein the heterodimeric protein comprises IL-15Rα or a functional fragment thereof, an IL-15 cytokine and two Fc domains, wherein in one monomer, the IL-15Rα or a functional fragment thereof is connected to the first Fc domain via a peptide linker, and the IL-15 cytokine is also connected to the first Fc domain; and, the other monomer comprises only the second Fc domain. In some embodiments, the peptide linker between the Fc domain and IL-15Rα or a functional fragment thereof comprises 3 to 40 amino acid residues; preferably, 3 to 25 amino acid residues; more preferably, 20 to 25 amino acid residues; further more preferably, 25 amino acid residues. In some embodiments, the monomer is a polypeptide.

[0189] In some embodiments, a heterodimeric protein of IL-15 and IL-15Rα comprising two monomers is provided, wherein the heterodimeric protein comprises IL-15Rα or a functional fragment thereof, an IL-15 cytokine and two Fc domains, wherein in one monomer, the IL-15Rα or a functional fragment thereof is connected to the first Fc domain via a peptide linker, and the IL-15 cytokine is connected to IL-15Rα or a functional fragment thereof; and, the other monomer comprises only the second Fc domain. In some embodiments, the peptide linker between the Fc domain and IL-15Rα or a functional fragment thereof comprises 3 to 40 amino acid residues; preferably, 3 to 25 amino acid residues; more preferably, 20 to 25 amino acid residues; further more preferably, 25 amino acid residues. In some embodiments, the IL-15 cytokine is covalently linked to IL-15Rα or a functional fragment thereof, optionally, via a peptide linker. In some embodiments, the IL-15 cytokine is non-covalently linked to IL-15Rα or a functional fragment thereof and forms an IL-15 / IL-15Rα complex.

[0190] In some embodiments, the IL-15 and IL-15Rα heterodimeric proteins described herein, wherein the peptide linker connecting IL-15Rα or its functional fragment and the Fc domain comprises 3 to 40 amino acid residues. In some embodiments, the peptide linker comprises 3 to 25 amino acid residues. In some embodiments, the peptide linker comprises 20 to 25 amino acid residues. In some embodiments, the peptide linker comprises 25 amino acid residues. In some embodiments, the peptide linker is rich in G and S amino acid residues. In some embodiments, the peptide linker consists of G and S amino acid residues.

[0191] In some embodiments, the IL-15 and IL-15Rα heterodimeric protein described herein, wherein the IL-15Rα or a functional fragment thereof is linked to the N-terminus or C-terminus of the Fc domain.

[0192] In some embodiments, the IL-15 and IL-15Rα heterodimeric proteins described herein, wherein the Fc domain is selected from the group consisting of human IgG1 Fc domain, human IgG2 Fc domain, human IgG3 Fc domain, human IgG4 Fc domain, IgA Fc domain, IgD Fc domain, IgE Fc domain and IgM Fc domain, optionally, the Fc domain is a human IgG1 Fc domain. In some embodiments, the Fc domain is a human IgG1 Fc domain with L234A and L235A mutations, according to the EU numbering system. In some embodiments, the Fc domain comprises knobs-into-holes mutations (Fc knob and Fc hole). In some embodiments, the Fc knob is linked to the IL-15 cytokine and the Fc hole is linked to IL-15Rα or a functional fragment thereof; or the Fc knob is linked to IL-15Rα or a functional fragment thereof and the Fc hole is linked to the IL-15 cytokine. In some embodiments, the Fc knob comprises a T366W mutation in a first Fc domain and the Fc hole comprises T366S, L368A and Y407V mutations in a second Fc domain, according to the EU numbering system. In some embodiments, the Fc knob further comprises an S354C mutation and the Fc hole further comprises a Y349C mutation, according to the EU numbering system.

[0193] In some embodiments, the IL-15 and IL-15Rα heterodimeric protein described herein, wherein the IL-15Rα or a functional fragment thereof is selected from the extracellular domain or sushi domain or a functional analogue of IL-15Rα.

[0194] In some embodiments, an IL-15 and IL-15Rα heterodimeric protein comprising two polypeptide chains is provided, wherein one polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16 or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 16; and the other polypeptide chain comprises the amino acid sequence of SEQ ID NO: 17 or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 17.

[0195] In some embodiments, an IL-15 and IL-15Rα heterodimeric protein comprising two polypeptide chains is provided, wherein one polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 16; and the other polypeptide chain comprises the amino acid sequence of SEQ ID NO: 18 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 18.

[0196] IL-15 prodrug

[0197] One method of reducing drug side effects in treatment includes providing a conditionally activatable prodrug, wherein the drug (e.g., IL-15) is connected to a masking moiety (MM) via a cleavable moiety (CM). The masking moiety (e.g., a portion prepared from a polypeptide, i.e., a masking polypeptide) can act on the drug (e.g., IL-15) by steric hindrance. The cleavable moiety (CM) can be designed to be cleaved by proteases of specific tissues or pathological tissues, thereby enabling the prodrug to be preferentially activated at the desired site (e.g., a tumor) to overcome the dosage limitations of the drug (e.g., IL-15). In some embodiments,

[0198] In some embodiments, the IL-15 and IL-15Rα heterodimeric protein is in the form of a prodrug (hereinafter referred to as IL-15 prodrug), which further comprises a cleavable portion (CM) and a masking portion (e.g., a masking polypeptide, MP), and wherein the masking portion (MM) is connected to the heterodimeric protein via the cleavable portion (CM). In the present application, the IL-15 prodrug described herein refers to a heterodimeric protein form of IL-15 and IL-15Rα comprising a masking portion (MM) and a cleavable portion (CM).

[0199] Masking part

[0200] The masking moiety provided herein refers to a moiety that blocks the activity of a biologically active moiety. In some embodiments, the MM can be any moiety that inhibits the ability of a cytokine to bind and / or activate its receptor, for example, a polypeptide (referred to as a masking polypeptide, MP). In some embodiments, the MM can be any moiety that can hinder the binding of an antibody or antigen-binding fragment to its target, and / or hinder an antibody or antigen-binding fragment, for example, inhibit cell proliferation, regulate cell activity and interaction, regulate the human immune system or neutralize antigens, for example, a polypeptide (referred to as a masking polypeptide, MP).

[0201] In some embodiments, the masking moiety may be a moiety that inhibits the ability of a cytokine to bind and / or activate its receptor by steric hindrance and / or non-covalent binding to a cytokine. Examples of suitable masking moieties include full-length cytokine cognate receptors or cytokine binding fragments or mutants thereof. Antibodies and antigen-binding fragments thereof that can bind to cytokines may also be used, including polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain Fv (scFv), single-domain antibodies, such as heavy chain variable domains (V H ), light chain variable domain (V L ) and camel-type nanobodies with variable junctions (V HH ), dAb, etc. Further examples of suitable masking moieties include polypeptides that can sterically inhibit or block the binding of cytokines to their cognate receptors. For example, peptides modified by coupling with water-soluble polymers (e.g., PEG) can sterically inhibit or prevent cytokines from binding to their receptors. Polypeptides and fragments thereof with long serum half-lives, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin, etc., as well as fragments or mutants of the above polypeptides can also be used. Antibodies and antigen binding domains that can bind, for example, proteins with long serum half-lives (such as HSA, immunoglobulins or transferrin) or receptors that recycle to the plasma membrane (such as FcRn or transferrin receptor) can also inhibit cytokines, especially when bound to their receptors. Other suitable antigen binding domains that bind to cytokines may also be used, including non-immunoglobulins that mimic antibody binding and / or structure, such as anticalins, affilins, affbody molecules, affmers, affins, alphabodys, avimers, DARPins, fynomers, Kunitz domain peptides, monomers, and binding domains based on other engineered scaffolds (e.g., SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds).

[0202] In some embodiments, the masking moiety is a synthetic masking polypeptide (MP), and the masking polypeptide (MP) has a hydrodynamic radius larger than its actual molecular weight. In some embodiments, the masking polypeptide can only form a random coil lacking a secondary structure. In some embodiments, the masking polypeptide has a steric masking effect, that is, due to its relative size, it can generally inhibit or block the activity of the biologically active portion when it is close to the biologically active portion.

[0203] In some embodiments, the masking polypeptide (MP) comprises the amino acid sequence of SEQ ID NO:14 or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:14.

[0204] In some embodiments, the masking polypeptide (MP) comprises the amino acid sequence of SEQ ID NO:15 or a variant thereof, wherein the variant has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:15.

[0205] Cleavable fraction (CM)

[0206] The cleavable moiety (CM) is a cleavage site of an enzyme or protease or a polypeptide comprising the above site. In some embodiments, the protease includes but is not limited to urokinase-type plasminogen activator (uPA); matrix metalloproteinases (e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26 and / or MMP27); tobacco etch virus (TEV) protease; plasmin; thrombin; PSA; PSMA; ADAMS / ADAMTS (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM13, A DAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4 and / or ADAMTS5); caspases (e.g., Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13 and / or Caspase-14); aspartic proteases (e.g., RACE and / or Renin); aspartic cathepsins (e.g., Cathepsin D and / or Cathepsin E); cysteine ​​cathepsins (e.g., Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V / L2 and / or Cathepsin X / Z / P); cysteine ​​proteases (e.g., Cruzipain, Legumain and / or Otubain-2); KLKs (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and / or KLK14); metalloproteinases (e.g., Meprin, Neprilysin, PSMA and / or BMP-1); serine proteases (e.g., activated protein C, Cathepsin A, Cathepsin G, chymase and / or coagulation factor protease (such as FVIIa, FIXa, FXa, FXla, FXIIa); elastase; granzyme B; guanidinase; HtrA1; human neutrophil elastase; lactoferrin; trypsin; NS3 / 4A; PACE4; tPA; tryptase;Type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, Hepsin, Matriptase-2, MT-SP1 / Matriptase, TMPRSS2, TMPRSS3 and / or TMPRSS4), etc. ;

[0207] In some embodiments, the cleavable moiety (CM) comprises a substrate sequence for at least one matrix metalloproteinase (MMP). Exemplary MMPs include MMP1; MMP2; MMP3; MMP7; MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP16; MMP17; MMP19; MMP20; MMP23; MMP24; MMP26 and MMP27. In some embodiments, the CM comprises substrate sequences for MMP2, MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11 and MMP19. In some embodiments, the CM comprises a substrate sequence for MMP2. In some embodiments, the CM comprises a substrate sequence for MMP9. In some embodiments, the CM comprises substrate sequences for two or more MMPs. In some embodiments, the CM comprises at least substrate sequences for MMP2 and MMP9. In some embodiments, the CM comprises two or more substrates of the same MMP. In some embodiments, the CM comprises at least two or more MMP2 substrates. In some embodiments, the CM comprises at least two or more MMP9 substrates.

[0208] The terminology describing the specificity of proteases, i.e., their ability to cleave peptide bonds near specific amino acids, is based on the terminology originally coined by Schechter & Berger (1967, 1968) to describe the specificity of papain. According to this model, the amino acid residues that cleave the substrate are designated as P1, P2, P3, P4, etc., in the direction of the N-terminal end of the cleaved bond. Similarly, the residues in the C-terminal direction are designated as P1', P2', P3', P4', etc.

[0209] In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 6 or a variant thereof having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 6.

[0210] In some embodiments, the IL-15 and IL-15Rα heterodimeric protein without a masking polypeptide or the IL-15 prodrug, wherein the IL-15 cytokine or the IL-15Rα or a functional fragment thereof (e.g., IL-15Rα_sushi domain) has one or more conservative amino acid substitutions.

[0211] Inserting non-natural amino acids, including synthetic non-natural amino acids, substituted amino acids, or one or more D-amino acids, into a polypeptide (e.g., an IL-15 cytokine in an IL-15 cytokine prodrug described herein) can have a variety of benefits. Compared with polypeptides containing L-amino acids, polypeptides containing D-amino acids and the like exhibit higher stability in vitro and in vivo. Therefore, when better intracellular stability is required, it is particularly useful to construct a polypeptide by adding D-amino acids. In particular, D-peptides and their analogs are able to tolerate endogenous peptidase and protease activity, thereby increasing the bioavailability of the molecule and extending its life in vivo when needed. In addition, D-peptides and their analogs cannot be effectively processed because the process of presentation to helper T cells by the type II major histocompatibility complex (MHC) is limited, and therefore it is not easy to induce a humoral immune response in a subject.

