Bioactivatable cytokine-based drugs and methods of use thereof

Abstract

The present application relates to cytokine-based bioactivatable drugs and methods of use thereof. The present invention provides a bioactivatable drug construct platform based on cytokines, aimed at reducing systemic toxicity, enabling proteins and cytokines, such as IL-15 and IL-2, to have a broader therapeutic use in the treatment of cancer, autoimmune diseases, inflammatory diseases, viral infections, transplantation and other disorders. The invention relates to a bioactivator comprising a tissue or disease site targeting portion D1 domain, a bioactivatable portion D2 domain and a shielding portion D3 domain. The active moiety of VitoKine remains inert until it is locally activated by an upregulated protease in diseased tissue, which limits binding of the active moiety to receptors or targets in the periphery or on the cell surface of non-diseased cells and tissues, prevents pathway over-activation and reduces undesirable "extracorporeal" "in-target" toxicity. Prior to protease activation, the inertness of the VitoKine active moiety significantly reduces potential antigen or target silencing, extends in vivo half-life and improves biodistribution, bioavailability and therapeutic efficacy.

Description

[0001] This application is a divisional application of an application filed on June 20, 2019, with application number 201980055435.7, and invention name “Cytokine-based bioactivatable drugs and methods of use thereof”.

[0002] Related patent applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 689,053, filed June 22, 2018, which is incorporated herein by reference in its entirety. background

[0004] Many cytokines have been evaluated as potential therapeutic agents for treating diseases. However, their systemic overstimulation or oversuppression of the body's immune system has severely hampered their development and clinical use.

[0005] 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 the proliferation of T cells; induce the production of cytotoxic T lymphocytes (CTLs); promote the proliferation of B cells and immunoglobulin synthesis; and induce the production and persistence of natural killer (NK) cells. Based on extensive preclinical studies and multiple clinical evaluations, these two cytokines are considered potentially valuable therapeutic agents for 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. For IL-15, there are several ongoing clinical trials in oncology, but it has not yet been approved for use. In addition, both IL-2 and IL-15 have a third, unique, non-signaling receptor α-subunit: IL-2Rα (also known as CD25) or IL-15Rα, respectively, which may contribute to their different receptor specificities and biological functions.

[0006] Recombinant human IL-2 is an effective immunotherapy being used for metastatic melanoma and renal cancer, with approximately 10% of patients having a durable response. However, the short half-life and severe toxicity limit the optimal dosing of IL-2. In addition, IL-2 also binds with high affinity to its heterotrimeric receptor IL-2Rαβγ, preferentially amplifying immunosuppressive regulatory T cells (Tregs) that express high constitutive IL-2Rα levels. The expansion of Tregs may represent an undesirable effect of IL-2 on cancer immunotherapy. However, the ability of IL-2 to stimulate Treg cells even at low doses may be used to treat autoimmune disorders and chronic inflammatory disorders. It has recently been discovered that IL-2 can be modified to selectively stimulate cytotoxic effector T cells or Treg cells. Various approaches have led to the production of IL-2 variants with improved and selective immunomodulatory activity.

[0007] Both IL-2 and IL-15 are effective immune effector cell agonists, and crucially, cytotoxic immune cells are fully activated only when at or very near a disease site (e.g., a cancer site) to specifically destroy only tumor cells; or cytotoxic immune cells are fully activated only when at or very near an inflammatory problem site to specifically destroy only autoimmune and chronic inflammatory disorders. For all cytokines, chemokines, and growth factors, it is very important to improve the specificity and selectivity of the target and to keep healthy cells and tissues intact and undamaged.

[0008] Disclosure of the Invention

[0009] In one aspect, the present invention provides a cytokine-based bioactivatable drug ("VitoKine") platform designed to reduce toxicity based on systemic mechanisms and enable broader therapeutic use 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. The VitoKine platform consists of Figure 1 The constructs depicted in Figure 2 The proposed activation method is defined as depicted in . Figure 1The novel VitoKine constructs of the present invention comprise 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-function moiety domain, 2) a D2 domain ("D2"), which is the "active moiety domain," and 3) a D3 domain ("D3"), which is the "shielding moiety domain." Importantly, the D2 domain of the VitoKine construct remains nearly inert or minimally active until locally activated by proteases upregulated in diseased tissue or hydrolysis at the site of disease, which limits binding of the active moiety to receptors in the periphery or on the cell surface of non-diseased cells or normal tissues to prevent overactivation of the pathway and reduce undesirable "on-target" or "off-tissue" toxicity and undesirable target sink.

