Cytokine-based bioactivating agents and their use

Abstract

The present disclosure provides a cytokine-based bioactive drug construct ("VitoKine") platform aimed at reducing toxicity based on systemic mechanisms and broadening the therapeutic utility of proteins and cytokines, such as IL-15 and IL-2, in the treatment of cancer, autoimmune diseases, inflammatory diseases, viral infections, transplants, and various other disorders. The novel VitoKine constructs of the present invention comprise: 1) a tissue or disease-targeting moiety, the D1 domain ("D1"); 2) a bioactive moiety, the D2 domain ("D2"); and a shielding moiety, the D3 domain ("D3"). Importantly, the "active moiety" of the VitoKine construct remains inactive until locally activated by proteases upregulated in the diseased tissue, thereby restricting the active moiety from binding to receptors or targets on the cell surface of surrounding or non-diseased cells and tissues, preventing excessive activation of the pathway and reducing undesirable "on-target" but "off-target" toxicity. Furthermore, the inactivity of the VitoKine active moiety prior to protease activation greatly reduces the potential for antigen and target sinks, resulting in an extended in vivo half-life and improved biodistribution, bioavailability, and therapeutic efficacy.

Description

[Background technology] 【0001】 Many cytokines have been evaluated as potential therapeutic agents for disease treatment. However, systemic overstimulation or oversuppression of the body's immune system by cytokines has significantly hindered their development and clinical usefulness. 【0002】 Interleukin-2 (IL-2) and interleukin-15 (IL-15) share common receptor components (γc and IL-2Rβ) and signaling pathways, and have several similar functions. Both cytokines stimulate T cell proliferation; stimulate cytotoxic T lymphocyte (CTL) production; promote B cell proliferation and B cell immunoglobulin synthesis; and induce natural killer (NK) cell production and retention. Numerous preclinical studies, as well as multiple clinical evaluations, have shown that both cytokines have potential therapeutic value in cancer, autoimmune disorders, inflammatory disorders, transplantation, and various other disorders. Recombinant IL-2 has been approved for use in patients with metastatic renal cell carcinoma and malignant melanoma. While several oncological clinical trials are underway for IL-15, none have yet been approved for use. Furthermore, both IL-2 and IL-15 possess a third unique non-signaling receptor α subunit, IL-2Rα (also known as CD25) or IL-15Rα, which may contribute to their different receptor specificities and biological functions. 【0003】 Recombinant human IL-2 is a highly effective immunotherapy used for metastatic melanoma and renal cell carcinoma, with sustained responses observed in approximately 10% of patients. However, its short half-life and high toxicity limit the optimal dose of IL-2. Furthermore, IL-2 binds with greater affinity to its heterotrimeric receptor, IL-2Rαβγ, leading to the preferential proliferation of immunosuppressive regulatory T cells (Tregs) that constitutively express high levels of IL-2Rα. Treg proliferation can be an undesirable effect of IL-2 in cancer immunotherapy. However, IL-2's ability to stimulate Treg cells even at low doses suggests potential applications in the treatment of autoimmune chronic inflammatory disorders. More recently, it has been discovered that IL-2 can be modified to selectively stimulate either cytotoxic effector T cells or Treg cells. Through various approaches, IL-2 variants with improved selective immunomodulatory activity have been generated. 【0004】 Both IL-2 and IL-15 are potent agonists of immune effector cells, and it is important that cytotoxic immune cells are fully activated only at the site of injury, such as cancer or its vicinity, to destroy tumor cells very specifically; or fully activated at or near inflammatory tissue sites, to act locally as anti-autoimmune and anti-chronic inflammatory disorders. For all cytokines, chemokines, and growth factors, improving the specificity and selectivity for targets while leaving healthy cells and tissues intact is of great interest. [Overview of the Initiative] 【0005】 In one aspect, the present invention provides a cytokine-based bioactivating agent ("VitoKine") platform for reducing toxicity based on systemic mechanisms and expanding the therapeutic utility of cytokines, chemokines, hormones, and growth factors such as IL-15 and IL-2 in the treatment of cancer, autoimmune disorders, inflammatory disorders, and various other disorders. This VitoKine platform is defined by the constructs shown in FIG. 1 and the proposed activation methods shown in FIG. 2. Referring to FIG. 1, the novel VitoKine construct of the present invention includes the following three domains: 1) a D1 domain ("D1") selected from the group consisting of a tissue targeting domain; a half-life extension domain; or a dual-functional partial domain, 2) a D2 domain ("D2") which is an "active partial domain", and 3) a D3 domain ("D3") which is a "shielding partial domain". Importantly, the D2 domain of the VitoKine construct remains substantially inactive or minimally active until it is locally activated by proteases whose expression is increased in the diseased tissue or by hydrolysis at the diseased site, so that the active portion is restricted from binding to receptors on the cell surface of surrounding or non-diseased cells or normal tissue cells, thereby suppressing excessive activation of that pathway and reducing undesirable "off-target" but "outside the target tissue" toxicity and unwanted target sinks. 【0006】 In various embodiments, the VitoKine construct of the present invention includes a targeting moiety D1, such as an antibody or antibody fragment that binds to a tumor-associated antigen (TAA), an immune checkpoint regulator, a tissue-specific antigen, a cell surface molecule or extracellular matrix protein or protease (one or more), or any post-translational modification residue (one or more). In various embodiments, the VitoKine construct of the present invention includes a targeting moiety D1, such as a protein or peptide that exhibits binding affinity to affected cells or tissues. Exemplary antibodies intended for use as D1 in the VitoKine construct of the present invention include various PD-1 antagonist antibodies, the PD-L1 blocking antibody Tecentriq, the anti-CTLA4 antibody ipilimumab, the agonist CD40 antibody RO7009789, tumor antigen targeting antibodies such as L19 against the extra-domain of fibronectin, rituximab against CD20, Herceptin against Her-2, cetuximab against EGFR, anti-FAP antibodies for tumor targeting and retention, and anti-inflammatory antibodies such as vedolizumab against integrin α4β7 and Humira against TNFα. 【0007】 In various embodiments, D1 is an antibody that is an antagonist anti-fibroblast-activating protein (FAP) antibody or antibody fragment. In various embodiments, the antibody is a humanized anti-FAP antibody containing the amino acid sequences described in SEQ ID NOs: 193 and 194. In various embodiments, D1 is an antibody or antibody fragment against an immune checkpoint regulator. In various embodiments, the antibody is an antagonist PD-1 antibody or antibody fragment. In various embodiments, the antibody is an antagonist humanized PD-1 antibody containing the amino acid sequences described in SEQ ID NOs: 195 and 196. In various embodiments, the antibody is an antagonist humanized PD-1 antibody containing the amino acid sequences described in SEQ ID NOs: 197 and 198. In various embodiments, the antibody is an antagonist humanized PD-1 antibody containing the amino acid sequences described in SEQ ID NOs: 275 and 276. In various embodiments, the antibody is an antagonist human PD-1 antibody containing the amino acid sequences described in SEQ ID NOs: 277 and 278. In various embodiments, the antibody is an antagonist PD-L1 antibody or antibody fragment. In various embodiments, the antibody is an antagonist human PD-L1 antibody containing the amino acid sequences described in SEQ ID NOs. 279 and 280. In various embodiments, the VitoKine construct contains the amino acid sequences described in SEQ ID NOs. 128-142, 180-181, 281-286, 296-297, and 303-306. 【0008】 In various embodiments, the VitoKine construct of the present invention comprises a modified protein or peptide, such as a glycan-modified protein or glycan-modified peptide, D1, which exhibits binding affinity for specific receptors, such as C-type lectin receptors, expressed on diseased cells or diseased tissues. In various embodiments, the VitoKine construct of the present invention comprises D1, which has the function of retaining cytokines at tissue sites. In various embodiments, the VitoKine construct of the present invention comprises D1, which is bifunctional, such as tissue targeting and tissue retention. In various embodiments, the VitoKine construct of the present invention comprises a D1 domain that is a polymer. In various embodiments, the VitoKine construct of the present invention comprises a D1 domain that is a half-life extension moiety. In various embodiments, the VitoKine construct of the present invention comprises a D1 domain that is an Fc domain or a functional fragment thereof. 【0009】 An "Fc domain" refers to a dimer consisting of two Fc domain monomers, typically containing all or part of a hinge region. In various embodiments, the Fc domain is selected from the group consisting of the Fc domains of human IgG1, human IgG2, human IgG3, human IgG4, IgA, IgD, IgE, IgG, and IgM, or any combination thereof. In various embodiments, the Fc domain includes amino acid changes that result in an Fc domain with altered complement-binding or Fc receptor-binding properties. Amino acid changes known to result in an Fc domain with altered complement-binding or Fc receptor-binding properties are known in the art. In various embodiments, the Fc domain sequence used in the production of the VitoKine construct is the human IgG1-Fc domain sequence described in SEQ ID NO: 13. In various embodiments, the Fc domain sequence used to produce the VitoKine construct is the sequence described in SEQ ID NO: 14, which includes amino acid substitutions that break FcγR or C1q binding. In various embodiments, the Fc domain includes amino acid changes that result in a further extension of the in vivo half-life. Amino acid changes known to result in an Fc domain with a further extended half-life are known in the art. In various embodiments, the Fc domain sequence used to produce the VitoKine construct is the sequence described in SEQ ID NO: 156 or SEQ ID NO: 166, which includes amino acid substitutions that break FcγR or C1q binding and extend the in vivo half-life. In various embodiments, the Fc domain sequence used to produce the VitoKine construct is derived from the Knob-Fc domain sequence described in SEQ ID NO: 15. In various embodiments, the Fc domain sequence used to produce the VitoKine construct is derived from the Hole-Fc domain sequence described in SEQ ID NO: 16. In various embodiments, the Fc domain sequence used to produce the VitoKine construct is derived from the Knob-Fc domain sequence with an extended in vivo half-life described in Sequence ID No. 167.In various embodiments, the Fc domain sequence used to produce the VitoKine construct is derived from the Hole-Fc domain sequence with an extended in vivo half-life described in Sequence ID No. 168. 【0010】 In various embodiments, the VitoKine construct of the present invention comprises a D2 domain which is a protein. In various embodiments, the VitoKine construct of the present invention comprises a D2 domain which is a cytokine selected from the group including, but not limited to, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-15, IL-23, and ligands of the transforming growth factor β (TGFβ) superfamily, e.g., TGFβ (SEQ ID NO: 24). In various embodiments, the VitoKine construct of the present invention comprises a D2 domain which is IL-15. In various embodiments, the VitoKine construct of the present invention comprises a D2 domain which is an IL-15 variant (or mutant) comprising one or more amino acid substitutions, amino acid deletions, or amino acid insertions in the IL-15 polypeptide. In various embodiments, the VitoKine construct of the present invention comprises a D2 domain which is IL-2. In various embodiments, the VitoKine construct of the present invention comprises a D2 domain which is an IL-2 variant (or mutant) comprising one or more amino acid substitutions, amino acid deletions, or amino acid insertions in the IL-2 polypeptide. 【0011】 In various embodiments, the D2 domain of the VitoKine construct is an IL-15 domain containing the sequence of the mature human IL-15 polypeptide described in SEQ ID NO: 2 (hereinafter also referred to as huIL-15 or IL-15 wild-type (wt)). In various embodiments, the IL-15 domain may be an IL-15 variant (or mutant) containing a sequence derived from the sequence of the mature human IL-15 polypeptide described in SEQ ID NO: 2. In various embodiments, the IL-15 domain may be an IL-15 variant (or mutant) containing a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology with SEQ ID NO: 2. IL-15 variants (or mutants) are named using the original amino acid, the position of the original amino acid in the mature sequence, and the variant-type amino acid. For example, huIL-15 "S58D" represents human IL-15 with an S-to-D substitution at position 58 of SEQ ID NO: 2. In various embodiments, the IL-15 variant functions as an IL-15 agonist, as indicated by increased binding activity to the IL-15Rβγc receptor compared to the native IL-15 polypeptide. In various embodiments, the IL-15 variant functions as an IL-15 antagonist, as indicated by decreased binding activity to the IL-15Rβγc receptor compared to the native IL-15 polypeptide, or by similar or increased binding activity to the IL-15Rβγc receptor but decreased or absent signaling activity. In various embodiments, the IL-15 variant has increased binding affinity or decreased binding activity to the IL-15Rβγc receptor compared to the native IL-15 polypeptide. In various embodiments, the sequence of the IL-15 variant has at least one (i.e., one, two, three, four, five, six, seven, eight, nine, ten, or more) amino acid changes compared to the native IL-15 sequence.The amino acid changes can include one or more amino acid substitutions, deletions, and insertions in the IL-15 polypeptide, such as the domain of IL-15 that interacts with IL-15Rβ and / or IL-15Rβγc. In various embodiments, the amino acid changes are one or more amino acid substitutions at positions 30, 31, 32, 58, 62, 63, 67, 68, or 108 of SEQ ID NO: 2. In various embodiments, the amino acid change is a substitution of D to T at position 30, V to Y at position 31, H to E at position 32, S to D or G or H or R or Q or I or P at position 58, T to D at position 61, V to F or A or K or R at position 63, I to V at position 67, I to F or H or D or K or Q or G at position 68, Q to A or M or S or E or K at position 108, or any combination of these substitutions. In various embodiments, the amino acid change is a deletion of one, two, three, four, five, or six amino acids at the N-terminus of SEQ ID NO: 2. In various embodiments, the amino acid change is the deletion of one, two, three, four, five, six, seven, eight, nine, or ten amino acids at the C-terminus of SEQ ID NO: 2. In various embodiments, the amino acid change is the insertion of "GS" (SEQ ID NO: 116), "GGSGG" (SEQ ID NO: 119), or "GSSGGSGGS" (SEQ ID NO: 110) after the N95 position of SEQ ID NO: 2. In various embodiments, the IL-15 domain has any combination of amino acid substitutions, amino acid deletions, and amino acid insertions. In various embodiments, the VitoKine construct utilizes an IL-15 variant with optimally attenuated potency, and therefore reduces the intrinsic base activity of the corresponding VitoKine construct. In various embodiments, the IL-15 variant includes the amino acid sequences described in SEQ ID NO: 3, 182-192, and 199-215. 【0012】 In various embodiments, the D2 domain of the VitoKine construct of the present invention contains an IL-2 polypeptide. In various embodiments, the VitoKine construct of the present invention contains a D2 domain which is an IL-2 variant (or mutant) comprising one or more amino acid substitutions, amino acid deletions, or amino acid insertions. In various embodiments, the VitoKine construct contains an IL-2 domain which is the mature human IL-2 polypeptide described in Sequence ID No. 8 (hereinafter also referred to as huIL-2 or IL-2 wild type (wt)). The D2 domain includes the sequence of . In various embodiments, the IL-2 domain may be an IL-2 variant (or variant) containing a sequence derived from the sequence of the mature human IL-2 polypeptide described in SEQ ID NO: 8. In various embodiments, the IL-2 domain may be an IL-2 variant (or variant) containing a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology to SEQ ID NO: 8. In various embodiments, the IL-2 variant functions as an IL-2 agonist. In various embodiments, the IL-2 variant functions as an IL-2 antagonist. In various embodiments, the amino acid change is one or more amino acid substitutions at positions 19, 20, 38, 41, 42, 44, 62, 65, 68, 88, 107, 125, or 126 of SEQ ID NO: 8.In various embodiments, the amino acid changes are substitutions of the mature human IL-2 sequence: L at position 19 to D, H, N, P, Q, R, S, or Y; D at position 20 to E, I, N, Q, S, T, or Y; R at position 38 to E, or A; T at position 41 to A, G, or V; F at position 42 to A; F at position 44 to G, or V; E at position 62 to A, F, H, or L; and P at position 65 to G, E, or H Alternatively, substitutions include R, A, K, N, or Q; substitution of E at position 68 to A, F, H, L, or P; substitution of N at position 88 to D, E, G, I, M, Q, T, or R; substitution of Y at position 107 to G, H, L, or V; substitution of S at position 125 to E, H, K, I, L, V, or W; substitution of Q at position 126 to D, E, K, L, M, or N; or any combination of these substitutions. In various embodiments, the VitoKine construct will contain an IL-2 moiety designed to reduce / eliminate binding to IL-2Rα. In various embodiments, the IL-2 variant has reduced binding activity to the IL-2Rβγc receptor compared to the native IL-2 polypeptide. The IL-2 variant has reduced / eliminated binding to IL-2Rα and altered binding activity to the IL-2Rβγc receptor compared to the native IL-2 polypeptide. In various embodiments, IL-2 variants with reduced / absent binding to IL-2Rα include the amino acid sequences described in SEQ ID NOs. 232-247. In various embodiments, IL-2-containing VitoKine constructs were designed for the selective expansion of Teff cells. Examples include those containing the amino acid sequences described in SEQ ID NOs. 59-61, 271-274, and 286-291. In various embodiments, the IL-2 variants in the VitoKine constructs can be modified to achieve an optimal balance between desired antitumor effects and undesirable systemic toxicity, thereby tunicating the inherent basic activity of IL-2VitoKine. 【0013】In various embodiments, the VitoKine construct of the present invention comprises a "shielding partial domain" (D3) which is a cognitive receptor / binding partner for the D2 protein or cytokine, or any identified binding partner. In various embodiments, the D3 domain is a variant of the cognitive receptor / binding partner to the D2 domain. In various embodiments, the D3 domain has enhanced binding affinity to the D2 domain compared to the wild-type cognitive receptor / binding partner. In various embodiments, the D3 domain has reduced or absent binding affinity to the D2 domain compared to the wild-type cognitive receptor / binding partner. In various embodiments, the D3 domain is a protein or peptide, or an antibody or antibody fragment that can shield the activity of D2. In various embodiments, the D3 domain is a polymer such as DNA, RNA fragment, or PEG. In various embodiments, the VitoKine construct of the present invention comprises a D3 domain which is an IL-15Rα extracellular domain or a functional fragment thereof. In various embodiments, the VitoKine construct of the present invention comprises a D3 domain which is an IL-15Rα Sushi domain. In various embodiments, the VitoKine construct of the present invention comprises an IL-2Rα extracellular domain or a D3 domain which is a functional fragment thereof. In various embodiments, the VitoKine construct of the present invention comprises a D3 domain which is an IL-2Rα Sushi domain. In various embodiments, the VitoKine construct of the present invention comprises a D3 domain which is a variant (mutant) of the IL-2Rα Sushi domain. In various embodiments, the variant (or mutant) of the IL-2Rα Sushi domain comprises an amino acid change to a sequence derived from the sequence described in SEQ ID NO: 10. In various embodiments, the amino acid change is one or more amino acid substitutions at positions 36, 38, 42, or 43 of SEQ ID NO: 10. In various embodiments, the amino acid change is a substitution of R to A at position 36, a substitution of K to E at position 38, a substitution of L to G at position 42, and a substitution of Y to A at position 43.In various embodiments, the D3 domain can block the functional activity of D2 until it is activated at the target therapeutic site. In various embodiments, the D3 domain is designed to promote cleavage by proteolysis and subsequent dissociation and diffusion after activation. 【0014】 In various embodiments, the D1, D2, and D3 domains of the VitoKine construct are linked by a protease-cleavable polypeptide linker sequence. In various embodiments, the D1, D2, and D3 domains of the VitoKine construct are linked by a polypeptide linker sequence that cannot be cleaved by a protease. In various embodiments, both L1 and L2 of the VitoKine construct of the present invention are protease-cleavable peptide linkers. In various embodiments, L1 of the VitoKine construct of the present invention is a protease-cleavable peptide linker, and L2 is an incleavable peptide linker. In various embodiments, L1 of the VitoKine construct of the present invention is an incleavable peptide linker, and L2 is a protease-cleavable peptide linker. In various embodiments, both L1 and L2 of the VitoKine construct of the present invention are incleavable linkers. In various embodiments, the non-cleavable linker is rich in G / S content (for example, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more amino acids in the linker are G or S). Each peptide linker sequence can be independently selected. In various embodiments, the protease-cleavable linker is selected from the group of sequences described in SEQ ID NOs. 71-96 and SEQ ID NOs. 157-161. In various embodiments, the protease-cleavable linker may have additional peptide spacers of variable length at the N-terminus, the C-terminus, or both ends of the cleavable linker. In various embodiments, the non-cleavable linker is selected from the group of sequences described in SEQ ID NOs. 107-127. In various embodiments, the linker is flexible or rigid and has a variety of lengths. 【0015】 In various embodiments, the D2 and D3 domains of the VitoKine construct are located at the N-terminus of the D1 domain, as shown in Figure 1A. In various embodiments, the D2 and D3 domains of the VitoKine construct are located at the C-terminus of the D1 domain, as shown in Figure 1B. 【0016】 In various embodiments, the D1, D2, and D3 domains of the VitoKine construct may be monomers, dimers, or combinations of dimers and monomers, such as D1 being a dimer and D2 and D3 being monomers. 【0017】 In another embodiment, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject in need thereof. In one embodiment, the subject is a human subject. In various embodiments, the cancer is selected from pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, rhabdomyosarcoma, or any other cancer. 【0018】 In another embodiment, the Disclosure provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the Invention in combination with a second therapeutic method selected from the group consisting of: cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiotherapy, stem cell transplantation, cell therapies including CAR-T, CAR-NK, iPS-induced CAR-T or iPS-induced CAR-NK, and vaccines such as Calmette-Guérin bacillus (BCG). In various embodiments, the combination therapy may include administering a therapeutically effective amount of immunotherapy to the subject, where the immunotherapy is a depleting antibody against a specific tumor antigen. Therapy using antibodies; therapy using antibody-drug conjugates; therapy using agonist antibodies, antagonist antibodies, or inhibitory antibodies against costimulatory or coinhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, PD-L1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, Siglec-7, Siglec-8, Siglec-9, Siglec-15, and VISTA; therapy using bispecific T-cell inducing antibodies (BiTE®) such as blinatumomab; therapy including administration of biological response modifiers such as IL-12, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ; therapy using therapeutic vaccines such as cyproisel T; dendritic Treatments include, but are not limited to, those using cell vaccines or tumor antigen peptide vaccines; treatments using chimeric antigen receptor (CAR)-T cells; treatments using CAR-NK cells; treatments using tumor-infiltrating lymphocytes (TILs); treatments using adoptive transplant antitumor T cells (T cells grown in vitro and / or TCR transgenic); treatments using TALL-104 cells; treatments using immunostimulants such as the Toll-like receptor (TLR) agonist CpG and imiquimod; and treatments using vaccines such as BCG; the above combination therapies increase the killing of tumor cells by effector cells, meaning that a synergistic effect exists between the VitoKine construct and immunotherapy when administered simultaneously. 【0019】 In another embodiment, the present disclosure provides a method for treating a viral infection in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject in need thereof. In one embodiment, the subject is a human subject. In various embodiments, the virus is HIV. 【0020】 In another embodiment, the present disclosure provides a method for treating a viral infection in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention in combination with a second therapeutic method, which includes but is not limited to acyclovir, Epclusa, Mavyret, zidovudine, and enfuvirtide. 【0021】 In another embodiment, the present disclosure provides a method for treating an autoimmune disease in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject in need thereof. In one embodiment, the subject is a human subject. In various embodiments, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), pemphigus vulgaris, myasthenia gravis, hemolytic anemia, thrombocytopenic purpura, Graves' disease, Sjögren's disease, dermatomyositis, Hashimoto's disease, polymyositis, inflammatory bowel disease, multiple sclerosis (MS), diabetes mellitus, rheumatoid arthritis, and scleroderma. 【0022】 In another embodiment, the present disclosure provides a method for treating an inflammatory disease in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject in need thereof. In one embodiment, the subject is a human subject. In various embodiments, the inflammatory disease is selected from the group consisting of Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, vacant colitis, Behçet's syndrome, and unclassifiable colitis. 【0023】 In various embodiments, inflammatory diseases include achalasia, adult Still's disease, agammaglobulinemia, amyloidosis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune inner ear disease, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuronal neuropathy, Barlow's disease, Behçet's disease, benign mucosal pemphigoid, Castleman disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy, chronic relapsing polymyelitis, Churg-Strauss syndrome, scarring pemphigoid, Cogan syndrome, Coxsackie myocarditis, Crest syndrome, herpetiform dermatitis, Devic's disease / neuromyelitis optica, lupus discoid, Dressler syndrome, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, fibrotic alveolitis, giant cell arteritis, giant cell myocarditis, Henoch-Schönlein purpura, herpes zoster of pregnancy or bullous pemphigoid of pregnancy, IgA nephropathy, IgG4-related sclerosing disease, immune-related adverse events, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile myositis, Lambert-Eaton syndrome, leukocytosis-destructive vasculitis, lichen planus, lichen sclerosing Sclerosis, woody conjunctivitis, linear IgA disease, chronic Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Mohren's ulcer, Mucha-Habermann disease, multifocal motor neuropathy, optic neuritis, relapsing rheumatoid arthritis, PANDAS, paraneoplastic cerebellar degeneration, Parry-Romberg syndrome, ciliary body squamous cellulitis, Personage-Turner syndrome, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polyglandular syndrome, polymyalgia rheumatica, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary sclerosing cholangitis, progesterone dermatitis Dematitis, psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum, Raynaud's phenomenonThe disease is selected from a group consisting of Phenomenon, reflex sympathetic dystrophy, relapsing polychondritis, retroperitoneal fibrosis, scleritis, sperm and testicular autoimmunity, generalized rigidus syndrome, subacute bacterial endocarditis, Suzak syndrome, sympathetic ophthalmitis, Takayasu arteritis, thrombocytopenic purpura, Trosa Hunt syndrome, transverse myelitis, undifferentiated connective tissue disease, and other autoimmune inflammatory diseases such as Vogt-Koyanagi-Harada disease. 【0024】 In another embodiment, the disclosure provides the use of a VitoKine construct for preparing a pharmaceutical product for the treatment of cancer. 【0025】 In another embodiment, the disclosure provides the use of a VitoKine construct for preparing a pharmaceutical product for the treatment of a viral infection. 【0026】 In another embodiment, the disclosure provides the use of a VitoKine construct for preparing a pharmaceutical product for the treatment of an autoimmune disease. 【0027】 In another embodiment, the present disclosure provides the use of a VitoKine construct for preparing a pharmaceutical product for the treatment of inflammation. 【0028】 In another embodiment, the present disclosure provides the use of the VitoKine construct of the present invention in combination with a second therapeutic agent or cell therapy capable of treating cancer, viral infections, autoimmune diseases, or inflammation. 【0029】 In another embodiment, the Disclosure provides isolated nucleic acid molecules comprising polynucleotides encoding the VitoKine constructs of the Disclosure. In another embodiment, the Disclosure provides vectors comprising the nucleic acids described herein. In various embodiments, the vectors are expression vectors. In another embodiment, the Disclosure provides isolated cells comprising the nucleic acids of the Disclosure. In various embodiments, the cells are host cells comprising the expression vectors of the Disclosure. In another embodiment, a method for producing VitoKine constructs is provided by culturing the host cells under conditions that promote the expression of the proteins or polypeptides described herein. 