[0212] Table A is a conservative substitution. More substantial substitutions are provided under the heading "Exemplary Substitutions" in Table A, as further described in detail below in the section on amino acid side chain categories. Amino acids can be classified according to common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain direction: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions require replacing one member of these categories with a member of another category. Amino acid substitutions can be introduced into protein constructs and screened for products that meet the desired activity described above.

[0213] Table A. Amino Acid Substitutions

[0214]

[0215] In some embodiments, the IL-15 prodrugs provided herein comprise the constructs shown in Table 1.

[0216] In some embodiments, the IL-15 prodrug provided herein comprises two polypeptide chains, wherein one polypeptide chain comprises the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 19; and the other polypeptide chain comprises the amino acid sequence of SEQ ID NO: 20 or a variant thereof having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0217] In some embodiments, the IL-15 prodrug provided herein comprises two polypeptide chains, wherein one polypeptide chain comprises the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 19; and the other polypeptide chain comprises the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 18.

[0218] In some embodiments, the IL-15 prodrug provided herein comprises two polypeptide chains, wherein one polypeptide chain comprises the amino acid sequence of SEQ ID NO: 21 or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 21; and the other polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22 or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22.

[0219] In some embodiments, the IL-15 prodrug provided herein comprises two polypeptide chains, wherein one polypeptide chain comprises the amino acid sequence of SEQ ID NO: 23 or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 23; and the other polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22 or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22.

[0220] In some embodiments, the IL-15 prodrug provided herein comprises two polypeptide chains, wherein one polypeptide chain comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 24; and the other polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22 or a variant thereof, which has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22.

[0221] Binding affinity

[0222] The binding affinity of a molecule (e.g., IL-15 or a functional fragment thereof) to its binding partner (e.g., IL-2 / IL-15Rβγ) can be determined by any suitable ligand binding assay or antibody / antigen binding assay known in the art, such as Western blot, enzyme-linked immunosorbent assay (ELISA), Meso Scale Discovery (MSD) electrochemiluminescence, bead-based multiplex immunoassay (MIA), RIA, surface plasmon resonance (SPR), ECL, IRMA, EIA, Biacore assay, Octet assay, peptide scanning, etc. For example, by using IL-15 or a functional fragment thereof or its receptor (e.g., IL-2 / IL-15Rβγ), or a subunit thereof labeled with various labeling agents, a simple analysis can also be performed using BiacoreX (Amersham Biosciences), which is an over-the-counter measurement kit or a similar kit, which can be operated according to the user manual and experimental operation method attached to the kit.

[0223] In some embodiments, protein microarrays are used to analyze the interaction, function and activity of IL-15 or its functional fragments described herein with its receptors on a large scale. The protein microarray has a support surface bound to a series of capture proteins (e.g., IL-15 receptors or their subunits). Fluorescently labeled probe molecules (e.g., IL-15 or its functional fragments described herein) are then added to the array and interact with the bound capture proteins, releasing fluorescent signals and reading them by a laser scanner.

[0224] Binding affinity can also be measured using SPR (Biacore T-200). For example, an anti-human IgG antibody is coupled to the surface of a CM-5 sensor chip using EDC / NHS chemistry. Human IL-2 / IL-15Rβγ-Fc fusion protein is then used as a capture ligand on the surface. Binding of a series of gradient dilutions of the IL-15 and IL-15Rα heterodimeric proteins described herein to the capture ligand allows real-time monitoring of the binding and dissociation of IL-15 and IL-15Rα heterodimeric proteins with IL-2 / IL-15Rβγ. The dissociation constant (K d ) and dissociation rate constants can be determined by kinetic analysis using BIA evaluation software.

[0225] Pharmacokinetics (PK)

[0226] Pharmacokinetics (PK) refers to the absorption, distribution, metabolism, and excretion of a drug (e.g., the IL-15 and IL-15Rα heterodimeric proteins described herein) after administration to a subject. Pharmacokinetic parameters that can be used to determine clinical efficacy include, but are not limited to, serum / plasma concentration, serum / plasma concentration over time, maximum serum / plasma concentration (C max ), time to reach maximum concentration (T max ), half-life (t 1 / 2 ), the area under the concentration-time curve during the dosing interval (AUC τ )wait.

[0227] Techniques for obtaining drug PK curves are known in the art, for example, IL-15 and IL-15Rα heterodimeric proteins described herein. See Heller et al., Annu Rev Anal Chem, 11, 2018; and Ghandforoush Sattari et al., J Amino Acids, Article ID 346237, Volume 2010. In some embodiments, the PK curves of IL-15 and IL-15Rα heterodimeric proteins as described herein are measured in individual blood, plasma or serum samples. In some embodiments, mass spectrometry techniques (e.g., LC-MS / MS or ELISA) are used to measure the PK curves of IL-15 and IL-15Rα heterodimeric proteins as described herein in individuals. PK analysis can be performed on the PK curve by any method known in the art, for example, non-compartmental analysis, using PKSolver V2 software (Zhang Y. et al., "PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel," Comput Methods Programs Biomed. 2010; 99(3): 306-1).

[0228] "C" represents the concentration of a drug or prodrug (e.g., IL-15 and IL-15Rα heterodimer protein) in the subject's plasma, serum, or any suitable body fluid or tissue, usually expressed as mass per unit volume, such as nanograms per milliliter. For convenience, the drug concentration in serum or plasma is referred to herein as "serum concentration" or "plasma concentration". The serum / plasma concentration at any time after administration (e.g., IL-15 and IL-15Rα heterodimer protein, such as intravenous, intraperitoneal, or subcutaneous injection) is referred to as C time or C t The maximum serum / plasma drug concentration during dosing is called C max ; C min Refers to the minimum serum / plasma drug concentration at the end of the dosing interval; C ave Refers to the average concentration during the dosing interval.

[0229] The term "bioavailability" refers to the extent or rate at which a drug or prodrug (eg, IL-15 and IL-15Rα heterodimeric protein) passes through the systemic circulation and thereby reaches the site of action.

[0230] "AUC" is the area under the serum / plasma concentration-time curve and is considered the most reliable measure of bioavailability, such as the area under the concentration-time curve during the dosing interval (AUC τ ), “total exposure” or “total drug exposure over time” (AUC 0-last or AUC 0-inf ), the area under the concentration-time curve (AUC 0-t )wait.

[0231] Peak serum / plasma concentration time (T max ) is the time to reach peak serum / plasma concentration (Cmax) following administration of a drug or prodrug (e.g., IL-15 and IL-15Rα heterodimeric protein).

[0232] Half-life (t 1 / 2 ) refers to the time required for the concentration of a drug or prodrug (e.g., IL-15 and IL-15Rα heterodimeric protein) measured in plasma or serum (or other biological matrix) to fall to half of its concentration or amount at a specific time point. For example, after intravenous administration, the drug concentration in plasma or serum decreases due to distribution and elimination of the drug. In the curve of plasma or serum drug concentration change over time after intravenous administration, the first phase or rapid decline is considered to be mainly due to distribution, while the later decline is usually slower and mainly due to elimination, although both processes occur in both phases. After sufficient time, drug distribution is completed. In general, the elimination half-life is determined by the terminal phase or elimination (primary) phase of the plasma / serum concentration-time curve. See Michael Schrag and Kelly Regal, "Chapter 3 - Pharmacokinetics and Toxicokinetics" of "A Comprehensive Guide to Toxicology in Preclinical Drug Development", 2013.

[0233] stability

[0234] In some embodiments, the fusion protein or heterodimeric protein described herein (e.g., IL-15 and IL-15Rα heterodimeric protein) has excellent stability, such as physical stability, chemical stability and / or biological stability. In some embodiments, the IL-15 and IL-15Rα heterodimeric protein described herein has excellent stability under accelerated stress (e.g., high temperature), such as less or no fragmentation, aggregate formation and / or aggregate increment.

[0235] The stability of proteins, especially the sensitivity to aggregation, depends mainly on the conformation and colloidal stability of protein molecules. It is generally believed that the first step of non-native protein aggregation, i.e., the most common form of aggregation, is a slight disturbance of the molecular structure, for example, partial unfolding of the protein, i.e., conformational change. This is determined by the conformational stability of the protein. In the second step, partially unfolded molecules approach and form aggregations driven by diffusion and random Brownian motion. The second step is mainly determined by the colloidal stability of the molecule (see Chi et al., Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony stimulating factor. Protein Science, 2003 May; 12 (5): 903-913). As described herein, the term "stability" generally refers to maintaining the integrity of a biologically active substance (such as a protein) or minimizing its degradation, denaturation, aggregation or unfolding. As used herein, "improved stability" generally means that the target protein (e.g., the IL-15 and IL-15Rα heterodimeric protein described herein) maintains better stability under conditions known to cause degradation, denaturation, aggregation or unfolding compared to a control protein (e.g., other IL-15 and IL-15Rα heterodimeric proteins).

[0236] Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) are well known techniques in the art for predicting the stability of protein formulations. Specifically, these techniques can be used to determine the unfolding temperature (T m ). The high T of a protein in a given preparation is m It is standard practice in the art to correlate measurements to more reliable and stable protein formulations that can be used for long-term, stable storage.

[0237] A "stable" heterodimeric protein or prodrug (or formulation), e.g., the IL-15 and IL-15Rα heterodimeric proteins described herein, substantially maintains its physical stability and / or chemical stability and / or biological activity during manufacturing and / or storage. There are a variety of analytical techniques for measuring protein stability in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29-90. 10: 29-90. For example, in one embodiment, the stability of a protein is determined based on the percentage of monomeric protein in a solution, wherein the percentage of degraded (e.g., fragmented) and / or aggregated protein is low. Preferably, the protein (or formulation) is stable at room temperature (about 30°C) or 40°C for at least 1 month and / or at about 2-8°C for at least 6 months, or at least 1 year or at least 2 years. In addition, the protein (or formulation) is preferably stable after freezing (e.g., -70°C) and thawing, hereinafter referred to as "freeze / thaw cycle".

[0238] A heterodimeric protein, e.g., an IL-15 and IL-15Rα heterodimeric protein as described herein, "retains its physical stability" in a formulation if it exhibits substantially no signs of instability, such as aggregation, precipitation, and / or denaturation, when measured by visual inspection of color and / or clarity or by ultraviolet light scattering or size exclusion chromatography. Aggregation is the process by which individual protein molecules or complexes covalently or non-covalently associate to form aggregates. Aggregation may proceed to the extent that a visible precipitate forms.

[0239] A heterodimeric protein, e.g., an IL-15 and IL-15Rα heterodimeric protein as described herein, "maintains its chemical stability" in a formulation if the chemical stability over a given time is such that the protein still maintains its biological activity (e.g., as described in the "Biological Activity" section above). Chemical stability can be assessed, for example, by detecting and quantifying different chemically altered forms of the protein. Chemical changes may involve size changes (e.g., shearing) and can be assessed using size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption ionization / time of flight mass spectrometry (MALDI / TOF MS). Other types of chemical changes include charge changes (e.g., changes due to deamidation or oxidation), which can be assessed, for example, by ion exchange chromatography.

[0240] A heterodimeric protein, e.g., an IL-15 and IL-15Rα heterodimeric protein described herein, "retains its biological activity" in a pharmaceutical formulation if the protein in the formulation is biologically active for its intended purpose. For example, a protein in a formulation retains its biological activity if the biological activity of the protein is within 30%, 20%, or 10% (within the analytical error range) of the biological activity exhibited when the formulation was prepared.

[0241] It is known to those skilled in the art that the stability of heterodimeric proteins (e.g., IL-15 and IL-15Rα heterodimeric proteins described herein) depends on other properties in addition to the composition of the formulation. For example, stability may be affected by temperature, pressure, humidity, pH, and external radiation. The stability of proteins (e.g., IL-15 and IL-15Rα heterodimeric proteins described herein) in protein formulations can be determined by a variety of methods. In some embodiments, protein stability is determined by size exclusion chromatography (SEC). SEC separates analytes according to the hydrodynamic size, diffusion coefficient, and surface properties of the analytes (e.g., macromolecules such as proteins). Thus, for example, SEC can separate the IL-15 and IL-15Rα heterodimeric proteins described herein in the native three-dimensional conformation from proteins in various denatured states and / or degraded proteins. In SEC, the stationary phase is typically composed of inert particles filled in a dense three-dimensional matrix in a glass or steel column. The mobile phase can be pure water, an aqueous buffer, an organic solvent, a mixture thereof, or other solvents. Stationary phase particles have pores and / or channels that only allow entry of material below a certain size. Large particles are therefore excluded from these pores and channels, but smaller particles are transported from the mobile phase. The amount of time that particles remain fixed in the stationary pores depends in part on how deeply they can penetrate into the pores. Their transport from the mobile phase stream causes them to take longer to elute from the column, thus separating the particles based on their size differences.