[0010] In various embodiments, the VitoKine construct of the present invention comprises D1, which is a targeting moiety, such as an antibody or antibody fragment that binds to a tumor-associated antigen (TAA) or a tissue-specific antigen, a cell surface molecule or an extracellular matrix protein or a protease or any post-translationally modified residue. In various embodiments, the VitoKine construct of the present invention comprises D1, which is a targeting moiety, such as a protein or peptide that exhibits binding affinity to diseased cells or tissues. In various embodiments, the VitoKine construct of the present invention comprises D1, which is a modified protein or peptide, such as a glycan-modified one, that exhibits binding affinity to specific receptors expressed on diseased cells or tissues, such as c-type lectin receptors. In various embodiments, the VitoKine construct of the present invention comprises a D1 domain, which is an antibody for an immune checkpoint regulator. In various embodiments, the VitoKine construct of the present invention comprises D1, which acts to keep cytokines at tissue sites. In various embodiments, the VitoKine construct of the present invention comprises D1, which is dual-functional, such as tissue targeting and maintenance. In various embodiments, the VitoKine constructs of the present invention comprise a D1 domain that is a polymer. In various embodiments, the VitoKine constructs of the present invention comprise a D1 domain that is a half-life extending moiety. In various embodiments, the VitoKine constructs of the present invention comprise a D1 domain that is an Fc domain (or a functional fragment thereof).

[0011] "Fc domain" refers to a dimer of two Fc domain monomers, typically comprising all or part of the hinge region. In various embodiments, the Fc domain is selected from the group consisting of a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, an IgAFc domain, an IgD Fc domain, an IgE Fc domain, an IgGFc domain, and an IgM Fc domain, or any combination thereof. In various embodiments, the Fc domain comprises amino acid changes that result in the Fc domain having altered complement binding properties or Fc receptor binding properties. Amino acid changes that are known to produce Fc domains with altered complement binding properties or Fc receptor binding properties are known in the art. In various embodiments, the Fc domain sequence used to prepare the VitoKine construct is the human IgG1-Fc domain sequence set forth in SEQ ID NO: 13. In various embodiments, the Fc domain sequence used to prepare the VitoKine construct is the sequence set forth in SEQ ID NO: 14, which contains amino acid substitutions that ablate FcγR and C1q binding. In various embodiments, the Fc domain includes amino acid changes that result in further extension of half-life in vivo. Amino acid changes known to produce Fc domains with further extended half-life are known in the art. In various embodiments, the Fc domain sequence used to prepare the VitoKine construct is the sequence set forth in SEQ ID NO: 156 or 166, both of which contain amino acid substitutions that ablate FcγR and C1q binding and extend half-life in vivo. In various embodiments, the heterodimeric Fc domain sequence used to prepare the VitoKine construct is derived from the Knob-Fc domain sequence set forth in SEQ ID NO: 15. In various embodiments, the heterodimeric Fc domain sequence used to prepare the VitoKine construct is derived from the Hole-Fc domain sequence set forth in SEQ ID NO: 16. In various embodiments, the heterodimeric Fc domain sequence used to prepare the VitoKine construct is derived from the Knob-Fc domain with an in vivo half-life extending sequence set forth in SEQ ID NO: 167. In various embodiments, the heterodimeric Fc domain sequence used to prepare the VitoKine construct is derived from the Hole-Fc domain with an in vivo half-life extending sequence set forth in SEQ ID NO: 168.

[0012] In various embodiments, the VitoKine constructs of the present invention comprise a D2 domain, which is a protein. In various embodiments, the VitoKine constructs of the present invention comprise 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 a ligand of the transforming growth factor beta (TGFβ) superfamily, such as TGFβ (SEQ ID NO: 24). In various embodiments, the VitoKine constructs of the present invention comprise a D2 domain, which is IL-15. In various embodiments, the VitoKine constructs of the present invention comprise a D2 domain, which is an IL-15 variant (or mutant) comprising one or more amino acid substitutions, deletions, or insertions to an IL-15 polypeptide. In various embodiments, the VitoKine constructs of the present invention comprise a D2 domain, which is IL-2. In various embodiments, the VitoKine constructs of the present invention comprise a D2 domain that is an IL-2 variant (or mutant) comprising one or more amino acid substitutions, deletions, or insertions to an IL-2 polypeptide.