【0030】 In another embodiment, the disclosure provides a pharmaceutical composition comprising an isolated VitoKine construct mixed with a pharmaceutically acceptable carrier. [Brief explanation of the drawing] 【0031】 [Figure 1] Figure 1 shows typical types of VitoKine constructs of the present invention. Figure 1A shows a VitoKine construct in which the D2 and D3 domains are located at the C-terminus of the D1 domain. Figure 1B shows a VitoKine construct in which the D2 and D3 domains are located at the N-terminus of the D1 domain. Figures 1C-1E show VitoKine constructs in which the D2 and D3 domains are located at the C-terminus of the D1 domain, where the D1 domain is an antibody. (1C) VitoKine construct having D2 and D3 domains located at the C-terminus of an antibody heterodimer weight chain. (1D) VitoKine construct having D2 and D3 domains located at the C-terminus of an antibody homodimer weight chain. (1E) VitoKine construct having D2 and D3 domains located at the C-terminus of an antibody light chain. Similarly, the D2 and D3 domains can be located at the N-terminus of the antibody heavy chain (both heterodimer and homodimer) and light chain. [Figure 2]Figure 2 shows the proposed activation mechanism of the VitoKine construct. An exemplary VitoKine contains two protease-cleavable linkers; activation of protease 1 by cleavage of the L1 linker results in active form 1; activation of protease 2 by cleavage of the L2 linker results in active form 2; and activation by both proteases by cleavage of both the L1 and L2 linkers results in active form 3. Figure 2A shows an example where the D3 domain remains non-covalently complexed with D2 after proteolysis. Figure 2B shows an example where D3 dissociates from D2 and diffuses after protease cleavage. [Figure 3] Figure 3 shows the protein profiles of exemplary IL-15 Fc VitoKine P-0315 after protein A purification, including A) SDS-PAGE in the absence and presence of a reducing agent, and B) size exclusion chromatogram. [Figure 4] Figure 4 shows the binding affinity and functional activity of IL-15 Fc VitoKine P-0172 compared to the highly active IL-15 fusion protein P-0198. (A) Binding activity to IL-2Rβ as measured by ELISA assay; (B-C) Induction of CD69 expression on human CD8+ T cells (B) and NK cells (C) of fresh human PBMCs by FACS analysis. [Figure 5] Figure 5 shows the functional activity of monomeric IL-15 Fc VitoKine P-0170 compared to the highly active IL-15 fusion protein P-0166. The induction of CD69 expression on human CD8+ T cells from fresh human PBMCs was measured and analyzed by FACS. [Figure 6] Figure 6 shows the induction of CD69 expression on human PBMCs A) CD8+ T cells and B) NK(CD56+) cells by exemplary IL-15 Fc VitoKine constructs with different linker lengths (P-0204, P-0205, and P-0206) compared to the highly active IL-15 / IL-15Rα Fc fusion protein P-0165. [Figure 7]Figure 7 shows the proliferation of NK (CD56+) cells in human PBMCs by exemplary IL-15 Fc VitoKine constructs (P-0202, P-0203, and P-0204) with different L1 and L2 linkers, compared to fully active IL-15 / IL-15Rα-Fc fusion proteins P-0207 and P-0217. [Figure 8] Figure 8 shows the proliferation of A) CD8+ T cells and B) NK (CD56+) cells in human PBMCs by exemplary IL-15 Fc VitoKine constructs (P-0351, P-0488, and P-0489) with different L2 linker sequence compositions, as measured by FACS, compared to the IL-15 / IL-15Rα-Fc fusion protein P-0156. [Figure 9] Figure 9 shows SDS-PAGE analysis of in vitro proteolysis of IL-15 Fc VitoKine P-0315 using various amounts of MMP-2. [Figure 10] Figure 10 shows SDS-PAGE analysis of in vitro proteolysis of IL-15 Fc VitoKine P-0203 using uPA under different conditions to determine reaction conditions suitable for complete cleavage. [Figure 11] Figure 11 shows (A) SDS-PAGE analysis of IL-15 Fc VitoKine P-0203 before and after in vitro proteolysis by uPA. (B) Protein profile of active VitoKine P-0203 after removal of Fc-containing fragments by uPA digestion and protein A purification. [Figure 12]Figure 12 shows A) SDS-PAGE analysis of IL-15 Fc VitoKine P-0315 before and after in vitro proteolysis by MMP-2. The gel shows the profile of P-0315 after MMP-2 digestion and protein A purification; B) the protein profile of active form 2 of VitoKine P-0315 obtained from protein A purification after MMP-2 digestion; and C) the protein profile of active form 3 of VitoKine P-0315 obtained from protein A purification in flow-through mode after biproteolysis by both MMP-2 and uPA. [Figure 13] Figure 13 shows the activity evaluation of IL-15 Fc VitoKine P-0203 activated by the protease (uPA) by analyzing the induction of the activation marker CD69 on A) CD56+ NK cells and B) CD8+ T cells. The highly active IL-15 fusion protein P-0165 was included as a positive control. [Figure 14] Figure 14 shows the activity evaluation of two forms of protease-activated IL-15 Fc VitoKine P-0315 by analyzing the induction of the activation marker CD69 on A) CD56+ NK cells and B) CD8+ T cells. Active form 2 of P-0315 was obtained from MMP-2 digestion, and active form 3 of P-0315 was obtained from biproteolysis by both MMP-2 and uPA. P-0313, a highly active IL-15 fusion protein structurally similar to active form 2 of P-0315, was included as a positive control. [Figure 15] Figure 15 shows the activity evaluation of MMP-2 activated IL-15 Fc VitoKine P-0315 (active form 2) by analyzing the induction of the proliferation marker Ki67 on A) CD56+ NK cells and B) CD8+ T cells. For comparison, P-0351, which contains both non-cleavable L1 and L2 linkers and shares the same L2 linker length as P-0315, was included. [Figure 16]Figure 16 shows the dose-dependent and time-dependent effects of cleavable IL-15 Fc VitoKine P-0315 and non-cleavable IL-15 Fc VitoKine P-0351 on the proliferation of (A) CD8+ T cells, (B) NK cells, and (C) leukocytes in peripheral blood after a single injection in Balb / C mice. The fully active IL-15-Fc fusion P-0313 was included for comparison. Blood was collected on days 1, 3, 5, and 7, and lymphocyte phenotyping was performed by FACS analysis. Data are expressed as mean ± SEM. For statistical analysis, two-way ANOVA was performed, followed by Tukey's post-hoc test. Compared to the PBS group at each time point, ****p<0.0001, ***p<0.001, and *p<0.05. [Figure 17] Figure 17 shows the inhibition of lung metastatic nodules in a mouse CT26 lung metastasis model one day after four doses of P-0315, P-0351, P-0313, or PBS control administered every five days. The first dose was initiated one day after CT26 cell injection. Unless otherwise noted, all comparisons are relative to the PBS group;****p<0.0001;**p<0.01;*p<0.05. [Figure 18] Figure 18 shows the percentage of A) CD8+ T cells (%) and B) NK cells (%) in the total blood lymphocytes of CT26 metastatic mice. Cell counts were determined by flow cytometry 4 days after three intraperitoneal injections of P-0315, P-0351, P-0313, or PBS control at 5-day intervals. All comparisons are relative to the PBS group;****p<0.0001;**p<0.01;*p<0.05. [Figure 19]Figure 19 shows the antitumor activity of IL-15 Fc VitoKine P-0315 compared to fully active IL-15-Fc fusion P-0313 in an established CT26 mouse colorectal cancer tumor model. Growth curves of subcutaneous CT26 tumors in individual mice after two Q5D treatments are plotted over time for A) solvent PBS group, B) 0.1 mg / kg P-0315 group, C) 0.1 mg / kg P-0313 group, and D) mean tumor volume ± SEM for each treatment group. All comparisons are relative to solvent treatment; n=11 / group; ****P<0.0001. [Figure 20] Figure 20 shows immunopharmacodynamic profiling of peripheral blood from mice treated with IL-15 Fc VitoKine P-0315 or the highly active IL-15-Fc fusion P-0313 in a CT26 mouse colorectal cancer tumor model. Treatment was started 11 days after tumor transplantation and administered twice at 5-day intervals. The increase in the proliferation marker Ki67 in A) NK cells and B) CD8+ T cells was determined by flow cytometry on day 19. ****P<0.0001 compared to PBS. [Figure 21] Figure 21 shows immunopharmacological profiling of peripheral blood from mice treated with P-0315 or P-0313 in a CT26 mouse colorectal cancer tumor model. Treatment was administered twice, 5 days apart, starting 11 days after tumor transplantation. On day 19, the increase in the number of circulating total leukocytes (A), NK cells (B), and CD8+ T cells (C) per μl of whole blood was determined by flow cytometry. ****P<0.0001 compared to PBS. [Figure 22] Figure 22 shows immunopharmacological profiling of the spleen after P-0315 or P-0313 treatment in a CT26 mouse colorectal cancer tumor model. Treatment was administered twice at 5-day intervals, starting 11 days after tumor transplantation. On day 19, increases in the number of total leukocytes (A), NK cells (B), and CD8+ T cells (C) in the spleen were determined by flow cytometry. Compared to PBS, ****P<0.0001 and *P<0.05. [Figure 23]Figure 23 shows the activity evaluation of various IL-15 / IL-15Rα Fc fusion proteins with one or two amino acid substitutions at positions V63, I68, and Q108 by analyzing the induction of the proliferation marker Ki67 on A) CD8+ T cells and B) CD56+ NK cells. For comparison, the potent IL-15 / IL-15Rα Fc fusion protein P-0313 was included. [Figure 24] Figure 24 shows the activity evaluation of P-0764, an IL-15Q108S / IL-15Rα Fc fusion protein, and its corresponding Fc VitoKine, named P-0682, by analyzing the induction of the proliferation marker Ki67 on A) CD8+ T cells and B) CD56+ NK cells. For comparison, P-0313, a potent IL-15 / IL-15Rα Fc fusion protein, was included. [Figure 25] Figure 25 shows a comparison of the activity of non-cleavable IL-15 Fc VitoKine P-0351 with a reference, by analyzing the induction of the proliferation marker Ki67 on A) CD56+ NK cells and B) CD8+ T cells. [Figure 26] Figure 26 shows the protein profiles of exemplary IL-2 VitoKine P-0320 after protein A purification, A) SDS-PAGE in the absence and presence of a reducing agent, and B) size exclusion chromatogram. [Figure 27] Figure 27 shows the activity evaluation of two IL-2 Fc VitoKines, P-0320 (IL-2 fused to the C-terminus of Fc) and P-0329 (IL-2 fused to the N-terminus of Fc), by analyzing pStat5 levels in A) CD4-positive Foxp3+ / CD25-high Treg and B) CD4-positive Foxp3- / CD25-low CD4-normal T cell subsets in fresh human PBMCs. P-0250, a highly active IL-2-Fc fusion protein, was included as a positive control. [Figure 28]Figure 28 shows, A) SDS-PAGE analysis of IL-2 VitoKine P-0382 and its activation after MMP-2 digestion and subsequent Ni-Excel purification, and B) the protein profile of MMP-2 activated P-0382 purified by protein A in bind-and-elute mode. [Figure 29] Figure 29 shows the activity evaluation of protease-activated IL-2 Fc VitoKine P-0382 by analyzing pStat5 levels in A) CD4+Foxp3+ / CD25 high Treg and B) CD4+Foxp3- / CD25 low CD4 normal T (Tconv) cell subsets in fresh human PBMCs. Two active samples were purified with Ni-Excel resin to remove the protease (P-0382 active form (activ.) 1), or with Protein A to remove both the protease and the IL-2Rα Sushi domain produced by proteolysis (P-0382 active form 2). The highly active IL-2-Fc fusion protein P-0250 was included as a positive control. [Figure 30] Figure 30 shows the evaluation of IL-2 Fc VitoKine P-0398 activity before and after MMP-2 proteolysis by analyzing pStat5 levels in A) CD4+Foxp3+ / CD25highTreg and B) CD4+Foxp3- / CD25lowCD4Tconv cell subsets in fresh human PBMCs. P-0382, which differs from P-0398 only in L2 linker length, and P-0250, a highly active IL-2-Fc fusion protein, were included for comparison. [Figure 31] Figure 31 shows the ELISA binding of P-0704, P-0707, P-0708, and P-0709, which are IL-2 variant Fc fusion proteins with different amino acid substitutions at position P65, to IL-2Rα. P-0689, the wild-type IL-2 counterpart, is included for comparison. [Figure 32]Figure 32 shows the activity evaluation of P-0704 and P-0689 by analyzing the induction of the proliferation marker Ki67 on CD8+ T cells in fresh human PBMCs. P-0704 is an IL-2P65R Fc fusion protein that has lost its binding activity to IL-2Rα, and P-0689 is the wild-type IL-2 counterpart. [Figure 33] Figure 33 shows the ELISA binding of various IL-2RαSushi variants, P-0751, P-0752, and P-0753, to the monovalent wild-type IL-2 Fc fusion protein P-0689. P-0757, which contains wild-type IL-2RαSushi, was included for comparison. [Figure 34] Figure 34 shows the activity evaluation of IL-2 Fc VitoKine having wild-type IL-2RαSushi as the D3 domain (P-0701) or one of the IL-2RαSushi variants as the D3 domain (P-0754, P-0755, and P-0756). P-0704, an IL-2P65R Fc fusion protein that lost its IL-2Rα binding activity but retained all IL-2Rβγ activity, was included as a control. Activity was evaluated by analyzing the induction of the proliferation marker Ki67 on A) CD8+ T cells and B) CD56+ NK cells, as measured by flow cytometry. [Figure 35] Figure 35 shows the activity evaluation of IL-2P65R Fc fusion protein P-0704 and its corresponding Fc VitoKine, which have either wild-type IL-2RαSushi as the D3 domain (P-0745) or one of the IL-2RαSushi variants as the D3 domain (P-0807, P-0808, and P-0809). Activity was evaluated by analyzing the induction of the proliferation marker Ki67 on A) CD8+ T cells and B) CD56+ NK cells, as measured by flow cytometry. [Figure 36]Figure 36 shows the activity evaluation of P-0755, an IL-2 Fc VitoKine with the IL-2RαSushiL42G variant as the D3 domain, before and after in vitro protease activation. P-0704, an IL-2P65R Fc fusion protein that fully retains IL-2Rβγ activity, was included as a control. Activity was evaluated by analyzing the induction of the proliferation marker Ki67 on A) CD8+ T cells and B) CD56+ NK cells in fresh human PBMCs. [Figure 37] Figure 37 shows the activity evaluation of IL-15 Fc VitoKine P-0315 compared to the antibody IL-15 VitoKine P-0485, by analyzing the induction of the proliferation marker Ki67 on A) CD8+ NK cells and B) CD56+ T cells in fresh human PBMCs by flow cytometry. [Figure 38] Figure 38 shows flow cytometry analysis of Ki67 expression on CD8+ T cells induced by various IL-15 VitoKines, compared to their respective non-VitoKine fusion counterparts. A) IL-15 Fc VitoKine P-0315 versus its corresponding Fc fusion in human PBMCs; B) IL-15 antibody VitoKine P-0875 versus its corresponding antibody fusion P-0870 and Fc fusion P-0773 in human PBMCs; C) P-0875 versus P-0773 in cynomolgus monkey PBMCs. [Figure 39] Figure 39 shows the activity evaluation of various IL-2 antibodies VitoKine P-0800, P-0830, P-0831, and P-0802 compared to the non-VitoKine IL-2 antibody fusion P-0782, by analyzing the induction of the proliferation marker Ki67 on A) CD8+ T cells and B) CD56+ NK cells, as measured by flow cytometry. The four IL-2 antibodies VitoKine differ only in their IL-2 moieties, which have varying levels of binding strength to IL-2Rα. [Figure 40]Figure 40 shows the protease cleavage and activation of the IL-2 antibody VitoKine P-0872 by dose-dependent induction of Ki67 expression on CD8+ T cells in human PBMCs, as measured by A) reduced SDS-PAGE gel and B) flow cytometry analysis. P-0872 contains a monovalent IL-2 moiety and a single protease-cleavable linker ligating the D2 and D3 domains. [Figure 41] Figure 41 shows the protease cleavage and activation of the IL-2 antibody VitoKine P-0929 by dose-dependent induction of Ki67 expression on CD8+ T cells in human PBMCs, as measured by A) reduced SDS-PAGE gel and B) flow cytometry analysis. P-0929 contains a bivalent IL-2 moiety and a biprotease-cleavable linker. [Figure 42] Figure 42 shows the binding of IL-2Rβ-based blocking peptides (L01, L02, L03, L04, and L05) to IL-15 in ELISA format. [Figure 43] Figure 43 shows the binding of IL-15 fusion proteins (P-0153, P-0159, P-0160, and P-0161) to plate-immobilized IL-2Rβ. P-0159, P-0160, and P-0161 contain various IL-2Rβ-based blocking peptides. [Figure 44] Figure 44 shows size exclusion chromatograms of four IL-2 VitoKines (P-0320, P-0382, P-0362, and P-0379) (Figures 40B-40E) in comparison with their corresponding Fc fusion protein, P-0250 (Figure 44A). For comparison, P-0531, which differs from P-0250 in that it has a single amino acid substitution S125I in IL-2, is included (Figure 44F). [Figure 45] Figure 45 shows SDS-PAGE gels of IL-15 Fc VitoKine P-0389(A) and P-0315(B) having different D3 domains. [Figure 46]Figure 46 shows the antitumor effects of the IL-2 PD-1 antagonist antibodies VitoKine P-0922A, P-0928A, and P-0929A compared to their non-resectable counterpart P-0877 in an established MC38 mouse colon cancer model. The tumor size of individual mice in each group at day 7 after monotherapy is illustrated. [Modes for carrying out the invention] 【0032】 This disclosure provides a novel “VitoKine” construct as a platform technology intended for use in the treatment of cancer, viral infections, autoimmune diseases, or inflammatory diseases, which reduces systemic on-target toxicity and increases the therapeutic index of cytokines. The VitoKine platform is defined by the construct shown in Figure 1 and the proposed activation method shown in Figure 2. Referring to Figure 1, the novel VitoKine construct of the present invention comprises the following three domains: 1) a D1 domain ("D1") selected from the group consisting of a tissue targeting domain; a half-life extension domain; an immune checkpoint regulator targeting domain; or a dual functional partial domain; 2) a D2 domain ("D2") which is an “active partial domain”; and 3) a D3 domain ("D3") which is a “shielding partial domain.” Importantly, the D3 domain is capable of shielding the functional activity of D2 until it is activated at the site of interest. 【0033】 These three domains are linked by a linker of variable length and rigidity, which contains a protease-cleavable sequence. This sequence acts as a peptide substrate for a specific protease subtype whose expression is elevated or dysregulated at the site of injury, allowing the functional D2 domain to be exposed or released at that site. Optimization of the linker's length and composition achieved optimal shielding of the D2 domain's reachability to its receptor, reducing systemic engagement while maintaining VitoKine's stability in the bloodstream and enabling efficient cleavage after encountering the specific protease at the target site. The design of "VitoKine" was also rationally based on insights into the intermolecular interactions between cytokines and their cognitive receptors. Cytokine receptors typically function as oligomeric complexes consisting of 2-4 receptor subunits. These different subunits perform specialized functions such as ligand binding and signal transduction. The α subunit of the cytokine receptor is a binding receptor that confers ligand specificity, enhances ligand interaction with signaling receptors, and converts signaling receptors from low affinity to high affinity. The D3 domain of VitoKine is preferably a cognitive binding receptor for the D2 domain. After cleavage, even if the D3 domain dissociates, it can reassociate with the D2 domain, potentially restoring the local binding and signaling activity of the D2 domain completely. In other words, the D3 domain may have a dual role in regulating the function of the D2 domain. The D3 domain keeps the D2 domain inactive when VitoKine is inactive, and can participate in D2 function when VitoKine is cleaved and activated. However, the D3 domain can be any protein, peptide, antibody, antibody fragment, polymer, or nucleotide that can shield D2 activity. 【0034】 In another embodiment, the addition of the D3 domain may result in a significant improvement in the development suitability profile of the VitoKine construct, enhanced expression yield, and reduced aggregation tendency. 【0035】 In addition to functioning as an adjunct domain that shields the functional activity of the D2 domain, the D1 domain may also be a half-life extension domain that extends the circulating half-life of VitoKine. The D1 domain is also a disease-targeting or tissue-targeting motif that specifically directs VitoKine to the target site, and by limiting VitoKine activation to the local area, the therapeutic index can be further improved. Therefore, the "VitoKine" platform has the advantage of enabling selective activation of cytokines at the target site, reducing systemic toxicity while increasing the therapeutic effect at the affected site and improving the therapeutic index. 【0036】 Although the D2 domain of the VitoKine construct is the active portion, it remains inactive until it is locally activated by a protease that is upexpressed in the affected tissue. This limits the binding of the active portion to receptors on the cell surface of surrounding or non-affected cells or tissues, suppressing excessive activation of the pathway and reducing undesirable "on-target" but "extra-tissue" toxicity. Furthermore, the inactivity of the VitoKine active portion before protease activation significantly reduces the possibility of antigen sinking, resulting in an extended in vivo half-life and improved bioavailability and efficacy at the therapeutic target site. Moreover, based on the present invention, the VitoKine platform can enhance the protein development suitability profile, including, but not limited to, improved expression levels and reduced aggregation tendencies, such as when a cognitive receptor α is used as the D3 domain. 【0037】 While cleavable links are preferable for most vitokines to limit systemic activation and release the active domain at the target site after administration, non-cleavable linkers may be desirable to allow sustained systemic exposure to pharmacologically active vitokines, albeit with lower potency, and to improve therapeutic effects. 【0038】 In exemplary embodiments, the VitoKine construct comprises an active moiety (D2) based on IL-15, an IL-15 variant, IL-2, or an IL-2 variant. In these IL-15-based and / or IL-2-based VitoKine constructs, the unique non-signaling α subunit of the receptor for each cytokine is used as one of the shielding subdomains (D3) via a protease-cleavable linker to reversibly shield cytokine activity. Depending on the contrasting properties of each receptor complex and the various requirements of the various disease indications to be treated by the VitoKine molecule, the shielding α subunit may preferably complex with the activated cytokine via non-covalent association after protease-cleavage of the linker (e.g., in the case of IL-15) or may preferably dissociate (e.g., in the case of IL-2). As a result, amino acid modifications of the α-receptor to regulate its binding affinity to cognitive cytokines may be necessary and beneficial. 【0039】 In exemplary embodiments, the VitoKine construct comprises an active moiety (D2) based on IL-15, an IL-15 variant, an IL-2, or an IL-2 variant. In these IL-15-based and / or IL-2-based VitoKine constructs, a receptor covalent β-subunit or receptor β-based blocking peptide is used as a shielding partial domain (D3) via a protease-cleavable linker to reversibly shield cytokine activity. 【0040】 This concept, which involves conjugating cognitive receptors, proteins, antibodies, antibody fragments, and binding peptides to cytokines via an activatable linker that shields their functional activity until activated at the target therapeutic site, can be adapted to various cytokines, such as, but not limited to, IL-4, IL-7, IL-9, IL-10, IL-12, IL-22, IL-23, and chemokines like TGFβ and CXCR3, or various growth factors like the TNF family, TGFα, and TGFβ, as well as hormones. The same concept can also be applied to other proteins to create proproteins, thereby enhancing targeting to the affected area and expanding therapeutic utility. 【0041】 definition In this specification, the terms “polypeptide,” “peptide,” and “protein” are used synonymously and refer to polymers of amino acid residues. In various embodiments, “peptide,” “polypeptide,” and “protein” are amino acid chains in which α-carbons are linked together by peptide bonds. The terminal amino acid at one end of the chain (amino-terminus) has a free amino group, while the terminal amino acid at the other end of the chain (carboxy-terminus) has a free carboxyl group. As used herein, the term “amino-terminus” (abbreviated as N-terminus) refers to the free α-amino group on the amino acid at the amino-terminus of a peptide, or to the α-amino group of any other amino acid within the peptide (an amino group if it is participating in a peptide bond). Similarly, the term “carboxy-terminus” (abbreviated as C-terminus) refers to the free carboxyl group at the carboxy-terminus of a peptide, or to the carboxyl group of any other amino acid within the peptide. Peptides also include substantially any polyamino acid, including, but not limited to, peptide mimetic compounds, such as those in which amino acids are linked by ethers rather than amide bonds. 【0042】 The polypeptides of this disclosure also include polypeptides that have been modified in any way and for any reason, such as (1) reduced sensitivity to proteolysis, (2) reduced sensitivity to oxidation, (3) alteration of binding affinity for the purpose of protein complex formation, (4) alteration of binding affinity, and (5) conferring or altering other physicochemical or functional properties. 【0043】 As used herein, an amino acid "substitution" refers to the substitution of a specific amino acid at a particular position within a parent polypeptide sequence with a different amino acid within the polypeptide. Amino acid substitutions can be produced using genetic or chemical methods well known in the art. For example, a single amino acid substitution (e.g., a conserved amino acid substitution) or multiple amino acid substitutions may be made in the natural sequence (e.g., in the polypeptide portion outside of domains forming intermolecular contacts). A "conserved amino acid substitution" refers to the substitution of an amino acid within a polypeptide with a functionally similar amino acid. The following six groups each contain amino acids that are conserved substitutions of each other. 1) Alanine (A), serine (S), and threonine (T) 2) Aspartic acid (D) and glutamic acid (E) 3) Asparagine (N) and glutamine (Q) 4) Arginine (R) and Lysine (K) 5) Isoleucine (I), leucine (L), methionine (M), and valine (V) 6) Phenylalanine (F), tyrosine (Y), and tryptophan (W) 【0044】 A "non-conservative amino acid substitution" refers to the substitution of a component of one class with a component of another class. In various embodiments, the hydrophobicity and hydrophilicity index of amino acids may be considered when making such changes. Each amino acid is assigned a hydrophobicity and hydrophilicity index based on its hydrophobicity and charge properties. The hydrophobic and hydrophilic indices for each are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). 【0045】 The importance of amino acid hydrophobicity and hydrophilicity indices in conferring interactive biological functions to proteins is well understood in the art (see, for example, Kyte et al., 1982, J.Mol.Biol., 157:105-131). It is known that similar biological activity can be maintained by substituting a particular amino acid with another amino acid having a similar hydrophobicity or hydrophilicity score. When inducing changes based on hydrophobicity and hydrophilicity indices, various embodiments include substitutions of amino acids whose hydrophobicity and hydrophilicity indices are within ±2. Various embodiments include substitutions within ±1, and various embodiments include substitutions within ±0.5. 【0046】 Furthermore, it is understood in the art that substitutions of similar amino acids can be efficiently performed based on hydrophilicity, especially when the biologically functional proteins or peptides thus produced are intended for use in the immunological embodiments disclosed herein. In various embodiments, the maximum local mean hydrophilicity of a protein, controlled by the hydrophilicity of adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with the biological properties of the protein. 【0047】 The following hydrophilic values ​​are assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). When similar changes occur based on hydrophilicity values, various embodiments include substitutions between amino acids with hydrophilicity values ​​of ±2 or less, substitutions of ±1 or less, and substitutions of ±0.5 or less. 【0048】 Examples of amino acid substitutions are listed in Table 1. TIFF0007872587000001.tif189170 【0049】 Those skilled in the art can determine preferred variants of the polypeptides described herein using well-known methods. In various embodiments, those skilled in the art can identify preferred regions of the molecule that can be altered without disrupting activity by targeting regions not considered important for activity. In other embodiments, those skilled in the art can identify residues and portions of the molecule that are conserved among similar polypeptides. In further embodiments, even regions that may be important for biological activity or structure can be subjected to conserved amino acid substitution without disrupting biological activity or adversely affecting the polypeptide structure. 【0050】 Furthermore, those skilled in the art can investigate structural-functional studies to identify residues within similar polypeptides that are important for their activity or structure. Taking such comparisons into account, those skilled in the art can predict the importance of amino acid residues within a polypeptide that correspond to amino acid residues that are important for the activity or structure of similar polypeptides. Those skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues. 【0051】 Furthermore, those skilled in the art can analyze the three-dimensional structure and amino acid sequence in relation to the structure of a similar polypeptide. Based on such information, they can predict the arrangement of amino acid residues in the polypeptide with respect to its three-dimensional structure. In various embodiments, those skilled in the art can select amino acid residues predicted to be present on the polypeptide surface in a way that avoids fundamental changes, as such residues may be involved in important interactions with other molecules. Furthermore, those skilled in the art can prepare test variants containing a single amino acid substitution for each of the desired amino acid residues. These variants can then be screened using activity quantification methods known to those skilled in the art. Using such variants, information on suitable variants can be gathered. For example, if it is found that a change to a particular amino acid residue results in disruption of activity, an undesirable reduction of activity, or inappropriate activity, variants with such changes can be avoided. In other words, based on information gathered from such conventional experiments, those skilled in the art can easily identify amino acids, alone or in combination with other mutations, that should be avoided for further substitution. 【0052】 The terms “polypeptide fragment” and “cleaved polypeptide,” as used herein, refer to polypeptides having deletions at the amino and / or carboxyl terminals compared to the corresponding full-length protein. In various embodiments, the fragments may have amino acid lengths of, for example, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more. In various embodiments, the fragments may also have amino acid lengths of, for example, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 450 or less, 400 or less, 350 or less, 300 or less, 250 or less, 200 or less, 150 or less, 100 or less, 50 or less, 25 or less, 10 or less, or 5 or less. The fragment may further contain one or more additional amino acids at one or both of its ends, for example, amino acid sequences derived from different natural proteins (e.g., Fc or leucine zipper domain) or artificial amino acid sequences (e.g., artificial linker sequences). 【0053】 The terms “polypeptide variant,” “hybrid polypeptide,” and “polypeptide variant,” as used herein, refer to a polypeptide comprising an amino acid sequence in which, compared to another polypeptide sequence, one or more amino acid residues are inserted into the amino acid sequence, one or more amino acid residues are deleted from the amino acid sequence, and / or one or more amino acid residues are substituted in the amino acid sequence. In various embodiments, the number of inserted, deleted, or substituted amino acid residues can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acid lengths. The hybrids of this disclosure encompass fusion proteins. 【0054】 A "derivative" of a polypeptide is a chemically modified polypeptide, such as one that has been attached to another chemical moiety, such as polyethylene glycol or albumin (e.g., human serum albumin), or that has been phosphorylated or glycosylated. 【0055】 In this specification, the term "% sequence identity" is used synonymously with the term "% identity" and refers to the value of amino acid sequence identity between two or more peptide sequences, or the value of nucleotide sequence identity between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same as 80% sequence identity measured by a specified algorithm, meaning that a given sequence has at least 80% identity with respect to another sequence of a different length. In various embodiments, % identity is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence identity with respect to a given sequence. In various embodiments, the percentage identity is, for example, within the range of about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%. 【0056】 In this specification, the term "% sequence homology" is used synonymously with the term "% homology" and refers to the value of amino acid sequence homology between two or more peptide sequences, or the value of nucleotide sequence homology between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% homology means the same as 80% sequence homology measured by a specified algorithm, i.e., homologs of a given sequence have more than 80% sequence homology over a certain length of the given sequence. In various embodiments, % homology is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, or more, with respect to a given sequence. In various embodiments, the percentage homology is, for example, in the range of about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%. 【0057】 Examples of computer programs that can be used to verify the identity between two sequences include, but are not limited to, a set of BLAST programs publicly available on the NCBI website, such as BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN. See also Altschul et al., J.Mol.Biol., vol. 215: pp. 403-10, 1990 (particularly in relation to the published initial settings, i.e., parameter w=4, parameter t=17), and Altschul et al., Nucleic Acids Res., vol. 25: pp. 3389-3402, 1997. When evaluating a given amino acid sequence by comparing it with amino acid sequences in GenBank Protein Sequences or other public databases, sequence searches are typically performed using the BLASTP program. The BLASTX program is preferred for searching for nucleic acid sequences translated in all read frames against amino acid sequences in GenBank Protein Sequences and other public databases. Using initial parameters of open gap penalty = 11.0 and gap extension penalty = 1.0, both BLASTP and BLASTX are performed, utilizing the BLOSUM-62 matrix. 【0058】 In addition to calculating percent sequence identity, the BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA, Vol. 90: pp. 5873–5787, 1993). One of the similarity measures provided by the BLAST algorithm is the smallest sum probability (P(N)), which indicates the probability that a match occurs by chance between two nucleotide sequences or two amino acid sequences. For example, if the smallest sum probability when comparing a test nucleic acid to a reference nucleic acid is, for example, less than approximately 0.1, less than approximately 0.01, or less than approximately 0.001, the nucleic acid is considered similar to the reference sequence. 【0059】 As used herein, the term "modification" refers to any manipulation of the peptide backbone (e.g., amino acid sequence) or post-translational modification of a polypeptide (e.g., glycosylation). 【0060】 The term “knob-into-hole modification,” as used herein, refers to a modification within the interface between two immunoglobulin heavy chains in the CH3 domain. In one embodiment, the “knob-into-hole modification” includes, in one antibody heavy chain, the amino acid substitution T366W and optionally S354C, and in the other antibody heavy chain, the amino acid substitutions T366S, L368A, Y407V and optionally Y349C. The knob-into-hole technique has been described in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prot Eng, Vol. 9, pp. 617-621 (1996); and Carter, J Immunol Meth, Vol. 248, pp. 7-15 (2001), among others. 【0061】 The terms "biologically activated agent" or "VitoKine," as used herein, mean a compound that is a drug precursor and, after administration to a subject, releases the agent in vivo through several chemical or physiological processes such that the bioactive agent is converted into a product active against the target tissue. A bioactive agent is any compound that exhibits its pharmacological effects after being activated in vivo. In other words, a bioactive agent can be considered an agent that contains a special non-toxic protecting group transiently used to alter or remove undesirable properties of the parent molecule. 【0062】 The term "fusion protein," as used herein, refers to a fusion polypeptide molecule containing two or more genes that originally encoded separate proteins, wherein its components are linked to one another by peptide bonds, either directly or via peptide linkers. The term "fused," as used herein, refers to components being linked by peptide bonds, either directly or via one or more peptide linkers. 【0063】 A "linker" is a molecule that connects two other molecules via covalent bonds, ionic bonds, van der Waals bonds, or hydrogen bonds. For example, a nucleic acid molecule that links two non-complementary sequences by hybridizing to the 5' end of one complementary sequence and then to the 3' end of another complementary sequence. A "cleavable linker" refers to a linker that can be cleaved by decomposition, digestion, or other means to separate the two components linked by the linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, and lipases. Cleavable linkers can also be cleaved by environmental factors, such as changes in temperature, pH, or salt concentration. 【0064】 The term "peptide linker," as used herein, refers to a peptide containing one or more amino acids, typically 1 to 30 amino acids. Peptide linkers are either known in the art or described herein. Suitable non-immunogenic linker peptides include (G4S) n Peptide linker, (SG4) n Peptide linker, or G4 (SG4) n Examples include peptide linkers. "n" is usually a number from 1 to 10, typically from 2 to 4. 【0065】 "Pharmaceutical composition" refers to a composition suitable for pharmaceutical use in animals. A pharmaceutical composition comprises a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. "Pharmacologically effective amount" refers to the amount of agent effective in obtaining the intended pharmacological effect. "Pharmacologically acceptable carrier" refers to any standard pharmaceutical carrier, solvent, buffer, and pharmaceutical additive, such as phosphate-buffered saline, 5% glucose aqueous solution, emulsions such as oil-in-water or water-in-oil emulsions, and various types of wetting agents and / or auxiliaries. Suitable pharmaceutical carriers and formulations are listed in Remington's Pharmaceutical Sciences, 21st edition, 2005, Mack Publishing Co., Easton. "Pharmacologically acceptable salt" refers to a salt that can be incorporated into a compound for pharmaceutical use, such as metal salts (sodium salts, potassium salts, magnesium salts, calcium salts, etc.), ammonia salts, and organic amine salts. 【0066】 As used herein, “treatment” (and its grammatical variations such as “to treat” and “to treat”) refers to a clinical intervention to alter the natural course of a disease in an individual receiving treatment, which may be performed preventively or during the course of clinicopathology. Desired therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, symptom relief, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, improvement or mitigation of the condition, and remission or improved prognosis. As used herein, “mitigating” a disease, disorder, or condition means reducing the severity and / or frequency of the symptoms of the disease, disorder, or condition. Furthermore, as used herein, “treatment” encompasses descriptions of curative, symptomatic, and preventive treatments. 【0067】 The terms “effective dose” or “therapeutic effective dose,” as used herein, refer to an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease, such as improving, alleviating, reducing, and / or delaying one or more of its symptoms. With respect to cancer or the growth of other undesirable cells, an effective dose includes an amount sufficient to: (i) reduce the number of cancer cells; (ii) reduce the size of the tumor; (iii) inhibit, delay, slow to some extent, preferably stop, cancer cell infiltration into peripheral organs; (iv) inhibit tumor metastasis (i.e., slow to some extent, preferably stop); (v) inhibit tumor growth; (vi) suppress or delay tumor development and / or recurrence; and / or (vii) reduce to some extent one or more of the symptoms associated with cancer. An effective dose may be administered in one or more doses. 【0068】 The expressions “to administer” or “cause to be administered” refer to an act performed by a healthcare professional (e.g., a physician) or a person responsible for the patient’s medical care who manages and / or authorizes the administration of the drug / compound in question to a patient. Causing administration may include making a diagnosis and / or determining an appropriate treatment regimen and / or prescribing a specific drug / compound to a patient. Such prescribing may include, for example, a draft prescription form or annotations in a medical record. Where administration is described herein, “cause to be administered” is also intended. 