[0242] In some embodiments, SEC is combined with an identification technique to identify or characterize a protein (e.g., IL-15 and IL-15Rα heterodimeric protein) or a fragment thereof. Protein identification and characterization can be accomplished by a variety of techniques, including, but not limited to, chromatographic techniques, such as high performance liquid chromatography (HPLC), capillary electrophoresis with sodium dodecyl sulfate (CE-SDS), immunoassays, electrophoresis, UV / visible / infrared spectroscopy, Raman spectroscopy, surface enhanced Raman spectroscopy, mass spectrometry, gas chromatography, static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding technique, intrinsic tryptophan fluorescence, differential scanning calorimetry, and / or ANS protein binding.

[0243] In some embodiments, optionally, sample preparations (e.g., containing IL-15 and IL-15Rα heterodimeric proteins described herein) and control preparations are assayed before the treatment stage to determine the content of monomers, aggregates and / or fragmented proteins (and / or percentage increase in fragments, percentage increase in aggregates, etc.). Subsequently, each protein preparation undergoes a treatment stage. For example, each protein preparation can be stored at a specific temperature (e.g., 40°C, 25°C, or 5°C) for a longer period of time (e.g., 3 months, 6 months, 12 months, or longer). In some embodiments, the protein preparation is subjected to a physical stress test, such as a stirring stress test. In some embodiments, the protein preparation is subjected to an accelerated stability test, such as treatment under accelerated stress, including high temperature (e.g., 40°C), high humidity, and / or low pH, etc. In some embodiments, the protein preparation undergoes a freeze and thaw cycle. In some embodiments, samples of the same protein preparation receive different treatments, such as storage at different temperatures for a period of time. After the treatment stage, the protein preparation is assayed to determine the content of protein monomers, aggregates, and / or fragments (and / or percentage increase in fragments, percentage increase in aggregates, etc.).

[0244] "Substantial protein aggregation" refers to protein aggregation levels in a protein formulation that are significantly higher than protein aggregation levels in a control protein formulation. A control protein formulation can be the same protein formulation before storage or before processing (e.g., before being subjected to unstable conditions, such as high temperature, humidity, pH and / or long-term storage).

[0245] "Substantially free of protein aggregation" refers to a protein (or formulation) of the invention whose level or percentage of protein aggregation is not significantly higher than a control formulation. In some embodiments, the stability is measured by SEC. In some embodiments, the stability is measured by CE-SDS.

[0246] In some embodiments, stability refers to reduced fragmentation of the IL-15 and IL-15Rα heterodimeric proteins described herein. The term "low to undetectable levels of fragmentation" as used herein refers to a sample containing equal to or greater than 80%, 85%, 90%, 95%, 98% or 99% of the total protein, for example, in a single peak if determined by HPSEC, or, if determined by reducing capillary gel electrophoresis (rCGE), in multiple peaks (e.g., the same number of peaks as the number of subunits), representing undegraded protein or its undegraded fragments, and does not contain other single peaks that account for more than 5%, more than 4%, more than 3%, more than 2%, more than 1% or more than 0.5% of the total protein peak. The term "reducing capillary gel electrophoresis" as used herein refers to capillary gel electrophoresis under reducing conditions, which are sufficient to reduce disulfide bonds in Fc-containing proteins, such as IL-15Rα and Fc fusion proteins or IL-15 prodrugs described herein.

[0247] Carrier

[0248] The application also relates to an isolated nucleic acid encoding any fusion protein, any heterodimeric protein or any prodrug (e.g., IL-15 prodrug) described herein, comprising a vector encoding a nucleic acid as described herein. It also relates to a host cell (e.g., CHO cell, HEK 293 cell, Hela cell or COS cell) comprising an isolated nucleic acid or vector encoding as described herein. Suitable nucleic acid constructs include, but are not limited to, constructs that can be expressed in eukaryotic cells or prokaryotic cells. The selection of expression constructs is generally for compatibility with the host cell used. In some embodiments, the vector encodes a masking polypeptide (e.g., MP80, MP96new, MP100, MP163 or MP240). In some embodiments, the vector encodes a cleavable portion (e.g., CM1, CM2 or CM4). In some embodiments, the vector encodes an indestructible linker (e.g., lk, lk1, lk2, lk3 or lk5). In some embodiments, the vector encodes a protein or prodrug (e.g., IL-15 and IL-15Rα heterodimeric protein).

[0249] In some embodiments, the vector comprising the IL-15 and IL-15Rα heterodimer protein or any protein or prodrug component encoding the present invention is suitable for replication and integration in eukaryotic cells, such as mammalian cells (e.g., CHO cells, HEK293 cells, Hela cells, COS cells). In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector, such as pTT5.

[0250] Many virus-based systems have been developed for transferring genes into mammalian cells. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, herpes simplex viral vectors and their derivatives. Viral vector technology is well known in the art, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals. Retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to engineered mammalian cells in vitro or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying construct protein coding sequences can be packaged using experimental methods known in the art. The resulting lentiviral vectors can be used to transduce mammalian cells using methods known in the art. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transduction because they allow long-term, stable integration of the transgene and propagation in daughter cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.

[0251] In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a pTT5 vector. In some embodiments, the vector is a transposon, such as the Sleeping Beauty (SB) transposon system or the PiggyBac transposon system. In some embodiments, the vector is a polymer-based non-viral vector, including, for example, poly(lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA), poly(ethyleneimine) (PEI) and dendrimers. In some embodiments, the vector is a cationic lipid-based non-viral vector, such as cationic liposomes, lipid nanoemulsions, and solid lipid nanoparticles (SLN). In some embodiments, the vector is a peptide-based non-viral gene vector, such as poly-L-lysine. Any known non-viral vector suitable for genome editing can be used to introduce nucleic acids encoding IL-15 prodrugs or anti-TNFR2 antibody prodrugs into host cells. See Yin H. et al., Nature Rev. Genetics (2014) 15: 521-555; Aronovich EL et al., "The Sleeping Beauty transposon system: a non-viral vector for gene therapy." Hum. Mol. Genet. (2011) R1: R14-20 and Zhao S et al., "PiggyBac transposon vectors: the tools of the human gene editing." Transl. Lung Cancer Res. (2016) 5 (1): 120-125, incorporated herein by reference. In some embodiments, any one or more nucleic acids or vectors encoding the prodrugs described herein are transferred into host cells (e.g., CHO, HEK 293, Hela or COS) by physical methods, including but not limited to electroporation, sonoporation, photoporation, magnetofection, and water poration.

[0252] In some embodiments, the vector comprises a selectable marker gene or a reporter gene for selecting cells expressing the prodrug described herein from a host cell population transfected with a vector (e.g., a lentiviral vector, a pTT5 vector). Both the selectable marker and the reporter gene may be surrounded by appropriate regulatory sequences to enable them to be expressed in the host cell. For example, the vector may comprise a transcription and translation terminator, a start sequence, and a promoter for regulating the expression of the nucleic acid sequence.

[0253] Can use any molecular cloning method known in the art, comprise, for example, use restriction endonuclease site and one or more selectable markers nucleic acid is cloned in vector.In certain embodiments, nucleic acid is operably connected on the promoter.A variety of promoters for carrying out gene expression in prokaryotic cells or eukaryotic cells (for example, mammalian cells) have been explored, and any promoter known in the art can be used for the present invention.Promoter can be roughly divided into constitutive promoter or regulated promoter, such as inducible promoter.

[0254] In some embodiments, the nucleic acid encoding the prodrug described herein is operably linked to a constitutive promoter. A constitutive promoter allows a heterologous gene (also referred to as a transgene) to be constitutively expressed in a host cell. Examples of promoters considered herein include, but are not limited to, CMV promoter (CMV), human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerol kinase promoter (PGK), simian virus 40 early promoter (SV40), chicken β-actin promoter coupled to CMV early enhancer (CAGG), Roche sarcoma virus (RSV) promoter, polyoma virus enhancer / herpes simplex thymidine kinase (MC1) promoter, β-actin (β-ACT) promoter, "myeloproliferative sarcoma virus enhancer, negative control region deletion, d1587rev primer binding site substitution (MND)" promoter. The efficiency of these constitutive promoters in driving transgenic expression has been widely compared in a large number of studies. In some embodiments, the nucleic acid encoding the prodrug described herein is operably linked to a CMV promoter.

[0255] In some embodiments, the nucleic acid encoding the prodrug described herein is operably linked to an inducible promoter. Inducible promoters belong to the category of regulatable promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of the host cell or the physiological state of the host cell, an inducer (i.e., an inducing agent) or a combination thereof. In some embodiments, the inducing conditions do not induce the expression of endogenous genes in the host cell. In some embodiments, the inducing conditions are selected from: inducers, radiation (such as ionizing radiation, light), temperature (such as heat), redox state, and activation state of the host cell. In some embodiments, the inducible promoter can be a NFAT promoter, Promoter or NFκB promoter.

[0256] Methods for increasing IL-15 activity

[0257] The present application also provides a method for improving the activity of IL-15Rα or its functional fragment and Fc fusion protein or IL-15 and IL-15Rα heterodimer protein described herein. In some embodiments, the method includes setting the peptide linker connecting IL-15Rα or its functional fragment to the Fc domain to more than 15 and less than 40 amino acid residues. In some embodiments, the peptide linker comprises 20 to 25 amino acid residues. In some embodiments, the peptide linker comprises 25 amino acid residues.

[0258] Preparation method

[0259] Also provided is a method for preparing any IL-15Rα and Fc fusion protein, IL-15 and IL-15Rα heterodimeric protein described herein. Therefore, in some embodiments, a method for preparing the IL-15Rα and Fc fusion protein, the IL-15 and IL-15Rα heterodimeric protein is provided, comprising: (a) culturing a host cell (e.g., CHO cell, HEK 293 cell, Hela cell or COS cell) containing any nucleic acid or vector encoding the IL-15Rα and Fc fusion protein, the IL-15 and IL-15Rα heterodimeric protein described herein under conditions that can effectively express the encoded prodrug; and (b) obtaining the expressed IL-15Rα and Fc fusion protein, IL-15 and IL-15Rα heterodimeric protein or prodrug from the host cell. In some embodiments, the method of step (a) further comprises preparing a host cell, wherein the host cell comprises the IL-15Rα and Fc fusion protein, the IL-15 and IL-15Rα heterodimeric protein described herein. The IL-15Rα and Fc fusion protein, the IL-15 and IL-15Rα heterodimeric protein described herein can be prepared using any method known in the art or described herein.

[0260] In some embodiments, the IL-15Rα and Fc fusion proteins described herein, the IL-15 and IL-15Rα heterodimer proteins are expressed in eukaryotic cells, such as mammalian cells. In some embodiments, the IL-15Rα and Fc fusion proteins described herein, the IL-15 and IL-15Rα heterodimer proteins are expressed in prokaryotic cells.

[0261] 1. Recombinant products of prokaryotic cells

[0262] a) Vector construction

[0263] The polynucleotide sequence encoding the protein construct described in the present application can be obtained using standard recombinant techniques. Polynucleotides can be synthesized using a nucleotide synthesizer or PCR technology. Once the sequence encoding the polypeptide is obtained, it is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors known and available in the art can be used for the present invention. The selection of a suitable vector depends mainly on the size of the nucleic acid to be inserted into the vector and the specific host cell transformed by the vector. Each vector contains various components, which depends on the function of the vector (amplification or expression of heterologous polynucleotides, or both) and the compatibility between the vector and the specific host cell in which it is located. Vector components generally include, but are not limited to: replication initiation site, selectable marker gene, promoter, ribosome binding site (RBS), signal sequence, heterologous nucleic acid insertion and transcription termination sequence.

[0264] In general, plasmid vectors contain replicons and control sequences from species compatible with host cells, which are used with these host cells. The vector usually carries a replication site, as well as a marker sequence that can provide phenotypic selection in the transformed cell. For example, Escherichia coli is usually transformed using pBR322, which is a plasmid derived from Escherichia coli. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, and therefore provides a simple method for identifying transformed cells. pBR322, its derivatives or other bacterial plasmids or phages may also contain or be modified to contain promoters that can be used by microorganisms to express endogenous proteins. Carter et al., US Pat. No. 5,648,237 details examples of pBR322 derivatives for expressing specific antibodies.