[0013] In various embodiments, the D2 domain of the VitoKine construct is an IL-15 domain comprising the sequence of the mature human IL-15 polypeptide as set forth in SEQ ID NO: 2 (also referred to herein as huIL-15 or IL-15 wild type (wt)). In various embodiments, the IL-15 domain is an IL-15 variant (or mutant) comprising a sequence derived from the mature human IL-15 polypeptide sequence as set forth in SEQ ID NO: 2. In various embodiments, the IL-15 domain is an IL-15 variant (or mutant) comprising a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology to SEQ ID NO: 2. Natural amino acids, the positions of natural amino acids in the mature sequence, and variant amino acids are used herein to refer to variants (or mutants) of IL-15. For example, huIL-15 "S58D" refers to a human IL-15 comprising an S to D substitution at position 58 of SEQ ID NO: 2. In various embodiments, the IL-15 variant acts as an IL-15 agonist, as demonstrated by, for example, increased binding activity to the IL-15Rβγc receptor compared to the native IL-15 polypeptide. In various embodiments, the IL-15 variant acts as an IL-15 antagonist, as demonstrated by, for example, reduced binding activity to the IL-15Rβγc receptor compared to the native IL-15 polypeptide, or similar or increased binding activity to the IL-15Rβγc receptor, but reduced or eliminated 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., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid change compared to the native IL-15 sequence. Amino acid changes can include one or more amino acid substitutions, deletions, or insertions in the IL-15 polypeptide, such as in the domain of IL-15 that interacts with IL-15Rβ and / or IL-15Rβγc. In various embodiments, the amino acid change is one or more amino acid substitutions or deletions 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 D to T substitution at position 30, a V to Y substitution at position 31, an H to E substitution at position 32, an S to D substitution at position 58, a T to D substitution at position 61, a V to F substitution at position 63, an I to V substitution at position 67, an I to F or H or D or K substitution at position 68, or a Q to A or M or S substitution at position 108 of the mature human IL-15 sequence, or any combination of these substitutions.In various embodiments, the amino acid change is an S to D substitution at position 58 of the mature human IL-15 sequence. In various embodiments, the IL-15 polypeptide comprises the IL-15 variant SEQ ID NO: 3. In various embodiments, the IL-15 domain has any combination of amino acid substitutions, deletions, and insertions.

[0014] In various embodiments, the D2 domain of the VitoKine construct of the present invention comprises an IL-2 polypeptide. 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, deletions, or insertions. In various embodiments, the VitoKine construct comprises a D2 domain, wherein the IL-2 domain comprises the sequence of a mature human IL-2 polypeptide as set forth in SEQ ID NO: 8 (also referred to herein as huIL-2 or IL-2 wild type (wt)). In various embodiments, the IL-2 domain is an IL-2 variant (or mutant) comprising a sequence derived from a mature human IL-2 polypeptide sequence as set forth in SEQ ID NO: 8. In various embodiments, the IL-2 domain is an IL-2 variant (or mutant) comprising 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 acts as an IL-2 agonist. In various embodiments, the IL-2 variant acts 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, 88, 107, 125, or 126 of SEQ ID NO:8. In various embodiments, the amino acid change is an L to D or H or N or P or Q or R or S or Y substitution at position 19 of the mature human IL-2 sequence, a D to E or I or N or Q or S or T or Y substitution at position 20, an R to E or A substitution at position 38, a T to A or G or V substitution at position 41, an F to A substitution at position 42, an F to G or V substitution at position 44, an N to D, E or G or I or M or Q or T or R substitution at position 88, a Y to G or H or L or V substitution at position 107, an S to E, H, K, I or W substitution at position 125, a Q to D or E or K or L or M or N substitution at position 126, or any combination of these substitutions.