【0069】 The terms “patient,” “individual,” and “subject” may be used synonymously and refer to mammals, preferably humans or non-human primates, but may also refer to domesticated mammals (e.g., dogs or cats), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., horses, cattle, pigs, sheep). In various embodiments, a patient may be a human being (e.g., adult male, adult female, adolescent male, adolescent female, boy, girl) receiving treatment from a physician or other healthcare professional in a hospital, psychiatric medical facility, as an outpatient, or in other clinical settings. In various embodiments, a patient may also be an immunocompromised or immune-impaired patient, such as, but not limited to, patients with primary immunodeficiency, AIDS; cancer patients and transplant patients taking certain immunosuppressants; and patients with genetic diseases affecting the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In various embodiments, the patient has immunogenic cancer, including, but is not limited to, bladder cancer, lung cancer, melanoma, and other cancers reported to have a high mutation rate (Lawrence et al., Nature, Vol. 499 (No. 7457): pp. 214-218, 2013). 【0070】 The term "immunotherapy" refers to cancer treatment, including, for example, treatment using depleted antibodies against specific tumor antigens; treatment using antibody-drug conjugates; and treatment using costimulatory or coinhibitory molecules (immune checkpoints), such as CTLA-4, PD-1, PDL-1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, SIRPα, CD47, GITR, ICOS, CD27, Siglec7, Siglec8, Siglec9, Siglec15, and V Therapy with agonist antibodies, antagonist antibodies, or inhibitor antibodies against ISTA, CD276, CD272, TIM-3, B7-H4; therapy with bispecific T cell inducing antibodies (BiTE®) such as blinatumomab; biological response modifiers, e.g., IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21, IL-22, GM-CSF, IFN-α, IFN-β, and IFN-γ, TGF-β antagonists or Therapies include: the administration of TGF-β traps; therapies using therapeutic vaccines such as cyproisel T; therapies using therapeutic viruses, including but not limited to oncolytic viruses such as T-vec; therapies using dendritic cell vaccines, or tumor antigen peptides or neoantigen vaccines; therapies using NK cells; therapies using chimeric antigen receptor (CAR)-T cells; therapies using CAR-NK cells; therapies using DC or T cells; therapies using iPS-induced NK cells; therapies using iPS-induced T cells, and therapies using vaccines such as Calmette-Guérin bacillus (BCG); therapies using tumor-infiltrating lymphocytes (TILs); therapies using adoptive transplant antitumor T cells (cells grown in vitro and / or TCR transgenic); therapies using TALL-104 cells; and therapies using immunostimulants, such as Toll-like receptor (TLR) agonists CpG, TLR7, TLR8, TLR9, and imiquimod. 【0071】 "Resistant or refractory cancer" refers to tumor cells or cancer that do not respond to conventional anti-cancer treatments, including chemotherapy, surgery, radiation therapy, stem cell transplantation, and immunotherapy. Tumor cells may be resistant or refractory from the start of treatment, or they may become resistant or refractory during treatment. Refractory tumor cells include tumors that do not respond to the start of treatment, or that show a short initial response but do not respond to treatment. Refractory tumor cells also include tumors that respond to anti-cancer therapy but do not respond to subsequent treatment. For the purposes of this invention, refractory tumor cells also include tumors that appeared to be inhibited by anti-cancer therapy but recurred within 5 years, in some cases within 10 years, or longer, after discontinuation of treatment. Anti-cancer treatments may include chemotherapy alone, radiation alone, targeted therapy alone, surgery alone, or a combination of these. For the sake of simplicity and not to limit the scope of explanation, it should be understood that the above-mentioned refractory tumor cells are interchangeable with resistant tumors. 【0072】 The term “tumor-associated antigen” (TAA) refers to a cell surface antigen that is selectively expressed by cancer cells or overexpressed in cancer cells compared to most normal cells. The terms “TAA variant” and “TAA variant,” as used herein, refer to a TAA comprising an amino acid sequence in which one or more amino acid residues are inserted, one or more amino acid residues are deleted, and / or one or more amino acid residues are substituted in the amino acid sequence, compared to another TAA sequence. In various embodiments, the number of inserted, deleted, or substituted amino acid residues can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acid lengths. 【0073】 The term "neoantigen" refers to cell surface antigens that the immune system has not previously been exposed to, particularly those selectively expressed by cancer cells or overexpressed in cancer cells compared to most normal cells, resulting from changes in host antigens due to radiation, chemotherapy, viral infection, tumor transformation / mutation, drug metabolism, etc. 【0074】 The term "antibody," as used herein, is used in its broadest sense and encompasses various antibody structures (IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgE), including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies or bifunctional antibodies), and antibody fragments insofar as they exhibit the desired antigen-binding activity. 【0075】 The term "antibody fragment," as used herein, refers to a molecule other than an intact antibody, which includes a portion of an intact antibody, and which binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′)2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. 【0076】 The term "Fab fragment," as used herein, refers to an immunoglobulin fragment comprising a light chain VL domain and a constant domain (CL), and a heavy chain VH domain and a primary constant domain (CH1). 【0077】 The terms “variable region” or “variable domain,” as used herein, refer to a domain of the heavy or light chain of an immunoglobulin or antibody, which is typically involved in the binding of the immunoglobulin or antibody to an antigen. The variable domains of the heavy and light chains of an immunoglobulin or antibody (VH and VL, respectively) typically have similar structures, and each domain comprises four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). 【0078】 As used herein, "human immunoglobulin" refers to an amino acid sequence that matches the amino acid sequence of an immunoglobulin, produced by a human or human cell, or derived from a non-human source utilizing the human immunoglobulin repertoire or other human immunoglobulin coding sequences. This definition of human immunoglobulin explicitly excludes humanized immunoglobulins that contain non-human antigen-binding residues. 【0079】 The terms “Fc domain” or “Fc region,” as used herein, are used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term encompasses both native and variant Fc regions. The Fc region of IgG consists of the CH2 domain and the CH3 domain. The CH3 region may, as used herein, be either the native CH3 domain or a variant CH3 domain (e.g., a CH3 domain in which a “knob” is introduced on one chain and a “hole” corresponding to the other chain; see U.S. Patent No. 5,821,333, as expressly incorporated herein by reference). Such variant CH3 domains may facilitate heterodimerization of two non-identical immunoglobulin heavy chains as described herein. Unless otherwise specified herein, amino acid residue numbering in the Fc region or constant region follows the EU numbering scheme. 【0080】 As used herein, the term "effector function" refers to the biological activity resulting from the Fc region of an immunoglobulin, which varies depending on the immunoglobulin isotype. Examples of effector functions of immunoglobulins include complement-dependent cell-mediated cytotoxicity (CDC) through binding to C1q, binding to Fc receptors, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), cytokine secretion, antigen uptake via immune complexes by antigen-presenting cells, decreased expression of cell surface receptors (e.g., B cell receptors), and B cell activation. 【0081】 As used herein, the terms “regulatory T cells” or “Treg cells” refer to a specific type of CD4+ T cell capable of suppressing the response of other T cells (effector T cells). Treg cells are characterized by the expression of CD4, the α subunit of the IL-2 receptor (CD25), and the transcription factor forkheadbox P3 (FOXP3) (Sakaguchi, Annu Rev Immunol, Vol. 22, pp. 531-62 (2004)), and play a crucial role in inducing and maintaining peripheral self-tolerance to antigens, including those expressed by tumors. 【0082】 The term "ordinary CD4+ T cells," as used herein, refers to CD4+ T cells other than regulatory T cells. 【0083】 As used herein, the term "selective activation of Treg cells" means the activation of Treg cells without the activation of other T cell subsets (such as CD4+ helper T cells, CD8+ cytotoxic T cells, or NKT cells) or natural killer (NK) cells. 【0084】 As used herein, “specific binding” means that binding is selective to the antigen and can be distinguished from undesirable or nonspecific interactions. The binding ability of an immunoglobulin to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or by other methods well known to those skilled in the art, such as surface plasmon resonance (SPR). 【0085】 The terms "affinity" or "binding affinity," as used herein, refer to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Generally, the affinity of molecule X for its partner Y can be expressed by the dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Surface plasmon resonance (SPR) is a specific method for measuring affinity. 【0086】 The term "decreased binding affinity," as used herein, refers to a decrease in affinity for each interaction, as measured by methods such as SPR. Conversely, "increased binding affinity" refers to an increase in binding affinity for each interaction. 【0087】 The term "polymer," as used herein, typically includes, but is not limited to, homopolymers; copolymers such as block copolymers, graft copolymers, random copolymers, and alternating copolymers; as well as terpolymers; and mixtures and modifications thereof. Furthermore, unless otherwise specified, the term "polymer" encompasses all possible geometric configurations of the substance. These configurations include, but are not limited to, isotactic symmetry, syndiotactic symmetry, and random symmetry. 【0088】 A "polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include not only natural nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), but also nucleic acid analogs. Nucleic acid analogs include those containing non-natural bases, which are nucleotides that participate in bonding with nucleotides other than natural phosphodiester bonds, and those containing bases added through bonding other than phosphodiester bonds. In other words, nucleic acid analogs include, but are not limited to, phosphorothioates, phosphorodithioates, phosphorotryesters, phosphoramidates, boranophosphates, methylphosphonic acid, chiral-methylphosphonic acid, 2-O-methylribonucleotides, and peptide-nucleic acid (PNA). Such polynucleotides can be synthesized using automated DNA synthesizers, etc. The term "nucleic acid" usually refers to large polynucleotides. The term "oligonucleotide" usually refers to short polynucleotides, typically about 50 nucleotides or less. It should be understood that when a nucleotide sequence is represented as a DNA sequence (i.e., A, T, G, C), the RNA sequence in which "U" is replaced by "T" (i.e., A, U, G, C) is also included. 【0089】 This specification describes polynucleotide sequences using conventional notation. The left end of a single-stranded polynucleotide sequence is the 5' end; the left-handed direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The addition of nucleotides from the 5' direction to the 3' direction to a nascent RNA transcript is referred to as the transcription direction. The DNA strand having the same sequence as mRNA is called the "coding strand"; the sequence on the DNA strand having the same sequence as the mRNA transcribed from DNA and located on the 5' side of the 5' end of the RNA transcript is called the "upstream sequence"; and the sequence on the DNA strand having the same sequence as RNA and located on the 3' side of the 3' end of the coding RNA transcript is called the "downstream sequence". 【0090】 "Complementary" refers to topological compatibility, that is, the agreement between the interacting surfaces of two polynucleotides. In other words, these two molecules can be described as complementary, and furthermore, their contact surface features are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide is hybridizable to the second polynucleotide under stringent hybridization conditions. 【0091】 "Specific hybridization" or "selective hybridization" refers to the preferential binding, double-stranding, or hybridization of a nucleic acid molecule to a specific nucleotide sequence under stringent conditions, when that sequence is present in a mixture of (e.g., whole-cell) DNA or RNA. The term "stringent conditions" refers to conditions under which a probe will preferentially hybridize to its target substance but less to other sequences, or not hybridize at all. In relation to nucleic acid hybridization experiments such as Southern and Northern hybridization, "stringent hybridization" and "stringent hybridization washing conditions" are sequence-dependent and will differ under different environmental parameters. Detailed guidelines for nucleic acid hybridization can be found in Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3rd edition, New York; and Ausubel et al. (eds.), Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, New York. 【0092】 Typically, highly stringent hybridization and highly stringent washing conditions are selected to be approximately 5°C lower than the melting temperature (Tm) of a particular sequence at specified ionic strength and pH. Tm is the temperature at which 50% of the target sequence hybridizes to a perfect match probe (under specified ionic strength and pH). Very stringent conditions are selected to be the same as the Tm of a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids with more than approximately 100 complementary residues on a filter in Southern or Northern blotting is 50% formalin + 1 mg heparin, 42°C, and overnight hybridization. An example of highly stringent washing conditions is 0.15 M NaCl, 72°C, and approximately 15 minutes. An example of stringent washing conditions is 0.2 × SSC washing, 65°C, and 15 minutes. For a description of SSC buffers, see Sambrook et al. A less stringent wash before a highly stringent wash can remove background probe signals. For example, a moderately stringent wash for double helix of over approximately 100 nucleotides is 1×SSC, 45°C, 15 minutes. A less stringent wash for double helix of over approximately 100 nucleotides is 4–6×SSC, 40°C, 15 minutes. Typically, in a particular hybridization assay, a signal-to-noise ratio (or more) that is twice (or greater) the signal-to-noise ratio observed with an unrelated probe indicates the detection of a specific hybridization. 【0093】 A "primer" refers to a polynucleotide capable of specifically hybridizing to a designated polynucleotide template, thereby providing a synthesis initiation site for a complementary polynucleotide. Such synthesis occurs when a polynucleotide primer is placed under conditions that induce synthesis, i.e., in the presence of nucleotides, a complementary polynucleotide template, and a polymerization agent such as DNA polymerase. Primers are typically single-stranded, but may also be double-stranded. Primers are typically deoxyribonucleic acid, but a wide variety of synthetic and natural primers are useful in many applications. Primers are complementary to the template and are designed to hybridize to the template to act as a synthesis initiation site, but they do not need to strictly reflect the template sequence. In such cases, the specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with chromogenic, radioactive, or fluorescent moieties and used as detectable portions. 【0094】 When used to refer to a polynucleotide, a "probe" is a polynucleotide capable of specifically hybridizing to a specified sequence of another polynucleotide. While a probe specifically hybridizes to the complementary polynucleotide of a target, it does not need to strictly reflect the complementary sequence of the template. In such cases, the specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. Probes can be labeled with chromogenic, radioactive, or fluorescent moieties, and these can be used as detectable regions. If the probe provides a synthesis starting point for the complementary polynucleotide, it can also be used as a primer. 【0095】 A "vector" is a polynucleotide that can be used to introduce another nucleic acid, to which it is ligated, into a cell. One type of vector is a "plasmid," which is a linear or circular double-stranded DNA molecule to which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-deficient retrovirus, replication-deficient adenovirus, and replication-deficient adeno-associated virus), into which additional DNA segments can be introduced. Certain vectors can autonomously replicate within the host cell into which they are introduced (e.g., bacterial vectors containing bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are replicated together with the host genome by being incorporated into the host cell's genome after introduction into the host cell. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide. 【0096】 A “regulatory sequence” is a nucleic acid that affects the expression of the nucleic acid to which it is functionally linked (e.g., expression level, timing, or site of expression). Regulatory sequences can exert their effects, for example, directly on the controlled nucleic acid or through the action of one or more other molecules (e.g., the regulatory sequence and / or polypeptides that bind to the nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, California, and Baron et al., 1995, Nucleic Acids Res., Vol. 23, pp. 3605-06. When a regulatory sequence affects the expression of a nucleotide sequence (e.g., expression level, timing, or site of expression), the nucleotide sequence is “functionally linked” to the regulatory sequence. 【0097】 A “host cell” is a cell that can be used for the expression of the polynucleotides of this disclosure. The host cell may be a prokaryote, for example, *E. coli*, or it may be a eukaryote, for example, a single-celled eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., human, monkey, hamster, rat, mouse, or insect cell), or a hybridoma. Typically, the host cell is a cultured cell capable of transformation or transfection with polypeptide-coding nucleic acids, enabling the expression of these polypeptide-coding nucleic acids. The term “recombinant host cell” may be used to refer to a host cell that has been transformed with or transfected with the nucleic acid of interest. The host cell may also be a cell that contains the nucleic acid but does not express it at the desired level, unless a regulatory sequence has been introduced into the host cell to functionally link with the nucleic acid. The term “host cell” is understood to refer not only to a specific target cell but also to the offspring and potential offspring of such a cell. Because certain modifications may occur in later generations due to mutations or environmental influences, such offspring may not actually be identical to the parent cells; however, even in such cases, they are still included within the scope of the terms used herein. 【0098】 The term "isolated molecule" (where the molecule is a polypeptide or polynucleotide, etc.) is defined, based on its origin or source of origin, as a molecule that (1) is not accompanied by its naturally occurring confoundings, (2) substantially does not contain other molecules from the same species, (3) is expressed by cells of a different species, or (4) does not occur naturally. In other words, a chemically synthesized molecule, or a molecule expressed in a cell line different from the cells in which it naturally occurs, is "isolated" from its naturally occurring confoundings. A molecule may be made substantially free of naturally occurring confoundings by isolation using purification methods known in the art. The purity or homogeneity of a molecule can be evaluated by several means known in the art. For example, the purity of a polypeptide sample can be evaluated by visualizing the polypeptide by polyacrylamide gel electrophoresis and gel staining using methods known in the art. For a particular purpose, higher resolution may be obtained by using HPLC or other purification methods known in the art. 【0099】 A protein or polypeptide is considered "substantially pure," "substantially homogeneous," or "substantially purified" if at least about 60% to 75% of the sample exhibits a single polypeptide species. This polypeptide or protein may be monomeric or polymeric. Substantially pure polypeptides or proteins typically constitute about 50% (W / W), 60% (W / W), 70% (W / W), 80% (W / W), or 90% (W / W) of a protein sample, more frequently about 95%, and preferably over 99% pure. The purity or homogeneity of a protein can be demonstrated by several means known in the art, such as visualizing single polypeptide bands by gel staining using staining methods well known in the art after subjecting the protein sample to polyacrylamide gel electrophoresis. For certain purposes, higher resolution may be obtained by using HPLC or other purification methods well known in the art. 【0100】 As used herein, the terms "label" or "labeling" refer to incorporating another molecule into an antibody. In one embodiment, the label is a detectable marker, for example, incorporation of a radio-labeled amino acid, or addition of a biotinyl moiety to a polypeptide that can be detected by labeled avidin (e.g., a fluorescent marker or streptavidin containing enzyme activity detectable by optical or calorimetric methods). In another embodiment, the label or marker can be a therapeutic label or marker, for example, a drug conjugate or toxin. Various labeling methods for polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131I) Fluorescent labels (e.g., FITC phosphors, rhodamine phosphors, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites to secondary antibodies, metal-binding domains, epitope labeling), magnetic drugs (e.g., gadolinium chelates), toxins (e.g., pertussis toxin, taxol), Cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues or homologues. In various embodiments, the labeling is added by spacer arms of varying lengths to suppress any steric hindrance. 【0101】 As used herein, the term "heterogeneous" refers to a composition or state that is not natural or does not exist in nature, for example, a composition or state that can be achieved by replacing an existing natural composition or state with a composition or state from another source. Similarly, the expression of a protein in an organism other than the one in which it is naturally expressed constitutes a heterogeneous expression system and a heterogeneous protein. 【0102】 The aspects and embodiments of the Disclosure described herein are understood to encompass "consisting of" and / or "essentially consisting of" the aspects and embodiments. 【0103】 In this specification, any mention of a value or parameter "about" includes (and describes) the variation of that value or parameter itself. For example, a mention of "about X" includes the mention of "X". 【0104】 Where used herein and in the appended claims, singular nouns such as “a,” “or,” and “the” are plural unless otherwise explicitly indicated by the context. The aspects and variations of the disclosure described herein are understood to include “consisting of” and / or “essentially consisting of” the aspects and variations. 【0105】 Description of the VitoKine platform The present invention provides a cytokine-based bioactivator ("VitoKine") platform aimed at reducing systemic mechanism-based toxicity and expanding the therapeutic utility of proteins such as cytokines. Referring to Figure 1, the novel VitoKine construct of the present invention comprises a D1 domain which is a targeting domain, an immune checkpoint regulator targeting domain, a half-life extension domain, or a bifunctional or multifunctional partial domain, an "active partial domain" (D2), and a "shielding partial domain" (D3). The proposed method of activating the VitoKine D2 domain is shown in Figure 2. Importantly, the D2 of the VitoKine construct remains inactive or attenuated until it is locally activated by a protease that is upexpressed in the affected tissue. This limits the binding of the active portion to receptors on the cell surface of surrounding or non-affected cells, suppressing excessive activation of the pathway and reducing undesirable "on-target" but "extra-target" toxicity. Furthermore, because the VitoKine active portion is inactive before activation by protease, the possibility of antigen sinking and target sinking is greatly reduced, resulting in an extended in vivo half-life and improved bioavailability in the body and at the target site of treatment. 【0106】 D1 domain ("Targeting domain, half-life extension domain, or dual-function or multi-function subdomain") In various embodiments, the VitoKine construct of the present invention includes a D1 domain which is a targeting moiety in the form of an antibody or antibody fragment or a protein or peptide against a tumor-associated antigen. In various embodiments, the VitoKine construct of the present invention includes a D1 domain which is an antibody, antibody fragment, protein, or peptide against an immune checkpoint modifier. In various embodiments, the VitoKine construct of the present invention includes a D1 domain which is an antibody or antibody fragment or a protein or peptide as an autoimmune modulator. In various embodiments, the VitoKine construct of the present invention includes D1 which functions to retain the D2 domain in a tissue site such as the tumor microenvironment (TME) or an inflammatory tissue site. In various embodiments, the VitoKine construct of the present invention includes a D1 which is bifunctional, such as tissue targeting and tissue retention. In various embodiments, the VitoKine construct of the present invention includes a D1 domain which is a polymer. In various embodiments, the VitoKine construct of the present invention includes a D1 domain which is a half-life extension moiety. In various embodiments, the VitoKine construct of the present invention includes a D1 domain which is an Fc domain. 【0107】 FC Domain IgG class immunoglobulins are the most abundant proteins in human blood. The circulating half-life of IgG class immunoglobulins can reach as long as 21 days. Fusion proteins have been reported, combining the Fc region of IgG with domains of other proteins, such as various cytokines and receptors (see, e.g., Capon et al., Nature, Vol. 337: pp. 525-531, 1989; Chamow et al., Trends Biotechnol., Vol. 14: pp. 52-60, 1996); U.S. Patents No. 5,116,964 and 5,541,087). The prototype of a fusion protein is a homodimer protein, a molecule similar to an IgG molecule but lacking the variable region of the heavy chain, the CH1 domain, and the light chain, linked via a cysteine ​​residue in the hinge region of the Fc domain of IgG. The dimer properties of fusion proteins containing the Fc domain can be advantageous in achieving higher-order interactions (i.e., bivalent or bispecific binding) with other molecules. Due to structural homology, Fc fusion proteins exhibit an in vivo pharmacokinetic profile comparable to human IgG with a similar isotype. 【0108】 The term "Fc" refers to a molecule or sequence containing a sequence of non-antigen-binding fragments of a full-length antibody, which may be in monomeric or polymeric form. The original immunoglobulin source for native Fc is preferably human, and may be any immunoglobulin, but IgG1 and IgG2 are preferred. Native Fc consists of monomeric polypeptides that can be linked by covalent (i.e., disulfide) and non-covalent bonds to form dimeric or polymeric forms. The number of intermolecular disulfide bonds between monomeric subunits of a native Fc molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgM, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgGA2). One example of native Fc is the disulfide-linked dimer resulting from the papain degradation of IgG (Ellison et al., (1982), Nucleic Acids Res., Vol. 10: pp. 4071-9). The term "native Fc," as used herein, refers to the monomeric, dimeric, and polymeric forms. It includes Fc domains containing binding sites for protein A, protein G, various Fc receptors, and complement proteins. 【0109】 In various embodiments, the term “Fc variant” refers to a molecule or sequence that has been modified from native Fc but still contains a binding site to the salvage receptor FcRn. International Publication No. 97 / 34631 (published September 25, 1997) and International Publication No. 96 / 32478 describe exemplary Fc variants and their interactions with salvage receptors, which are incorporated herein by reference. Furthermore, native Fc may include sites that confer structural features or biological activity not required for the fusion molecule of the present invention and may therefore be removed. In other words, in various embodiments, the term “Fc variant” includes molecules or sequences lacking one or more native Fc sites or residues that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with selected host cells, (3) heterogeneity of the N or C terminus after expression in selected host cells, (4) glycosylation, (5) interaction with complement, such as CDC, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cell-mediated cytotoxicity (ADCC). 【0110】 The term “Fc domain” encompasses the molecules and sequences of native Fc and Fc variants as defined above. Similar to Fc variants and native Fc, the term “Fc domain” encompasses molecules in monomeric or multimeric form, digested from full-length antibodies or produced by recombinant gene expression or other means. In various embodiments, “Fc domain” refers to a dimer consisting of two Fc domain monomers (SEQ ID NO: 13), typically containing all or part of the hinge region. In various embodiments, the Fc domain may be mutated to lack effector function. In various embodiments, each of the Fc domain monomers of the Fc domain contains amino acid substitutions in the CH2 antibody constant domain to reduce interaction or binding between the Fc domain and the Fcγ receptor. In various embodiments, each subunit of the Fc domain contains two amino acid substitutions (L234A and L235A) to reduce binding to the activating Fc receptor and / or effector function. In various embodiments, each subunit of the Fc domain contains three amino acid substitutions (L234A, L235A, and G237A) that reduce binding to and / or effector function to the activating Fc receptor (SEQ ID NO: 14). 【0111】 In various embodiments, the Fc domain may be mutated to further extend its in vivo half-life. In various embodiments, each subunit of the Fc domain contains three amino acid substitutions (M252Y, S254T, and T256E) that enhance binding to human FcRn, as disclosed in U.S. Patent No. 7,658,921 (SEQ ID NO: 156). In various embodiments, each subunit of the Fc domain contains one amino acid substitution (N434A) that enhances binding to human FcRn, as disclosed in U.S. Patent No. 7,371,826 (SEQ ID NO: 166). In various embodiments, each subunit of the Fc domain contains one amino acid substitution (M428L and N434S) that enhances binding to human FcRn, as disclosed in U.S. Patent No. 8,546,543. In various embodiments, half-life extension mutations can be combined with amino acid substitutions that reduce binding to the activating Fc receptor and / or effector function. 【0112】 In various embodiments, the two Fc domain monomers of the Fc domain each contain amino acid substitutions that promote heterodimerization of these two monomers. In various other embodiments, heterodimerization of the Fc domain monomers can be promoted by introducing different but compatible substitutions (such as a "knob-into-hole" residue pair) into the two Fc domain monomers. This "knob-into-hole" technique is also disclosed in U.S. Patent No. 8,216,805. In yet another embodiment, one Fc domain monomer contains the knob-type mutation T366W, and the other Fc domain monomer contains the hole-type mutations T366S, L358A, and Y407V. In various embodiments, two Cy residues that form a stabilizing disulfide bridge (S354C on the "knob" side and Y349C on the "hole" side) are introduced (SEQ ID NOs. 15 and 16). By using heterodimer Fc, monovalent VitoKine constructs can be obtained. 【0113】 Antibodies and protein / peptide conjugates against disease-related targets or tumor-related antigens In various embodiments, D1 may be a targeting moiety in the form of an antibody against a tumor-associated antigen (TAA), or it may be a targeting moiety in the form of another protein or peptide that exhibits binding affinity to affected cells or tissues. The TAA may be any molecule, macromolecule, or combination of molecules against which an immune response is desired. The TAA may be a protein comprising one or more polypeptide subunits. For example, the protein may be a dimer, trimer, or higher-order multimer. In various embodiments, two or more subunits of the protein may be linked by covalent bonds, such as disulfide bonds. In various embodiments, the subunits of the protein may be held together by non-covalent interactions. Thus, the TAA may be any peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate, or small organic molecule, or any combination thereof, against which a person skilled in the art would like to induce an immune response. In various embodiments, TAA is a peptide containing about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 amino acids. In various embodiments, the peptide, polypeptide, or protein is a molecule that is typically administered to a target by injection. In various embodiments, after administration, tumor-specific antibodies or binding proteins act as targeting portions that guide VitoKine to the affected area, such as a cancer site, where the active domain can be released and interact with its cognitive receptor on the affected cells or tissue. 【0114】 Any of the above markers can be used as disease-related targets or TAA targets for the VitoKine constructs of the present invention. In various embodiments, one or more disease-related targets or variants thereof, or TAAs, TAA variants, or TAA variants intended for use in the VitoKine constructs and methods of this disclosure are selected from or derived from the list shown in Table 2. TIFF0007872587000002.tif186170TIFF0007872587000003.tif213170TIFF0007872587000004.tif140170 【0115】 Further examples of tumor-associated antigens include TRP-1, TRP-2, MAG-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-BSO(LAGE), SCP-1, Hom / Mel-40, H-Ras, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA19-9, CA72-4, CAM17.1, Numa, K-ras, and β-ka. Examples include tenin, CDK4, Muni-1, p16, TAGE, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, β-HCG, BCA225, BTAA, CA15-3 (CA27.29 / BCAA), CA195, CA242, CA-50, CAM43, CD68 / KF1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophyllin C-related protein), TAAL6, TAG72, TLP, and TPS. 