[0265] In addition, phage vectors containing replicons and control sequences that are compatible with the host microorganism can be used as transformation vectors with these host cells. For example, phage such as GEM TM -11 can be used to prepare a recombinant vector, and the recombinant vector can be used to transform susceptible host cells, such as Escherichia coli LE392.

[0266] A promoter is a non-translated regulatory sequence located upstream (5') of a cistron that regulates downstream gene expression. Prokaryotic promoters are generally divided into two categories, inducible and constitutive. An inducible promoter is a promoter that can respond to changes in culture conditions (e.g., the presence or absence of nutrients or changes in temperature) to initiate and increase transcription levels of a cistron.

[0267] Many promoters that can be recognized by potential host cells are all known.Promoter is removed from source DNA by restriction enzyme and the promoter sequence of separation is inserted in the carrier of the present application, and selected promoter is operably connected on the cistron DNA of coded polypeptide.Natural promoter sequence and many heterologous promoters can be used for instructing amplification and / or expression of target gene.In certain embodiments, utilize heterologous promoter, because compared with natural target polypeptide promoter, heterologous promoter usually allows larger transcription, and the output of expressing target gene is higher.

[0268] Promoters suitable for prokaryotic hosts include PhoA promoter, beta-galactosidase and lactose promoter systems, tryptophan (trp) promoter systems and hybrid promoters, such as tac or trc promoters. However, other promoters that are functional in bacteria (such as other known bacterial or phage promoters) are also suitable. Their nucleic acid sequences have been published, so that technicians can use linkers or adapters to provide any required restriction sites to connect them to the cistrons encoding the light and heavy chains of interest (Siebenlist et al., (1980) Cell 20: 269).

[0269] In some embodiments, each cistron in the recombinant vector contains a secretion signal sequence component, which directly guides the expressed polypeptide to transfer across the membrane. In general, the signal sequence can be a component of the vector or a part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the present invention should be a sequence that can be recognized and processed by the host cell (i.e., cut by a signal peptidase). For prokaryotic host cells that cannot recognize and process the intrinsic signal sequence of heterologous polypeptides, the signal sequence is replaced by a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, Ipp or heat-stable enterotoxin II (STII) leader, LamP, PhoE, PelB, OmpA and MBP.

[0270] In some embodiments, production of the protein construct of the present application can occur in the cytoplasm of the host cell, so there is no need for a secretion signal sequence to be present in each cistron. In some embodiments, the polypeptide components are expressed, folded, and assembled to form a protein construct in the cytoplasm. Certain host strains (e.g., E. coli trxB- strains) provide cytoplasmic conditions that are conducive to disulfide bond formation, thereby allowing the expressed protein subunits to fold and assemble appropriately. See Proba and Pullthun, Gene, 159: 203 (1995).

[0271] b) Prokaryotic host cells

[0272] Prokaryotic host cells suitable for expressing the protein of the present application include archaea and true bacteria, such as gram-negative bacteria or gram-positive bacteria. Examples of available bacteria include Escherichia coli (e.g., Escherichia coli), Bacillus (e.g., Bacillus subtilis), Enterobacter, Pseudomonas (e.g., Pseudomonas aeruginosa), Salmonella typhimurium, Serratia marcescens, Klebsiella, Proteus, Shigella, Rhizobium, Vitreoscilla or Paracoccus. In some embodiments, gram-negative cells are used. In some embodiments, Escherichia coli cells are used as hosts of the present invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, including strain 33D3 (US Pat. No. 5,639,635) having the genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompT A (nmpc fepE) degP41 kanR. Other strains and their derivatives, such as E. coli 294 (ATCC 31446), E. coli B, E. coli 1776 (ATCC 31537) and E. coli RV308 (ATCC 31608), are also suitable. These examples are illustrative and not limiting. Methods for constructing bacterial derivatives of any of the above-mentioned known genotypes are known in the art and are described in detail, for example, in Bass et al., Proteins, 8:309-314 (1990). Considering the replicability of the replicon in the bacterial cell, it is usually necessary to select suitable bacteria. For example, when using known plasmids such as pBR322, pBR325, pACYC177 or pKN410 to provide the replicon, Escherichia coli, Serratia or Salmonella are suitable for use as hosts.

[0273] Generally, the host cells should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors should be appropriately included in the cell culture.

[0274] c) Protein production

[0275] Host cells are transformed with the above expression vectors and cultured in conventional nutrient media modified appropriately to induce promoters, select transformants, or amplify genes encoding the desired sequences. Transformation refers to the introduction of DNA into a prokaryotic host so that the DNA can replicate as an extrachromosomal element or by chromosomal integration. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. Calcium treatment using calcium chloride is commonly used for bacterial cells that contain a large cell wall barrier. Another transformation method uses polyethylene glycol / dimethyl sulfoxide. Another technique is electroporation.

[0276] Prokaryotic cells used to produce the protein constructs of the present application are known in the art and are suitable for growing in a culture medium for cultivating the selected host cells. Suitable culture medium includes luria broth (LB) and necessary nutritional supplements. In some embodiments, the culture medium also includes a selection agent selected based on the structure of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the culture medium for the growth of cells expressing an ampicillin resistance gene.

[0277] In addition to carbon sources, nitrogen sources and inorganic phosphate sources, any necessary supplements may also be added at appropriate concentrations, either alone or as a mixture with other supplements or media (such as a complex nitrogen source). Optionally, the culture medium may contain one or more reducing agents selected from glutathione, cysteine, cystamine, thioglycine, dithioerythritol and dithiothreitol. Prokaryotic host cells are cultured at a suitable temperature. For example, for the growth of Escherichia coli, the preferred temperature range is 20°C to 39°C, more preferably 25°C to 37°C, and even more preferably 30°C. The pH value of the culture medium can be any pH value between 5 and 9, depending primarily on the host organism. For Escherichia coli, the pH value is preferably 6.8 to 7.4, and more preferably 7.0.

[0278] If an inducible promoter is used in the expression vector of the present application, protein expression is induced under conditions suitable for promoter activation. In one aspect of the present application, the PhoA promoter is used to control transcription of the polypeptide. Therefore, the transformed host cells are cultured in a phosphate-limited medium for induction. Preferably, the phosphate-limited medium is CRAP medium (see Simmons et al., J. Immunol. Methods (2002), 263: 133-147). Depending on the vector structure used, a variety of other inducing agents known in the art can be used, which is known in the art.

[0279] The protein construct expressed in the present application is secreted into the periplasm of the host cell and recovered therefrom. Protein recovery generally involves destroying microorganisms, generally by means of osmotic shock, ultrasonic treatment or cracking. Once the cells are destroyed, cell debris or whole cells can be removed by centrifugation or filtration. For example, the protein can be further purified by affinity resin chromatography. Alternatively, the protein can be transported to the culture medium and separated therein. Cells can be removed from the culture medium, and the culture supernatant can be filtered and concentrated to further purify the protein produced. The expressed polypeptide can be further separated and identified by common methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot tests.

[0280] Alternatively, proteins are produced on a large scale by fermentation processes. Various large-scale fed-batch fermentation procedures can be used to produce recombinant proteins. Large-scale fermentations have a capacity of at least 1,000 liters, preferably 1,000 to 100,000 liters. These fermenters use agitator impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon / energy source). Small-scale fermentation generally refers to fermentation in fermenters with a capacity volume of no more than 100 liters, ranging from 1 liter to 100 liters.

[0281] During fermentation, protein expression is usually induced after cells have grown to a desired density under appropriate conditions, e.g., at OD 550 The induction time is about 180-220 hours, when the cells are in the early stationary phase. Depending on the vector structure used, a variety of inducing agents known in the art and described above can be used. The cells can be grown for a shorter time before induction. The cells are usually induced for about 12-50 hours, although longer or shorter induction times can be used.

[0282] In order to improve the yield and quality of the protein constructs of the present application, various fermentation conditions can be improved. For example, in order to improve the correct assembly and folding of secreted polypeptides, additional vectors that overexpress chaperone proteins can be used to co-transform host prokaryotes, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD or DsbG) or FkpA (a peptide prolyl cis-trans isomerase with chaperone activity). Chaperone proteins have been shown to help promote the correct folding and dissolution of heterologous proteins produced in bacterial host cells. Chen et al., (1999) J Bio Chem 274:19601-19605; Georgiou et al., US Pat. No. 6,083,715; Georgiou et al. al., US Pat. No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275: 17106-17113; Arie et al. al., (2001) Mol. Microbiol. 39:199-210.

[0283] In order to minimize the hydrolysis of expressed heterologous proteins (especially proteolytically sensitive proteins), certain host strains lacking proteolytic enzymes can be used in the present invention. For example, the host cell strain can be modified to cause genetic mutations in genes encoding known bacterial proteases, such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof. Some E. coli protease deletion strains can be used, as described in detail in Joly et al., (1998), supra; Georgiou et al., US Pat. No. 5,264,365; Georgiou et al., US Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2: 63-72 (1996).

[0284] E. coli strains lacking proteolytic enzymes and transformed with a plasmid overexpressing one or more chaperone proteins can be used as host cells in expression systems encoding the protein constructs described in this application.

[0285] d) Protein purification

[0286] The protein constructs produced herein are further purified to obtain substantially uniform preparations for further analysis and use. Standard protein purification methods known in the art can be used. The following procedures are examples of applicable purification procedures: fractionation on immunoaffinity columns or ion exchange columns, ethanol precipitation, reversed phase liquid chromatography HPLC, silica or cation exchange resin (such as DEAE) chromatography, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation and gel filtration, for example, Sephadex G-75.

[0287] In some embodiments, protein A immobilized on a solid phase is used for immunoaffinity purification of a protein construct comprising an Fc region as described herein. Protein A is a 42 kDa surface protein from Staphylococcus aureus that has a high binding affinity to an Fc-containing construct, for example, an IL-15Rα and Fc fusion protein, an IL-15 and IL-15Rα heterodimeric protein or a prodrug as described herein. Lindmark et al., (1983) J. Immunol. Meth. 62: 1-1. The solid phase to which protein A is fixed preferably comprises a column having a glass or silica surface, more preferably a controlled pore glass column or a silicic acid column. In certain applications, the chromatographic column is coated with a reagent, such as glycerol, to prevent nonspecific adhesion of contaminants. The solid phase is then washed to remove contaminants that are nonspecifically bound to the solid phase. Finally, the target protein construct is recovered from the solid phase by elution.

[0288] 2. Recombinant products of eukaryotic cells

[0289] For eukaryotic expression, vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0290] a) Signal sequence elements

[0291] The carrier used for eukaryotic hosts can also be an insert, and this insert encoding signal sequence or has other polypeptides of specific cleavage site at the N-terminal of mature protein or polypeptide.The heterologous signal sequence selected is preferably a sequence recognized and processed (that is, cut by signal peptidase) by host cells.In mammalian cell expression, mammalian signal sequence and virus secretion leader can be obtained, for example, herpes simplex gD signal, all are useful.The DNA in this precursor region is connected with the DNA of the protein construct of encoding the application in reading frame.

[0292] b) Origin of replication

[0293] Generally, mammalian expression vectors do not require an origin of replication element (the SV40 origin is often used only because it contains the early promoter).

[0294] c) Selecting genetic elements

[0295] Expression and cloning vectors may contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that confer (a) resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic proteins, or (c) provide key nutrients that are not available in complex culture media, such as the gene encoding Bacillus D-alanine racemase.

[0296] An example of a selection scheme is the use of a drug to stop the growth of host cells. Those cells that are successfully transformed with the heterologous gene produce a protein that confers drug resistance and thus survive the selection scheme. Examples of this type of dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0297] Another example of a selectable marker suitable for mammalian cells is one that can identify cells that are competent to carry the nucleic acid encoding the protein construct described in the present application, such as DHFR, thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

[0298] For example, cells transformed with the DHFR selection gene are first cultured in a medium containing methotrexate (Mtx) for identification, and methotrexate is a competitive antagonist of DHFR. When wild-type DHFR is used, a suitable host cell is a Chinese hamster ovary (CHO) cell line lacking DHFR activity (e.g., ATCC CRL-9096).

[0299] Alternatively, host cells transformed or co-transformed with a DNA sequence encoding a polypeptide, a wild-type DHFR protein, and another selection marker, such as aminoglycoside 3′-phosphotransferase (APH), especially wild-type hosts containing endogenous DHFR, can be selected by growing cells in a medium containing a selection marker, such as an aminoglycoside antibiotic, for example, kanamycin, neomycin or G418. See US Pat. No. 4,965,199.