[0015] In various embodiments, the VitoKine constructs of the present invention comprise a "masking domain" (D3), which is a cognate receptor / binding partner or any binding partner identified for the D2 protein or cytokine. In various embodiments, the D3 domain is a variant of the cognate receptor / binding partner of the D2 domain. In various embodiments, the D3 domain has enhanced binding to the D2 domain compared to the wild-type cognate receptor / binding partner. In various embodiments, the D3 domain has reduced or eliminated binding to the D2 domain compared to the wild-type cognate receptor / binding partner. In various embodiments, the D3 domain is a protein, or peptide, or antibody, or antibody fragment that is capable of masking the activity of D2. In various embodiments, the D3 domain is a DNA, RNA fragment, or polymer, such as PEG. In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain, which is the extracellular domain of IL-15Rα or a functional fragment thereof. In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is an IL-15Rα Sushi domain. In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is an IL-2Rα extracellular domain or a functional fragment thereof. In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is an IL-2Rα Sushi domain. In various embodiments, the D3 domain is capable of shielding the functional activity of D2 until activated at the intended treatment site.

[0016] In various embodiments, the D1 domain, D2 domain, and D3 domain of the VitoKine construct are connected by a protease-cleavable polypeptide linker sequence. In various embodiments, the D1 domain, D2 domain, and D3 domain of the VitoKine construct are connected by a non-cleavable polypeptide linker sequence. In various embodiments, L1 and L2 of the VitoKine construct of the present invention are both 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 a non-cleavable peptide linker. In various embodiments, L1 of the VitoKine construct of the present invention is a non-cleavable peptide linker, and L2 is a protease-cleavable peptide linker. In various embodiments, L1 and L2 of the VitoKine construct of the present invention are both non-cleavable linkers. In various embodiments, the linker is rich in G / S content (e.g., at least about 60%, 70%, 80%, 90% or more of the amino acids in the linker are G or S). Each peptide linker sequence can be selected independently. In various embodiments, the protease cleavable linker is selected from the group of sequences listed in SEQ ID NOs: 71-96 and 157-161. In various embodiments, the protease cleavable linker can have additional peptide spacers of variable length at the N-terminus of the cleavable linker or at the C-terminus of the cleavable linker or at both ends of the cleavable linker. In various embodiments, the non-cleavable linker is selected from the group of sequences listed in SEQ ID NOs: 107-127. In various embodiments, the linker is flexible or rigid and has various lengths.

[0017] In various embodiments, the D2 and D3 domains of the VitoKine construct are placed N-terminally to the D1 domain, e.g. Figure 1 In various embodiments, the D2 and D3 domains of the VitoKine construct are placed C-terminally to the D1 domain, as shown in FIG. Figure 1 Depicted in.

[0018] In various embodiments, the D1 domain, D2 domain, and D3 domain of the VitoKine construct can be monomers or dimers or a combination of dimers and monomers, such as D1 is a dimer, and D2 and D3 are monomers.

[0019] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention. 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, or rhabdomyosarcoma, or any cancer.

[0020] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention in combination with a second therapy selected from the group consisting of: cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiotherapy, stem cell transplantation, cell therapy including CAR-T, CAR-NK, iPS-induced CAR-T or iPS-induced CAR-NK, and vaccines such as Bacille Calmette-Guerine (BCG). In various embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of an immunotherapy, including, but not limited to, treatment with depleting antibodies directed against specific tumor antigens; treatment with antibody-drug conjugates; treatment with agonistic, antagonistic, or blocking antibodies directed against co-stimulatory or co-inhibitory 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; treatment with bispecific T cell-engaging antibodies; treatments such as blinatumomab; treatments involving administration of biological response modifiers (such as IL-12, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ); treatments using therapeutic vaccines such as sipuleucel-T; treatments using dendritic 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 adoptively transferred anti-tumor T cells (ex vivo expanded T cells and / or TCR transgenic T cells); treatments using TALL-104 cells; and treatments using immunostimulants such as the Toll-like receptor (TLR) agonists CpG and imiquimod; and treatments using vaccines such as BCG; wherein the combination therapy provides increased effector cell killing of tumor cells, i.e., there is a synergistic effect between the VitoKine construct and the immunotherapy when co-administered.

[0021] In another aspect, the present disclosure provides a method for treating a viral infection in a subject, the method comprising administering a therapeutically effective amount of a 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.

[0022] In another aspect, the present disclosure provides a method for treating a viral infection in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention in combination with a second therapy including, but not limited to, acyclovir, Epclusa, Mavyret, zidovudine, and enfuvirtide.

[0023] On the other hand, present disclosure provides a method for treating an autoimmune disease in a subject, the method comprising administering a therapeutically effective amount of a 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 syndrome, dermatomyositis, Hashimoto's disease, polymyositis, inflammatory bowel disease, multiple sclerosis (MS), diabetes, rheumatoid arthritis, and scleroderma.