【0116】 Immune checkpoint regulators Several immune checkpoint protein antigens have been reported to be expressed on various immune cells, including, for example, CD152 (expressed by activated CD8+ T cells, CD4+ T cells, and regulatory T cells), CD279 (expressed on tumor-infiltrating lymphocytes, and by activated T cells (both CD4 and CD8), regulatory T cells, activated B cells, activated NK cells, immune-unresponsive T cells, monocytes, and dendritic cells), CD274 (expressed on T cells, B cells, dendritic cells, macrophages, vascular endothelial cells, and pancreatic islet cells), and CD223 (expressed by activated T cells, regulatory T cells, immune-unresponsive (angergic) T cells, NK cells, NKT cells, and plasmacytoid dendritic cells) (see, e.g., Pardoll, D., Nature Reviews Cancer, Vol. 12: pp. 252-264, 2012). Antibodies that bind to antigens identified as immune checkpoint proteins are known to those skilled in the art.For example, various anti-CD276 antibodies have been reported in the art (see, for example, U.S. Patent Application Publication No. 20120294796 (Johnson et al.) and its cited references); various anti-CD272 antibodies have been reported in the art (see, for example, U.S. Patent Application Publication No. 20140017255 (Mataraza et al.) and its cited references); and various anti-CD152 / CTLA-4 antibodies have been reported in the art (see, for example, U.S. Patent Application Publication No. 20130136749 (Korman et al.) See (al) and its references); various anti-LAG-3 / CD223 antibodies have been reported in the art (e.g., see U.S. Patent Application Publication No. 20110150892 (Thudium et al.) and its references); various anti-CD279 / PD-1 antibodies have been reported in the art (e.g., see U.S. Patent No. 7,488,802 (Collins et al.) and its references); various anti-PD-L1 antibodies have been reported in the art (e.g., see U.S. Patent Application Publication No. 20130122014 (Korman et al.) and its references); various anti-TIM-3 antibodies have been reported in the art (e.g., see U.S. Patent Application Publication No. 20140044728 (Takayanagi et al.) and its references); various anti-B7-H4 antibodies have been reported in the art (e.g., see U.S. Patent Application Publication No. 20110085970 (Terrett et al.) and its references). Each of these references is incorporated herein by reference in its entirety with respect to the specific antibody and sequence taught therein. 【0117】 In various embodiments, D1 may include an antibody, antibody fragment, or protein or peptide that binds to an immune checkpoint protein antigen present on the surface of immune cells. In various embodiments, the immune checkpoint protein antigen is selected from, but is not limited to, the group consisting of CD276, CD272, CD152, CD223, CD279, CD274, CD40, SIRPα, CD47, OX-40, GITR, ICOS, CD27, 4-1BB, TIM-3, B7-H4, Siglec-7, Siglec-8, Siglec-9, Siglec-15, and VISTA. 【0118】 In various embodiments, D1 may include antibodies against immune checkpoint protein antigens selected from, but not limited to, the group consisting of PD-L1, B7-H3, and B7-H4, which are present on the surface of tumor cells. 【0119】 In various embodiments, D1 is an antibody that is an antagonist anti-fibroblast-activating protein (FAP) antibody or antibody fragment. In various embodiments, the antibody is a humanized anti-FAP antibody containing the amino acid sequences described in SEQ ID NOs: 193 and 194. In various embodiments, D1 is an antibody or antibody fragment against an immune checkpoint regulator. In various embodiments, the antibody is an antagonist PD-1 antibody or antibody fragment. In various embodiments, the antibody is an antagonist humanized PD-1 antibody containing the amino acid sequences described in SEQ ID NOs: 195 and 196. In various embodiments, the antibody is an antagonist humanized PD-1 antibody containing the amino acid sequences described in SEQ ID NOs: 197 and 198. In various embodiments, the antibody is an antagonist humanized PD-1 antibody containing the amino acid sequences described in SEQ ID NOs: 275 and 276. In various embodiments, the antibody is an antagonist human PD-1 antibody containing the amino acid sequences described in SEQ ID NOs: 277 and 278. In various embodiments, the antibody is an antagonist PD-L1 antibody or antibody fragment. In various embodiments, the antibody is an antagonist human PD-L1 antibody containing the amino acid sequences described in SEQ ID NOs. 279 and 280. In various embodiments, the antibody VitoKine construct contains the amino acid sequences described in SEQ ID NOs. 128-142, 180-181, 281-286, 296-297, and 303-306. 【0120】 Modulators of autoimmune and inflammatory disorders Any of the above proteins, which are highly expressed on various inflammatory tissues or immune cells, can be used as targets for autoimmune / inflammatory diseases for the VitoKine constructs of the present invention. In various embodiments, one or more targets for autoimmune / inflammatory diseases, their variants, or their variants / isoforms intended for use in the VitoKine constructs and methods of this disclosure are selected from or derived from the list shown in Table 3. These targets may also be applicable as cancer targets. TIFF0007872587000005.tif166170TIFF0007872587000006.tif134170 【0121】 In various embodiments, the D1 targeting portion can be an inflammatory tissue-specific antibody, antibody fragment, another protein, or peptide that binds to affected cells or the affected microenvironment, such as TNF, TNFR, integrin A4β7, IL-6Rα, BLYS, or TSLP. In various embodiments, the antibody VitoKine construct contains the amino acid sequences described in SEQ ID NOs. 143-146. 【0122】 polymer In various embodiments, D1 can be a polymer such as polyethylene glycol (PEG). In various embodiments, polymers such as PEG can be covalently attached to the N-terminus, C-terminus, or internal position using conventional chemical methods such as chemical bonding. In various embodiments, polymers such as PEG can be covalently attached to the N-terminus of the D2 domain via site-directed binding or via other amino acids of cytokines or specific manipulated amino acid substitutions. 【0123】 Half-life extension portion Other half-life extending portions that can be used as the D1 domain in the present invention to extend the blood half-life of VitoKine in various embodiments. Examples of half-life extending portions include, but are not limited to, Fc domains, Fc variants, antibodies, antibody fragments (Fab, ScFv), and EXTEN (Schellenberger et al., Nat. Biotechnol. 27: pp. 1186-1192, 2009), and human serum albumin protein. 【0124】 D2 domain ("active partial domain") D2 is the active portion of the VitoKine construct, whose activity is reversibly shielded within the construct and can be restored upon protease cleavage at the site of injury. This active portion can be any protein, including but not limited to any native or variant interleukin or cytokine polypeptide. Importantly, the "active portion" of the VitoKine construct remains inactive or attenuated until it is locally activated by a protease upregulated in the affected tissue. This limits the binding of the active portion to receptors on the cell surface of surrounding or non-affected cells, suppressing excessive activation of the pathway and reducing undesirable "on-target" but "extra-tissue" toxicity. Furthermore, the inactivity of the VitoKine active portion before protease activation significantly reduces the possibility of antigen and target sinking, resulting in an extended in vivo half-life and improved distribution and exposure at the target site of treatment. 【0125】 IL-15 Interleukin-15 (IL-15) is a cytokine identified by two separate groups based on its ability to stimulate the proliferation of IL-2-dependent CTLL-2 T cell lines in the presence of anti-IL-2 neutralizing antibodies (Steel et al., Trends in Pharmacological Sciences, Vol. 33 (No. 1): pp. 35-41, 2012). IL-15 and IL-2 are receptor (R) signaling components (IL-2 / 15Rβγ). cAs seen in the sharing of IL-2, they possess similar biological properties in vitro. However, the difference in specificity of IL-15 compared to IL-2 is brought about by unique individual α-chain receptors that complete the heterotrimeric high-affinity receptor complexes IL-15Rαβγ and IL-2Rαβγ, enabling differences in responsiveness depending on the ligand and expressed high-affinity receptor. Interestingly, both IL-15 transcripts and IL-15Rα transcripts have a considerably broader tissue distribution than IL-2 / IL-2Rα. Furthermore, multiple complex post-transcriptional regulatory mechanisms tightly control IL-15 expression. That is, the complex regulation of IL-15 and IL-15Rα expression, and even the differences in expression patterns, suggest that the important in vivo functions of this receptor / ligand pair may differ from those of IL-2 and IL-2Rα. Studies investigating the biological properties of IL-15 have identified several important non-redundant roles, including its importance in the development and function of natural killer (NK) cells, NK-T cells, and intestinal intraepithelial lymphocytes. The role of IL-15 in autoimmune processes such as rheumatoid arthritis and in malignancies such as adult T-cell leukemia suggests that IL-15 dysregulation may have adverse effects on the host (Fehniger et al., Blood, Vol. 97: pp. 14-32, 2001). 【0126】 As used herein, the terms “native IL-15” and “native interleukin-15” refer to any natural mammalian interleukin-15 amino acid sequence, encompassing immature or precursor forms and mature forms, within the context of a protein or polypeptide. Non-limiting examples of GenBank accession numbers for various native mammalian interleukin-15 amino acid sequences include NP_032383 (mouse (Mus musculus), immature form), AAB60398 (rhesus monkey (Macaca mulatta), immature form), NP_000576 (human, immature form), CAA62616 (human, immature form), AAI00964 (human, immature form), and AAH18149 (human). In various embodiments of the present invention, native IL-15 is an immature or precursor form of natural mammalian IL-15. In other embodiments, native IL-15 is the mature form of natural mammalian IL-15. In various embodiments, native IL-15 is a precursor of natural human IL-15. In various embodiments, native IL-15 is the mature form of natural human IL-15. In various embodiments, the native IL-15 protein / polypeptide is isolated or purified. In various embodiments, the IL-15-based domain D2 is derived from the amino acid sequence of the human IL-15 precursor sequence described in Sequence ID No. 1 below: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(Sequence ID 1) 【0127】 In various embodiments, the IL-15-based domain D2 comprises the amino acid sequence of the mature human IL-15 sequence described in Sequence ID No. 2 below: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(Sequence ID 2) 【0128】 In various embodiments, the IL-15-based domain D2 may be an IL-15 variant (or mutant) containing a sequence derived from the sequence of the mature human IL-15 polypeptide described in SEQ ID NO: 2. IL-15 variants (or mutants) are designated using the original amino acid, the position of the original amino acid in the mature sequence, and the variant-type amino acid. For example, "huIL-15S58D" represents human IL-15 with an S-to-D substitution at position 58 of SEQ ID NO: 2. In various embodiments, the D2 domain of the present invention contains an IL-15 domain that is an IL-15 variant (hereinafter also referred to as the IL-15 mutant domain). In various embodiments, the IL-15 variant contains an amino acid sequence different from that of the native (or wild-type) IL-15 protein. In various embodiments, the IL-15 variant binds to the IL-15Rα polypeptide and functions as an IL-15 agonist or antagonist. In various embodiments, an IL-15 variant having agonist activity has superagonist activity. In various embodiments, IL-15 variants can function as IL-15 agonists or antagonists, independently of their binding to IL-15Rα. IL-15 agonists are exemplified by equivalent or increased bioactivity compared to wild-type IL-15. IL-15 antagonists are exemplified by decreased bioactivity compared to wild-type IL-15, or by their ability to inhibit IL-15-mediated responses. In various embodiments, IL-15 variants bind to the IL-15RβγC receptor with increased or decreased activity. In various embodiments, the sequence of the IL-15 variant has at least one amino acid change, e.g., substitution or deletion, compared to the native IL-15 sequence, and such change results in either IL-15 agonist or IL-15 antagonist activity. In various embodiments, the amino acid substitution / deletion affects IL-15Rβ and / or γ CIt is located within the domain of IL-15 that interacts with it. In various embodiments, amino acid substitutions / deletions do not affect binding to the IL-15Rα polypeptide or the ability to produce IL-15 variants. Suitable amino acid substitutions / deletions for producing IL-15 variants can be identified based on the known IL-15 structure by comparing IL-15 with structurally known similar molecules such as IL-2, through rational or random mutagenesis and functional analysis, as provided herein, or other empirical methods. Furthermore, suitable amino acid substitutions may be conservative changes and additional amino acid insertions, or non-conservative changes and additional amino acid insertions. In various embodiments, the IL-15 variants of the present invention contain one or more amino acid deletions or one or more amino acid substitutions at positions 30, 31, 32, 58, 62, 63, 67, 68, or 108 of the mature human IL-15 sequence described in SEQ ID NO: 2. In various embodiments, D30T (where "D30" refers to the amino acid and residue position in the native mature human IL-15 sequence, and "T" refers to the substituted amino acid residue at that position in the IL-15 variant), V31Y, H32E, S58H, S58I, S58P, S58R, S58Q, D62T, V63A, V63F, V63K, V63R, I67V, I68H, I68F, I68Q, I68G, I68K, I68D, Q108A, Q108S, Q108E, Q108K, or Q108M ​​substitutions result in IL-15 variants with antagonist activity, while the S58D substitution results in IL-15 variants with agonist activity. In various embodiments, the IL-15 variant contains one, two, three, four, five, or six amino acid deletions at the N-terminus of SEQ ID NO: 2. In various embodiments, the IL-15 variant contains one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions at the C-terminus of SEQ ID NO: 2. In various embodiments, the IL-2 variant contains an amino acid insertion of "GS" (SEQ ID NO: 12), "GGSGG" (SEQ ID NO: 153), or "GSSGGSGGS" (SEQ ID NO: 154) after the N95 position of SEQ ID NO: 2.In various embodiments, the IL-15 variant includes the amino acid sequence described in SEQ ID NOs: 3, 182-192, and 199-215. 【0129】 Table 4 shows an example of an IL-15 Fc VitoKine construct. TIFF0007872587000007.tif203170 【0130】 In various embodiments, each molecule of the IL-15 antibody VitoKine or IL-15 Fc fusion will contain two or more heterodimer chains, as shown in Table 5. TIFF0007872587000008.tif192170 【0131】 In various embodiments, the IL-15-based D2 domain will comprise an IL-15 construct containing an IL-2Rβ-based blocking peptide selected from the IL-2Rβ construct described in SEQ ID NO: 12 or the constructs having the amino acid sequences described in SEQ ID NOs: 66-70. 【0132】 In various embodiments, the IL-15-based D2 domain may include an IL-15 construct comprising an IL-2Rβ-based blocking peptide and having two or more heterodimer chains as described in Table 6. TIFF0007872587000009.tif64170 【0133】 IL-2 Interleukin-2 (IL-2) is a classic Th1 cytokine produced by post-activation T cells via the T cell antigen receptor and the co-stimulating molecule CD28. IL-2 regulation occurs through activation of signaling pathways and transcription factors that act on the IL-2 promoter to revitalize gene transcription, but also involves the regulation of IL-2 mRNA stability. IL-2 binds to a highly regulated multi-chain receptor containing α, β, and γ chains, mediating signaling via the Jak-STAT pathway. IL-2 delivers activation, proliferation, and differentiation signals to T cells, B cells, and NK cells. IL-2 also plays a crucial role in mediating activation-induced T cell death, a function essential for terminating the immune response. Aldesleukin, a commercially available non-glycosylated human recombinant IL-2 product (available from Prometheus Laboratories Inc. in San Diego, California, under the trademark PROLEUKIN® as des-alanil-1, serine-125 human interleukin-2), is approved for administration to patients with metastatic renal cell carcinoma and metastatic melanoma. IL-2 has also been suggested for use in patients with hepatitis C virus (HCV), human immunodeficiency virus (HIV), acute myeloid leukemia, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, juvenile rheumatoid arthritis, atopic dermatitis, breast cancer, and bladder cancer. Unfortunately, its short half-life and high toxicity limit the optimal dose of IL-2. 【0134】 As used herein, the terms “native IL-2” and “native interleukin-2” refer to any natural mammalian interleukin-2 amino acid sequence, encompassing immature or precursor forms and mature forms, within the context of proteins or polypeptides. Non-limiting examples of GenBank accession numbers for amino acid sequences of various native mammalian interleukin-2 species include NP_032392.1 (mouse, immature), NP_001040595.1 (rhesus monkey, immature), NP_000577.2 (human, precursor), CAA01199,1 (human, immature), AAD48509.1 (human, immature), and AAB20900.1 (human). In various embodiments of the present invention, native IL-2 is an immature or precursor form of natural mammalian IL-2. In other embodiments, native IL-2 is a mature form of natural mammalian IL-2. In various embodiments, native IL-2 is a precursor of natural human IL-2. In various embodiments, native IL-2 is a mature form of natural human IL-2. In various embodiments, the IL-2-based domain D2 is derived from the amino acid sequence of the human IL-2 precursor sequence described in Sequence ID No. 6 below: MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(Sequence ID 6) 【0135】 In various embodiments, the IL-2-based domain D2 comprises the amino acid sequence of the mature wild-type human IL-2 sequence described in Sequence ID No. 8 below, which includes a cysteine-to-serine substitution at position 125, but does not alter the binding affinity to the IL-2 receptor compared to natural IL-2: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 8) 【0136】 In various embodiments, the IL-2-based domain D2 may be an IL-2 variant (or mutant) containing a sequence derived from the sequence of the mature human IL-2 polypeptide described in Sequence ID No. 8. In various embodiments, the IL-2 variant contains an amino acid sequence different from that of the native (or wild-type) IL-2 protein. In various embodiments, the IL-2 variant binds to the IL-2Rα polypeptide and functions as an IL-2 agonist or antagonist. In various embodiments, an IL-2 variant having agonist activity has superagonist activity. In various embodiments, the IL-2 variant can function as an IL-2 agonist or antagonist independently of its binding to IL-2Rα. IL-2 agonists are exemplified by equivalent or increased biological activity compared to wild-type IL-2. IL-2 antagonists are exemplified by decreased biological activity compared to wild-type IL-2, or by their ability to inhibit IL-2-mediated reactions. In various embodiments, the IL-2 variant sequence has at least one amino acid change, e.g., substitution or deletion, compared to the native IL-2 sequence, and such change results in either IL-2 agonist or IL-2 antagonist activity. In various embodiments, the IL-2 variant has reduced / absent binding to IL-2Rα and an amino acid sequence derived from SEQ ID NO: 8 that selectively activates and proliferates effector T cells (Teff) for cancer treatment. Exemplary amino acid substitutions are listed in Table 7. In various embodiments, the IL-2 variant with reduced / absent binding to IL-2Rα includes the amino acid sequences described in SEQ ID NOs: 232-247. In various embodiments, the IL-2 variant has reduced binding to IL-2Rβ and / or γc and an amino acid sequence derived from SEQ ID NO: 8 that enhances selectivity in the activation and proliferation of regulatory T cells (Treg) for the treatment of autoimmune diseases. Exemplary amino acid substitutions are listed in Table 7. As will be understood by those skilled in the art, all of these mutations can be optionally combined independently to achieve optimal regulation of affinity and activation. TIFF0007872587000010.tif106170 【0137】 Table 8 shows exemplary IL-2-based VitoKine constructs. TIFF0007872587000011.tif159170TIFF0007872587000012.tif139170 【0138】 In various embodiments, the active moiety is selected from the group consisting of the amino acid sequences of interleukin-4 (IL-4) (SEQ ID NO: 17), interleukin-7 (IL-7) (SEQ ID NO: 18), interleukin-9 (IL-9) (SEQ ID NO: 19), interleukin-10 (IL-10) (SEQ ID NO: 20), interleukin-12α (IL-12α) (SEQ ID NO: 21), interleukin-12β (IL-12β) (SEQ ID NO: 22), interleukin-23α (IL-23α) (SEQ ID NO: 23), and TGFβ (SEQ ID NO: 24), but is not limited to these. In various embodiments, the active moiety is a heterodimer human IL-12 cytokine comprising SEQ ID NO: 21 as the first chain and SEQ ID NO: 22 as the second chain. In various embodiments, the active moiety is a heterodimer human IL-23 cytokine comprising SEQ ID NO: 23 as the first chain and SEQ ID NO: 22 as the second chain. 【0139】 D3 domain ("shielding subdomain") The D3 domain is a “shielding subdomain” primarily used to reversibly shield the activity of the D2 domain in certain VitoKine constructs. The D3 domain can shield the functional activity of D2 until it is activated at the target therapeutic site. In various embodiments, the VitoKine constructs of the present invention include a “shielding subdomain” (D3) which is a cognitive receptor / binding partner for the D2 protein or cytokine. In various embodiments, the D3 domain is a variant of the cognitive receptor / binding partner to the D2 domain, or a specific conjugate such as a peptide or antibody fragment. In various embodiments, the D3 domain has enhanced binding affinity to the D2 domain compared to the wild-type cognitive receptor / binding partner. In various embodiments, the D3 domain has reduced or absent binding affinity to the D2 domain compared to the wild-type cognitive receptor / binding partner. In various embodiments, the D3 domain is a protein, or peptide, or antibody, or antibody fragment that can shield the activity of D2. In various embodiments, the D3 domain is a polymer such as DNA, RNA fragment, or PEG, cleavable linker. In various embodiments, the VitoKine construct of the present invention comprises a D3 domain which is an IL-15Rα extracellular domain or a functional fragment or variant thereof. In various embodiments, the VitoKine construct of the present invention comprises a D3 domain which is an IL-15Rα Sushi domain (amino acids 1-65 of SEQ ID NO: 5). In various preferred embodiments, the VitoKine construct of the present invention comprises a D3 domain which is an IL-15Rα Sushi+ domain (e.g., SEQ ID NO: 5) which contains 1-30 additional IL-15Rα residues at the C-terminus of the Sushi domain. In various embodiments, the VitoKine construct of the present invention comprises a D3 domain which is an IL-2Rα extracellular domain or a functional fragment thereof. In various preferred embodiments, the VitoKine construct of the present invention comprises a D3 domain which is an IL-2Rα Sushi domain.In various preferred embodiments, the VitoKine construct of the present invention comprises a D3 domain which is a variant of the IL-2Rα Sushi domain. In various embodiments, the VitoKine construct of the present invention comprises a D3 domain which is either an IL-2Rβ extracellular domain or an IL-2Rβ-derived blocking peptide. In various embodiments, the D3 domain can shield the functional activity of D2 until it is activated at the target therapeutic site. 【0140】 IL-15 receptor alpha The IL-15 receptor is a type I cytokine receptor consisting of a beta (β)-gamma (γ) subunit (shared with the IL-2 receptor) and an alpha (α) subunit (which binds to IL-15 with high affinity). Full-length human IL-15Rα is a type 1 transmembrane protein with a 32-amino acid signal peptide, a 173-amino acid extracellular domain, a 21-amino acid transmembrane region, a 37-amino acid cytoplasmic end, and multiple N-linked or O-linked glycosylation sites (Anderson et al., J. Biol Chem, vol. 270: pp. 29862-29869, 1995). Previously, it was revealed that the naturally soluble form of the IL-15Rα chain, which corresponds to the entire extracellular domain of IL-15Rα, acts as a high-affinity IL-15 antagonist. However, in stark contrast to that finding, it was revealed that the recombinant soluble sushi domain of IL-15Rα, which accounts for the majority of the binding affinity to IL-15, acts as a potent IL-15 agonist by enhancing its binding affinity and biological effects (proliferation and protection from apoptosis) via the IL-15Rβ / γ heterodimer, while not affecting the IL-15 binding affinity and function of the trimolecular IL-15Rα / β / γ membrane receptor. These results suggest that, if naturally produced, such a soluble sushi domain may be involved in the transpresentation mechanism of IL-15 (Mortier et al., J. Biol Chem, Vol. 281 (No. 3): pp. 1612-1619, 2006). 【0141】 As used herein, the terms “native IL-15Rα” and “native interleukin-15 receptor α” refer to any native mammalian interleukin-15 receptor α ("IL-15Rα") amino acid sequence, encompassing immature or precursor forms, mature forms, and native isoforms, within the context of proteins or polypeptides. Non-limiting examples of GenBank accession numbers for various native mammalian IL-15Rα amino acid sequences include NP_002180 (human), ABK41438 (rhesus monkey), NP_032384 (mouse), Q60819 (mouse), and CA141082 (human). In various embodiments, native IL-15Rα is the immature form of the native mammalian IL-15Rα polypeptide. In various embodiments, native IL-15Rα is the mature form of the native mammalian IL-15Rα polypeptide. In various embodiments, native IL-15Rα is a form of the natural mammalian IL-15Rα polypeptide. In various embodiments, native IL-15Rα is the full-length form of the natural mammalian IL-15Rα polypeptide. In various embodiments, native IL-15Rα is the immature form of the natural human IL-15Rα polypeptide. In various embodiments, native IL-15Rα is the mature form of the natural human IL-15Rα polypeptide. In various embodiments, native IL-15Rα is the full-length form of the natural human IL-15Rα polypeptide. In various embodiments, the native IL-15Rα protein or polypeptide is isolated or purified. In various embodiments, the IL-15Rα domain is derived from the amino acid sequence of the human IL-15Rα sequence described in Sequence ID No. 4 below: MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL(Sequence ID 4) 【0142】 In various embodiments, the VitoKine construct of the present invention comprises a D3 domain, which is an IL-15RαSushi+ domain containing the amino acid sequence of the mature human IL-15Rα polypeptide described in Sequence ID No. 5 below: ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP(Sequence ID 5) 【0143】 In various embodiments, IL-15RαSushi+ (SEQ ID NO: 5), a cleaved cognitive coreceptor of IL-15 that replicates most of the binding affinity of full-length IL-15Rα (SEQ ID NO: 4), was used as a D3 domain to shield IL-15 activity in order to construct IL-15 VitoKine by adjusting the cleavable or non-cleavable linker connecting IL-15 and IL-15RαSushi+. As will be understood by those skilled in the art, the length of the D3 domain may differ from the sequence described in SEQ ID NO: 5, as long as it replicates most of the binding activity of full-length IL-15Rα (SEQ ID NO: 4), i.e., as long as it is a functional fragment. What distinguishes the IL-15 VitoKine design from others is that it fully utilizes the unique characteristics of the IL-15 pathway, including the exceptionally high affinity (30 pM) between IL-15 and IL-15α, and that the complex formation of IL-15α enhances IL-15 activity in vivo. Even after the linker connecting IL-15 and IL-15αSushi+ is cleaved by a protease whose expression is elevated at the site of infection, IL-15RαSushi+ or any functional fragment derived from IL-15RαECD is expected to retain non-covalent association of IL-15 and enhance IL-15 activity. 【0144】 IL-2 receptor The IL-2 receptor (IL-2R) is a heterotrimeric protein expressed on the surface of certain immune cells, such as lymphocytes, and binds to and responds to a cytokine called IL-2. IL-2R has the following three subunits: α (CD25), β (CD122), and γ c (CD132, co-chained with five other cytokine receptors (IL-4R, IL-7R, IL-9R, IL-15R, and IL-21R)). The α-chain of the human receptor (also known as the Tac antigen or p55) is encoded by the IL-2RA gene on chromosomes 10p14-15. The gene for the human β-chain of this receptor (IL-2RB, CD122) is located on chromosomes 22q11.2-12, while the common IL-2Rγ CThe gene for the IL-2R chain (IL-2RG) is located on chromosome Xq13. The assembly of all three subunits of this receptor is crucial for signaling to B and T cells. IL-2R is found (ephemerally or constitutively) on the cell surface of almost all hematopoietic cells, including lymphoid T cells, B cells, and NK cells, as well as myeloid cells such as macrophages, monocytes, and neutrophils. This signal is transmitted intracellularly via the Janus kinases Jak1 and Jak3. Phosphorylation of the cytosolic portion of the receptor β chain enables homodimerization of STAT-3 and STAT-5 factors. The STAT-3 and STAT-5 homodimers exhibit increased affinity for the nucleus, where they bind to specific DNA elements that enhance the transcription of IL-2-dependent genes. 【0145】 As used herein, the terms “native IL-2Rα” and “native interleukin-2 receptor α” refer to any native mammalian interleukin-2 receptor α ("IL-2Rα") amino acid sequence, encompassing immature or precursor forms, mature forms, and native isoforms, within the context of proteins or polypeptides. Non-limiting examples of GenBank accession numbers for various native mammalian IL-2Rα amino acid sequences include NP_032393.3 (mouse), CAK26553.1 (human), and NP_000408.1 (human). In various embodiments, native IL-2Rα is the immature form of the native mammalian IL-2Rα polypeptide. In various embodiments, native IL-2Rα is the mature form of the native mammalian IL-2Rα polypeptide. In various embodiments, native IL-2Rα is a form of the native mammalian IL-2Rα polypeptide. In various embodiments, native IL-2Rα is the full-length form of the native mammalian IL-2Rα polypeptide. In various embodiments, native IL-2Rα is the immature form of the natural human IL-2Rα polypeptide. In various embodiments, native IL-2Rα is the mature form of the natural human IL-2Rα polypeptide. In various embodiments, native IL-2Rα is the full-length form of the natural human IL-2Rα polypeptide. In various embodiments, the native IL-2Rα protein or polypeptide is isolated or purified. In various embodiments, the IL-2Rα domain is derived from the amino acid sequence of the human IL-2Rα sequence described in Sequence ID No. 9 below: MDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI(Sequence ID 9) 【0146】 In various embodiments, the VitoKine construct of the present invention comprises a D3 domain, which is an IL-2Rα Sushi domain containing the amino acid sequence of the mature human IL-2Rα polypeptide described in Sequence ID No. 10 below: ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG (Sequence ID 10) 【0147】 In various embodiments, IL-2RαSushi (SEQ ID NO: 10) is used to shield IL-2 activity and to construct IL-2 VitoKine. Unlike IL-15Rα, which contains a single sushi domain, IL-2Rα contains two sushi domains separated by a linker region. In various embodiments, IL-2 VitoKine includes IL-2RαSushi variants containing amino acid substitutions to reduce the binding affinity of IL-2Rα to IL-2 by breaking a specific non-covalent interaction between IL-2Rα and IL-2. Native IL-2Rα binds to IL-2 with a moderate affinity of 30 nM, but there is a possibility that IL-2Rα will not dissociate even after linker cleavage. The association of IL-2Rα with IL-2 may reduce IL-2 activity and / or shift the balance of T cell subgroups in an unfavorable direction. Affinity reduction mutations (one or more) introduced into IL-2RαSushi, such as R36A, K38E, L42G, or Y43A, or any combination of their substitutions, make the IL-2Rαsushi domain highly likely to dissociate from IL-2 after linker protease cleavage. 【0148】 In various embodiments, the VitoKine construct of the present invention contains a D3 domain, which is one of the IL-2RαSushi domain variants containing the amino acid sequences described in SEQ ID NOs. 267-270. 【0149】 L1 linker and L2 linker Cuttable linker A cleavable linker, i.e., a linker sensitive to disease-related enzymes, may contain a portion such as a protein substrate that can be specifically cleaved by a protease that is present in high levels in the affected area compared to non-affected tissue. Enzymes with known substrates that are elevated in various types of cancer (e.g., solid tumors) have been reported in the literature. See, for example, La Rocca et al., Brit. J. Cancer, vol. 90: pp. 1414–1421, and Ducry et al., Bioconjug. Chem., vol. 21: pp. 5–13, 2010, each cited herein by reference in its entirety. In various embodiments, proteases capable of cleaving protease-cleavable linkers are selected from the group consisting of metalloproteinases, e.g., matrix metalloproteinases (MMPs) 1-28, serine proteases, e.g., urokinase-type plasminogen activator (uPA) and matryptase, cysteine ​​proteases, e.g., regmine, aspartate protease, and cathepsin proteases. Exemplary protease substrate peptide sequences are shown in Table 9. TIFF0007872587000013.tif234170 【0150】 Table 10 shows example peptide sequences of protease substrates that can be used as protease-cleavable linkers, with or without peptide spacers of varying lengths at the C-terminus, N-terminus, or both ends of the cleavable linker. TIFF0007872587000014.tif141170 【0151】 In various embodiments, the protease is MMP-9 or MMP-2. In a further specific embodiment, the protease is uPA. In a further specific embodiment, the protease is MMP-14. In a further specific embodiment, the protease is legmine. In various embodiments, one VitoKine molecule contains two different proteases. In various embodiments, the protease-cleavable linker contains the protease recognition sequence "GPLGMLSQ" (SEQ ID NO: 77). In various embodiments, the protease-cleavable linker contains the protease recognition sequence "LGGSGRSANAILE" (SEQ ID NO: 80). In various embodiments, the protease-cleavable linker contains the protease recognition sequence "SGRSENIRTA" (SEQ ID NO: 157). In various embodiments, the protease-cleavable linker contains the protease recognition sequence "GPTNKVR" (SEQ ID NO: 158). In various embodiments, the linker (e.g., the cleavable linker) may be cleaved with a tumor-associated protease. In various embodiments, the cleavable linker may be cleaved by other disease-specific proteases in non-cancer diseases such as inflammatory diseases. 【0152】 In various embodiments, the peptide spacer may be inserted on either side of the protease-cleavable sequence, or adjacent to both sides of the protease-cleavable sequence, or may be inserted as a non-cleavable linker without a protease substrate site. The peptide spacer serves to position the cleavable linker so that the enzymes involved in cleavage can reach it more easily. The length of the spacer may be modified or optimized to balance the reachability for enzymatic cleavage with the spatial constraints required to reversibly block the expression of the biological activity of the D2 domain. The spacer may contain 1 to 100 amino acids. Suitable peptide spacers are known in the art and include, but are not limited to, peptide linkers containing mobile amino acid residues such as glycine and serine. In various embodiments, the spacer may contain 1 to 12 amino acids including the motifs G, S, GS, GSGS (SEQ ID NO: 116), GGS (SEQ ID NO: 117), GSGS (SEQ ID NO: 121), GSGSGS (SEQ ID NO: 122), GSGSGSGS (SEQ ID NO: 123), GSGSGSGSGS (SEQ ID NO: 124), or GSGSGSGSGSGS (SEQ ID NO: 125). In other embodiments, the spacer may contain (GGGGS) (SEQ ID NO: 118). n The spacer may contain the motif, where n is an integer from 1 to 10. In other embodiments, the spacer may also contain amino acids other than glycine and serine. 【0153】 Table 11 shows exemplary protease-cleavable linkers in which the spacer peptide is adjacent to the protease substrate peptide (underlined). TIFF0007872587000015.tif98170 【0154】 In various embodiments, the cleavable linker can be activated by mechanisms other than proteolysis, including but not limited to hydrolysis, such as a free-release pegylated polymer that can be released via a controlled release mechanism under various pH levels. 【0155】 Linker that cannot be cut Non-cleavable linkers provide covalent bonds and additional structural and / or spatial mobility between protein domains. As is known in the art, peptide linkers containing mobile amino acid residues such as glycine and serine can be used as non-cleavable linkers. In various embodiments, a non-cleavable linker may contain 1 to 100 amino acids. In various embodiments, the spacer may contain GS, GSGS (SEQ ID NO: 116), GGS (SEQ ID NO: 117), GGGGS (SEQ ID NO: 118), GGSG (SEQ ID NO: 119), or SGGG (SEQ ID NO: 120). In other embodiments, the linker may contain the motif (GGGGS)(SEQ ID NO: 118)n, where n is an integer from 1 to 10. In other embodiments, the linker may contain amino acids other than glycine and serine. In another embodiment, the non-cleavable linker may be a simple chemical bond, for example, an amide bond (e.g., by the chemical bonding of PEG). The non-cuttable linker is stable not only under physiological conditions but also in affected areas such as cancer sites and inflammatory disease sites. 