[0300] d) Promoter element

[0301] Expression and cloning vectors usually contain a promoter, which is recognized by the host and operably linked to the nucleic acid encoding the desired polypeptide sequence. Almost all eukaryotic genes have an AT-rich region located about 25 to 30 bases upstream of the transcription start point. Another sequence found 70 to 80 bases upstream of the transcription start point of many genes is the CNCAAT region, in which N can be any nucleotide. There is an AATAAA sequence at the 3' end of most eukaryotic organisms, which may be a signal for adding a poly A tail to the 3' end of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors. See "Vector" section above.

[0302] Transcription of the polypeptide in a mammalian host cell vector is controlled by a promoter, for example, by promoters obtained from viral genomes, such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably Simian Virus 40 (SV40), promoters from heterologous mammals, for example, the actin promoter from a heat shock promoter, or immunoglobulin promoters, provided that such promoters are compatible with the host cell systems.

[0303] The SV40 virus early and late promoters are conveniently obtained as SV40 restriction fragments that also contain the SV40 virus replication origin. The immediate early promoter of human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. US Pat. No. 4,419,446 discloses a system for expressing DNA in a mammalian host using bovine papilloma virus as a vector. Improvements to this system are detailed in US Pat. No. 4,601,978. See Reyes et al., Nature 297: 598-601 (1982) for expression of human interferon cDNA in mouse cells under the control of the herpes simplex virus thymidine kinase promoter. Alternatively, the Rous sarcoma virus long terminal repeat sequence can be used as a promoter.

[0304] e) Enhancer elements

[0305] The transcription of the DNA encoding the protein construct of the present application by higher eukaryotes is usually increased by inserting an enhancer sequence in the vector. Many enhancer sequences have been found in mammalian genes (globin, elastase, albumin, alpha-fetoprotein and insulin). However, enhancers of eukaryotic cell viruses are generally used. Examples include the SV40 enhancer at the end of the replication origin (100-270bp), the cytomegalovirus early promoter enhancer, the polyoma enhancer at the end of the replication origin, and the adenovirus enhancer. See Yaniv, Nature 297:17-18 (1982), for enhancing elements for activating eukaryotic promoters. The enhancer can be spliced ​​to the vector at 5' or 3' of the polypeptide coding sequence, but is preferably located at 5' of the promoter.

[0306] f) Transcription termination element

[0307] Expression vectors used in eukaryotic host cells (nucleated cells of yeast, fungi, insects, plants, animals, humans or other multicellular organisms) also contain sequences necessary for transcription termination and stabilization of mRNA. These sequences are usually available from the 5' untranslated region of eukaryotic or viral DNA or cDNA, and occasionally the 3' end. These regions contain nucleotide fragments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the polypeptide. A suitable transcription termination element is the bovine growth hormone polyadenylated region. See WO94 / 11026 and the expression vectors disclosed therein.

[0308] g) Host cell selection and transformation

[0309] Suitable host cells for cloning or expressing the DNA in the vectors described herein include higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) is routine procedure. Examples of useful mammalian host cell lines are SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); COS fibroblast-like cell line derived from monkey kidney tissue; human embryonic kidney line (293 or 293 cell subclones for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC-ccl2); canine kidney cells (MDCK, ATCC-ccl34); buffalo-rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells and human hepatoma cell line (Hep G2).

[0310] Host cells are transformed with the above-described expression vectors or cloning vectors to produce the protein construct and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants, or amplify genes encoding the desired sequences.

[0311] h) Cultivating host cells

[0312] Host cells used to produce the protein constructs of the present application can be cultured in a variety of culture media. Commercial culture media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing host cells. In addition, any culture medium described in Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), US Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655 or 5,122,469, WO 90 / 03430, WO 87 / 00195 or US Pat. Re. 30,985 can be used as a culture medium for host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPE), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamicin), TM Drugs), trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be added at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those previously used for host cell expression and will be apparent to the skilled artisan.

[0313] i) Protein purification

[0314] When using recombinant technology, the protein construct of the present invention can be produced in the cell, in the periplasm, or directly secreted into the culture medium. If the protein construct is produced in the cell, the first step is to remove particulate debris (i.e., host cells or cleavage fragments) by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) details the procedure for separating antibodies secreted into the periplasm of E. coli. In short, the cell body is melted for about 30 minutes under the conditions of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. When the protein construct is secreted into the culture medium, the supernatant of such expression system is usually first concentrated using a commercial protein concentration filter, for example, Amicon or Millipore Pellicon ultrafiltration device. Protease inhibitors such as PMSF can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0315] The protein composition prepared from the cell can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, wherein affinity chromatography is a preferred purification technique. The suitability of protein A as an affinity ligand depends on the type and subtype of any immunoglobulin Fc domain present in the Fc-containing protein construct. Protein A can be used to purify Fc-containing proteins based on human immunoglobulins containing 1, 2 or 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse subtypes and human type 3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is connected is usually agarose, but there are other matrices. Compared with agarose, mechanically stable matrices (such as controlled pore glass or poly (styrene-divinyl) benzene) can achieve faster flow rates and shorter processing times. Bakerbond ABXTM resin can be used to purify C-containing proteins. H A 3-domain protein construct (JT Baker, Phillipsburg, NJ). Other protein purification techniques are also applicable, such as ion exchange column fractionation, ethanol precipitation, reversed phase liquid chromatography HPLC, silica gel chromatography, heparin SEPHAROSE TM Chromatography, anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, depending on the protein construct to be recovered.

[0316] Following any preliminary purification steps, the mixture comprising the protein construct of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography, with the elution buffer having a pH of about 2.5-4.5, preferably at low salt concentration (eg, from 0-0.25 M salt).

[0317] Pharmaceutical composition

[0318] Further provided are pharmaceutical compositions comprising any IL-15Rα and Fc fusion proteins, IL-15 and IL-15Rα heterodimeric proteins described herein, and optional pharmaceutically acceptable carriers. Pharmaceutical compositions can be prepared by mixing the prodrugs described herein with the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of lyophilized preparations or aqueous solutions.

[0319] The recombinant formulation can be prepared by dissolving the lyophilized protein in a diluent to uniformly disperse the protein. Examples of pharmaceutically acceptable (safe and non-toxic to human administration) diluents suitable for use in the present application include, but are not limited to, sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate-buffered saline), sterile saline, Ringer's solution or glucose solution, or aqueous solutions of salts and / or buffers.

[0320] In some embodiments, the pharmaceutical composition comprises a homogeneous population of IL-15Rα and Fc fusion proteins, IL-15 and IL-15Rα heterodimeric proteins described herein. A homogeneous population means that the proteins are exactly the same as each other, for example, the same fusion protein structure, the same heterodimeric protein structure, the same IL-15 prodrug structure, the same IL-15 cytokine, the same IL-15Rαsushi domain, the same masking polypeptide, the same cleavable portion, the same non-cleavable linker (if any) and the same Fc domain. In some embodiments, at least 70% (such as at least any one of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) of the proteins in the pharmaceutical composition are homogeneous.

[0321] The pharmaceutical composition is preferably stable, wherein the protein contained therein substantially maintains its physical and chemical stability and integrity during storage. There are various analytical techniques for measuring protein stability in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature and a selected time period. For accelerated screening, the formulation can be stored at 40° C. for 2 weeks to 1 month, during which stability is measured. For example, the degree of aggregation during storage can be used as an indicator of protein stability.

[0322] In some embodiments, the pharmaceutical composition has a shelf life of at least 15 days, such as at least 20 days, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years or longer, for example, at 2-25° C. (such as 2-8° C.). As used herein, "shelf life" refers to the storage period during which the active ingredient in the pharmaceutical formulation, such as a therapeutic protein (e.g., an IL-15Rα and Fc fusion protein, an IL-15 and IL-15Rα heterodimeric protein or a prodrug described herein), undergoes minimal degradation (e.g., no more than 5% degradation, such as no more than 4%, 3%, or 2% degradation) when the pharmaceutical formulation is stored under specific storage conditions, such as 2-8° C. Exemplary techniques for assessing protein or formulation stability include size exclusion chromatography (SEC)-HPLC to detect, for example, aggregation, reverse phase liquid chromatography (RP)-HPLC to detect, for example, protein fragments, ion exchange HPLC to detect, for example, changes in protein charge, mass spectrometry, fluorescence spectroscopy, circular dichroism (CD) spectroscopy, Fourier transform infrared spectroscopy (FT-IR) and Raman spectroscopy to detect protein conformational changes. All of these techniques can be used alone or in combination to assess the degradation of proteins in pharmaceutical formulations and determine the shelf life of the formulation.

[0323] Acceptable carriers, excipients or stabilizers are nontoxic to subjects at the dosages and concentrations used, and include buffers, antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives, isotonic agents (such as sodium chloride), stabilizers, metal complexes (e.g., zinc-protein complexes); chelating agents such as EDTA and / or nonionic surfactants.

[0324] Examples of physiologically acceptable carriers include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than 10 residues) polypeptides. ; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as Tween TM , polyethylene glycol (PEG) and PLURONICS TM .

[0325] Buffers are used to control the pH value within a range that optimizes the therapeutic effect, especially when the stability depends on the pH value. Buffers suitable for the present application include organic acids and inorganic acids and their salts. For example, citrates, phosphates, succinates, tartrates, fumarates, gluconates, oxalates, lactates, acetates. In addition, the buffer may include histidine and trimethylamine salts, such as Tris.

[0326] Preservatives are added to prevent microbial growth. For example, the addition of preservatives can facilitate the production of multiple-use (multiple-dose) formulations. Preservatives suitable for use in the present application include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl alcohol or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol and m-cresol.

[0327] Tonicity agents, sometimes called "stabilizers," are used to adjust or maintain the tonicity of a liquid in a composition. When used with large charged biomolecules such as proteins, they are often referred to as "stabilizers" because they can interact with the charged groups of the amino acid side chains, thereby reducing the likelihood of intermolecular and intramolecular interactions. Taking into account the relative amounts of the other ingredients, tonicity agents may be present in any amount between 0.1% and 25% (by weight), preferably 1% to 5%. Preferred tonicity agents include polyols, preferably triols or high sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0328] Other excipients include one or more of the following: (1) fillers, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to the container wall. Such excipients include: polysaccharide alcohols (as described above); amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbitol, xylose, ribose, ribitol, inositol, inositol, galactose, galactitol, glycerol, cyclohexanol (such as inositol), polyethylene glycol, etc. ; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0329] The presence of a nonionic surfactant or detergent (also called a "wetting agent") helps solubilize the protein and protect it from aggregation caused by agitation, which also allows the formulation to be exposed to shear surface stresses without causing denaturation of the active protein.

[0330] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamate (184, 188, etc.), Polyols, Polyoxyethylene sorbitan monoether ( -20, -80, etc.), lauryl alcohol 400, polyoxyethylene 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0331] In order for pharmaceutical compositions to be useful for in vivo administration, they must be sterile. Pharmaceutical compositions can be sterilized by filtration through a sterile filter membrane. The pharmaceutical compositions are typically placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle.

[0332] Sustained release formulations may be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, for example, films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (US Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid.

[0333] Depending on the needs of the specific indication to be treated, the pharmaceutical compositions described herein may also contain more than one active compound, preferably compounds with complementary activities that do not adversely affect each other. Such molecules are combined together in an effective amount to achieve the intended purpose.

[0334] The active ingredient can also be encapsulated in microcapsules prepared, for example, by gelling techniques or interfacial polymerization, for example, hydroxymethylcellulose microcapsules or gelatin microcapsules and polymethylmethacrylate microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Remington's Pharmaceutical Sciences discloses these techniques.

[0335] In some embodiments, the pharmaceutical composition is contained in a disposable vial, such as a disposable sealed vial. In some embodiments, the pharmaceutical composition is contained in a vial that can be used multiple times. In some embodiments, the pharmaceutical composition is bulked in a container. In some embodiments, the pharmaceutical composition is stored frozen.

[0336] Treatment of the disease

[0337] Further provided are methods for treating a subject who is ill or at risk of illness, such as a proliferative disease, a neoplastic disease, an inflammatory disease, an immune disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, or a graft-versus-host disease. The methods disclosed herein administer an effective amount of an activatable prodrug to a subject in need thereof, typically as a pharmaceutical composition, wherein the prodrug is activated after being cleaved by an enzyme. In some embodiments, the method further comprises selecting a subject who is suffering from or at risk of suffering from such a disease or condition. In some embodiments, the prodrug is activated in a tumor microenvironment. The prodrug has therapeutic activity after cleavage from the masking polypeptide. Therefore, in some embodiments, the active agent is a cleavage product. In some embodiments, the prodrug can rely on the binding of an antigen to an antigen binding domain to treat a disease.