[0024] In another aspect, the present disclosure provides a method for treating an inflammatory disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention. 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, divertic colitis, Behcet's syndrome, and indeterminate colitis.

[0025] In various embodiments, the inflammatory disease is selected from the group consisting of other autoimmune diseases and inflammatory diseases such as achalasia, adult-onset Still's disease, agammaglobulinemia, amyloidosis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune inner ear disease, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuronal neuropathy, Barlow's disease, Behcet's disease, benign mucous membrane pemphigoid, Castleman's disease, Chagas' disease, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal osteomyelitis, allergic granulomatosis with polyangiitis, cicatricial pemphigoid, Cochrane syndrome coxsackievirus myocarditis, CREST syndrome, dermatitis herpetiformis, Devic disease / neuromyelitis optica, discoid lupus, Dressler syndrome, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, mixed cryoglobulinemia, fibrosing alveolitis, giant cell arteritis, giant cell myocarditis, Henoch-Schönlein purpura, herpes gestationis or pemphigoid gestationis, IgA nephropathy, IgG4-related sclerosing disease, immune-related adverse events, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile myositis, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, woody conjunctivitis, linear IgA disease, chronic Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Mooren's ulcer Ulcer), Mucha-Habermann disease, multifocal motor neuropathy, optic neuritis, relapsing rheumatic disease, PANDAS, paraneoplastic cerebellar degeneration, Parry-Romberg syndrome, pars planitis, Parsonage-Turner syndrome, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polyglandular syndrome, polymyalgia rheumatica, post-myocardial infarction syndrome, postpericardiotomy syndrome, primary sclerosing cholecystitis, angitis, progesterone dermatitis, psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum, Raynaud's phenomenon, reflex sympathetic dystrophy, relapsing polychondritis, retroperitoneal fibrosis, scleritis, sperm and testicular autoimmunity, stiff-man syndrome, subacute bacterial endocarditis, Susac's syndrome, sympathetic ophthalmia, Takayasu arteritis, thrombocytopenic purpura, painful ophthalmoplegia syndrome, transverse myelitis, undifferentiated connective tissue disease, Vogt-Koyonagi-Harada disease.

[0026] In another aspect, the present disclosure provides the use of a VitoKine construct for the preparation of a medicament for the treatment of cancer.

[0027] In another aspect, the present disclosure provides the use of a VitoKine construct for the preparation of a medicament for the treatment of a viral infection.

[0028] In another aspect, the present disclosure provides the use of a VitoKine construct for the preparation of a medicament for the treatment of an autoimmune disease.

[0029] In another aspect, the present disclosure provides the use of a VitoKine construct for the preparation of a medicament for the treatment of inflammation.

[0030] In another aspect, the present disclosure provides for the use of a VitoKine construct of the present invention in combination with a second therapeutic agent or cell therapy capable of treating cancer, viral infection, or autoimmune disease or inflammation.

[0031] In another aspect, the present disclosure provides an isolated nucleic acid molecule comprising a polynucleotide encoding a VitoKine construct of the present disclosure. In another aspect, the present disclosure provides a vector comprising a nucleic acid described herein. In various embodiments, the vector is an expression vector. In another aspect, the present disclosure provides an isolated cell comprising a nucleic acid of the present disclosure. In various embodiments, the cell is a host cell comprising an expression vector of the present disclosure. In another aspect, a method of preparing a VitoKine construct is provided by culturing a host cell under conditions that promote expression of a protein or polypeptide.

[0032] In another aspect, the present disclosure provides pharmaceutical compositions comprising an isolated VitoKine construct admixed with a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Representative VitoKine construct formats of the invention are depicted.

[0034] Figure 2 The proposed activation mechanism of the VitoKine construct is depicted. The exemplary VitoKine comprises two protease-cleavable linkers; activation by protease 1, generated by cleavage of the L1 linker, produces active form 1; activation by protease 2, generated by cleavage of the L2 linker, produces active form 2; and activation by both proteases, generated by cleavage of the L1 and L2 linkers, produces active form 3.

[0035] Figures 3A-3B Depicted are the protein profiles of an exemplary IL-15 VitoKine P-0315 after Protein A purification, A) SDS-PAGE in the absence and presence of a reducing agent, B) size exclusion chromatogram.