【0156】 Table 12 shows an example of a linker that cannot be cut. TIFF0007872587000016.tif132170 【0157】 A combination of a severable linker and a non-severable linker. In various embodiments, the L1 and L2 linkers can be made cleavable, non-cleavable, or a combination of cleavable and non-cleavable linkers, allowing the active portion of the D2 domain to take on various forms, thereby achieving various therapeutic objectives, balancing risk-benefit ratios, and confirming various cytokine properties. Figure 2 illustrates the active forms released by linker cleavage. Active form 1, resulting from L1 cleavage, and active form 3, resulting from cleavage of both L1 and L2, are short-acting cytokines with varying degrees of functional activity depending on the higher-order structure of D3. Cleavage and release from D1, which is the half-life extension portion or the affected tissue targeting portion, will increase the local concentration of the active D2 domain. After local action, this short-acting active form can be rapidly eliminated from systemic circulation, potentially reducing toxicity. In contrast, the secondary active form resulting from L2 cleavage is a fully functional, long-acting, tissue-targeting, conserved cytokine that persists in the affected area, exhibiting longer-lasting and enhanced efficacy. 【0158】 Polynucleotides In another embodiment, the Disclosure provides isolated nucleic acid molecules comprising polynucleotides encoding IL-15, IL-15 variants, IL-15Rα, IL-15Rα variants, IL-2, IL-2 variants, IL-2Rα, IL-2Rα, Fc, Fc variants, antibodies targeting TAA or immune checkpoint regulators, antibody fragments, or VitoKine constructs of the Disclosure. The nucleic acids of the subject may be single-stranded or double-stranded. Such nucleic acids may be DNA molecules or RNA molecules. Examples of DNA include cDNA, genomic DNA, synthetic DNA, PCR-amplified DNA, and combinations thereof. Genomic DNA encoding VitoKine constructs can be obtained from genome libraries corresponding to many species. Synthetic DNA can be obtained by chemically synthesizing duplicate oligonucleotide fragments, assembling those fragments, and reconstructing some or all of the coding region and adjacent sequences. RNA can be obtained from RNA polymerase and prokaryotic expression vectors that direct high levels of mRNA synthesis, such as vectors using a T7 promoter. The DNA molecules disclosed herein include not only full-length genes but also polynucleotides and their fragments. Full-length genes may also include sequences encoding N-terminal signal sequences. Such nucleic acids can be used in methods for constructing novel VitoKine constructs, among other applications. 【0159】 In various embodiments, the isolated nucleic acid molecule comprises a polynucleotide described herein, and further comprises a polynucleotide encoding at least one heterologous protein described herein. In various embodiments, the nucleic acid molecule further comprises a polynucleotide encoding a linker or hinge linker described herein. 【0160】 In various embodiments, the recombinant nucleic acids of this disclosure may be functionally ligated to one or more regulatory nucleotide sequences within an expression construct. The regulatory sequences are those approved in the art and selected to direct the expression of the VitoKine construct. That is, the term regulatory sequence includes promoters, enhancers, and other expression regulatory elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, California (1990). Typically, one or more of the above regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription start and termination sequences, translation start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art are contemplated in this disclosure. The promoter may be a natural promoter or a hybrid promoter combining two or more promoter elements. The expression construct may be intracellular or episomal, such as a plasmid, or it may be inserted into a chromosome. In various embodiments, the expression vector includes a selection marker gene to enable the selection of transformed host cells. The selection marker gene is well known in the art and varies depending on the host cell used. 【0161】 In another aspect of this disclosure, the nucleic acid of the subject is provided in an expression vector containing a nucleotide sequence functionally linked to at least one regulatory sequence encoding a VitoKine construct. The term “expression vector” refers to a plasmid, phage, virus, or vector for expressing a polypeptide from a polynucleotide sequence. Vectors suitable for expression in host cells are readily available, and insertion of nucleic acid molecules into the vector is performed using standard recombinant DNA techniques. Such vectors may contain a variety of regulatory sequences that, when functionally linked, regulate the expression of a DNA sequence and can be used in these vectors to express the DNA sequence encoding a VitoKine construct. Such useful regulatory sequences include, for example, the early and late promoters of SV40, the tet promoter, the pre-early promoter of adenovirus or cytomegalovirus, the RSV promoter, the lac system, the trp system, the TAC system or TRC system, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of λ phage, regulatory regions for fd coat proteins, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, promoters for acid phosphatases (e.g., PhoS), promoters for yeast α-conjugation factors, polyhedron promoters of baculovirus systems, and other sequences known to regulate the expression of genes in prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof. It will be understood that the design of expression vectors may differ depending on factors such as the selection of the host cell to be transformed and / or the type of protein to be expressed. Furthermore, the copy number of the vector, its ability to regulate copy number, and the expression of any other proteins encoded by the vector (such as antibiotic markers) should also be considered. Examples of suitable expression vectors for VitoKine include pDSRa containing VitoKine polynucleotide and any additional suitable vectors known in the art or described below, and derivatives thereof. 【0162】 The recombinant nucleic acids of this disclosure can be produced by ligating the cloned gene or a portion thereof into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast cells, avian cells, insect cells, or mammalian cells), or both. The expression solvent for producing the recombinant VitoKine construct includes plasmids and other vectors. Suitable vectors include, for example, the following plasmids for expression in prokaryotic cells such as E. coli: pBR322 plasmid, pEMBL plasmid, pEX plasmid, pBTac plasmid, and pUC plasmid. 【0163】 Some mammalian expression vectors contain both prokaryotic sequences that promote vector growth in bacteria and one or more eukaryotic transcription units expressed in eukaryotic cells. Examples of mammalian expression vectors suitable for eukaryotic cell transfection include pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg. Some of these vectors have been modified with sequences derived from bacterial plasmids, such as pBR322, to promote replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, for transient protein expression in eukaryotic cells, derivatives of viruses such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, derived from pREP, and p205) can be used. Examples of other viral (including retrovirus) expression systems can be found in the description of gene therapy delivery systems below. Various methods used in plasmid preparation and host organism transformation are well known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, as well as general recombination methods, see Chapters 16 and 17 of Molecular Cloning: A Laboratory Manual, 2nd edition, Sambrook, Fritsch, and Maniatis (eds.) (Cold Spring Harbor Laboratory Press, 1989). In some cases, it may be desirable to express recombinant polypeptides using baculovirus expression systems. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as B-gal-containing pBlueBacIII). 【0164】 In various embodiments, vectors are designed to produce the subject VitoKine construct in CHO cells, such as the Pcmv-Script vector (Stratagene, La Jolla, California), the pcDNA4 vector (Invitrogen, Carlsbad, California), and the pCI-neo vector (Promega, Madison, Wisconsin). As will be described later, the subject gene construct can be used to induce expression of the subject VitoKine construct in cells grown in culture medium, thereby producing, for example, a protein (including a fusion protein or variant protein) which can then be purified. 【0165】 This disclosure also relates to host cells transfected with recombinant genes containing nucleotide sequences encoding amino acid sequences of one or more subjective VitoKine constructs. The host cells may be any prokaryotic or eukaryotic cells. For example, the VitoKine constructs of this disclosure may be expressed in bacterial cells such as Escherichia coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells known to those skilled in the art include Chinese hamster ovary (CHO) cells or human embryonic kidney 293 (HEK293) cells. 【0166】 In response to this, the disclosure further relates to a method for producing the subject VitoKine construct. For example, the VitoKine construct can be expressed by culturing host cells transfected with an expression vector encoding the VitoKine construct under appropriate conditions. After secretion, the VitoKine construct can be isolated from a mixture of cells and culture medium containing the VitoKine construct. Alternatively, the VitoKine construct may be retained in the cytoplasm or membrane fraction, the cells may be recovered and lysed, and the protein isolated. The cell culture medium includes host cells, culture medium, and other by-products. Suitable culture media for cell culture are well known in the art. 【0167】 The polypeptides and proteins of this disclosure can be purified according to protein purification methods well known to those skilled in the art. These methods involve crude fractionation of proteinaceous and nonproteinaceous fractions at a certain level. After separating the peptide polypeptide from other proteins, the peptide or polypeptide of interest can be further purified by chromatography and electrophoresis to achieve partial or complete purification (i.e., purification to homogeneity). The terms “isolated polypeptide” or “purified polypeptide,” as used herein, are intended to refer to a composition that can be isolated from other components, in which the polypeptide has been purified to any degree relative to its naturally occurring state. A purified polypeptide also refers to a polypeptide that has been isolated from the environment in which it may occur naturally. Generally, “purified” refers to a polypeptide composition that has been fractionated and various other components removed, substantially retaining its expressed biological activity. When the term "substantially purified" is used, this designation refers to a peptide composition or polypeptide composition in which polypeptides or peptides form the main components of the composition, for example, constituting about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, or about 90% or more of the proteins in the composition. 【0168】 Various methods suitable for purification are well known to those skilled in the art. These methods include, for example, precipitation using ammonium sulfate, PEG, antibodies (immunoprecipitation), or precipitation by thermal denaturation followed by centrifugation; chromatography such as affinity chromatography (protein A column), ion exchange chromatography, gel filtration chromatography, reversed-phase chromatography, hydroxyapatite chromatography, and hydrophobic interaction chromatography; isoelectric focusing; gel electrophoresis; and combinations of these methods. As is well known in the art, it is considered that even if the order in which various purification steps are performed is changed, or even if certain steps are omitted, a method suitable for preparing substantially purified polypeptides can still be obtained. 【0169】 Pharmaceutical composition In another embodiment, the Disclosure provides a pharmaceutical composition comprising a VitoKine construct mixed with a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are well known and understood by those skilled in the art and have been extensively described (see, for example, Remington's Pharmaceutical Sciences, 18th edition, ARGennaro (ed.), Mack Press, 1990). The pharmaceutically acceptable carrier may be included for the purpose of altering, maintaining, or preserving the pH, molar osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate, release rate, adsorption, or permeability of the composition. Such a pharmaceutical composition may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the polypeptide.Suitable pharmaceutically acceptable carriers include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffering agents (such as borates, bicarbonates, tris-HCl, citrates, phosphates, and other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants; flavorings and diluting agents. Agent; emulsifier; hydrophilic polymer (e.g., polyvinylpyrrolidone); low molecular weight polypeptide; salt-forming counterion (e.g., sodium); preservative (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvent (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohol (e.g., mannitol or sorbitol); suspending agent; surfactant or wetting agent (e.g., Pluronic acid, PEG, sorbitan ester, polysorbate, e.g., polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapal); stabilizer (e.g., sucrose or sorbitol); isotonic enhancing agent (e.g., alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, sorbitol); delivery carrier (delivery Examples include, but are not limited to, vehicles; diluents; excipients; and / or pharmaceutical adjuvant. 【0170】 The primary solvent or carrier in the pharmaceutical composition may be essentially aqueous or non-aqueous. For example, a suitable solvent or carrier may be distilled water for injection, physiological saline, or artificial cerebrospinal fluid, and other substances commonly found in parenteral compositions may be added. Further exemplary solvents include neutral buffered saline or saline mixed with serum albumin. Other exemplary pharmaceutical compositions include Tris buffer at approximately pH 7.0–8.5, or acetate buffer solution at approximately pH 4.0–5.5, the buffer of which may further contain sorbitol or a suitable substitute. In one embodiment of this disclosure, a selected composition having the desired purity may be mixed with any formulation agent (Remington's Pharmaceutical Sciences, above) to prepare the composition for storage in the form of a lyophilized cake or aqueous solution. Furthermore, the therapeutic composition may be formulated as a lyophilized product with appropriate pharmaceutical additives such as sucrose. The optimal pharmaceutical composition is determined by those skilled in the art based on the intended route of administration, mode of delivery, and target dose, etc. 【0171】 When parenteral administration is intended, the therapeutic pharmaceutical composition may be in the form of a pyrogenically acceptable aqueous solution for parenteral administration, containing the desired VitoKine construct in a pharmaceutically acceptable solvent. A particularly preferred solvent for parenteral injection is sterile distilled water, in which the polypeptide is formulated as a properly preserved sterile isotonic solution. In various embodiments, pharmaceutical formulations suitable for injection may be formulated in aqueous solutions, but it is preferable that they be formulated in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological buffered saline. The aqueous injection suspension may contain a substance that increases the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Furthermore, the suspension of the active compound may be prepared as a suitable oily injection suspension. If desired, the suspension may contain a suitable stabilizer, i.e., a drug that increases the solubility of the compound, thereby enabling the preparation of a highly concentrated solution. 【0172】 In various embodiments, therapeutic pharmaceutical compositions can be formulated for targeted delivery using colloidal dispersion systems. Examples of colloidal dispersion systems include polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Examples of lipids useful for liposome formation include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine, as well as sphingolipids, cerebrosides, and gangliosides. Exemplary phospholipids include egg yolk phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. Liposome targeting is also possible, for example, based on organ specificity, cell specificity, and organelle specificity, and is known in the art. 【0173】 In various embodiments, the pharmaceutical composition is intended to be administered orally. The pharmaceutical composition administered in this manner may be formulated with or without carriers, which are commonly used in the preparation of solid dosage forms such as tablets and capsules. In solid dosage forms for oral administration (capsules, tablets, pills, sugars, powders, granules, etc.), one or more therapeutic compounds of the present disclosure may be mixed with one or more pharmaceutically acceptable carriers, for example, sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or bulking agents such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants such as glycerol; (4) disintegrants such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents such as paraffin. (6) Absorption enhancers such as quaternary ammonium compounds; (7) Wetting agents such as cetyl alcohol and glycerol monostearate; (8) Adsorbents such as kaolin clay and bentonite clay; (9) Lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) Coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical composition may contain buffering agents. Similar types of solid compositions may be used as fillers in soft gelatin capsules and hard gelatin capsules, using pharmaceutical additives such as lactose and high molecular weight polyethylene glycol. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, aqueous solutions, suspensions, syrups, and elixirs.In addition to the active ingredient, the liquid dosage form may contain inert excipients commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol sorbitan fatty acid esters, and mixtures thereof. 【0174】 In various embodiments, topical administration of the pharmaceutical composition to the skin or mucous membranes is intended. Topical formulations may further contain one or more of various agents known to be effective as skin penetration enhancers or stratum corneum penetration enhancers. Examples of these include 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl alcohol, isopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents may be further included to make the formulation cosmetically acceptable. Examples of these include fats, waxes, oils, pigments, fragrances, preservatives, stabilizers, and surfactants. Keratinophages, such as those known in the art, may also be included. Examples include salicylic acid and sulfur. Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and, if necessary, with any preservatives, buffers, or propellants. In addition to the compounds of the subject matter of this disclosure (e.g., VitoKine constructs), ointments, pastes, creams, and gels may contain pharmaceutical additives such as animal fats, vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof. 【0175】 Additional pharmaceutical compositions intended for use herein include formulations comprising polypeptides in sustained-release or controlled-delivery formulations. In various embodiments, the pharmaceutical composition may be incorporated into nanoparticles as a sustained-release hydrogel or incorporated into an oncolytic virus. Methods for such nanoparticles include, for example, encapsulation into nanoparticles consisting of polymers having a hydrophobic backbone and hydrophilic branches as drug carriers, encapsulation into microparticles, insertion into liposomes in emulsions, and binding to other molecules. Examples of nanoparticles include mucosal-adhering nanoparticles coated with chitosan or Carbopol (Takeuchi et al., Adv. Drug Deliv. Rev. 47(1):39-54, 2001) and nanoparticles containing charged combination polyesters, poly(2-sulfobutyl-vinyl alcohol) and poly(D,L-lactic acid-coglycolic acid) (Jung et al., Eur. J. Pharm. Biopharm. 50(1):147-160, 2000). Albumin-based nanoparticle compositions have been developed as drug delivery systems for delivering hydrophobic drugs such as taxanes. See, for example, U.S. Patents 5,916,596, 6,506,405, 6,749,868, 6,537,579, 7,820,788, and 7,923,536. Abraxane®, an albumin-stabilized nanoparticle formulation of paclitaxel, was approved in the United States in 2005 for the treatment of metastatic breast cancer and has since been approved in various other countries. 【0176】 Methods for formulating various other sustained or controlled delivery means, such as liposome carriers, biodegradable microparticles or porous beads, and depot agents, are also known to those skilled in the art. 【0177】 The effective dose of a therapeutically used pharmaceutical composition varies depending on the nature and purpose of the treatment. That is, as will be understood by those skilled in the art, the appropriate dose level for treatment will vary in part depending on the molecule being delivered, the indication for which the polypeptide is intended to be used, the route of administration, and the patient's size (body weight, body surface, or organ size) and condition (age, and overall health). Therefore, clinicians may adjust the dose setting and route of administration to achieve the optimal therapeutic effect. Typical doses can range from approximately 0.0001 mg / kg to approximately 100 mg / kg or higher, depending on the factors mentioned above. Preferably, the polypeptide composition may be injected or administered intravenously. A sustained-release pharmaceutical composition may be administered every 3-4 days, weekly, or bi-weekly, depending on the half-life and clearance rate of the particular formulation. The frequency of administration will depend on the pharmacokinetic parameters of the polypeptide in the formulation used. Typically, the composition is administered until a dose achieving the desired effect is reached. The composition may therefore be administered as a single dose, as multiple doses over a long period (at the same or different concentrations per dose), or as a continuous intravenous infusion. Further improvements to the appropriate dosage will be made periodically. The appropriate dosage may be confirmed using appropriate dose-response data. 【0178】 The routes of administration of the pharmaceutical composition are according to known methods, for example, by injection via oral, intravenous, intraperitoneal, intratumoral, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intrafocal, intramedullary, subarachnoid, intraventricular, intravesical, percutaneous, subcutaneous, or intraperitoneal route; as well as by intranasal, enteral, topical, sublingual, transurethral, ​​transvaginal, or transrectal means, continuous release system, or implantable device. If desired, the composition may be administered by bolus, continuous infusion, or by implantable device. Alternatively, the composition may be administered topically by implantation of a membrane, sponge, or other suitable material on which the target molecule is adsorbed or encapsulated. When an implantable device is used, the device can be implanted in any suitable tissue or organ, and the target molecule may be delivered via diffusion, a timed-release bolus, or continuous administration. 【0179】 therapeutic use This disclosure provides a method for treating cancer cells in a subject, comprising administering to the subject a therapeutically effective amount (as a monotherapy regimen or in a combination therapy regimen) of the VitoKine construct of this disclosure in a pharmaceutically acceptable carrier, wherein such administration inhibits the growth and / or proliferation of cancer cells. In particular, the VitoKine construct of this disclosure is useful in treating disorders characterized as cancer. Such disorders include, but are not limited to, solid tumors such as breast cancer, airway cancer, brain cancer, genital cancer, gastrointestinal cancer, urinary tract cancer, eye cancer, liver cancer, skin cancer, head and neck cancer, thyroid cancer, parathyroid cancer, and their distant metastases, lymphoma, sarcoma, multiple myeloma, and leukemia. Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, intraductal carcinoma in situ, and non-invasive lobular carcinoma. Examples of airway cancers include, but are not limited to, small cell lung cancer, non-small cell lung cancer, bronchial adenoma, and pleuroblastoma. Examples of brain cancers include, but are not limited to, brainstem glioma, hypothalamic glioma, cerebellar astrocytoma, cerebral astrocytoma, neuroblastoma, medulloblastoma, ependymoma, neuroectodermal tumor, and pineal gland tumor. Examples of male reproductive organ tumors include, but are not limited to, prostate cancer and testicular cancer. Examples of female reproductive organ tumors include, but are not limited to, endometrial cancer, cervical cancer, ovarian cancer, vaginal cancer, and vulvar cancer, as well as uterine sarcoma. Examples of gastrointestinal tract tumors include, but are not limited to, anal cancer, colon cancer, colorectal cancer, esophageal cancer, gallbladder cancer, stomach cancer, liver cancer, breast cancer, pancreatic cancer, rectal cancer, small intestine cancer, and salivary gland cancer. Tumors of the urinary tract include, but are not limited to, bladder cancer, penile cancer, kidney cancer, renal pelvis cancer, ureteral cancer, and urethral cancer. Tumors of the eye include, but are not limited to, intraocular melanoma and retinoblastoma. Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (hepatocellular carcinoma with or without lamellar dysplasia), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed types of hepatocellular carcinoma and cholangiocarcinoma.Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell carcinoma, and non-melanoma skin cancer. Head and neck cancers include, but are not limited to, nasopharyngeal cancer, as well as cancers of the lips and oral cavity. Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and lymphomas of the central nervous system. Sarcomas include, but are not limited to, soft tissue sarcomas, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and pilocytic cell leukemia. In various embodiments, cancers are characterized by high expression levels of TGF-β family members such as activin A, myostatin, TGF-β, and GDF15, and include, for example, pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, lung cancer, prostate cancer, brain cancer, bladder cancer, and head and neck cancer. 【0180】 In various embodiments, VitoKine constructs can be used as monotherapy for the treatment of all types of cancer, including but not limited to non-small cell lung cancer, small cell lung cancer, melanoma, renal cell carcinoma, urothelial carcinoma, liver cancer, breast cancer, pancreatic cancer, colorectal cancer, gastric cancer, prostate cancer, and sarcoma. 【0181】 In another embodiment, the Disclosure provides a method for treating an autoimmune disease in a subject, comprising administering to the subject a therapeutically effective amount (as a monotherapy regimen or in a combination therapy regimen) of the VitoKine construct of the Disclosure in a pharmaceutically acceptable carrier. “Autoimmune disease” means a non-malignant disease or disorder that originates from and is directed toward an individual’s own tissues. Examples of autoimmune diseases or disorders include, but are not limited to, inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); reactions associated with inflammatory bowel disease (e.g., Crohn’s disease and ulcerative colitis); dermatitis; allergic conditions, such as eczema and asthma; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including, but not limited to, lupus nephritis and cutaneous lupus); diabetes mellitus (e.g., type 1 diabetes mellitus or insulin-dependent diabetes mellitus); multiple sclerosis and juvenile-onset diabetes mellitus. 【0182】 In another embodiment, the present disclosure provides a method for treating an inflammatory disease in a subject, comprising administering to the subject a therapeutically effective amount (as a monotherapy regimen or in a combination therapy regimen) of the VitoKine construct of the present disclosure in a pharmaceutically acceptable carrier. “Inflammatory disease” encompasses all diseases involving acute or chronic inflammation. Acute inflammation is the body’s initial response to a harmful stimulus, resulting from increased migration of plasma and leukocytes (e.g., granulocytes) from the blood to the injured tissue. Numerous biochemical events, including the local vascular system, immune system, and various cells within the injured tissue, propagate and mature the inflammatory response. Persistent inflammation, also known as chronic inflammation, is characterized by a gradual change in the types of cells present at the site of inflammation, and simultaneous tissue destruction and recovery from the inflammatory process. Examples of inflammatory diseases are well known in the art. In various embodiments, inflammatory diseases are selected from the group consisting of inflammatory bowel disease, psoriasis, and bacterial sepsis. As used herein, the term “inflammatory bowel disease” refers to a group of inflammatory conditions of the colon and small intestine, including, for example, Crohn’s disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, empty colitis, Behçet’s syndrome, and unclassified colitis. 【0183】 In another embodiment, the Disclosure provides a method for treating a viral infection in a subject, comprising administering to the subject a therapeutically effective amount (as a monotherapy regimen or in a combination therapy regimen) of the VitoKine construct of the Disclosure in a pharmaceutically acceptable carrier. In various embodiments, the viral infection of the subject to be treated may be caused by an infectant including, but not limited to, bacteria, fungi, protozoa, and viruses. Viral diseases that can be prevented, treated, and / or managed according to the methods described herein include, but are not limited to, hepatitis A, hepatitis B, hepatitis C, influenza, varicella, adenovirus, herpes simplex virus type I (HSY-I), herpes simplex virus type II (HSY-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papillomavirus, papovavirus, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsackievirus, mumps virus, measles virus, rubella virus, poliovirus, smallpox, Epstein-Barr virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), and viral diseases caused by vectors of viral diseases such as viral meningitis, encephalitis, dengue fever, or smallpox. 【0184】 Bacterial diseases caused by bacteria (e.g., Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Candida albicans, Proteus vulgaris, Staphylococcus viridians, and Pseudomonas aeruginosa) that can be prevented, treated, and / or controlled according to the methods described herein include mycobacteria such as Rickettsia, Mycoplasma, Neisseria, Streptococcus pneumoniae, Borrelia burgdorferi (Lyme disease), and Bacillus anthrosis. This includes, but is not limited to, Bacillus anthracis (bacteria that cause anthrax), Bacillus tetanus, Streptococcus, Staphylococcus, Mycobacterium, pertussis, cholera, plague, diphtheria, Chlamydia, Staphylococcus aureus (S. aureus), and Legionella. 【0185】 Protozoan diseases caused by protozoa that can be prevented, treated, and / or controlled according to the methods described herein include, but are not limited to, leishmania, cokzidioa, trypanosoma, or malaria. 【0186】 Parasitic diseases caused by parasites that can be prevented, treated, and / or controlled according to the methods described herein include, but are not limited to, Chlamydia and Rickettsia. 【0187】 The term "therapeutic dose" refers to the amount of medication to be administered that will alleviate, to some extent, one or more symptoms of the disorder being treated. 【0188】 First, IC 50The effective therapeutic dose can be evaluated from the cell culture assay by measurement. Next, in an animal model, the above IC measured in the cell culture medium 50 Doses can be formulated to achieve a circulating plasma concentration range that includes [specific component]. Using such information, useful doses in humans can be determined more accurately. Plasma levels can be measured by HPLC or other methods. Depending on the patient's condition, the appropriate composition, route of administration, and dose can be selected by individual physicians. 【0189】 The administration plan can be adjusted to obtain the optimal desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus dose may be administered, followed by several divided doses (multiple doses, repeated doses, or maintenance doses) over a long period, with the dose increasing or decreasing in proportion to the needs of the treatment situation. For ease of administration and dose uniformity, it is particularly advantageous to formulate parenteral compositions in drug units. As used herein, a drug unit refers to a physically separated unit suitable as a uniform dose for the mammalian subject being treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in combination with the necessary pharmaceutical carrier. The specifications of the drug unit in this disclosure are determined primarily by the unique characteristics of the antibody and the specific therapeutic or prophylactic effect to be achieved. 【0190】 In other words, as will be understood by those skilled in the art, based on the disclosures provided herein, doses and administration plans are adjusted according to methods well known in the therapeutic field. That is, the maximum tolerated dose can be easily established, the effective dose to give a detectable therapeutic effect to the subject can be determined, and similarly, the time requirements for administering each drug to give a detectable therapeutic effect to the subject can be determined. Thus, although certain doses and administration plans are illustrated herein, these examples do not in any way limit the doses and administration plans that may be given to a subject when implementing this disclosure. 【0191】 Furthermore, dose values ​​will vary depending on the type and severity of the condition to be improved, and may include single doses or repeated doses. It should also be understood that for any particular subject, a specific administration plan should be adjusted over a long period according to the individual needs and the professional judgment of the person managing or supervising the administration of the composition, and that the dose ranges described herein are for illustrative purposes only and are not intended to limit the scope or practice of the claimed composition. Moreover, administration plans using the compositions of this disclosure may be based on a variety of factors, including the type of disease, the subject's age, weight, sex, medical condition, severity of the condition, route of administration, and the specific antibody used. That is, administration plans can be diverse but can be routinely determined using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include toxic effects and / or clinical effects such as laboratory values. In other words, this disclosure includes intra-subject dose escalation as required by those skilled in the art. Determining appropriate doses and administration plans is well known in the relevant art and should be understood as achievable by those skilled in the art if the teachings disclosed herein are provided. 【0192】 The exemplary and non-limiting daily dose range of a therapeutic or prophylactic effective dose of VitoKine or VitoKine variant of this disclosure is 0.0001–100 mg / kg body weight, 0.0001–90 mg / kg body weight, 0.0001–80 mg / kg body weight, 0.0001–70 mg / kg body weight, 0.0001–60 mg / kg body weight, 0.0001–50 mg / kg body weight, 0.0001–40 mg / kg body weight, 0.0001–30 mg / kg body weight, 0.0001–20 mg / kg body weight, 0.00 01~10mg / kg body weight, 0.0001~5mg / kg body weight, 0.0001~4mg / kg body weight, 0.0001~3mg / kg body weight, 0.0001~2mg / kg body weight, 0.0001~1mg / kg body weight, 0.001~50mg / kg body weight, 0. 