[0338] In some embodiments, a method for treating an individual (e.g., human) disease (e.g., tumor, viral infection, or bacterial infection) is provided, comprising administering to the individual an effective dose of any of the IL-15Rα and Fc fusion proteins described herein, the IL-15 and IL-15Rα heterodimeric proteins, or a pharmaceutical composition thereof. In some embodiments, the IL-15Rα and Fc fusion proteins, the IL-15 and IL-15Rα heterodimeric proteins (or their pharmaceutical compositions) are administered by intravenous, intramuscular, or subcutaneous injection. In some embodiments, the treatment method further comprises administering an additional therapeutic drug in combination with the prodrug (before, after, or simultaneously). The additional drug may be an antibody or an antigen-binding fragment thereof, a small molecule drug, or other type of therapeutic drug.

[0339] In some embodiments, the IL-15Rα and Fc fusion protein, the IL-15 and IL-15Rα heterodimer protein is used to treat cancer or tumor in a subject, comprising administering a therapeutically effective amount of the IL-15Rα and Fc fusion protein, the IL-15 and IL-15Rα heterodimer protein to the subject. As described herein, in some embodiments, the term "tumor or cancer" refers to all types of cancer, tumors or malignancies in mammals, including leukemia, lymphoma, melanoma, neuroendocrine tumors, carcinomas and sarcomas. Exemplary cancers that can be treated with the masked cytokines, pharmaceutical compositions, or methods provided herein include lymphoma, sarcoma, bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g., triple negative, ER positive, ER negative, chemotherapy resistant, Herceptin resistant, HER2 positive, doxorubicin resistant, tamoxifen resistant, ductal carcinoma, small cell carcinoma, leukemia, leukemia, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g., triple negative, ER positive, ER negative, chemotherapy resistant, Herceptin resistant, HER2 positive, doxorubicin resistant, tamoxifen resistant, ductal carcinoma, small cell carcinoma, leukemia ... lobar carcinoma, primary, metastatic), ovarian cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer, squamous cell lung cancer, adenocarcinoma, large cell lung cancer, small cell lung cancer, carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple-negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head and neck or esophageal), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma.Other examples include thyroid cancer, endocrine system cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, stomach cancer, uterine or medulloblastoma, Hodgkin's disease ( Disease), Non-Hodgkin's Lymphoma, Multiple Myeloma, Neuroblastoma, Glioblastoma Multiforme, Ovarian Cancer, Rhabdomyosarcoma, Essential Thrombocytosis, Essential Macroglobulinemia, Primary Brain Tumor, Cancer, Malignant Insuloma, Malignant Carcinoid, Bladder Cancer, Precancerous Skin Lesions, Testicular Cancer, Lymphoma, Thyroid Cancer, Neuroblastoma, Esophageal Cancer, Genitourinary Tract Cancer, Malignant Hypercalcemia, Endometrial Cancer, Adrenal Cortical Carcinoma, Endocrine or Exocrine Pancreatic Tumors, Medullary Thyroid Cancer, Medullary Thyroid Cancer, Melanoma, Colorectal Cancer, Papillary Thyroid Cancer, Hepatocellular Carcinoma, Paget's Disease of the Nipple, Phyllodes Tumors, Lobular Carcinoma, Ductal Carcinoma Carcinoma, pancreatic stellate cell carcinoma, hepatic stellate cell carcinoma, or prostate cancer.

[0340] In some embodiments, the IL-15Rα and Fc fusion protein, the IL-15 and IL-15Rα heterodimer protein are used to treat bacterial infections, such as sepsis. In some embodiments, the bacteria causing the bacterial infection are drug-resistant bacteria. In some embodiments, the antigen binding portion binds to a bacterial antigen.

[0341] In some embodiments, the IL-15Rα and Fc fusion protein, the IL-15 and IL-15Rα heterodimer protein are used to treat viral infection. In some embodiments, the virus causing viral infection is hepatitis C (HCV), hepatitis B (HBV), human immunodeficiency virus (HIV) or human papillomavirus (HPV). In some embodiments, the antigen binding portion binds to a viral antigen.

[0342] The administration of the prodrugs or their pharmaceutical compositions described herein can be carried out in any convenient manner, including by injection or infusion. The route of administration is in accordance with known and recognized methods, such as by single or multiple push injections or infusion for a long time in an appropriate manner. The prodrug or its pharmaceutical composition can be administered orally, subcutaneously, intravenously, intracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonaryly, vaginally, rectally, intraocularly, topically, intraarterially, intradermally, intrasegmentally, intracavitarily, or intramedullary, intrathecally, intraventricularly, intracerebrally, intraspinally, intrathecally, intralesionally or intraocularly. In some embodiments, the prodrug or its pharmaceutical composition is administered systemically. In some embodiments, the prodrug or its pharmaceutical composition is administered to an individual by infusion (such as intravenous infusion). The infusion techniques used for immunotherapy are known in the art (see Rosenberg et al., New Eng. J. of Med. 319: 1676 (1988)). In some embodiments, the IL-15Rα and Fc fusion protein, the IL-15 and IL-15Rα heterodimeric protein, or a pharmaceutical composition thereof is administered to an individual by intradermal or subcutaneous (i.e., under the skin) injection. For subcutaneous injection, a syringe can be used to inject the prodrug or its pharmaceutical composition. However, there are other devices for administering the prodrug or its pharmaceutical composition, such as injection devices; injection pens; automatic injector devices, needle-free devices; and subcutaneous patch delivery systems. In some embodiments, the prodrug or its pharmaceutical composition is administered by intravenous injection. In some embodiments, the prodrug or its pharmaceutical composition is injected directly into the brain or spine. In some embodiments, the prodrug or its pharmaceutical composition is administered by sustained release or extended release technology.

[0343] The dosage and required drug concentration of the pharmaceutical composition of the present invention may vary depending on the specific intended use. Determining the appropriate dosage or route of administration is well within the skill of those skilled in the art. Animal experiments provide reliable guidance for determining effective doses for human treatment. Interspecies analogies of effective doses can be made based on the principles of Mordenti, J. and Chappell, W. "The Use of Interspecies Scaling in Toxicokinetics," In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp. 42-46.

[0344] When using the prodrug or its pharmaceutical composition for in vivo administration, the dosage is different according to the route of administration and the type of mammal. Within the scope of the present application, different preparations will be effective for different treatments and different diseases, and the mode of administration intended to treat a specific organ or tissue may be different from the mode for another organ or tissue. In addition, the dosage can be administered by one or more separate administrations or continuous infusions. For repeated administrations of several days or longer, depending on the condition, the treatment continues until the disease symptoms reach the desired degree of inhibition. However, other dosage regimens may be useful. The progress of this treatment is easily monitored by conventional techniques and analysis.

[0345] In some embodiments, the IL-15Rα and Fc fusion protein, the IL-15 and IL-15Rα heterodimeric protein, or a pharmaceutical composition thereof is administered once (e.g., bolus injection). In some embodiments, the IL-15Rα and Fc fusion protein, the IL-15 and IL-15Rα heterodimeric protein, or a pharmaceutical composition thereof is administered multiple times (e.g., 2, 3, 4, 5, 6 or more times). If administered multiple times, it may be performed by the same or different routes and may be performed at the same site or other sites. The prodrug or its pharmaceutical composition may be administered once a day to once a year. The interval between two administrations may be from 24 hours to any time of the year. The interval may also be irregular (e.g., as the tumor progresses). In some embodiments, there is no interruption in the dosing plan. The optimal dose and treatment regimen for a particular patient can be determined by a technician in the medical field by monitoring the patient's disease signs and adjusting accordingly.

[0346] In some embodiments, the IL-15Rα and Fc fusion protein, the IL-15 and IL-15Rα heterodimer protein or a pharmaceutical composition thereof is administered in divided doses, such as 2, 3, 4, 5 or more doses. In some embodiments, divided doses are administered for more than 1 week, 1 month, 2 months, 3 months or longer. In some embodiments, the dose is divided equally. In some embodiments, the divided doses are 20%, 30% and 50% of the total dose. In some embodiments, the intervals between consecutive divided doses are 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months or longer. For repeated administration for several days or longer, depending on the condition, treatment continues until the expected degree of suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this treatment is easily monitored by conventional techniques and analysis.

[0347] Products and kits

[0348] Kits, unit doses and articles of manufacture comprising any prodrug described herein are further provided. In some embodiments, a kit comprising any prodrug pharmaceutical composition described herein is provided, and preferably instructions for use thereof are provided, such as for treating a disease described herein (eg, a tumor).

[0349] The kit of the present invention includes one or more containers containing the prodrugs described herein, for example, for treating a disease. For example, instructions describing the administration of the prodrug to treat a disease (such as a tumor) are included. The kit may further include a description of selecting an individual (e.g., human) suitable for treatment based on identifying whether the individual suffers from a disease and the stage of the disease. The instructions related to the use of the prodrug generally include information about the dose, dosing schedule, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package) or a subunit dose. The instructions provided in the kit of the present invention are generally written instructions on a label or drug instruction sheet (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions stored on a disk or CD) are also acceptable. The kit of the present application uses suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. It is also contemplated to use packaging in conjunction with a specific device, such as an infusion device such as a micropump. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an IL-15Rα and Fc fusion protein, a heterodimeric protein of IL-15 and IL-15Rα as described herein. The container may further contain a second pharmaceutically active agent. The kit may optionally provide additional components, such as a buffer and interpretative information. Generally, the kit comprises a container and a label or drug instructions on or associated with the container.

[0350] Therefore, the present application also provides articles, including vials (such as sealed vials), bottles, cans, soft packages, etc. The article comprises a container and a label or drug instructions on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be made of a variety of materials, such as glass or plastic. In general, the composition contained in the container can effectively treat the diseases or conditions described herein (such as tumors), and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). The label or drug instructions indicate that the composition is used to treat a specific condition of an individual. The label or drug instructions further include instructions for administering the composition to an individual. The label may indicate instructions for reconstruction and / or use. The container for holding the pharmaceutical composition can be a vial for multiple uses, allowing the reconstituted preparation to be repeatedly administered (for example, 2-6 administrations). Drug instructions refer to instructions usually contained in the commercial packaging of therapeutic products, which contain indications, usage, dosage, administration, contraindications and / or warning information about the use of such therapeutic products. In addition, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. From a commercial and user perspective, other required materials may be further included, including other buffers, diluents, filters, needles and syringes.

[0351] The kit or article of manufacture includes multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies. Example

[0352] The following examples are intended merely to be exemplary of the present invention and therefore should not be construed as limiting the present invention in any way.The following examples and detailed description are provided by way of illustration and not by way of limitation.

[0353] Example 1: Construction, expression and purification of Fc-IL-15 / Fc-IL-15Rα_sushi heterodimer protein

[0354] To extend the half-life of the IL-15 / IL-15Rα_sushi complex, IL-15 is connected to the first Fc domain and IL-15Rα_sushi is connected to the second Fc domain, respectively, to prepare Fc-IL-15 / Fc-IL-15Rαsushi heterodimeric proteins, and the half-life is extended by FcRn-mediated circulation. To reduce the toxicity of the heterodimeric protein, the Fc-IL-15 / Fc-IL-15Rα_sushi heterodimeric protein form (hereinafter referred to as IL-15 prodrug) in the embodiment is designed by connecting the cleavable part (CM) to the masking polypeptide (MP). In the exemplary IL-15 prodrug, the masking polypeptide (MP) masks IL-15 and reduces the activity of IL-15 in non-target tissues, and the cleavable part (CM) contains a substrate sequence of a target tissue (e.g., tumor) protease (e.g., MMP2 or MMP9) for releasing the masking polypeptide (MP). Once the masked polypeptide is released from the IL-15 prodrug in the target tissue, the protein is activated and fully exhibits its activity. The constructs of the Fc-IL-15 / Fc-IL-15Rα_sushi heterodimer protein and its prodrug form are shown in Table 1. Figure 1 and Figure 2 Exemplary schematic diagrams of the Fc-IL-15 / Fc-IL-15Rα_sushi heterodimer protein and its prodrug form are shown respectively. All proteins were constructed and recombinantly expressed in HEK293 cells.