001~40mg / kg body weight, 0.001~30mg / kg body weight, 0.001~20mg / kg body weight, 0.001~10mg / kg body weight, 0.001~5mg / kg body weight, 0.001~4mg / kg body weight, 0.001~3mg / kg body weight, 0.00 1-2 mg / kg body weight, 0.001-1 mg / kg body weight, 0.010-50 mg / kg body weight, 0.010-40 mg / kg body weight, 0.010-30 mg / kg body weight, 0.010-20 mg / kg body weight, 0.010-10 mg / kg body weight, 0.010-5 mg / kg body weight, 0.010-4 mg / kg body weight, 0.010-3 mg / kg body weight, 0.010-2 mg / kg body weight, 0.010-1 mg / kg body weight, 0.1-50 mg / kg body weight, 0.1-40 mg / kg body weight, 0.1-30 mg / kg body weight The dosage can be g, 0.1-20 mg / kg body weight, 0.1-10 mg / kg body weight, 0.1-5 mg / kg body weight, 0.1-4 mg / kg body weight, 0.1-3 mg / kg body weight, 0.1-2 mg / kg body weight, 0.1-1 mg / kg body weight, 1-50 mg / kg body weight, 1-40 mg / kg body weight, 1-30 mg / kg body weight, 1-20 mg / kg body weight, 1-10 mg / kg body weight, 1-5 mg / kg body weight, 1-4 mg / kg body weight, 1-3 mg / kg body weight, 1-2 mg / kg body weight, or 1-1 mg / kg body weight. Note that the dosage value may vary depending on the type and severity of the condition to be improved.Furthermore, it should be understood that for any particular subject, the specific administration plan should be adjusted over a long period of time according to the individual needs and the professional judgment of the person managing or supervising the administration of the composition, and that the dose ranges described herein are for illustrative purposes only and are not intended to limit the scope or practice of the claimed composition. 【0193】 The toxicity and therapeutic index of the pharmaceutical compositions disclosed herein is LD50. 50 (A lethal dose for 50% of the population) or ED 50 This can be determined using standard pharmaceutical methods in cell culture media or experimental animals to find the dose that is effective in treating 50% of the population. The dose ratio between the toxic dose and the therapeutic dose is the therapeutic index, or LD50. 50 / ED 50 It can be expressed as a ratio. Compositions exhibiting a large therapeutic index are generally preferred. 【0194】 The frequency of administration of the VitoKine construct pharmaceutical composition will depend on the treatment method and the nature of the specific disease being treated. The patient may be treated at regular intervals, such as weekly or monthly, until the desired treatment outcome is achieved. Examples of administration frequencies, but not limited to, include: once a week; once a week every other week; once every two weeks; once every three weeks; once a week for two weeks, then once a month; once a week for three weeks, then once a month; once a month; once every two months; once every three months; once every four months; once every five months; or once every six months, or once a year. 【0195】 Combination therapy As used herein, the terms “concurrent administration,” “concurrently administered,” and “in combination with” referring to a VitoKine construct of the Disclosure and one or more other therapeutic agents are intended to mean, and include: concurrent administration of such combination of a VitoKine construct of the Disclosure and a therapeutic agent to a subject in need of treatment, wherein such components are combined into a single dosage form and each component is released to the subject substantially simultaneously; substantially concurrent administration of such combination of a VitoKine construct of the Disclosure and a therapeutic agent to a subject in need of treatment, wherein such components are formulated separately into separate dosage forms and are released to the subject substantially simultaneously when ingested substantially simultaneously by the subject. Substantially simultaneous administration; continuous administration of such combination of the VitoKine constructs and therapeutic agents of the present disclosure to a subject in need of treatment, wherein the components are formulated separately to form separate dosage forms, and when they are ingested by the subject in a continuous period of time with a considerable time interval between each administration, the components are released to the subject at substantially different times; and continuous administration of such combination of the VitoKine constructs and therapeutic agents to a subject in need of treatment, wherein the components are combined to form a single dosage form, and when the components are released in a sustained-release manner, they are released to the subject simultaneously and / or at different times in a concurrent, continuous, and / or overlapping manner, and each portion may be administered via the same or different routes. 【0196】 In another embodiment, the Disclosure provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the Invention in combination with a second treatment method, which includes but is not limited to immunotherapy, cytotoxic chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiotherapy, and stem cell transplantation. For example, such a method can be used prophylactically, for the prevention of cancer, and for the prevention of cancer recurrence and metastasis after surgery, as an adjunct to other conventional cancer treatments. As permitted by the Disclosure, the effectiveness of conventional cancer treatments (e.g., chemotherapy, radiotherapy, phototherapy, immunotherapy, and surgery) can be enhanced through the use of the combination methods described herein. 【0197】 A wide range of conventional compounds have been shown to possess antitumor activity. These compounds are used as drugs in chemotherapy to regress solid tumors, inhibit metastasis and further growth, or reduce the number of malignant T cells in leukemic or myeloid malignancies. While chemotherapy has been effective in treating various types of malignancies, many antitumor compounds induce undesirable side effects. It has been shown that when two or more different therapies are combined, they may work synergistically, allowing for dose reductions of each therapy and thereby reducing the adverse side effects that each compound may exhibit at higher doses. In other cases, treatment-resistant malignancies may respond to combination therapy of two or more different therapies. 【0198】 In various embodiments, a second anticancer agent, such as a chemotherapeutic agent, is administered to the patient. A list of exemplary chemotherapeutic agents includes: daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, bendamustine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin, carboplatin, and oxaliplatin. Examples of such chemotherapeutic agents include, but are not limited to, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fludarabine, ifosfamide, hydroxyurea, taxanes (such as paclitaxel and docetaxel), and / or anthracycline antibiotics, as well as combinations of agents such as DA-EPOCH, CHOP, CVP, or FOLFOX. In various embodiments, the dose of such chemotherapeutic agents may be, but is not limited to, approximately 10 mg / m². 2 , about 20mg / m 2 , about 30mg / m 2 , about 40mg / m 2 , about 50mg / m 2 , about 60mg / m 2 , about 75mg / m 2 , about 80mg / m 2 , about 90mg / m 2 , about 100mg / m 2 , about 120mg / m 2 , about 150mg / m 2 , about 175mg / m 2 , about 200mg / m 2 , about 210mg / m 2 , about 220mg / m 2 , about 230mg / m 2 , about 240mg / m 2 , about 250mg / m 2 , about 260mg / m 2, and approximately 300 mg / m² 2 One of the following. 【0199】 In various embodiments, the combination therapy of the present disclosure may further include administering a therapeutically effective dose of immunotherapy to a target, such as: therapy with depleted antibodies against specific tumor antigens; therapy with antibody-drug conjugates; and antibodies against CTLA-4, PD-1, PDL-1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, SIRPα, CD47, GITR, ICOS, CD27, Siglec7, Siglec8, Siglec9, Siglec15, and VISTA, CD276, CD272, TIM-3, B7-H4. Treatments include, but are not limited to, those using agonist antibodies, antagonist antibodies, or inhibitory antibodies against costimulatory or coinhibitory molecules (immune checkpoints); treatments using bispecific T cell inducing antibodies (BiTE®) such as blinatumomab; treatments involving the administration of biological response modifiers such as IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, IL-22, GM-CSF, IFN-α, IFN-β, IFN-γ, TGF-β antagonists, or TGF-β traps; and tumor lysis such as T-vec. Therapies using therapeutic vaccines, including but not limited to detoxifying viruses or therapeutic vaccines such as ciproisel T; dendritic cell vaccines, or tumor antigen peptide vaccines or neoantigen vaccines; chimeric antigen receptor (CAR)-T cells; CAR-NK cells; NK cells; iPS-induced NK cells; iPS-induced T cells; iPS-induced CAR-T cells or iPS-induced CAR-NK cells; tumor-infiltrating lymphocytes (TILs); adoptive transfer Treatments include, but are not limited to, those using grafted anti-tumor T cells (extra vivo-grown and / or TCR-T cells); treatments using TALL-104 cells; and treatments using immunostimulants, such as Toll-like receptor (TLR) agonists CpG, TLR7, TLR8, TLR9, and vaccines such as Calmette-Guérin bacillus (BCG), and imiquimod; the above combination therapies increase the killing of tumor cells by effector cells, i.e., a synergistic effect exists between the VitoKine construct and immunotherapy when administered simultaneously. 【0200】 In various embodiments, the combination therapies of the present disclosure may further comprise administering therapeutically effective doses of anti-inflammatory agents to autoimmune diseases, inflammatory diseases, and other immunodeficiencies, for example, therapy with depletion antibodies against specific immune cells; IL-1α, IL-1β or IL-1R, IL-4 or IL-4R, IL-5 or IL-5R, IL-6 or IL-6R, IL-8 or IL-8R, IL-7 or IL-7R, IL-10 or IL-10R, IL-11 or IL-11R, IL-12 or IL-12R, IL-17 or IL-17R, IL-18 or IL-18R, IL-21 or IL-18R, IL-22 or IL-22R, IL-23 or IL-23R, MCSF or MCSF-R, GM-CSF or GM-CSFR, IFN-α, IFN-β, IFN-γ, TGF-α, TGF-β or TGF-β, TNF family or related receptors, integrin family (for example) Treatment using modulated antibodies (agonist antibodies, antagonist antibodies, or blocking antibodies) as immune response target modifiers against targets (ligands or their receptors), including but not limited to α4β7, TSLP, complement C5 (C5) or C5a, IgE, APRIL, TACI, BCMA, CD20, CD22, CD40 / CD40L, B7H1, B7H2, ICOS, BAFF, BCR, Blys, B7RP1, TLR7, TLR8, and TLR9; NFκB Treatment with regulatory small molecules (agonists or antagonists) as immune response target modifiers against targets including, but not limited to, Jak1, Jak2, Jak3, Tyk2, Syk, BTK, PIK3, cyclooxygenase 2, and NMDA receptors; the above combination therapies increase the effectiveness of immune response modulation, i.e., a synergistic effect exists between VitoKine constructs and anti-inflammatory therapies when administered simultaneously. 【0201】 In various embodiments, combination therapy involves simultaneously administering a VitoKine construct and a second drug composition, either in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, the VitoKine construct composition and the second drug composition are administered sequentially, i.e., the VitoKine construct composition is administered before or after the administration of the second drug composition. In various embodiments, the administration of the VitoKine construct composition and the second drug composition is simultaneous, i.e., the timing of administration of the VitoKine construct composition and the timing of administration of the second drug composition overlap. In various embodiments, the administration of the VitoKine construct composition and the second drug composition is not simultaneous. For example, in various embodiments, the administration of the VitoKine construct composition is completed before the administration of the second drug composition. In various embodiments, the administration of the second drug composition is completed before the administration of the VitoKine construct composition. [Examples] 【0202】 The following embodiments are provided to better illustrate the present disclosure and are not intended to limit the scope of the present disclosure. 【0203】 Example 1 Construction and fabrication of IL-15 Fc VitoKine constructs The goal was to design an IL-15 VitoKine construct that remains inactive until locally activated by a protease upregulated in cancer or affected tissue. VitoKines described herein in which wild-type IL-15 (SEQ ID NO: 2) or an IL-15 mutant protein (e.g., SEQ ID NO: 3) as the active moiety is reversibly shielded between the Fc domain and IL-15RαSushi+ (SEQ ID NO: 5). These constructs contain one or two cleavable linkers recognized by tumor-specific proteases. In the presence of tumor cells expressing the protease, IL-15 activity is restored by cleavage of the linker linking Fc to the IL-15 mutant protein and / or the linker linking IL-15 to IL-15αSushi+. Notably, the free IL-15αSushi+ after proteolysis exhibits an exceptionally high affinity (K) between IL-15 and IL-15Rα. D At 30 pM, it is expected that a non-covalent association state with IL-15 will be maintained. IL-15 Fc VitoKine constructs were created by combining linkers and peptide spacers in various ways. Schematic diagrams are shown in Figure 1, and the sequences of each are listed in the table as SEQ ID NOs: 25-43, 162-165, and 169-174. 【0204】 All genes were codon-optimized for expression in mammalian cells, synthesized, and subcloned into recipient mammalian expression vectors (GenScript). Protein expression is driven by a CMV promoter, and a synthetic SV40 poly(A) signal sequence is present at the 3' end of the CDS. A leader sequence is provided at the N-terminus of the above construct, which enables appropriate signal transduction and processing for secretion. 【0205】 The above construct was produced by co-transfecting HEK293-F cells growing in suspension with the mammalian expression vector using polyethyleneimine (PEI, molecular weight 25,000, linear, Polysciences). If two or more expression vectors are present, the vectors will be transfected in a 1:1 ratio. For transfection, HEK293 cells were cultured in serum-free FreeStyle® 293 Expression Medium (Thermo Fisher Scientific). When producing in a 1000 ml shaking flask (working volume 330 mL), HEK293 cells were 0.8 × 10⁶ 24 hours before transfection. 6 Cells were seeded at a density of cells / ml. An expression vector containing a total of 330 μg of DNA was mixed with 16.7 ml of Opti-MeM medium (Thermo Fisher Scientific). 0.33 mg of PEI diluted in 16.7 ml of Opti-MeM medium was added, the mixture was vortexed for 15 seconds, and then allowed to stand at room temperature for 10 minutes. This DNA / PEI solution was then added to the cells and incubated at 37°C in an incubator under an 8% CO2 atmosphere. Sodium butyrate (MilliporeSigma) at a final concentration of 2 mg / L was added to the cell culture medium on day 4 to support the maintenance of protein expression. After 6 days of incubation, the supernatant was collected and purified by centrifugation at 2200 rpm for 20 minutes. This solution was filtered through a sterile filter (0.22 μm filter, Corning). Secretory proteins were purified from the cell culture supernatant using protein A affinity chromatography. 【0206】 In affinity chromatography, the supernatant was loaded onto a HiTrap MabSelectSure Protein A FF column (CV=5mL, GE Healthcare) equilibrated with 25 ml of phosphate-buffered saline (pH 7.2) (Thermo Fisher Scientific). Unbound proteins were removed by washing with 5 column volumes of PBS (pH 7.2), and the target protein was eluted with 25 mM sodium citrate and 25 mM sodium chloride (pH 3.2). The protein solution was neutralized by adding 3% 1M Tris (pH 10.2). The target protein was concentrated using an Amicon® Ultra-15 concentrator 10 kDa NMWC (Merck Millipore). 【0207】 The purity and molecular weight of the purified constructs were analyzed by SDS-PAGE in and out of the presence of a reducing agent, as well as staining with Coomassi (Imperial® Stain). The NuPAGE® Pre-Cast gel system (4-12% or 8-16% Bis-Tris, Thermo Fisher Scientific) was used according to the manufacturer's instructions. The protein concentration of the purified protein sample was determined by measuring the UV absorbance at 280 nm (Nanodrop spectrophotometer, Thermo Fisher Scientific) and dividing by the molar extinction coefficient calculated based on the amino acid sequence. The amount of aggregate of the constructs was analyzed using an Agilent 1200 high-performance liquid chromatography (HPLC) system. The sample was injected into an AdvanceBio size exclusion column (300 Å, 4.6 × 150 mm, 2.7 μm, LC column, Agilent) using 150 mM sodium phosphate (pH 7.0) as the mobile phase at 25°C. 【0208】 P-0315 is a dimeric C-terminal IL-15 Fc VitoKine containing uPA and MMP cleavage sequences in the L1 and L2 linkers, respectively. IL-15 is an S58D variant protein. As an example of the protein profile of IL-15 Fc VitoKine, the SDS-PAGE analysis of P-0315 (SEQ ID NO: 33) is shown in Figure 3A. Size exclusion chromatogram (Figure 3B). 【0209】 Example 2 In the VitoKine form, the in vitro activity of IL-15 was effectively shielded. IL-15 VitoKine P-0172 (SEQ ID NO: 27) contains an IL-15 / IL-15RαSushi+ fusion polypeptide linked by a short GS peptide linker. This fusion polypeptide is in a homodimeric fusion form, bound to the C-terminus of the homodimeric Fc domain via a linker cleavable with uPA. P-0198 is a dimeric C-terminal Fc-IL-15 fusion protein with non-covalently complexed IL-15RαSushi. These two molecules have similar conformations between the Fc and IL-15 fusions, with the main difference being the incorporation of IL-15RαSushi. One is fused via a short GS linker (P-0172), while the other is free due to non-covalent linkage (P-0198). The binding activity of P-0172 to IL-2Rβ was determined by enzyme-linked immunosorbent assay (ELISA) and compared with that of P-0198 (including SEQ ID NOs. 45, 44, and 5), a highly active IL-15 / IL-15Rα-Fc fusion protein. 【0210】 In short, IL-2Rβ-ECD (SEQ ID NO: 12) was coated at 1 μg / well in each well of a Nunk 96-well microplate Maxisorp. After incubation at 4°C overnight and blocking with SuperBlock (Thermo Fisher Scientific), IL-15 compound was added to each well at a rate of 100 μl / well, starting at 100 nM and serially diluted 3-fold. After incubation at room temperature for 1 hour, 100 μl / well of goat anti-human IgG Fc-HRP (diluted 1:5000 with diluent) was added to each well, and incubation at room temperature for 1 hour. After each step, the wells were thoroughly aspirated and washed three times with PBS / 0.05% Tween-20. Finally, 100 μl of TMB substrate (Thermo Fisher) was added to each well, the plates were allowed to develop color in the dark at room temperature for 10 minutes, and 100 μl / well of stop solution (2N sulfuric acid, Ricca Chemical) was added. The absorbance at 450 nm was determined, and the curves were fitted using Prism software (GraphPad). As shown in Figure 4A, the binding activity of VitoKine P-0172 to IL-2Rβ was significantly reduced compared to P-0198 (12.2 nM vs. 0.21 nM), which may be due to spatial constraints caused by the short covalent bond between IL-15 and IL-15RαSushi. This suggests that IL-15RαSushi in the VitoKine platform effectively shielded the IL-15 domain that binds to the receptor. 【0211】 The functional activity of IL-15 VitoKine P-0172, compared to P-0198, was further evaluated by examining IL-15-mediated induction of CD69 expression on human NK cells and human CD8+ T cells derived from fresh human peripheral blood mononuclear cells (PBMCs) using FACS analysis. CD69 is a cell surface glycoprotein that is initially induced during lymphoid system activation, including NK cells and T cells. 【0212】 In short, human PBMCs were isolated by Ficol-Highpack centrifugation from buffy coat purchased from the Oklahoma Blood Institute. Purified human PBMCs were treated with serial dilutions of each IL-15 test compound and incubated at 37°C for 48 hours. Cells were collected by centrifugation at 300×g and resuspended in FACS buffer. After blocking the Fc receptor with Human TruStain FcX (1:50 dilution), cells were stained with anti-human CD56-FITC antibody, anti-human CD69-PE antibody, and anti-human CD8-APC antibody (1:50 dilution). After incubation with the antibodies at room temperature for 30 minutes, cells were collected, washed, resuspended in FACS buffer, and analyzed by flow cytometry. CD69 expression was measured by gateding CD56+ NK cells and CD8+ T cells. Data are expressed as the percentage of CD69-positive cells in the gated population. 【0213】 As shown in Figures 4B and 4C, CD69 activation on CD8+ T cells and NK cells by VitoKine P-0172 was drastically reduced, measurable only at the highest test concentration, and the binding strength was at least 2-3 logarithm lower than that of P-0198. This indicates efficient shielding of IL-15 activity in the VitoKine form. This shielding effect was more pronounced in the PBMC CD69 activation assay than in the IL-2Rβ ELISA binding assay, suggesting that the severity of IL-15 activity impairment is more evident under physiological conditions than under reconstituted ELISA conditions in vitro. Due to spatial constraints, VitoKine was not effective against IL-2Rβ and γ expressed on lymphocytes. C The binding of IL-15 to the complex was severely impaired, resulting in insufficient activation of the pathway and a significant reduction in its activity. 【0214】 The biological activity of monomeric IL-15 Fc VitoKine was also investigated. P-0170 (SEQ ID NO: 26 and SEQ ID NO: 15) is the monomeric counterpart of P-0172, having the same linker and fusion configuration. Compared to the highly active IL-15 Fc fusion protein P-0166, P-0172 showed significantly reduced ability to activate CD69 on CD8+ T cells (Figure 5), suggesting that the monomeric VitoKine platform also efficiently shielded the biological activity of IL-15 in the D2 domain. 【0215】 Example 3 Comparison of shielding efficiency of IL-15 Fc VitoKine between IL-15 and IL-15RαSushi+, and between Fc and IL15, when linker length and composition differ. IL-15 VitoKine is constructed by fusing human IL-15 between two distinct domains, such as the half-life extension Fc domain and its cognitive high-affinity coreceptor α domain, via peptide linkers L1 and L2, as shown in Figure 1. The differences in the effects of the two linkers on the biological activity of IL-15—the linker connecting Fc and IL-15, and the linker connecting IL-15 and the 15RαSushi domain—as well as the differences in the effects of the length and composition of each linker, were investigated for desired activity inhibition. 【0216】 FACS analysis of CD69, an activation marker for immune cell subpopulations in fresh human PBMCs, was performed to evaluate IL-15 VitoKine with different lengths of the non-cleavable linker (L2) between IL-15 and IL-15RαSushi+. The same protocol as in Example 2 was followed. 【0217】 P-0204 (sequence number 30), P-0205 (sequence number 31), and P-0206 (sequence number 32) are IL-15 Fc VitoKines that share the same uPA cleavable linker sequence (L1) between Fc and IL-15. However, the linkers (L2) that connect IL-15 and the IL-15RαSushi+ domain in these three VitoKines differ in length, being (GGGGS)3 (sequence number 127), (GGGGS)2 (sequence number 126), and GGGGS (sequence number 118), respectively. 【0218】 As shown in Figure 6, IL-15 Fc VitoKines with linker lengths varying by 5 to 15 amino acids between IL-15 and IL-15RαSushi+ all exhibited significantly reduced activation ability against CD8+ T cells (Figure 6A) or NK cells (Figure 6B). Comparing the activities of P-0206, P-0205, and P-0204, it was revealed that the shorter the linker length connecting IL-15 and the IL-15RαSushi+ domain, the more inactive the VitoKine became. This suggests that the degree of activity reduction can be further adjusted by the length of the L2 linker. In summary, it was shown that by adjusting the length of the linker (L2) between IL-15 and IL-15Rα to create an appropriate level of spatial constraint, IL-15 can be shielded between the Fc domain and its cognitive high-affinity coreceptor α, resulting in almost complete abolition of its activity. 【0219】 The effect of the linker (L1) connecting Fc and IL-15 on the biological activity of VitoKine was also investigated (shown in Figure 7). P-0204 and P-0203 (SEQ ID NO: 29) share the same 15-amino acid length flexible (G4S)3 linker (SEQ ID NO: 112) (L2) between IL-15 and IL-15Rα, but their L1 linker lengths differ. P-0203 contains a peptide spacer that is longer than P-0204 by the amount of 7 GS-rich residues that sandwich the uPA substrate peptide linking Fc and IL-15. Despite the difference in the length of the L1 linker linking Fc and IL-15, the biological activity of P-0204 and P-0203 is similar (Figure 7), suggesting that the impact on the impairment of IL-15 activity is minimized when the L1 linker linking Fc and IL-15 is between 13 and 35 amino acid residues in length. However, L1 linkers shorter than 13 amino acids, longer than 35 amino acids, or associated with different cytokines may affect the shielding activity of the D2 domain. In the same study, P-0202 was included, which shared the same L1 linker linking Fc and IL-15 as P-0203, but had an L2 linker linking IL-15 and the IL-15RαSushi domain that was 13 amino acids shorter. P-0202 showed lower biological activity than P-0203, confirming that linker L2 is more important than linker L1 in shielding VitoKine activity. 【0220】 The effect of linker composition, i.e., linker peptide sequence, on VitoKine activity was investigated by measuring Ki67 expression in the nuclei of NK cells and CD8 T cells after IL-15 VitoKine treatment. Ki67 is a marker of cell proliferation. An extracellular human PBMC assay was established. Briefly, purified human PBMCs were treated with a serial dilution series of IL-15 VitoKine compounds and incubated at 37°C for 5 days. On day 5, cells were washed once with FACS buffer (1% FBS / PBS) and stained for the first time with an Fc blocker and surface marker antibodies including anti-human CD56-FITC and anti-human CD8-APC (1:50 dilution). After 30 minutes of incubation and washing, the cell pellet was completely resuspended in 200 μl / well of 1×Foxp3 fixation and permeabilization working solution and incubated in the dark at room temperature for 30 minutes. After centrifugation, 200 μl of 1× permeabilization buffer was added to each well for further washing. The cell pellet was resuspended in permeabilization buffer containing anti-human Ki67-PE (1:25 dilution). After incubation at room temperature for 30 minutes, cells were collected, washed, resuspended in FACS buffer, and analyzed by flow cytometry. Data are expressed as the percentage of Ki67-positive cells in a gated population. 【0221】 Since the L2 linker has a more significant impact on the activity of IL-15 VitoKine compared to the L1 linker, the effects of various L2 linker sequence compositions on the biological activity of IL-15 VitoKine were investigated. P-0351 (SEQ ID NO: 25), P-0488 (SEQ ID NO: 163), and P-0489 (SEQ ID NO: 164) all share the same (G4S)3 linker (L1) that links Fc and IL-15. The linkers linking IL-15 and IL-15Rα are all 10 amino acid long, but their sequences differ. The linker is (G4S)2 in P-0351, the MMP-14 substrate peptide (SEQ ID NO: 157) in P-0488, or the regmine substrate peptide (SEQ ID NO: 160) in P-0489. 【0222】 As shown in Figure 8, all three IL-15 VitoKines had a significantly impaired ability to proliferate CD8+ T cells (Figure 8A) or NK cells (Figure 8B) compared to the highly active IL-15 / IL-15Rα Fc fusion protein P-0156 (SEQ ID NOs: 175 and 176). Even when the peptide linker sequences were different, there was only a slight effect on the biological activity of each VitoKine (Figure 8A and Figure 8B), which may be due to the structural flexibility of each linker peptide. The more rigid the L2 linker peptide becomes, the greater the structural constraints imposed on the VitoKine molecule, and the more pronounced the impairment of activity can be. However, since the effect of the L2 linker sequence composition on VitoKine activity was slight, this data supports that various cleavable linkers can be incorporated as the L2 linker to efficiently shield the activity of the D2 domain, thereby broadening the scope of VitoKine design and utility. 【0223】 Taken together, these data indicated that the L2 linker connecting the IL-15 domain (D2) and the IL-15Rα Sushi+ domain (D3) plays an important role in shielding D2 activity to obtain an inactive VitoKine. The level of inactivity could be further adjusted by modulating the length of the L2 linker and changing the linker sequence / flexibility. The selection of the length and sequence of the cleavable L2 linker should balance the presence of specific proteases at the target disease site of interest, the accessibility of the substrate peptide to the protease, and the desired proteolysis rate. 【0224】 Example 4 Determination of reaction conditions suitable for complete in vitro protease cleavage The first in vitro protease cleavage experiments were performed using the IL-15 Fc VitoKine constructs P-0315 and P-0203 to determine the protease cleavage and optimal cleavage conditions for MMP-2 and uPA, respectively. P-0315 (SEQ ID NO: 33) contains a uPA-cleavable linker (L1) linking the Fc domain and the IL-15 domain, and an MMP-2 / 9-cleavable linker (L2) linking the IL-15 domain and the IL-15RαSushi+ domain. P-0203 (SEQ ID NO: 29) contains a single protease-cleavable linker (uPA) (L1) linking the Fc domain and the IL-15 domain. The linker between the IL-15 domain and the IL-15RαSushi+ domain in P-0203 is a flexible (G4S)3 linker. Recombinant human uPA and recombinant human MMP-2 were purchased from BioLegend. The MMP-2 was supplied in latent form and activated with p-aminophenylmercury acetate (APMA, Millipore Sigma) according to the manufacturer's instructions. 【0225】 For protein cleavage with MMP-2, 4 μg of P-0315 was incubated with 30 ng, 100 ng, or 300 ng of APMA-activated MMP-2 in the manufacturer's recommended assay buffer (100 mM Tris, 20 mM CaCl2, 300 mM NaCl, 0.1% (w / v) Brij35, pH 7.5) at 37°C for 3 hours. To stop the reaction, an SDS-PAGE loading dye was added to the reaction mixture, and the mixture was heated at 95°C for 5 minutes. To evaluate cleavage, the digested samples were separated on 4–12% Tris-Bis SDS-PAGE gels. Comparison of untreated and treated samples showed that IL-15 VitoKine was completely cleaved after MMP-2 treatment at all test concentrations. This was indicated by a size shift and the appearance of a sharp band of approximately 9 kDa in the SDS page gel (Figure 9), which was identified as the IL-15RαSushi+ domain cleaved from P-0315. 【0226】 The cleavage ability of uPA was evaluated using P-0203. First, various amounts of uPA were added to 2 μg of P-0203 in 20 μl of PBS (pH 7.2) buffer, and the reaction mixture was incubated at 37°C for 2 hours. Figure 10A shows the cleavage performed using 0, 25 ng, 50 ng, 100 ng, and 300 ng of uPA. The three arrows in Figure 10A are for the non-reducing (NR) sample and indicate the changes in the Fc chain due to uPA proteolysis. In "partial cleavage," the IL-15 / IL-15RαSushi+ fusion polypeptide was cleaved from only one of the two Fc chains, while in "total cleavage," the IL-15 / IL-15RαSushi+ fusion polypeptide was cleaved from both Fc chains. In Figure 10A, the smear-like band circled is the IL-15 / IL-15RαSushi+ fusion polypeptide cleaved from Fc, and this smear-like appearance is most likely due to glycosylation. In the reduced (R) sample, the upper band was identified as the Fc chain linked to the IL-15 / IL-15RαSushi+ fusion polypeptide, and the sharp lower band was identified as the Fc chain from which the IL-15 / IL-15RαSushi+ fusion polypeptide was cleaved. 【0227】 The SDS-PAGE gel clearly shows that the amount of fully cleaved protein in the non-reduced samples gradually increased as the amount of uPA increased. Similarly, the amount of cleaved Fc chains increased in the reduced samples, indicating an increase in the level of cleavage. However, no condition achieved complete cleavage. To achieve complete digestion, similar uPA digestion reaction solutions were incubated for longer periods. Figure 10B shows the cleavage of 2 μg of P-0203 with 50 ng, 100 ng, and 300 ng of uPA at 37°C for 24 hours. From the data, it can be seen that nearly complete cleavage was achieved with 100 ng of uPA and 24 hours of incubation. 【0228】 Example 5 Protease cleavage of IL-15 Fc VitoKine P-0203 to obtain the active IL-15 product. VitoKine P-0203 (SEQ ID NO: 29) contains a uPA substrate peptide linker (SEQ ID NO: 90) flanked by spacer peptides linking Fc and IL-15, with a second 15-amino acid flexible linker (GGGGS)3 (SEQ ID NO: 127) linking the IL-15 domain and the IL-15RαSushi+ domain. In vitro protease cleavage was achieved by incubating 100 μg of VitoKine P-0203 with 5 μg of recombinant human uPA (Biolegend) in 500 μl of PBS (pH 7.2) buffer at 37°C for 24 hours. To stop the reaction, 25 μl of Ni-Excel resin (50% slurry equilibrated in PBS, GE Healthcare) was added to remove the 6×His-tagged uPA from the solution. On the other hand, 50 μl of MabSelectSure Protein A resin (50% slurry equilibrated with PBS, GE Healthcare) was added to the reaction mixture to remove the cleaved Fc fraction and uncleaved or incompletely digested P-0203. After incubation with both affinity resins at room temperature for 15 minutes, the resins were removed by centrifugation, and the flow-through containing protease-active P-0203, i.e., IL-15 / IL-15αSushi+ fusion polypeptide (schematically shown as active form 1 in Figure 2), was recovered. As can be seen from Figures 11A and 11B, the active P-0203 fragments migrated while forming smear-like bands, and this smear-like band formation is most likely due to glycosylation. 【0229】 Example 6 Protease cleavage of IL-15 Fc VitoKine P-0315 to obtain the active IL-15 product. VitoKine P-0315 (SEQ ID NO: 33) contains a uPA substrate peptide linker (SEQ ID NO: 92) linking Fc and IL-15, and a second 10-amino acid MMP-2 / 9 cleavable linker (SEQ ID NO: 95) linking the IL-15 domain and the IL-15RαSushi+ domain. The IL-15 domain of P-0315 contains an S58D substitution to enhance binding to the receptor β subunit. Two active forms of P-0315 were prepared by protease digestion. 【0230】 One of the active forms of P-0315 (schematically shown as active form 2 in Figure 2) was obtained by in vitro protease cleavage using MMP-2. Briefly, 660 ng of latent MMP-2 (BioLegend) was activated with APMA (Millipore Sigma) according to the manufacturer's instructions, the buffer was changed, and P-0315 (80 μg) was added to 0.4 ml of the manufacturer's recommended assay buffer (100 mM Tris, 20 mM CaCl2, 300 mM NaCl, 0.1% (w / v) Brij35, pH 7.5). After incubation at 37°C for 3 hours, 50 μl of MabSelectSure Protein A resin (50% slurry equilibrated in PBS, GE Healthcare) was added to the reaction mixture. The target active form 1 was eluted with 25 mM sodium citrate and 25 mM sodium chloride (pH 3.2). The protein was neutralized by adding 3% 1 M Tris (pH 10.2). To evaluate cleavage, the samples were separated on a 4-12% Tris-Bis SDS-PAGE gel (Figure 12A). Lane 1 shows P-0315 before MMP-2 digestion in the presence of a reducing agent. Lanes 2 and 3 show the unreduced and reduced forms of P-0315 after MMP-2 proteolysis but before protein A purification. The IL-15Rα-sushi+ domain appeared as a sharp band at the 9 kDa position on the gel, confirming efficient MMP-2 cleavage in the MMP-2 / 9 substrate peptide linker. After protein A purification, each sample (lanes 4 and 5) showed the same migration pattern. This data suggests that the IL-15RαSushi+ domain, freed from covalent bonding as shown in Figure 2 as active form 2, remained non-covalently associated with IL-15 fused to Fc; this association was strong enough to withstand the low pH conditions during protein A elution. Figure 12B further shows the two non-covalently associated components of this active form. 