[0355] Plasmid construction: The nucleic acid sequences of polypeptides, IL-15 and IL-15Rα_sushi were commercially synthesized (GenscriptsUSA) and digested with restriction enzymes accordingly. Human IgG1 Fc (hole) or Fc (knob) was amplified by PCR and digested with restriction enzymes. The forward and reverse nucleotide single strands with corresponding restriction enzyme sites at both the 5′ and 3′ ends were annealed at 50°C to synthesize the cleavable part CM. All synthesized gene fragments and PCR fragments were run on 1% agarose gel, purified using a commercial kit and cloned into plasmid pcDNA3.1 (Invitrogen) using a T4 ligation kit. The ligation products were transformed into competent cells. After transformation and plating, colonies were picked and cultured overnight at 37°C in LB medium containing carbenicillin. The recombinant plasmid was extracted using a commercial kit (Qiagen, Cat No. 27104) and sequenced using T7 forward and BGH reverse primers. The entire coding sequence was verified by DNA sequencing.

[0356] Exemplary sequences of non-cleavable linkers (lk, lk1, lk2, lk3 and lk5) are shown in Table 2, exemplary sequences of cleavable moieties CM1 are shown in Table 3, exemplary sequences of human IL-15 (mature form or precursor form) and IL-15Rα_sushi (long form or short form) are shown in Table 4, exemplary sequences of human IgG1 Fc (hole) and Fc (knob) are shown in Table 5, exemplary sequences of masking polypeptides (MP) are shown in Table 6, and exemplary sequences of Fc-IL-15 / Fc-IL-15Rα_sushi heterodimeric polypeptides and prodrug forms thereof are shown in Table 7. In Table 7, human IL-15 or IL-15Rα_sushi domains are marked in italics, non-cleavable linkers are marked in bold, and cleavable moieties are marked in Single underline Labeling, masking peptides Double underscore The introduced restriction endonuclease recognition sites are marked with dotted lines.

[0357] Linker between Fc and IL-15Rα_sushi:

[0358] In fusion protein engineering, it is crucial to select a suitable linker to connect protein domains. Peptide linkers not only provide spatial distance between fusion protein domains and enable them to fold independently of each other, but also directly affect the structural stability and functional properties of fusion proteins. This paper explores the effects of linker flexibility and length on the bioactivity of Fc-IL-15 / Fc-IL-15Rα_sushi heterodimer protein and IL-15 prodrug.

[0359] In some embodiments, IL-15Rα_sushi and Fc are connected by a peptide linker. In some embodiments, the linker includes, for example, GSG and (GGGGS)n, wherein n is a positive integer of at least 1 (optionally, from 1 to 5), and the linker allows the two domains to have sufficient length and flexibility so that each domain retains its biological function.

[0360] As shown in Table 1, Group 1 is an exemplary Fc-IL-15 / Fc-IL-15Rα_sushi heterodimeric protein without a masking polypeptide: SB1902-C1 and SB1902-C1-variant1, the only difference between SB1902-C1 and SB1902-C1-variant1 is the linker between Fc and IL-15Rα_sushi. SB1902-C1 has a linker "lk5" (GGGGS)5 between Fc and IL-15Rα_sushi, and SB1902-C1-variant1 has a linker "lk" (GSG) between Fc and IL-15Rα_sushi.

[0361] Group 2 is an exemplary IL-15 prodrug with masking polypeptide MP80: SB1902-C2 and SB1902-C2-variant0, the only difference between SB1902-C2 and SB1902-C2-variant0 is the linker between Fc and IL-15Rα_sushi. SB1902-C2 has a linker "lk5" (GGGGS) 5 between Fc and IL-15Rα_sushi, and SB1902-C2-variant0 has a linker "lk" (GSG) between Fc and IL-15Rα_sushi.

[0362] Group 3 is an exemplary IL-15 prodrug with masking polypeptide MP163: SB1902-C9-variant1, SB1902-C9-variant2 and SB1902-C9-variant3, the only difference between SB1902-C9-variant1, SB1902-C9-variant2 and SB1902-C9-variant3 is the linker between Fc and IL-15Rα_sushi. SB1902-C9-variant1 has a linker "lk5" (GGGGS) 5 between Fc and IL-15Rα_sushi, SB1902-C9-variant2 has a linker "lk2" (GGGGS) 2 between Fc and IL-15Rα_sushi, and SB1902-C9-variant3 has a linker "lk1" (GGGGS) 1 between Fc and IL-15Rα_sushi.

[0363] Table 1 Fc-IL-15 / Fc-IL-15Rαsushi heterodimeric protein and its prodrug form

[0364]

[0365]

[0366] Table 2

[0367] Peptide linker SEQ ID NO. sequence lk 1 GSG lk1 2 (G4S)1 lk2 3 (G4S)2 lk3 4 (G4S)3 lk5 5 (G4S)5

[0368] Table 3

[0369] Cleavable part SEQ ID NO. sequence CM1 6 MVPSALTASG

[0370] Table 4

[0371]

[0372]

[0373] Table 5

[0374]

[0375] Table 6

[0376]

[0377] Table 7

[0378]

[0379]

[0380]

[0381]

[0382]

[0383] Expression and purification: Transient transfection encoding Fc-IL-15 / Fc-IL-15Rα_sushi heterodimer protein and IL-156 prodrug vectors were used to express proteins in Expi293 cells (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's recommended method. The culture supernatant medium was clarified by centrifugation and 0.2 μm membrane filtration. According to the method recommended by the manufacturer, exemplary IL-15 prodrugs (SB1902-C2, SB1902-C2-variant0, SB1902-C9 variant1, SB1902-C9-variant2 and SB1902-C9-variant3) and exemplary Fc-IL-15 / Fc-IL-15Rα_sushi heterodimer proteins (SB1902-C1 and SB1902-C1-variant1) were purified by a two-step purification process, including the use of pre-made MabSelect SuRe pcc columns (Cytiva lifescience, Cat No. 17549112) and size exclusion chromatography (Superdex200, Cytiva, USA). The purity of the purified molecules was analyzed by SDS-PAGE (with and without reducing drugs) and SEC-HPLC (partial data not shown). Figure 3 and Figure 4 Shown are the results of SDS-PAGE and SEC-HPLC analysis of SB1902-C1, respectively. The exemplary data indicate that the Fc-IL-15 / Fc-IL-15Rα_sushi heterodimeric protein was successfully prepared and exhibited good purity and homogeneity.

[0384] Example 2: Receptor Binding Affinity

[0385] ELISA was used to detect the binding affinity of Fc-IL-15 / Fc-IL-15Rα_sushi heterodimer protein and IL-15 prodrug to IL-2 / IL15Rβγ-Fc fusion protein. ELISA plates were coated with 1μg / ml recombinant IL15Rβγ-Fc at 4°C overnight. After blocking and washing the plates with PBS containing 1% BSA, serial gradient dilutions of SB1902-C1 or SB1902-C2 were added to the plates and incubated for 2 hours. Human IgG1 subtype antibody MOPC21, Figure 5 hIgG1 (see Hamlyn PH, Gait MJ, Milstein C. (1981) Complete sequence of an immunoglobulin mRNA using specific priming and the dideoxynucleotide method of RNA sequencing. Nucleic Acids Res. 9 (18): 4485-4494) was used as a negative control. After washing the plate, anti-human IgG-Fc-AP conjugated antibody diluted 1:2000 was added to each well of the plate and incubated for 45 min. After washing the plate, AP substrate pNPP (Thermo, USA) was added and the optical density was obtained at 405 nm using a spectrophotometer (BioTec, USA). GraphPad Prism8 software was used to analyze and plot the data.

[0386] like Figure 5 As shown, the Fc-IL-15 / Fc-IL-15Rα_sushi heterodimeric protein SB1902-C1 and its prodrug form SB1902-C2 both exhibited binding affinity to IL-15Rβγ, indicating that these proteins are able to exhibit IL-15 activity, and the prodrug SB1902-C2 has a lower binding affinity to IL-15Rβγ than SB1902-C1. Other Fc-IL-15 / Fc-IL-15Rα_sushi heterodimeric proteins or their prodrug forms also exhibited activity binding to the IL-15Rβγ receptor (data not shown).

[0387] Example 3: In vitro functional assay: CD8+ T cell activity assay

[0388] According to the literature, IL-15 promotes the proliferation, survival and balance of CD8+ memory T cells, natural killer cells (NK) and NKT cells. IL-15 causes T cell activation, which can be indicated by the up-regulated expression of CD69 on the cell membrane surface and the release of cytokines including IFNγ. CD69 is a marker for early activation of T cells. The percentage of CD8+T cell activation is reflected by the percentage of CD69 expressed on the cell surface. In the CD8+T cell activity test, various forms of Fc-IL-15 / Fc-IL-15Rα_sushi heterodimeric proteins and IL-15 prodrugs as described above were tested.

[0389] The basic operation of this experiment is as follows: human PBMC (Stem Cell Technologies, Cat No. 70500) was cultured at a cell number of 2×10 5 / well was added to a 96-well round-bottom cell culture plate containing RPMI-1640 culture medium, and 10% FBS, 1% penicillin and streptomycin were added to the culture medium. Fc-IL-15 / Fc-IL-15Rα_sushi heterodimer protein and IL-15 prodrug were diluted 3 times with culture medium, and 5 μL was added to the well. Each concentration was repeated three times, and the blank well (only culture medium was added) was used as a blank control. The cell culture dish was incubated in an incubator for 3 days. The cells were then centrifuged, and the cell pellet was stained with anti-CD3 Ab (biolgend, cat#317306), anti-CD8 Ab (biolgend, cat#344722) and anti-CD69 Ab (biolgend, cat#310906) in FACS buffer for 30 minutes. Wash twice with FACS buffer and obtain cells with Attune (ThermoFisherScientific) flow cytometry. Data were analyzed using FlowJo software. Graphpad Prism 8 software was used to plot the percentage of CD69+ cells to CD8+ T cells.

[0390] To compare the effects of different linker lengths between Fc and IL-15Rα_sushi on IL-15 activity, the biological activity was determined by measuring the induced expression of CD69 on CD8+ T cells in the above-mentioned in vitro human PBMC-based FACS assay.

[0391] In the SB1902-C1 construct of group 1, the linker between Fc and IL-15Rα_sushi is a GS-rich flexible linker with a length of 25 amino acids (GGGGS) 5 (SEQ ID NO: 5), and in the SB1902-C1-variant1 construct of group 1, the linker between Fc and IL-15Rα_sushi is a GS-rich flexible linker with a length of 3 amino acids GSG (SEQ ID NO: 1). The only difference between SB1902-C1 and SB1902-C1-variant1 is the linker between Fc and IL-15Rα_sushi. Figure 6 As shown, in the CD8+T cell activity assay, SB1902-C1 with a linker "lk5" (GGGGS)5 between Fc and IL-15Rα_sushi had better activity in inducing CD69 expression than SB1902-C1-variant1 with a linker "lk" (GSG) between Fc and IL-15Rα_sushi.

[0392] In the SB1902-C2 construct of group 2, the linker between Fc and IL-15Rα_sushi is a GS-rich flexible linker with a length of 25 amino acids (GGGGS) 5 (SEQ ID NO: 5), and in the SB1902-C2-variant0 construct of group 2, the linker between Fc and IL-15Rα_sushi is a GS-rich flexible linker with a length of 3 amino acids GSG (SEQ ID NO: 1). The only difference between SB1902-C2 and SB1902-C2-variant0 is the linker between Fc and IL-15Rα_sushi. Figure 7 As shown, in the CD8+T cell activity assay, SB1902-C2 with a linker "lk5" (GGGGS)5 between Fc and IL-15Rα_sushi had better activity in inducing CD69 expression than SB1902-C2-variant0 with a linker "lk" (GSG) between Fc and IL-15Rα_sushi.

[0393] In the SB1902-C9-variant1 construct of group 3, the linker between Fc and IL-15Rα_sushi is a GS-rich flexible linker with a length of 25 amino acids (GGGGS)5 (SEQ ID NO:5); in the SB1902-C9-variant2 construct of group 3, the linker between Fc and IL-15Rα_sushi is a GS-rich flexible linker with a length of 15 amino acids (GGGGS)3 (SEQ ID NO:4); and in the SB1902-C9-variant3 construct of group 3, the linker between Fc and IL-15Rα_sushi is a GS-rich flexible linker with a length of 5 amino acids (GGGGS)1 (SEQ ID NO:2). The only difference between SB1902-C9-variant1, SB1902-C9-variant2 and SB1902-C9-variant3 is the linker between Fc and IL-15Rα_sushi. Figure 8 As shown, in the CD8+T cell activity assay, SB1902-C9-variant2 having a linker "lk3" (GGGGS) 3 between Fc and IL-15Rα_sushi and SB1902-C9-variant3 having a linker "lk1" (GGGGS) 1 between Fc and IL-15Rα_sushi showed comparable activity to each other, however, SB1902-C9-variant1 having a linker "lk5" (GGGGS) 5 between Fc and IL-15Rα_sushi had better activity in inducing CD69 expression than SB1902-C9-variant2 and SB1902-C9-variant3.