【0231】 Other active forms of P-0315 (schematically shown as active form 3 in Figure 2) were obtained by protease cleavage of P-0315 using both uPA and MMP-2. Briefly, 100 μg of P-0315 was incubated with 5 μg of uPA in 400 μl of PBS (pH 7.2) buffer for 20 hours. Then, an equal volume of buffer (pH 7.5) containing 200 mM Tris, 40 mM CaCl2, 450 mM NaCl, and 0.2% (w / v) Brij35 was added to the reaction mixture to adjust the buffer to approximate the manufacturer's recommended MMP-2 assay buffer (100 mM Tris, 20 mM CaCl2, 300 mM NaCl, 0.1% (w / v) Brij35, pH 7.5). Latent MMP-2 (660 ng) was activated with APMA, the buffer was replaced with the assay buffer described above, and the mixture was added to the reaction solution and incubated at 37°C for 3 hours. Ni-Excel resin (50 μl 50% slurry equilibrated in PBS, GE Healthcare) was added to remove His-tagged MMP-2 and uPA from the solution. Meanwhile, 100 μl of MabSelectSure Protein A resin (50% slurry equilibrated in PBS, GE Healthcare) was added to the reaction solution to remove the cleaved Fc fraction and uncleaved or incompletely digested P-0315. After incubation with both affinity resins at room temperature for 15 minutes, the resins were removed by centrifugation, and the flow-through containing active form 3 of P-0315, schematicly shown in Figure 2, was collected. As shown in Figure 12C, active form 3 of P-0315 contains an IL-15 / IL-15αSushi+ non-covalent complex, as expected from the biproteolytic reaction; IL-15 is migrating as smear band formation, and IL-15RαSushi+ is a sharp band at approximately 9 kDa, as seen in active form 2 (Figure 12B). 【0232】 Example 7 Activity evaluation of protease-active IL-15 Fc VitoKine using human PBMC assay. FACS analysis of the activation marker CD69 in the immune cell subfraction of fresh human PBMCs was performed as detailed in Example 2, and the activity of the protease-active IL-15 VitoKine was evaluated. A comparison of P-0203 and its corresponding active form obtained from uPA digestion (active form P-0203; schematically shown as active form 1 in Figure 2) is shown in Figure 13. The activity of VitoKine before protease activation was approximately 3 logarithms lower than that of the highly active IL-15 / IL-15Rα Fc fusion protein P-0165, which was consistent with the VitoKine activity described in Example 3. The activation ability of both CD56+ NK cells (Figure 13A) and CD8+ T cells (Figure 13B) was significantly restored by uPA digestion, but was still clearly lower than that of P-0165, which may be due to the covalent binding between IL-15 and the IL-15Rα domain. Extending the length of the flexible linker connecting IL-15 and IL-15Rα is expected to enhance the activity of the active form. Paradoxically, extending the linker length may also reduce the activity shielding efficiency of the D3 domain, potentially resulting in a VitoKine construct with higher basal activity. 【0233】 The biological activity of another IL-15 Fc VitoKine P-0315 and its two active forms was evaluated by measuring CD69 activation in the activation of the immune cell subfraction of fresh human PBMCs. As can be seen in Figure 14, the activity of the uncleaved P-0315 was almost unmeasurable at the tested concentrations, confirming effective shielding of the VitoKine-type active moiety. Active form 2 of P-0315, as shown in Figure 2, contains Fc-fused IL-15 non-covalently complexed with the IL-15RαSushi+ domain released by MMP-2 cleavage, and is structurally similar to the highly active IL-15 IL-15Rα Fc-fusion protein P-0313-positive control. Active form 3 of P-0315, as shown in Figure 2, contains a free IL-15 domain cleaved from the Fc domain by uPA and an IL-15RαSushi+ domain released by MMP-2 cleavage, these two forming a non-covalent complex. Both active forms of P-0315 showed complete or near-complete recovery of activity in both CD56+ NK cells (Figure 14A) and CD8+ T cells (Figure 14B); active form 3 was slightly more active than active form 2. The absence of the Fc domain in active form 3 may be beneficial when transient activation of the target pathway in the tumor microenvironment is desired. 【0234】 Furthermore, the activity of P-0315 before and after MMP-2 proteolysis was investigated by measuring Ki67 expression in the nuclei of treated NK cells (Figure 15A) and CD8+ T cells (Figure 15B). P-0351, containing two non-cleavable flexible linkers, was used for comparison. These data further demonstrated the absence of activity in VitoKine and the recovery of activity by approximately 3 logarithms in both NK cells and CD8+ T cells after in vitro proteolytic activation. The confirmation that P-0351 and P-0315 possessed identical activity suggests that the two cleavable linkers of P-0315 remained intact during production, expression, and storage, and were specific to each protease. 【0235】 In summary, cleavage of IL-15 VitoKine P-0315 by MMP-2 / 9 and / or uPA leads to activation of the molecule, and the activity of the cytokine is within the EC range of nM or less. 50 It recovered to a level similar to that of P-0313, a highly active IL-15 compound possessing [specific properties]. 【0236】 Example 8 Minimal systemic cytokine activity when IL-15 Fc VitoKine is used in healthy mice. The goal of VitoKine platform technology is to reduce systemic on-target toxicity and expand the therapeutic concentration range. VitoKine shields active cytokines in an inactive state, preventing them from binding to receptors on the cell surface of surrounding or non-affected cells. As a result, the VitoKine platform limits excessive activation of cytokine pathways and reduces undesirable "on-target" but "extra-tissue" toxicity. VitoKine is intended to be locally activated by proteases that are increased in expression in the affected tissue. To evaluate this premise, protease-cleavable and non-cleavable VitoKine were administered to healthy mice, and their systemic cytokine activity was evaluated compared to that of highly active IL-15 Fc fusion proteins. 【0237】 P-0313 (SEQ ID NO: 47 and SEQ ID NO: 5) is a fully active IL-15 / IL-15Rα Fc fusion molecule used as a positive control. P-0315 (SEQ ID NO: 33) is an IL-15 Fc VitoKine containing two protease-cleavable linkers. P-0351 (SEQ ID NO: 25) is an IL-15 Fc VitoKine containing two non-cleavable linkers. A solvent (PBS) was used as a negative control. The compounds were administered as a single intraperitoneal injection to healthy BALB / c mice (8-10 weeks old, n=6 mice / group) at doses of 0.1 mg / kg and 0.3 mg / kg. Blood samples were collected before administration (-1 day) or on days 3, 5, and 7 after administration and subjected to immunophenotyping. 【0238】 After lysing red blood cells with BD pharm lysis buffer, the total number of living mononuclear blood cells was counted using trypan blue dead cell elimination. Fc receptors were blocked with purified anti-mouse CD16 / CD32 (1:50 dilution), and then the cells were stained with anti-mouse CD3-FITC, anti-mouse CD49b-APC, and anti-mouse CD8-Percpcy 5.5 (1:50 dilution). After incubation for 30 minutes, cells were collected, washed, resuspended in FACS buffer, and analyzed by flow cytometry. 【0239】 As shown in Figure 16, the fully active IL-15 Fc fusion protein P-0313 dramatically increased cytotoxic CD8+ T cells (Figure 16A), NK cells (Figure 16B), and total leukocytes (Figure 16C) in peripheral blood in a dose-dependent manner at two test doses. Cell proliferation was observed on day 3, peaked on day 5, and returned to near baseline by day 7. In contrast, neither cleavable VitoKine (P-0315) nor non-cleavable VitoKine (P-0351) showed any increase in CD8 T cells throughout the entire 7-day study period. In NK cell proliferation, a small, delayed increase was observed in mice treated with high doses of cleavable VitoKine P-0315. P-0351 and low doses of P-0315 showed no signs of increase in any of the target cell populations tested. Overall, compared to the active molecule P-0313, the two VitoKine strains tested showed minimal systemic activation and proliferation of the target lymphocyte population, demonstrating their ability to effectively shield and delay IL-15 activity in the periphery. 【0240】 Example 9 Inhibition of lung metastasis of colon cancer cells using IL-15 Fc VitoKine in mice In a mouse CT26 lung metastasis model, the metastasis-suppressing effect and immune response of the IL-15 Fc VitoKine molecule were investigated. In short, 1 × 10⁻¹⁶ 5Individual mouse colon cancer cells CT26-WT (ATCC CRL-2638) were intravenously injected into female Balb / C mice (9-11 weeks old). From the next day (day 1), intraperitoneal injections were started, with a total of 4 treatments every 5 days. The treatment groups (6 groups in total, n = 7 mice / group) included the 0.3 mg / kg P-0315 group, the 0.3 mg / kg P-0351 group, and the 0.1 mg / kg P-0313 group. P-0315 (SEQ ID NO: 33) is an IL-15 Fc VitoKine containing two protease-cleavable linkers. P-0351 (SEQ ID NO: 25) is a non-cleavable IL-15 Fc VitoKine. P-0313 (SEQ ID NO: 47 and SEQ ID NO: 5) is a fully active IL-15 / IL-15Rα Fc fusion molecule. As a negative control, a solvent (PBS) was used. On day 17, all mice were sacrificed and tissues were excised. The lungs were inflated with 15% India ink and de-stained with Fekete's solution (10% formaldehyde, 5% glacial acetic acid, and 60% ethanol). Pulmonary tumor nodules were counted, and the inhibitory effect on metastasis was represented by the difference in the number of tumor nodules between the treatment groups and the solvent control. 【0241】 As shown in Figure 17, P-0313 showed a significant effect in suppressing the formation and growth of lung metastases. At 0.1 mg / kg, P-0313 treatment achieved nearly complete inhibition of lung metastases. The cleavable VitoKine P-0315 showed 70% inhibition of the development of pulmonary nodules; the anti-metastatic effect was equivalent at all three doses (0.3 mg / kg, 1 mg / kg, or 3 mg / kg). The non-cleavable VitoKine P-0351 showed a relatively weak but significant effect in reducing the occurrence of metastases, suggesting some intrinsic basal activity at high doses. However, P-0315 showed a clearly better anti-metastatic effect than P-0351 (p<0.05; Figure 17), suggesting that proteolytic cleavage of one or both linkers in P-0315 and subsequent release of active IL-15 may contribute to the superiority of P-0315 over P-0351 in in vivo efficacy. The occurrence of tumor metastasis can lead to an increase in proteolytic activity near the tumor microenvironment. 【0242】 The immune response after IL-15 compound treatment was examined by flow cytometry analysis of mouse peripheral blood on day 15 (four days after the third treatment). Compared to controls, CD8+ T cell proliferation was observed in mice treated with the active IL-15 Fc fusion P-0313, but not in mice treated with the cleavable VitoKine P-0315 or the non-cleavable VitoKine P-0351. This suggests that the inhibitory effect on colon cancer metastasis was observed when VitoKine did not result in a systemic increase in CD8+ T cells (Figures 17 and 18A). However, peripheral blood NK cells increased in all three IL-15 compound treatment groups, with the most significant increase observed in the non-cleavable VitoKine group after repeated administration (Figure 18B). In the VitoKine-treated group, systemic proliferation of NK cells increased, but systemic proliferation of CD8+ T cells did not. This suggests that NK cells are more responsive to IL-15 treatment than CD8+ T cells, and that the inherent basal activity of VitoKine may induce NK cell proliferation. In other words, adjusting the dosage of IL-15 VitoKine is important to reduce the remaining systemic effects. The significant increase in NK cells in the P-0351 group also suggests that this low-activity, non-cleavable VitoKine may weakly but persistently activate the pathway, leading to a long-term immune response. 【0243】 Example 10 IL-15 Fc VitoKine P-0315 inhibited the growth of established CT26 tumors in mice, with minimal systemic cytokine activation. The antitumor activity and immune response of IL-15 Fc VitoKine P-0315 were investigated in a CT26 mouse colorectal cancer tumor model, compared to the fully active IL-15 / IL-15Rα-Fc fusion protein P-0313. In short, 1 × 10¹⁶ cells were implanted in the right flank of female Balb / C mice (10-12 weeks old). 5 Individual CT26 cells were injected subcutaneously. On day 11, the average tumor volume was approximately 70 mm². 3At this point, the mice were randomly divided into three groups (n=11 mice / group), and on the same day of randomization, they received intraperitoneal injections of either the solvent (PBS) or 0.1 mg / kg of P-0315 or P-0313. On day 16, an additional intraperitoneal injection of each test drug was administered (two times at 5-day intervals). Tumors were measured three times a week using calipers, and tumor volume was calculated as follows: Volume = 0.5 × (width). 2 × (length). On day 19, non-terminal peripheral blood was collected in a heparinized tube for immune response study. On day 21, all mice were sacrificed and tissue was extracted. 【0244】 As shown in Figure 19A, PBS-treated mice rapidly developed large subcutaneous tumors, and treatment of mice with P-0315 or P-0313 was approximately equal in efficacy in delaying tumor growth (Figures 19B and 19C). 21 days after tumor inoculation, the mean tumor volume of control-treated mice was approximately 1000 mm². 3 However, in contrast, P-0315 treated mice and P-0313 treated mice measured approximately 450 mm. 3 This was the case (****P<0.0001; one-way ANOVA and Tukey's post-hoc test) (Figure 19D). Notably, P-0313 initially showed a greater reduction in tumor volume than P-0315, but this difference gradually decreased as treatment progressed. The delayed antitumor effect of P-0315 is thought to be due to the time it took to generate a sufficient amount of protease to access and cleave the substrate peptide linker and activate VitoKine. 【0245】 Next, the effect of P-0315 on the proliferation of CD8+ T cells and NK cells in peripheral blood was investigated by flow cytometry, compared with P-0313 and the solvent. The effects of P-0315 on total WBCs and lymphocyte subsets (CD8+ T cells and NK cells) in peripheral blood and spleen were similarly evaluated. 【0246】 Injection of tumor-bearing mice with the fully activated IL-15 / IL-15Rα Fc fusion P-0313 strongly induced lymphocyte proliferation and growth in both peripheral blood and spleen (Figures 20-22). Compared to the PBS group, P-0313 treatment resulted in a 4-fold increase in Ki67 proliferation in peripheral NK cells (61% vs. 15%; Figure 20A) and a 5.3-fold increase in CD8+ cells (46% vs. 8.6%; Figure 20B). Similarly, P-0313 treatment resulted in significant cell proliferation of total leukocytes, NK cells, and CD8+ T cells in both peripheral blood (Figures 21A-21C) and spleen (Figures 22A-22C). For example, the total peripheral WCB cell count increased sixfold, the CD8+ T cell count increased fivefold, and the NK cell count showed a dramatic 85-fold increase. In the spleen, the most significant cell proliferation was observed in NK cells (10-fold increase), followed by CD8+ T cells, which increased 2.9-fold. The total WBC in the spleen showed a modest increase of 1.7-fold. The robust activation of cytotoxic CD8+ T cells and NK cells is consistent with the overall immunomodulatory effect of IL-15, and this potent immune response is thought to be a major contributing factor to the antitumor activity of P-0313 in vivo. However, dramatic changes in the lymphocyte subset in the blood may cause toxicity and reduce the therapeutic index. 【0247】 In striking contrast to P-0313, treatment with IL-15 Fc VitoKine P-0315 resulted in minimal changes to the homeostasis of lymphocyte subsets in the blood. This finding is shown in Figure 20 by Ki67 proliferation of NK cells and CD8+ T cells in peripheral blood, and in Figure 21 by cell proliferation of total leukocytes, NK cells, and CD8+ T cells in peripheral blood. The only noteworthy immunopharmacological effect after P-0315 treatment was a fourfold increase in the number of NK cells in the spleen (Figure 22B). Since P-0315 was approximately equivalent in efficacy to P-0313 in delaying the growth of established CT26 tumors (Figures 19A-19D), the in vivo antitumor activity of P-0315 is thought to be due to the activation of VitoKine after proteolytic degradation of cleavable linkers near the tumor microenvironment. Because active VitoKine appears only near tumors, the response of peripheral lymphocytes to administration of inactive VitoKine molecules was much weaker than that to fully active P-0313. 【0248】 In summary, the IL-15 Fc VitoKine exemplified by P-0315 was able to efficiently delay tumor growth without causing significant alterations to the proliferative capacity and proliferation of lymphocyte subsets in the blood and spleen. Consequently, the VitoKine type was able to suppress or reduce the excessive activation of pathways, undesirable "on-target" but "extra-target" toxicity, and undesirable target sinking that are usually associated with fully active cytokines, without impairing the antitumor effect. 【0249】 Example 11 Adjustment of the potency of the IL-15 moiety to minimize the intrinsic basal activity of the corresponding VitoKine. Despite the more than three-to-one reduction in activity between the fully active divalent IL-15S58D / IL-15Rα Fc fusion P-0313 and its corresponding Fc VitoKine P-0315, P-0315 still exhibits an EC of 50-100 nM in vitro. 50It possesses an intrinsic basal activity that can stimulate effector cells (as illustrated in Figure 15). At high in vivo doses, the intrinsic basal activity of VitoKine may result in peripheral receptor stimulation accompanied by sustained in vivo pharmacodynamic effects, potentially leading to systemic toxicity. Therefore, we hypothesized that adjusting the potency of the IL-15 moiety could minimize the corresponding intrinsic basal activity of VitoKine. 【0250】 A panel of IL-15 mutant proteins containing amino acid substitutions that disrupt IL-15Rβγ was expressed as IL-15 / IL-15Rα (non-covalent) Fc fusion proteins and screened for potency reduction using human PBMC assays, as described above. P-0313 was used as a control molecule. Table 13 summarizes mutants with exemplary single or combined IL-15 amino acid changes at residues V63, I68, and Q108 that result in reduced CD8 T cell proliferation. Compared to P-0313, these IL-15 variant / IL-15Rα Fc fusion proteins showed a wide range of potency reductions, from 5-fold to approximately 6700-fold. Figures 23A and 23B further show the percentage of Ki67 expression on CD8 T cells and NK cells after treatment with a small number of representative IL-15 variants / / IL-15Rα Fc fusions, including P-0736, P-0772, P-0737, P-0768, P-0793, and P-0764. The changes in IL-15 amino acids in the fusion proteins shown in Figure 23 are summarized in Table 13. TIFF0007872587000017.tif133170 【0251】 Furthermore, IL-15 variants including amino acid deletions or insertions, or combinations of amino acid substitutions and deletions / insertions, as exemplified in SEQ ID NOs. 182-192, showed varying levels of potency reduction when expressed as IL-15 / IL-15RαSushi Fc fusions (data not shown). Such IL-15 moieties can similarly be used in IL-15 VitoKine forms to optimally tune the intrinsic basal activity. As will be understood by those skilled in the art, all mutations (amino acid substitutions, deletions, and insertions) can be combined arbitrarily and independently in any way to achieve optimal activity regulation. 【0252】 To test whether adjusting the potency of the IL-15 moiety truly minimizes the intrinsic basal activity of the corresponding VitoKine, IL-15Q108S, exhibiting significantly attenuated activity, was incorporated as the D2 domain into VitoKine, referred to as P-0682. In addition to containing a different IL-15 variant, P-0682 also differs from P-0315, which has a non-cleavable, flexible L1 linker. However, in other respects, these two VitoKines are identical. As shown in Figures 24A and 24B, P-0682 completely lost its activity to induce Ki67 expression on CD8 T cells or NK cells, even at the highest test concentration of 1 μM. P-0764 is the Fc fusion counterpart of P-0682 and is similar to the activated form derived from P-0682. The data from this invention, taken together, suggest that the IL-15 VitoKine platform generally causes a 1000 to 2000-fold attenuation of cytokine efficacy in extracellular assays, and therefore the EC of IL-15Q108S-based VitoKine P-0682 when inducing CD8 T cells and NK cells. 50 The values ​​are estimated to be 100 μM and 20 μM, respectively. Therefore, this data precisely supports the inference that the attenuation of IL-15 potency leads to a decrease in the intrinsic basal activity of the corresponding VitoKine. 【0253】 The inherent basal activity of IL-15 VitoKine is proportional to the activity of the cytokine moiety, and can therefore be adjusted by incorporating IL-15 moieties with different levels of potency, as listed in Table 13. For example, P-0806 (SEQ ID NO: 231), an IL-15 Fc VitoKine containing IL-15V63A / I68H as the D2 domain, induces CD8 T cells, and is a putative EC2. 50 At a concentration of 2-3 μM, it is expected to have intermediate basal activity between P-0315 and P-0682. Importantly, the tunable basal activity inherent to IL-15 VitoKine allows for achieving an optimal balance between VitoKine inactivation before cleavage and potency after activation, thus facilitating the achievement of the desired antitumor effect while minimizing undesirable systemic toxicity. 【0254】 Example 12 VitoKine, an uncleavable cytokine with reduced activity. It is known in the art that highly potent cytokines in vitro may not induce the strongest lymphocyte response in vivo. Highly active cytokines often lead to stronger receptor stimulation, internalization, and desensitization, resulting in attenuated signaling, proliferation, and function, and increased cell death and clonal depletion. Therefore, attenuated cytokines may be highly preferable to suppress excessively strong lymphocyte activation and achieve sustained and enhanced in vivo pharmacodynamic and antitumor activity. 【0255】 The cleavable IL-15 Fc VitoKine P-0351 showed significantly suppressed activity in vitro compared to fully active IL-15 compounds, but exhibited metastasis suppression and a remarkable NK cell response in a mouse CT26 lung metastasis model (Example 8). In other words, by using a cleavable VitoKine construct to function as an activity-reducing cytokine with sustained activity, the in vivo pharmacodynamic effect can be optimized. 【0256】 P-0351 exhibited the same activity as the equivalent reference molecule (SEQ ID NOs. 177 and 178) of XENP024306 in International Patent Publication No. 2018071919A1 in inducing Ki67 proliferation in both NK cells and CD8+ T cells (Figures 25A and 25B). XENP024306 is an IL-15 / IL-15Rα Fc fusion molecule containing an amino acid substitution (D30N / E64Q / N65D) in IL-15 and a half-life extension mutation in Fc. While a triple mutation in the IL-15 chain of XENP024306 has been reported to result in a 200-fold decrease in potency in vitro, XENP024306 was shown to be more active in vivo, likely due to optimized pharmacodynamics in vivo. 【0257】 Similarly, the attenuation of potency in P-0351 is expected to result in more sustained exposure for improved pharmacodynamics (PD) by avoiding or reducing undesirable target sinks associated with hyperactivation and generally fully active cytokines. In other words, P-0651 (SEQ ID NO: 170), the half-life extended counterpart of P-0351, promotes a longer half-life and further extends the pharmacodynamic effect in vivo. 【0258】 Example 13 Construction and production of IL-2 Fc VitoKine for selective proliferation of regulatory T cells (Treg IL-2 VitoKine). The goal is to design an IL-2 VitoKine construct that remains inactive until it is locally activated by a protease whose expression is elevated at the site of inflammation. It has been reported that low doses of wild-type IL-2 preferentially stimulate Tregs over effector T cells, and that IL-2 mutant proteins with reduced binding affinity to IL-2Rβ widen the selectivity window. These molecules can be developed as prophylactic therapies for autoimmune diseases. IL-2Rβ binding and / or γ C Other mutations that interfere with binding but do not affect interaction with IL-2Rα can also broaden the window of selectivity for Treg activation, even more so than for Teff activation. 【0259】 The active site used was IL-2 Fc VitoKine, which contains wild-type IL-2 or an IL-2 mutant protein with increased selectivity to stimulate Tregs more than effector T cells. This active site is reversibly shielded between the Fc domain and IL-2RαSushi (SEQ ID NO: 10). IL-2Rα (SEQ ID NO: 9) contains two sushi domains separated by a native peptide linker region. The IL-2 VitoKine construct contains one or two cleavable linkers recognized by proteases that have been reported to be increasedly expressed at inflammatory injury sites. The linker linking Fc and the IL-2 / mutant protein can be either cleavable or non-cleavable, but it is preferable that the linker linking IL-2 and IL-2αSushi be specifically cleavable by a protease. 【0260】 The activity of the IL-2 mutant protein that selectively stimulates Treg cells is expected to be restored after the release and diffusion of IL-2Rα from IL-2 following protease cleavage. Due to the nM binding affinity between IL-2Rα and IL-2, IL-2RαSushi continues to associate non-covalently with IL-2 even after linker cleavage; consequently, the interaction of IL-2 with IL-2Rα on Treg cells may remain blocked. To address this potential issue, we designed an IL-2Rα mutant protein with amino acid substitutions on the contact surface with IL-2 to weaken its binding to IL-2. That is, after protease cleavage of the linker, the IL-2RαSushi mutant dissociates from IL-2 and then diffuses, and this activation mechanism (schematically illustrated in Figure 2B) differs slightly from that illustrated in Figure 2A. 【0261】 Representative amino acid substitutions were made at positions 38 (i.e., K38E) and 43 (i.e., Y43A) of the IL-2Rα domain. Other IL-2Rα variants with substitutions at IL-2 interaction residues are expected to disrupt the interaction between IL-2 and IL-2Rα and can be incorporated accordingly. As will be understood by those skilled in the art, all of these mutations can be optionally combined, independently, or otherwise to achieve optimal affinity regulation. IL-2 VitoKine molecules were constructed containing different linker combinations and wild-type or variant IL-2, as well as wild-type or variant IL-2RαSushi. Their respective sequences are listed as SEQ ID NOs. 49–65. 【0262】 Gene synthesis, expression vector construction, and protein production, purification, and characterization were performed according to the same procedures detailed in Example 1. As an example of the protein profile of IL-2 VitoKine, the SDS-PAGE analysis of P-0320 is shown in Figure 26A. The size exclusion chromatogram in Figure 26B showed less than 5% aggregation after the initial protein A capture step without polishing. This low aggregation tendency suggests a favorable development suitability profile for IL-2 VitoKine. 【0263】 Example 14 In vitro activity evaluation of Treg IL-2 Fc VitoKine The physiological activity of IL-2 VitoKine on T cells was determined by measuring phosphorylated STAT5 (pStat5) levels in specific T cell subsets of fresh human PBMCs. Stat5 is a transmembrane molecule that inhibits IL-12Rβγ CIt is known to be involved in downstream intracellular signaling induced by IL-2 binding to the complex. pStat5 levels were measured by flow cytometry in cells fixed and permeabilized using an antibody against the pStat5 peptide. Briefly, human PBMCs were isolated by Ficol-Hypak centrifugation from the buffy coat of healthy donors purchased from the Oklahoma Blood Institute. 2 × 10⁻⁶ 5 Each PBMC was treated with serial dilutions of the test compound at 37°C for 30 minutes. The cells were then treated with Foxp3 / Transcription Factor Staining Buffer Set (EBIO) according to the manufacturer's instructions. Next, the cells were fixed with Cytofix buffer, permeabilized with Perm Buffer III (BD Biosciences), and washed. After blocking the Fc receptor by adding Human TruStain FcX (1:50 dilution), the cells were stained at room temperature for 60 minutes with a mixture of anti-CD25-PE antibody, anti-FOXP3-APC antibody, anti-pSTAT5-FITC antibody, and anti-CD4-PerCP-Cy5.5 antibody at concentrations recommended by the manufacturer. The cells were then collected, washed, resuspended in FACS buffer, and analyzed by flow cytometry. Flow cytometry data were gated to analyze Foxp3+ / CD25 for the Treg cell subset and the CD4+ conventional T cell subset, respectively. high Group and Foxp3- / CD25 low The data was divided into groups. The data is expressed as the percentage of pStat5-positive cells in the gated population. 【0264】 The pStat5 activation of IL-2 VitoKine proteins P-0320 (SEQ ID NO: 49) and P-0329 (SEQ ID NO: 62) was evaluated compared to P-0250 (SEQ ID NO: 48). P-0320 contains a wild-type IL-2 domain, its N-terminus fused to an Fc domain via a uPA-cleavable linker, and its C-terminus linked to the IL-2RαSushi domain via a flexible (GGGGS)3 (SEQ ID NO: 127) linker. P-0329 contains a wild-type IL-2 domain, its C-terminus fused to an Fc domain via a uPA-cleavable linker, and its N-terminus linked to the IL-2RαSushi domain via a flexible (GGGGS)3 (SEQ ID NO: 127) linker. P-0250 is a highly active IL-2 Fc fusion protein. The percentage of pStat5-positive cells in the Treg cell subset and the CD4+ normal T cell (Tconv) subset for each test compound is shown in Figure 27. Compared to fully active IL-2 fusion proteins, both IL-2 VitoKine forms dramatically reduced pStat5 activation in Treg cells. In CD4+ Tconv cells, pStat5 activation was barely detectable. This data demonstrates efficient shielding of IL-2 activity in VitoKine form. 【0265】 Example 15 Evaluation of IL-2 Fc VitoKine's protease activation and in vitro activity. IL-2 Fc VitoKine P-0382 (SEQ ID NO: 51) contains a flexible GGGSGGGS linker (SEQ ID NO: 115) linking Fc and IL-2, and a 10-amino acid MMP-2 / 9 cleavable linker (SEQ ID NO: 77) linking the IL-2 domain and the IL-2RαSushi domain. The IL-2RαSushi domain of P-0382 contains an amino acid substitution (K38E) designed to reduce its binding affinity to IL-2, allowing it to dissociate from IL-2 after protease cleavage of the linker and subsequently diffuse. 【0266】 P-0382 was activated by in vitro protease cleavage using MMP-2. Briefly, 3.3 μg of latent MMP-2 (BioLegend) was first activated with APMA (Millipore Sigma) according to the manufacturer's instructions, then the buffer was changed and 120 μg of P-0382 was added to 0.4 ml of the manufacturer's recommended assay buffer (100 mM Tris, 20 mM CaCl2, 300 mM NaCl, 0.1% (w / v) Brij 35, pH 7.5). After incubation at 37°C for 20 hours, half of the reaction mixture was purified using MabSelectSure Protein A resin, and the active VitoKine was eluted with 25 mM sodium citrate and 25 mM sodium chloride (pH 3.2). The protein was neutralized by adding 3% 1M Tris (pH 10.2). The other half of the sample was incubated with Ni-Excel resin to stop the reaction by removing the His-tagged MMP-2 protein, and the Ni resin was removed by centrifugation to collect the active VitoKine. Protein A purification was performed to confirm that the IL-2RαSushi domain did not covalently associate with IL-2 after polypeptide chain cleavage, as schematically illustrated in Figure 2B. The samples were evaluated on a 4-12% Tris-Bis SDS-PAGE gel shown in Figure 28. Compared with structurally similar IL-15 VitoKine (e.g., P-0315), the reaction did not result in complete cleavage despite increased protease levels and extended reaction time. Comparison of MMP-2 treated samples with and without protein A purification (Figures 29A and 29B) confirmed that the IL-2RαSushi domain detached covalently and was not co-purified with the Fc-IL-2 fusion polypeptide. 【0267】 Although the cleavage was incomplete, two types of MMP-2 activated samples were evaluated using the pStat5 activation assay described in Example 14, one as Ni-Excel flow-through (active form 1) and the other as protein A eluent (active form 2). The data are shown in Figure 29. The activity of P-0382 was very low in Treg cells and almost undetectable in CD4+ Tconv cells, reaffirming the effective shielding of the active portion in IL-2 VitoKine form. Both activated samples showed almost complete recovery of activity. The slightly lower activity compared to P-0250 was thought to be due to incomplete proteolysis. 【0268】 Since activated forms 1 and 2 exhibit equivalent activity in inducing pStat5 phosphorylation in both Treg and Tconv cells (Figures 28A and 28B), the presence of the IL-2RαSushi domain cleaved by MMP-2 in the activated form 1 sample was not thought to alter the activity of activated IL-2 VitoKine. The data suggested that the IL-2RαSushi domain produced by MMP-2 cleavage did not associate with IL-2, and therefore should not interfere with the binding of IL-2 to the receptor complex expressed on lymphocytes. 【0269】 MMP-2 proteolysis of P-0382 did not result in complete cleavage, suggesting that extending the cleavable linker could make the substrate peptide more accessible to the cleavage-carrying protease. By replacing the 10-amino acid linker of P-0382 (SEQ ID NO: 95) with a 15-amino acid MMP-2 / 9 cleavable linker (SEQ ID NO: 94) containing an extra adjacent residue, a novel VitoKine construct P-0398 (SEQ ID NO: 52) was obtained. P-0398 was activated by in vitro protease cleavage using MMP-2, following the same protocol detailed above. Even with three times the amount of MMP-2 (1.5 μg of MMP-2 for 180 μg of P-0398 compared to 3.3 μg of MMP-2 for 120 μg of P-0382), P-0398 was completely digested, as evidenced by the presence of only a "total cleavage" band on the SDS-PAGE gel (data not shown). 【0270】 The physiological activity of active P-0398, from which the IL-2RαSushi domain was removed by protein A purification, was measured using the pStat5 assay (Figures 29A and 29B). Active P-0398 is similar in sequence and structure to the IL-2 Fc fusion molecule P-0250, and they exhibit nearly identical activity in inducing Stat5 phosphorylation in both Treg and Tconv cells. Both VitoKine P-0382 and P-0398 have significantly impaired physiological activity (by four-to-fourths) due to covalent linkage to the IL-2RαSushi domain, but P-0398, containing a longer L2 linker, appeared to tend to be slightly more active. Similar to what was observed with IL-15 Fc VitoKine, the level of inactivity of IL-2 VitoKine could be further adjusted by modifying the L2 linker length. Similarly, the selection of the length and sequence of cleavable L2 linkers should balance the presence of a specific protease at the target disease site, the reachability of the substrate peptide to that protease, and the desired rate of proteolysis. 【0271】 In summary, compared to IL-15 VitoKine, IL-2 VitoKine required a longer L2 linker for optimal enzymatic reach to achieve complete proteolysis. Cleavage of exemplary IL-2 VitoKine constructs P-0382 and P-0398 by MMP-2 resulted in complete activation of each molecule. These activated IL-2 VitoKines achieved similar physiological activity to the highly active IL-2 Fc fusion compound P-0250. 【0272】 Example 16 Construction and production of IL-2 Fc VitoKine (Teff IL-2 VitoKine) for selective proliferation of effector T cells. The goal is to design IL-2 VitoKine constructs that remain inactive until locally activated by proteases present only in tumor sites or whose expression is elevated in tumor sites. Since Tregs can suppress effector T cell responses, preferential proliferation of Tregs by IL-2 is an undesirable effect of IL-2 in cancer immunotherapy. High and constitutive expression of IL-2Rα on Tregs, in addition to the signaling receptor βγ subunit, leads to preferential Treg proliferation by IL-2. To overcome these limitations, IL-2 variants designed to no longer bind to IL-2Rα are expected to activate only Tregs at concentrations that also activate CD8+ T and NK cells, rather than preferentially activating Tregs. Following this concept, we designed a panel of IL-2 variants containing one or more amino acid substitutions in residues that interact with IL-2Rα. Residues R38, T41, F42, F44, E62, P65, E68, and Y107 are located at the interface with IL-2Rα and form either hydrogen bonds / salt bridges or hydrophobic interactions with numerous IL-2Rα residues (Mathias Rickert, et al. (2005) Science 308, 1477-80). Amino acid substitutions at these sites were expected to disrupt the interaction with IL2Rα, resulting in IL2 variants with reduced or absent binding to IL2Rα. 【0273】 A series of IL-2 mutant proteins were expressed as C-terminal fusions of Fc heterodimers to Fc homodimers, and their binding to IL-2Rα was screened using enzyme-linked immunosorbent assay (ELISA). Briefly, IL-2Rα-ECD was coated onto wells at a rate of 0.1 μg / well. After incubation at 4°C overnight and blocking, serial dilutions of IL-2 Fc fusion proteins were added to each well at a rate of 100 μl / well. After incubation at room temperature for 1 hour, 100 μl / well of goat anti-human IgG Fc-HRP (diluted 1:5000 with diluent) was added to each well, and incubation at room temperature for 1 hour. After adding 100 μl of TMB substrate for 10 minutes, the plates were allowed to develop color in the dark at room temperature, and 100 μl / well of stop solution was added. The absorbance at 450 nm was determined, and the curves were fitted using Prism software (GraphPad). P-0531 (SEQ ID NO: 248) and P-0689 (SEQ ID NOs: 249 and 168), which are S125I equivalents of the wild-type IL-2 Fc fusion protein with bivalent and monovalent IL-2 moieties, respectively, were included as controls. 