[0394] Combination Figure 6 , 7 and 8. The results showed that the peptide linker connecting Fc and IL-15Rα_sushi affected the biological activity of IL-15 in the Fc-IL-15 / Fc-IL-15Rα_sushi heterodimer and its prodrug form within a certain range. When the length of the linker was shorter than 15 amino acids, the length of the linker had little effect on the activity of IL-15. However, when the length of the linker was longer than 15 amino acids and shorter than 25 amino acids, the longer the linker, the higher the activity of IL-15 in the Fc-IL-15 / Fc-IL-15Rα_sushi heterodimer and the prodrug.

[0395] Example 4: In vivo tumor model to evaluate the activity of IL-15 / IL-15Rα_sushi-Fc heterodimer protein

[0396] The ability of Fc-IL-15 / Fc-IL-15Rα_sushi heterodimer to promote tumor clearance and inhibit metastasis was evaluated in vivo using the mouse WEHI-164 tumor model. Human IgG1 subtype antibody MOPC21 was used as a control in this experiment. A. In vivo activity of the prodrug in the WEHI-164 subcutaneous tumor model

[0397] Animals and husbandry : Female mice aged 7-9 weeks were used in this study. Mice were fed irradiated Harlan 2918.15 mouse diet and water was available ad libitum. For ease of identification, the animals were ear-tagged and the left dorsal region was shaved in preparation for cell implantation. Animals were housed in Innovive disposable ventilated cages with corncob bedding and 60 complete air changes per hour. The environment was controlled within a temperature range of 70°±2°F and a humidity range of 30-70%. All procedures in this experiment were performed by technicians who adhered to all laws, regulations, and guidelines of the National Institutes of Health (NIH) and were approved by Biomere's Animal Care and Use Committee (Richmond, CA).

[0398] Cell preparation and implantation : WEHI-164 cells were obtained from ATCC (CAT#: CRL-1751 TM ). WEHI-164 cells were cultured and expanded in Dulbecco's Modified Eagles Medium (DMEM) containing 2mM L-glutamine, 10% fetal bovine serum (FBS) and 1% 100× penicillin / streptomycin (PS). The growth environment was maintained at 37°C and 5% CO2 in an incubator. When the expansion was completed, the cells were trypsinized using 0.25% trypsin-EDTA solution, washed and counted. The cell viability was >95% before implantation. The cells were resuspended in Dulbecco's phosphate buffered saline (DPBS). The experimental animals were disinfected with alcohol cotton at the implantation site and 0.1mL was implanted subcutaneously using a 25-gauge needle and a 1ml syringe on day 0.

[0399] Measurement and processing : Allow tumors to grow to 70-150mm 3 The mice were randomly divided into groups according to the range of tumor size and weight. The mice were grouped to ensure that the average weight of mice in all groups was within 10% of the overall average tumor burden in the study population. The mice were intravenously injected with 3 mg / kg doses of human IgG1 subtype antibody or Fc-IL-15 / Fc-IL-15Rα_sushi heterodimer protein twice a week for 2 weeks, and the tumor volume was monitored.

[0400] Side Effect Assessment : All animals were observed for clinical signs or toxicity at least once a day. Animals were weighed weekly. Individual animals were weighed daily if they showed obvious signs of distress or lost 15% of their body weight. Animals were euthanized if they lost more than 20% of their body weight or showed other clinical signs requiring euthanasia. Individual animals were euthanized when their tumor volume reached or exceeded 2000 mm. 3 euthanasia was performed on him.

[0401] result :Take SB1902-C1 as an example. Figure 9-10 As shown, WEHI-164 tumor animals were treated with human IgG1 subtype control antibody or SB1902-C1. Throughout the treatment, all animals in the study showed no obvious signs of systemic toxicity. Fig. 9 ) compared to tumor growth in SB1902-C1 treated animals ( Fig.10 The results showed that Fc-IL-15 / Fc-IL-15Rα_sushi heterodimer protein can play a role in tumor mice.

[0402] At the same time, other heterodimeric proteins or their prodrug forms were also tested in vitro. In other Fc-IL-15 / Fc-IL-15Rα_sushi heterodimeric proteins or their prodrug forms, the effect of the linker length between Fc and IL-15Rα_sushi was further verified, which was consistent with the trend confirmed in the CD8+T cell activity assay (data not shown).

Claims

1. An isolated fusion protein, wherein the fusion protein comprises: IL-15Rα or a functional fragment thereof, which is connected to an Fc domain via a peptide linker.

2. The isolated fusion protein according to claim 1, wherein the peptide linker comprises 3 to 40 amino acid residues; preferably 3 to 25 amino acid residues.

3. The isolated fusion protein according to claim 1 or 2, wherein the peptide linker comprises 20 to 25 amino acid residues; preferably 25 amino acid residues. 4 . The isolated fusion protein according to any one of claims 1 to 3 , wherein the IL-15Rα or a functional fragment thereof is linked to the N-terminus or C-terminus of the Fc domain.

5. The isolated fusion protein according to any one of claims 1 to 4, wherein the fusion protein further comprises an IL-15 cytokine.

6. The isolated fusion protein of claim 5, wherein the IL-15 cytokine is linked to the N-terminus or the C-terminus of the Fc domain. 7 . The isolated fusion protein according to claim 5 , wherein the IL-15 cytokine is linked to the N-terminus or C-terminus of IL-15Rα or a functional fragment thereof.

8. An isolated heterodimeric protein comprising two monomers, wherein one monomer comprises the isolated fusion protein according to any one of claims 1 to 4, and the other monomer comprises an IL-15 cytokine and / or an Fc domain. 9 . The isolated heterodimeric protein according to claim 8 , wherein the IL-15Rα or a functional fragment thereof or the IL-15 cytokine is linked to the N-terminus or the C-terminus of the Fc domain.

10. The isolated protein according to any one of claims 1-9, wherein the Fc domain is selected from the group consisting of a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, an IgAFc domain, an IgD Fc domain, an IgE Fc domain and an IgM Fc domain; optionally, the Fc domain is a human IgG1 Fc domain.

11. The isolated protein of claim 10, wherein the Fc domain is a human IgG1 Fc domain having L234A and L235A mutations, the mutation sites being numbered according to the EU numbering system.

12. The isolated heterodimeric protein according to any one of claims 8-11, wherein the Fc domain comprises a knobs-into-holes mutation (Fc knob and Fc hole).

13. The isolated heterodimeric protein according to claim 12, wherein the Fc knob is linked to IL-15 cytokine, and the Fc hole is linked to IL-15Rα or a functional fragment thereof; or, The Fc knob is connected to IL-15Rα or a functional fragment thereof, and the Fc hole is connected to IL-15 cytokine.

14. The isolated heterodimeric protein of claim 12 or 13, wherein the Fc knob comprises a T366W mutation in the Fc domain, and the Fc hole comprises T366S, L368A and Y407V mutations in the Fc domain, and the mutation sites are numbered according to the EU numbering system.

15. The isolated heterodimeric protein of claim 14, wherein the Fc knob further comprises an S354C mutation, and the Fc hole further comprises a Y349C mutation, and the mutation sites are numbered according to the EU numbering system. 16 . The isolated protein according to claim 1 , wherein the IL-15Rα or a functional fragment thereof is selected from the extracellular domain or sushi domain or a functional analogue of IL-15Rα.

17. The isolated protein according to any one of claims 1-16, wherein the IL-15Rα or a functional fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 9-11, or a variant thereof, wherein the variant has at least 90% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 9-11.

18. The isolated protein according to any one of claims 5-17, wherein the IL-15 cytokine comprises the amino acid sequence shown in any one of SEQ ID NOs: 7-8, or a variant thereof, wherein the variant has at least 90% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 7-8.

19. An isolated protein according to any one of claims 1-18, wherein the peptide linker is rich in G and S amino acid residues; preferably, the peptide linker consists of G and S amino acid residues; more preferably, the peptide linker comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-5, or a variant thereof, wherein the variant has at least 90% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs: 1-5.

20. The isolated protein according to any one of claims 1-19, further comprising a cleavable moiety (CM) and a masking polypeptide (MP), and wherein the masking polypeptide (MP) is linked to the protein via the cleavable moiety (CM).

21. The isolated protein according to claim 20, wherein the masking polypeptide (MP) comprises the amino acid sequence shown in any one of SEQ ID NOs: 14-15, or a variant thereof, wherein the variant has at least 90% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 14-15.

22. The isolated protein of claim 20, wherein the cleavable moiety (CM) comprises the amino acid sequence of SEQ ID NO: 6, or a variant thereof having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

6.

23. An isolated heterodimeric protein comprising two polypeptide chains, wherein: (i) one polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof, which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 16; and the other chain comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof, which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 17 has at least 80% sequence identity; or (ii) one polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof, which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 16; and the other chain comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof, which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 18 has at least 80% sequence identity; or (iii) one polypeptide chain comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof, which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 19; and the other chain comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof, which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 20 have at least 80% sequence identity; or (iv) one polypeptide chain comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof, which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 19; and the other chain comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof, which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 18 has at least 80% sequence identity; or (v) one polypeptide chain comprises the amino acid sequence of SEQ ID NO:21, or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:21; and the other chain comprises the amino acid sequence of SEQ ID NO:22, or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 22 has at least 80% sequence identity; or (vi) one polypeptide chain comprises the amino acid sequence of SEQ ID NO:23, or a variant thereof, which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:23; and the other chain comprises the amino acid sequence of SEQ ID NO:22, or a variant thereof, which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 22 has at least 80% sequence identity; or (vii) one polypeptide chain comprises the amino acid sequence of SEQ ID NO:24, or a variant thereof, which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:24; and the other chain comprises the amino acid sequence of SEQ ID NO:22, or a variant thereof, which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 22 have at least 80% sequence identity.

24. A method for improving the activity of a fusion protein or a heterodimeric protein, wherein the fusion protein or the heterodimeric protein comprises IL-15Rα or a functional fragment thereof and an Fc domain, which comprises setting the peptide linker connecting IL-15Rα or a functional fragment thereof and the Fc domain to more than 15 and less than 40 amino acid residues.

25. The method of claim 24, wherein the peptide linker comprises 20 to 25 amino acid residues.

26. The method of claim 24 or 25, wherein the peptide linker comprises 25 amino acid residues.

27. An isolated nucleic acid molecule encoding the isolated protein of any one of claims 1-23.

28. A vector comprising the isolated nucleic acid molecule of claim 27.

29. An isolated host cell comprising the isolated protein of any one of claims 1-23, the isolated nucleic acid molecule of claim 27, or the vector of claim 28.

30. A method for preparing the isolated protein of any one of claims 1-23, comprising: a) culturing the host cell of claim 29 under conditions that effectively express the fusion protein or heterodimeric protein; and b) obtaining the expressed fusion protein or heterodimer protein from the host cell.

31. A pharmaceutical composition comprising the isolated protein of any one of claims 1-23, the isolated nucleic acid molecule of claim 27, the vector of claim 28, the host cell of claim 29, the protein prepared by the method of claim 30, and a pharmaceutically acceptable carrier portion or excipient.

32. A method of treating a disease or condition in an individual in need thereof, comprising administering to the individual an effective amount of the isolated protein of any one of claims 1-23, the isolated nucleic acid molecule of claim 27, the vector of claim 28, the host cell of claim 29, the protein prepared by the method of claim 30, or the pharmaceutical composition of claim 31.

33. The method of claim 32, wherein the disease or condition is cancer or an infectious disease or stimulation of the immune system in a patient in need thereof.

34. The method of claim 33, wherein the cancer is selected from the group consisting of prostate cancer, colon cancer, kidney cancer, melanoma, lung cancer, breast cancer, thyroid cancer, bladder cancer, stomach and esophageal cancer, pancreatic cancer, liver cancer, brain cancer, head and neck cancer, neuroblastoma, soft tissue cancer, lymphoma, leukemia, multiple myeloma, or any metastasis thereof.

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