【0274】 Table 14 summarizes exemplary single or combined IL-2 amino acid changes that result in reduced or absent binding. Figure 31 shows ELISA binding curves for several representative IL-2 variant monovalent Fc fusion proteins, P-0704, P-0707, P-0708, and P-0709, to IL-2Rα, compared with P-0689. Table 14 Exemplary single or combined IL-2 amino acid substitutions disrupted the IL-2Rα interaction, resulting in IL-2 variants with reduced / absent binding to IL-2Rα. TIFF0007872587000018.tif134170 【0275】 In addition to containing amino acid substitutions (one or more) that disrupt IL-2Rα, all IL-2 variants in Table 14 also include S125I mutations to significantly enhance protein expression and reduce aggregation tendencies. As will be understood by those skilled in the art, any further combinational variants to modulate their affinity for IL-2Rα, whether or not they alter their affinity for specific components of the IL-2 receptor, are included in the spirit and scope of the invention. 【0276】 Furthermore, these IL-2 variants retain full binding and functional activity to the dimeric IL-2Rβγ receptor, albeit with reduced / absent CD25 binding, and can activate effector cells via retained IL-2Rβγ signaling. This was exemplified by P-0704 in a human PBMC assay. P-0704, a monovalent IL-2P65R Fc fusion protein with abolished IL-2Rα binding, is as potent as its wild-type IL-2 counterpart, P-0689 (Figure 32), in inducing Ki67 expression in CD8 T cells. In future examples, P-0704 and P-0689 will be used interchangeably as controls with full IL-2 Teff potency. 【0277】 The Teff IL-2 Fc VitoKine constructs contain an IL-2 variant in which binding to IL-2Rα as the active site is reduced / absent, reversibly shielded between the Fc domain and IL-2RαSushi (SEQ ID NO: 10). These constructs contain one or two cleavable linkers recognized by proteases whose increased expression has been reported in various types of cancer (e.g., solid tumors). The linker linking Fc to the IL-2 / mutant protein can be either cleavable or non-cleavable, but the linker linking IL-2 to IL-2αSushi is preferably specifically cleavable by a protease. Since the IL-2 portion of VitoKine is designed not to bind to IL-2Rα, the shielded D3 portion of VitoKine, IL-2Rα, is likely to diffuse after in vivo proteolytic cleavage (Figure 28), resulting in the restoration of IL-2 mutein activity. Figure 1 schematically shows IL-2 VitoKine molecules incorporating various IL-2 mutant proteins as the active site. Exemplary IL-2 Fc VitoKine molecules for selective proliferation of Teff cells were constructed and synthesized. Their respective sequences are listed as SEQ ID NOs. 59-61 and 271-274. 【0278】 Gene synthesis, expression vector construction, and protein production, purification, and characterization were carried out according to the same procedures detailed in Example 1. 【0279】 Example 17 VitoKine containing the IL-2Rα variant as the IL-2 shielding D3 portion. IL-2Rα variants were designed to reduce binding to IL-2 by incorporating mutations in residues that interact with IL-2, allowing the D3 moiety to easily diffuse during proteolytic degradation in vivo. Three exemplary IL-2RαSushi variants (SEQ ID NOs: 267-269) were expressed as monovalent Fc fusion proteins corresponding to P-0751(Y43A), P-0752(L42G), and P-0753(R36A), respectively. The effects of individual mutations on IL-2 binding in ELISA were evaluated by comparing the three IL-2RαSushi variant Fc fusions with a wild-type IL-2RαSushi counterpart fusion called P-0757. 【0280】 In short, IL-2Rα Sushi variant monovalent Fc fusion protein was coated at 1 μg / well on the wells of a Nunk Maxisorp 96-well microplate. After incubation at 4°C overnight and blocking with 1% BSA, serial dilutions of IL-2S125I monovalent Fc fusion protein P-0689 were added to each well at 100 μl / well. After incubation at room temperature for 1 hour, 100 μl / well of biotin anti-IL2 antibody clone B33-2 (BD Bioscience) was added to each well at 1 μg / ml and incubated at room temperature for 1 hour. Subsequently, 100 μl / well of avidin-HRP (BioLegend) was added to each well at a 1:500 dilution and incubated for 30 minutes. Finally, 100 μl of TMB substrate was added to each well, the plate was colored at room temperature, and 100 μl / well of stop solution was added. The absorbance at 450 nm was determined, and the curve was fitted using Prism software (GraphPad). 【0281】 As summarized in Table 15 and shown in Figure 33, the IL-2Rα amino acid substitutions Y43A, L42G, and R36A all consequently disrupted the interaction with IL-2. Y43A resulted in a moderate (8.1-fold) decrease in IL-2 binding, while the R36A mutation disrupted the binding of EC2. 50The L42G mutation resulted in a more dramatic 346-fold decrease, causing an intermediate or 35-fold decrease in IL-2 binding. TIFF0007872587000019.tif61170 【0282】 Four Teff IL-2 Fc VitoKine molecules were constructed using the three IL-2Rα variants described above, along with their wild-type counterparts. All of these molecules contain a monomeric IL-2S125I (wild-type equivalent) as the D2 domain and a 15-amino acid MMP2 / 9 cleavable L2 linker. Subsequently, the shielding efficiency of the IL-2Rα variants was evaluated by comparing their efficacy in inducing Ki67 expression in CD8T and NK cells in human PBMC assays with P-0704, an Fc fusion equivalent to P-0689, the Fc fusion counterpart of this group of VitoKines. The data are summarized in Table 16 and shown in Figures 34A and 34B. TIFF0007872587000020.tif68170 【0283】 As shown in Figure 34, wild-type IL-2RαSushi in VitoKine P-0701 as a shielding D3 domain resulted in a dramatic 3-logarithmic decrease in CD8+T and NK cell proliferation induction compared to its fully active Fc fusion counterpart, P-0704. It was predicted that the incorporation of the IL-2 binding disruption mutation in IL-2RαSushi would weaken the D3 domain shielding effect and thus reduce VitoKine activity inactivation. Furthermore, it was anticipated that the degree of reduction in shielding effect would correlate with the level of decrease in binding strength between IL-2 and the IL-2RαSushi variant. 【0284】 As shown in Figure 34A and Table 16, the IL-2RαSushi variants Y43A and R36A as D3 domains in each Fc VitoKine (P-0754 and P-0756) showed a weakened shielding effect compared to P-0701 in inducing CD8 T cell proliferation, and therefore reduced VitoKine activity inactivation. A similar activity trend was observed in NK cells (Figure 34B). Nevertheless, although the IL-2RαSushi variant Y43A slightly reduced IL-2 binding (8.1-fold), while the R36A substitution resulted in a much more significant 346-fold reduction in IL-2 binding, each of these VitoKines showed a similar weakened shielding effect, which contradicted the prediction that the degree of reduction in shielding effect should correlate with the level of reduction in binding strength between IL-2 and the IL-2RαSushi variant. Even more surprisingly, despite a 35-fold reduction in binding to IL-2, the IL-2RαSushiL42G variant retained its shielding effect compared to its wild-type counterpart, and therefore almost completely preserved the activity inactivity of its corresponding VitoKine, P-0755 (Figures 34A and 34B). The unexpected experimental observation that the incorporation of the IL-2 disruption mutation into IL-2Rα showed varying levels of impact on the shielding effect, and did not correlate with the degree of change in binding strength, was observed in multiple experiments. This may involve optimal spatial interaction between the IL-2 and IL-2Rα domains. Due to its retained shielding effect, the IL-2RαSushiL42G variant is selected as the preferred D3 domain for IL-2 VitoKine, maintaining the activity inactivity of the corresponding VitoKine while its weakened binding to IL-2 allows it to easily diffuse during proteolytic degradation in vivo and achieve full activity. On the other hand, if it is desirable to adjust the inherent basic activity of IL-2 VitoKine to achieve an optimal balance between the desired antitumor effect and undesirable systemic toxicity, an IL-2Rα variant, such as R36A, can be used as the D3 domain. 【0285】 Furthermore, IL-2 Fc VitoKines were constructed containing IL-2P65R as the D2 domain and wild-type IL-2RαSushi (P-0745), IL-2RαSushiY43A (P-0807), IL-2RαSushiL42G (P-0808), or IL-2RαSushiR36A (P-0809) as the D3 domain, and their activity in inducing CD8T and NK cell proliferation was evaluated. As shown in Figures 35A and 35B, all VitoKines showed a 10- to 20-fold decrease in activity compared to P-0704, the corresponding Fc fusion of these VitoKines. Since the P65R mutation inhibited the binding of IL-2 to IL-2Rα, the moderate shielding effect was thought to be due to spatial complementarity between IL-2 and IL-2RαSushi, and further IL-2 disruption mutations in IL-2Rα did not further affect the shielding effect. 【0286】 Furthermore, IL-2 Fc VitoKine P-0755, containing IL-2 as the D2 domain and IL-2RαSushiL42G as the D3 domain, was activated via in vitro MMP-2 cleavage according to the method detailed in Example 15 and evaluated by human PBMC assay. After cleavage and diffusion of the D3 domain, activated P-0755 is used as part of a protease substrate and is similar to its Fc fusion counterpart P-0689, with several extra residues remaining at the C-terminus of the IL-2 moiety. As shown in Figures 36A and 36B, P-0755 achieves a near 3-logarithmic decrease in activity as a VitoKine and can be activated to restore full potency in stimulated proliferation of effector cells, including CD8T and NK cells. 【0287】 In summary, exemplary Teff IL-2 Fc VitoKine containing the IL-2RαSushi variant as the shielding D3 domain was constructed and evaluated. The IL-2RαSushiL42G variant was selected as the preferred D3 domain due to its retained wild-type shielding effect in VitoKine and its weakened binding to IL-2, which facilitates diffusion to achieve full activation during proteolysis. If higher IL-2RαSushi intrinsic basal activity is desired to achieve the optimal balance between desired antitumor effect and undesirable systemic toxicity, other IL-2RαSushi mutations with reduced shielding ability can be employed. 【0288】 Example 18 Construction, expression, and purification of the antibody VitoKine The use of recombinant antibody-cytokine fusion proteins (immune cytokines) is expected to enhance the therapeutic index of cytokines by targeting them to the affected site. However, fusion of fully active cytokines with antibodies may result in peripheral activation or insufficient tumor targeting. By inactivating VitoKine before activation at the target therapeutic site, antibody VitoKine becomes a novel and progressive form of immune cytokine. In addition to tumor-targeting antibodies, antibody VitoKine can also be constructed using immune checkpoint blocking antibodies that bypass immunosuppressive effects in the tumor microenvironment, or immunostimulatory antibodies to enhance existing responses, thereby further enhancing the activity of the immune system against tumors. Furthermore, antibody VitoKine targeting inflammatory issue sites can be used to treat autoimmune chronic inflammatory disorders. 【0289】 Following this concept, we constructed an antibody VitoKine protein containing either wild-type or variant IL-15 or wild-type or variant IL-2 as the D2 domain. Examples of antibodies include various PD-1 antagonist antibodies, such as various human / humanized PD-1 antagonist antibodies (SEQ ID NOs. 195-198 and 275-278), the PD-L1 blocking antibody atezolizumab (SEQ ID NOs. 279-280), the anti-CTLA4 antibody ipilimumab, the agonist CD40 antibody RO7009789, tumor antigen targeting antibodies, such as L19 against the extra-domain of fibronectin, rituximab against CD20, Herceptin against Her-2, cetuximab against EGFR, anti-FAP antibodies for tumor targeting and retention (SEQ ID NOs. 193-194), and anti-inflammatory antibodies such as vedolizumab against integrin α4β7 and Humira against TNFα. The sequences of the exemplary antibody VitoKine are listed in SEQ ID NOs: 128-146, 180-181, 281-286, 296-297, and 303-306. 【0290】 Gene synthesis, expression vector construction, and protein production, purification, and characterization were performed according to the same procedures detailed in Example 1. Exemplary IL-15 antibody VitoKine and IL-2 antibody VitoKine exhibited similar expression profiles, e.g., productivity and aggregation tendencies, to their counterparts FcVitoKine. 【0291】 Example 19 In vitro functional and physiological activity evaluation of the IL-15 antibody VitoKine The physiological activity of exemplary anti-PDL1 antibody IL-15 VitoKine P-0485 (SEQ ID NO: 180 and SEQ ID NO: 181) was investigated by measuring Ki67 expression in CD8+ T cells (Figure 37A) and NK cells (Figure 37B) after treating human PBMCs with each IL-15 VitoKine compound. P-0485 shares the same L1 and L2 linkers and D2 and D3 domains as its Fc VitoKine counterpart, P-0315. P-0485 appeared to have slightly higher activity, which may be due to lymphocyte activation by PD-L1 blockade. 【0292】 As shown in Example 11, the intrinsic basal activity of IL-15 Fc VitoKine can be tuned by incorporating IL-15 moieties of varying potencies. Similarly, the IL-15 PD-1 antagonist antibody VitoKine, P-0875 (SEQ ID NOs. 196 and 284), was constructed with the IL-15V63A / I68H variant as the D2 domain. P-0875 was tested against its IL-15 / IL-15RαSushi antibody fusion counterpart P-0870 (SEQ ID NOs. 196, 297, and 5) and its IL-15 / IL-15RαSushi Fc fusion counterpart P-0773 (Figure 38B) by measuring Ki67 expression in CD8+ T cells after treatment with human PBMCs. P-0773 and P-0870 were equally potent in inducing Ki67 expression, and their EC50 was 18.5 nM, suggesting that IL-15 activity is not affected by the fusion form. Based on the data from this invention, which suggest that the IL-15 VitoKine platform generally causes approximately 1000-fold attenuation of activity, the EC50 of P-0875 inducing CD8 T cells is considered to be less potent. 50The concentration is estimated to be 18 μM. Such a low potency prediction is consistent with the lack of activity against P-0875 even at the highest test concentration of 1 μM (Figure 38B), however, the potency of P-0875 cannot be reliably extrapolated from the data. Next, P-0875 and P-0773 were further tested in cynomolgus monkey PBMCs prepared in the same manner as human PBMCs. Both compounds showed proportionally enhanced physiological activity compared to human cells, and EC 50 Activity curves were obtained within the test concentration range to ensure accurate deriving of values ​​(Figure 38C). EC of P-0773 and P-0875 induces Ki67 expression in cynomolgus monkey CD8+ T cells. 50 These were 0.259 nM and 254 nM, respectively. The 1000-fold decrease in potency was a characteristic of the IL-15 VitoKine platform consistently demonstrated for IL-15 Fc VitoKine, based on the prototype IL-15 VitoKine compounds, P-0315 vs. P-0313 (EC50 of 18.6 pM for P-0313 and 16.9 pM for P-0315 in induction of Ki67 expression in human CD8+ T cells, as shown in Figure 38A). 【0293】 In summary, the IL-15 antibody VitoKine retains the platform's characteristic attenuation of cytokine efficacy. Furthermore, by adjusting the efficacy of the IL-15 moiety, the intrinsic basal activity of the corresponding VitoKine can be minimized. 【0294】 Example 20 In vitro functional and physiological activity evaluation of the Teff IL-2 antibody VitoKine Exemplary Teff IL-2 antibody VitoKine was constructed, and its D3 domain shielding efficacy was evaluated for each IL-2 mutation scenario. All four exemplary IL-2 antibody VitoKine, P-0800, P-0830, P-0831, and P-0802, contain anti-mouse PD1 antibodies (SEQ ID NOs. 299 and 302) as the D1 domain and the IL-2RαSushiL42G variant as the D3 domain. The monovalent D2 domains, containing IL-2P65R in P-0800, IL-2P65N in P-0830, IL-2P65Q in P-0831, and the IL-2 wild-type equivalent in P-0802, are fused to the C-terminus of the heterodimer HC chain (SEQ ID NO: 301) via a non-cleavable (G4S)3 linker (SEQ ID NO: 112) and ligated to the N-terminus of the D3 domain with an MMP-2 / 9 cleavable linker (SEQ ID NO: 94). Each of the four IL-2 antibody VitoKine also contains two further polypeptides described in SEQ ID NOs: 300 and 302. The IL-2 moieties of each of the four VitoKine constructs retained full Teff potency, but the levels of binding strength to IL-2Rα varied. As shown in Table 14 of Example 16, the P65R mutation resulted in the loss of binding to IL-2Rα, while P65N and P65Q reduced the binding strength by 8.6 and 43 times, respectively. 【0295】 Four exemplary IL-2 antibodies, VitoKine, were evaluated for their respective ability to induce dose-dependent Ki67 expression in CD8+ T cells (Figure 39A) and NK cells (Figure 39B) in fresh human PBMCs. For comparison, P-0782, containing an anti-mouse PD1 antibody fused to a monovalent IL-2P65R fused to the C-terminus of a heterodimeric weight chain, was included. P-0782, P-0800, and P-0802 are antibody fusion counterparts of P-0704, P-0808, and P-0755, respectively. The data shown in Figures 34, 35, and 39 together clearly demonstrate that the morphology of the D1 domain, as either Fc or antibody as exemplified herein, does not affect the efficiency of the D3 domain in shielding cytokine efficacy. When the D2 domain is wild-type IL-2, the IL-2Rα-based D3 domain results in approximately 3 times the reduction in activity. However, when the D2 domain is an IL-2 variant in which binding to IL-2Rα has been lost, the D3 domain contributes only 10 to 20 times the shielding effect. 【0296】 Interestingly, when IL-2 variants with moderately reduced binding strength to IL-2Rα, exemplified by P65N and P65Q, were used as the D2 domain, the D3 domain yielded a similar shielding efficiency to that promoting wild-type IL-2. Data on dose-dependent induction of Ki67 expression on CD8+ T cells are shown in Figure 39A, and data on Ki67 expression on NK cells are shown in Figure 39B. EC of each compound in Ki67 expression induction on NK cells. 50 The values ​​are further summarized in Table 17. TIFF0007872587000021.tif68170 【0297】 The threshold binding affinity between the D2 and D3 domains and the optimal spatial arrangement of the binding interface were thought to play a role in determining the shielding efficiency of Teff IL-2 VitoKine. The IL-2P65Q variant significantly reduced its binding strength to IL-2Rα, but it was still efficiently masked by IL-2RαSushiL42G and remained inactive as a VitoKine. With complete recovery of protease cleavage and physiological activity in vivo, IL-2P65Q is expected to exhibit significantly impaired Treg cell stimuli compared to wild-type IL-2 (data not shown). Therefore, IL-2P65Q is selected as the preferred D2 domain for constructing Teff IL-2 VitoKine. Nevertheless, other mutant IL-2 variants can be used to achieve an optimal balance between desired antitumor effects and undesirable systemic toxicity. 【0298】 Various IL-2 antibody VitoKines, each containing a human PD-1 antagonist antibody as the D1 domain, an IL-2P65Q variant as the D2 domain, and an IL-2RαSushiL42G variant as the D3 domain, were constructed by varying the cytokine binding titer and linker combinations. Table 18 lists exemplary IL-2 PD-1 antibody VitoKines. TIFF0007872587000022.tif77170 【0299】 Exemplary IL-2 PD-1 antibody VitoKine, P-0872 and P-0929, were further evaluated for their protease cleavage efficiency in relation to the bulk D1 domain. P-0872 contains a single MMP-2 / 9 cleavable linker (SEQ ID NO: 94) that ligates the monovalent IL-2 moiety to the D2 and D3 domains. P-0872 was digested with MMP-2 protease according to the protocol detailed in Example 15. The digested samples were purified using Protein A in binding-elution mode, and the eluted samples were analyzed on a reduced SDS-PAGE gel to evaluate their biological function using in vitro functional assays. 【0300】 As shown in Figure 40A, the D3 domain of P-0872 was efficiently and completely cleaved to produce activated form 2 (P-0972-activated form) shown in Figure 2B, resulting in a complete restoration of activity, as exemplified by its potency equivalent to that of the non-VitoKine IL-2 PD-1 antibody fusion counterpart P-0879 (SEQ ID NOs. 285 and 296) in inducing dose-dependent Ki67 expression on CD8+ T cells in fresh human PBMCs (Figure 40B). 【0301】 Another representative IL-2 antibody, VitoKine, P-0929, contains a bivalent IL-2 moiety and a dual protease-cleavable linker comprising an MMP-2 / 9 cleavable linker linking the D1 and D3 domains and an MMP-14 cleavable linker (SEQ ID NO: 298) linking the D2 and D3 domains. P-0929 was cleaved with MMP-14 protease according to a similar protocol for MMP-2 digestion. The digested samples were purified using Protein A, and the flow-through and eluted samples were analyzed in reduced SDS-PAGE gel. 【0302】 The SDS-PAGE gel image shown in Figure 41A demonstrated that the MMP-14 protease could recognize and efficiently cleave both the MMP-2 / 9 and MMP-14 substrate peptides, generating active forms 1 and 3 in the absence of active form 2. This observation was consistent with the fact that MMP substrates have low specificity for one member of the MMP family. The presence of the cleaved D3 domain in the sample was due to the purification scheme and did not indicate that the D3 domain did not diffuse after cleavage. Protein A flow-through samples containing active forms 1 and 3 (P-0929-Activ) were then analyzed in human PBMCs. As shown in Figure 41B, activated P-0929 induces dose-dependent Ki67 expression on CD8+ T cells more potently than the monovalent non-VitoKine IL-2 PD-1 antibody fusion counterpart P-0879. In summary, the physiological activity of Teff IL-2, whose binding to IL-2Rα is impaired, can be efficiently shielded by the VitoKine type IL-2 antibody and readily restored by proteolysis. In the case of a dual protease-cleavable linker, the sequence and selection of the two cleavable linkers can be further optimized to suit different disease indications and / or stages. 【0303】 Example 21 Evaluation of the IL-15 variant antibody VitoKine in vivo The goal of IL-15 VitoKine, which has a bioactively attenuated IL-15 variant as its D2 domain, is to modulate the intrinsic basal activity of VitoKine to further minimize systemic on-target toxicity and undesirable antigen sink, thereby improving bioavailability and extending the therapeutic window. To evaluate this hypothesis, we are testing the IL-15 antibody VitoKine P-0869 in vivo. P-0869 contains a surrogate mouse PD-1 antibody (SEQ ID NOs. 299 and 302) as the D1 domain, an IL-15V63A / I68H variant (SEQ ID NOs. 213) as the D2 domain, an uncleavable L1 linker and an MMP-2 / 9 cleavable L2 linker (SEQ ID NOs. 95), and IL-15RaSushi (SEQ ID NOs. 5) as the D3 domain. As shown in Example 11 and Table 13, the IL-15 amino acid substitution V63A / I68H resulted in approximately a 2-logarithmic decrease in potency when inducing Ki67 expression on CD8+ T cells in vitro. Furthermore, P-0875, the human PD-1 antibody counterpart of P-0869, showed no detectable biological activity at the highest test concentration of 1 μM (Figure 38B) in fresh human PMBC, suggesting a significant decrease in the intrinsic basal activity of VitoKine. 【0304】 As a negative control, P-0869 was administered as a single intraperitoneal dose at doses of 1, 3, 5, and mg / kg to healthy BALB / c mice (8-10 weeks old, n=6 mice / group) with a solvent (PBS). P-0773 (SEQ ID NOs. 227 and 5), an IL-15V63A / I68H variant / IL-15RaSushi Fc fusion protein, was included as a positive control and administered as a single intraperitoneal injection at 0.5 mg / kg. Blood samples were collected pre-administration (-1 day) or post-administration (3, 5, and 7 days) and subjected to immunophenotyping. Based on the IL-15 VitoKine platform, P-0869 is expected to show only slight systemic activation and expansion of the target lymphocyte population even at very high dose levels, with a significant decrease in basal activity. 【0305】 P-0869 has been further tested in various mouse syngeneic models, including a mouse CT26 lung metastasis model, a colonized subcutaneous CT26 tumor model, and a colonized subcutaneous MC38 mouse colon cancer model. The experimental procedure is the same as that described in Examples 9 and 10. The IL-15 antibody VitoKine, with its attenuated D2 domain, is expected to exhibit tumor growth inhibition with minimal systemic cytokine activation at high doses. By inactivating VitoKine before activation at the target therapeutic site, the antibody VitoKine becomes a novel and progressive form of immune cytokine. The reduction in the basal activity of VitoKine by adjusting the potency of the D2 domain further promotes the establishment of a stoichiometric balance between the cytokine and the target antibody, enabling optimal drug delivery. 【0306】 Example 22 Evaluation of the Teff IL2 antibody VitoKine in vivo P-0831, a Teff IL2 antibody VitoKine, was evaluated in vivo in the same manner as described in Example 21. P-0831 contains an anti-mouse PD1 antibody (SEQ ID NOs. 300, 301, and 302) as the D1 domain, an IL-2P65Q / S125I variant (SEQ ID NOs. 240) as the D2 domain, and an IL-2RαSushiL42G variant (SEQ ID NOs. 268) as the D3 domain. P-831 also contains a non-cleavable L1 linker (SEQ ID NOs. 112) and a 15-amino acid MMP2 / 9 cleavable linker (SEQ ID NOs. 94). The monomeric IL-2 moiety retains full Teff potency, but its binding strength to IL-2Rα is significantly reduced (43-fold), which is expected to reduce stimulation of undesirable Treg subsets. As described in Example 20, the D3 domain of P-0831 efficiently shielded IL-2 activity, resulting in a nearly 1000-fold reduction in potency (Figure 39). 【0307】 P-0831 has been further tested in various mouse syngeneic models, including a mouse CT26 lung metastasis model, a grafted subcutaneous CT26 tumor model, and a grafted subcutaneous MC38 mouse colon cancer model. The experimental procedure is the same as that described in Examples 9 and 10. 【0308】 Several IL-2 antibodies, VitoKine, P-0922A, P-0928A, P-0929A, and their non-cleavable counterparts, P-0877, were further tested in a colonized subcutaneous MC38 mouse colon cancer model. All four VitoKine antibodies contain the IL-2P65Q / S125I variant (SEQ ID NO: 240) as the D2 domain and the IL-2RαSushiL42G variant (SEQ ID NO: 268) as the D3 domain. P-0922A and P-0929A contain surrogate mouse PD1 antagonist antibodies with homodimeric weight chains (SEQ ID NOs: 299 and 302) as the D1 domain, while P-0928A and P-0877 contain surrogate mouse PD1 antagonist antibodies with heterodimeric weight chains (SEQ ID NOs: 300, 301, and 302) as the D1 domain. The L1 and L2 linkers in P-0922A are non-cuttable (G4S)3 (sequence number 112) and cuttable (sequence number 94), respectively. Both the L1 and L2 linkers in P-0928A are cuttable, with sequence numbers 298 and 94, respectively. Both the L1 and L2 linkers in P-0929A are cuttable, with sequence numbers 94 and 298, respectively. P-0877 contains two non-cuttable (G4S)3 (sequence n...

Claims

[Claim 1] A physiologically active polypeptide drug construct, wherein the direction from the N-terminus to the C-terminus (D1-D2-D3), It comprises 1) a D1 domain ("D1") which is a tissue or affected area target portion, 2) a D2 domain ("D2") which is a physiologically activated portion, and 3) a D3 domain ("D3") which is a shielding portion, wherein D1 functions to target the physiologically activated portion to the target treatment site; D3 can shield the functional activity of D2 until it is activated at the target treatment site; or It comprises 1) a functional portion, the D1 domain ("D1"), 2) a physiologically active portion, the D2 domain ("D2"), and 3) a shielding portion, the D3 domain ("D3"), wherein D1 functions to target and retain the physiologically active portion at the target treatment site; and D3 can shield the functional activity of D2 until it is activated at the target treatment site; D2 is an IL-15 polypeptide selected from the group of polypeptides having the amino acid sequences described in SEQ ID NOs: 182-192 and 199-215. D1 is an Fc domain having an amino acid sequence selected from the group consisting of the amino acid sequences described in SEQ ID NOs: 14, 15, 16, 156, and 166-168. The D3 domain is a cognitive receptor / binding partner (or variant thereof) for IL-15, and includes the amino acid sequence described in SEQ ID NO: 4 or the amino acid sequence described in SEQ ID NO:

5. Bioactive polypeptide drug constructs. [Claim 2] A physiologically active polypeptide drug construct, wherein the direction from the N-terminus to the C-terminus (D1-D2-D3), It comprises 1) a D1 domain ("D1") which is a tissue or affected area target portion, 2) a D2 domain ("D2") which is a physiologically activated portion, and 3) a D3 domain ("D3") which is a shielding portion, wherein D1 functions to target the physiologically activated portion to the target treatment site; D3 can shield the functional activity of D2 until it is activated at the target treatment site; or It comprises 1) a functional portion, the D1 domain ("D1"), 2) a physiologically active portion, the D2 domain ("D2"), and 3) a shielding portion, the D3 domain ("D3"), wherein D1 functions to target and retain the physiologically active portion at the target treatment site; and D3 can shield the functional activity of D2 until it is activated at the target treatment site; D2 is an IL-2 variant polypeptide selected from the group of polypeptides having the amino acid sequences described in SEQ ID NOs: 232-247. D1 is an Fc domain having an amino acid sequence selected from the group consisting of the amino acid sequences described in SEQ ID NOs: 14, 15, 16, 156, and 166-168. The D3 domain is a cognitive receptor / binding partner (or variant thereof) for IL-2 and comprises an amino acid sequence selected from the group consisting of the amino acid sequences described in SEQ ID NOs: 10 and 267-270. Bioactive polypeptide drug constructs. [Claim 3] A physiologically active polypeptide drug construct, wherein the direction from the N-terminus to the C-terminus (D1-D2-D3), It comprises 1) a D1 domain ("D1") which is a tissue or affected area target portion, 2) a D2 domain ("D2") which is a physiologically activated portion, and 3) a D3 domain ("D3") which is a shielding portion, wherein D1 functions to target the physiologically activated portion to the target treatment site; D3 can shield the functional activity of D2 until it is activated at the target treatment site; or It comprises 1) a functional portion, the D1 domain ("D1"), 2) a physiologically active portion, the D2 domain ("D2"), and 3) a shielding portion, the D3 domain ("D3"), wherein D1 functions to target and retain the physiologically active portion at the target treatment site; and D3 can shield the functional activity of D2 until it is activated at the target treatment site; D2 is an IL-15 polypeptide selected from the group of polypeptides having the amino acid sequences described in SEQ ID NOs: 182-192 and 199-215. D1 is an antagonist humanized PD-1 antibody selected from antibodies containing the heavy chain and light chain amino acid sequences described in SEQ ID NOs: 195 and 196, SEQ ID NOs: 197 and 198, SEQ ID NOs: 275 and 276, or SEQ ID NOs: 277 and 278. The D3 domain is a cognitive receptor / binding partner (or variant thereof) for IL-15, and includes the amino acid sequence described in SEQ ID NO: 4 or the amino acid sequence described in SEQ ID NO:

5. Bioactive polypeptide drug constructs. [Claim 4] A physiologically active polypeptide drug construct, wherein the direction from the N-terminus to the C-terminus (D1-D2-D3), It comprises 1) a D1 domain ("D1") which is a tissue or affected area target portion, 2) a D2 domain ("D2") which is a physiologically activated portion, and 3) a D3 domain ("D3") which is a shielding portion, wherein D1 functions to target the physiologically activated portion to the target treatment site; D3 can shield the functional activity of D2 until it is activated at the target treatment site; or It comprises 1) a functional portion, the D1 domain ("D1"), 2) a physiologically active portion, the D2 domain ("D2"), and 3) a shielding portion, the D3 domain ("D3"), wherein D1 functions to target and retain the physiologically active portion at the target treatment site; and D3 can shield the functional activity of D2 until it is activated at the target treatment site; D2 is an IL-2 variant polypeptide selected from the group of polypeptides having the amino acid sequences described in SEQ ID NOs: 232-247. D1 is an antagonist humanized PD-1 antibody selected from antibodies containing the heavy chain and light chain amino acid sequences described in SEQ ID NOs: 195 and 196, SEQ ID NOs: 197 and 198, SEQ ID NOs: 275 and 276, or SEQ ID NOs: 277 and 278. The D3 domain is a cognitive receptor / binding partner (or variant thereof) for IL-2 and comprises an amino acid sequence selected from the group consisting of the amino acid sequences described in SEQ ID NOs: 10 and 267-270. Bioactive polypeptide drug constructs. [Claim 5] The construct according to any one of claims 1 to 4, wherein the D1 domain, D2 domain, and D3 domain of the construct are each in monomer form, each in dimer form, or in a form in which a dimer and a monomer are combined. [Claim 6] The construct according to any one of claims 1 to 5, wherein D2 is attached to D1 by a peptide linker ("L1") selected from the group consisting of protease-cleavable peptide linkers and non-cleavable peptide linkers. [Claim 7] The construct according to claim 6, wherein the protease-cleavable peptide linker is selected from the group consisting of sequences described in SEQ ID NOs: 71-96, 157-161, and 298. [Claim 8] The construct according to claim 6, wherein the non-cleavable peptide linker is selected from the group consisting of the sequences described in SEQ ID NOs: 107 to 127. [Claim 9] The construct according to any one of claims 1 to 5, wherein D2 is attached to D3 by a peptide linker ("L2") selected from the group consisting of a protease-cleavable peptide linker and a peptide linker that cannot be cleaved. [Claim 10] The construct according to claim 9, wherein the protease-cleavable peptide linker is selected from the group consisting of sequences described in SEQ ID NOs: 71-96, 157-161, and 298. [Claim 11] The construct according to claim 9, wherein the non-cleavable peptide linker is selected from the group consisting of the sequences described in SEQ ID NOs: 107 to 127. [Claim 12] D2 is attached to D1 by a peptide linker ("L1"), and D2 is attached to D3 by a peptide linker ("L2") Both L1 and L2 are peptide linkers that can be cleaved by proteases, or Both L1 and L2 are inclementable peptide linkers, or L1 is a peptide linker that can be cleaved by protease, and L2 is a peptide linker that cannot be cleaved, or L1 is a peptide linker that cannot be cleaved, and L2 is a peptide linker that can be cleaved by proteases. The construct according to any one of claims 1 to 5. [Claim 13] A pharmaceutical composition comprising a construct according to any one of claims 1 to 12, mixed with a pharmaceutically acceptable carrier. [Claim 14] The pharmaceutical composition according to claim 13, for use in the treatment of cancer or cancer metastasis in a subject. [Claim 15] The pharmaceutical composition according to claim 14, wherein the treatment further comprises a second therapeutic agent or treatment method capable of treating cancer or cancer metastasis in the subject. [Claim 16] A nucleic acid molecule encoding the construct according to any one of claims 1 to 12. [Claim 17] An expression vector comprising the nucleic acid molecule described in claim 16. [Claim 18] A host cell comprising the expression vector described in claim 17. [Claim 19] A method for producing a physiologically activated polypeptide drug construct according to any one of claims 1 to 12, comprising culturing the host cells according to claim 18 under conditions that promote the expression of the physiologically activated polypeptide drug construct and recovering the physiologically activated polypeptide drug construct protein. [Claim 20] An isolated physiologically active polypeptide drug construct protein produced by the method described in claim 19.

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