[0036] Figures 4A-4CFigure 2: Binding and functional activity of IL-15 VitoKine P-0172 compared to the highly active IL-15 fusion protein P-0198. (A) Binding activity to IL-2Rβ measured by ELISA assay; (B-C) Induction of CD69 expression on CD8+ T cells (B) and NK cells (C) of fresh human PBMCs analyzed by FACS.

[0037] Figure 5 Depicted are the functional activities of monomeric Fc IL-15 VitoKine P-0170 compared to the highly active IL-15 fusion protein P-0166. Induction of CD69 expression on human CD8+ T cells from fresh human PBMCs was measured and analyzed by FACS.

[0038] Figures 6A-6B Depicted are the effects of illustrative VitoKine constructs with varying linker lengths (P-0204, P-0205, and P-0206) on human PBMCs A) CD8 + Induction of CD69 expression on T cells and B) NK (CD56+) cells.

[0039] Figure 7 Depicted are the proliferation of NK (CD56+) cells in human PBMCs induced by illustrative VitoKine constructs with different L1 and L2 linkers (P-0202, P-0203, and P-0204) compared to the fully active IL-15 / IL-15RαFc fusion proteins P-0207 and P-0217.

[0040] Figures 8A-8B Depicted are A) NK (CD56+) cells and B) CD8 in human PBMCs elicited by illustrative Fc IL-15 VitoKine constructs with varying L2 linker sequence compositions (P-0351, P-0488, and P-0489) compared to the IL-15 / IL-15Rα Fc fusion protein P-0156, as measured by FACS. + Proliferation of T cells.

[0041] Figure 9 Depicted is SDS-PAGE analysis of Fc IL-15 VitoKine P-0315 proteolysis using varying amounts of MMP-2.

[0042] Figures 10A-10B Depicted is SDS-PAGE analysis of Fc IL-15 VitoKine P-0203 proteolysis using uPA under different conditions to determine the appropriate reaction conditions for complete cleavage.

[0043] Figures 11A-11B Depicted are A) SDS-PAGE analysis of Fc IL-15 VitoKine P-0203 before and after proteolysis by uPA. B) Protein profile of activated VitoKine P-0203 after uPA digestion and protein A purification to remove the cleaved Fc fragment.

[0044] Figures 12A-12C Depicted are A) SDS-PAGE analysis of Fc IL-15 VitoKine P-0315 before and after proteolysis by MMP-2. The gel also shows the profile of P-0315 digested with MMP-2 and purified by Protein A; B) the protein profile of active form 2 of VitoKine P-0315 produced by digestion with MMP-2 followed by purification by Protein A; C) the protein profile of active form 3 of VitoKine P-0315 produced by dual proteolysis by MMP-2 and uPA followed by purification by Protein A in flow-through mode.

[0045] Figures 13A-13B Depicted is the assessment of the activity of the protease (uPA) activated Fc IL-15 VitoKine P-0203 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.

[0046] Figures 14A-14B Depicted is the activity evaluation of two forms of protease-activated Fc IL-15 VitoKine P-0315 by analyzing the induction of the activation marker CD69 on A) CD56+ NK cells and B) CD8+ T cells. P-0315 active form 2 is generated by MMP-2 digestion, and P-0315 active form 3 is generated by dual proteolysis 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.

[0047] Figures 15A-15B Depicted is the evaluation of the activity of IL-15 VitoKine P-0315 (active form 2) on MMP-2 activation by analyzing the induction of the proliferation marker Ki67 on A) CD56+ NK cells and B) CD8+ T cells. P-0351, which contains non-cleavable L1 and L2 linkers and shares the same L2 linker length as P-0315, was included for comparison.

[0048] Figures 16A-16CDepict after single injection in Balb / C mice, the dose-dependent effect and time-dependent effect of the amplification of CD8+T (A), NK cells (B) and leukocytes (C) of cleavable Fc IL-15VitoKine P-0315, non-cleavable Fc IL-15VitoKine P-0351 on peripheral blood. Fully active IL-15Fc fusion protein P-0313 is included for comparison. Blood is collected at -1 day, 3 days, 5 days and 7 days to perform lymphocyte phenotyping analysis by FACS analysis. Data are expressed as mean ± SEM. Statistical analysis was performed by two-way ANOVA test, followed by Tukey's post hoc test. At respective time points, ****p<0.0001, ***p<0.001, *p<0.05 compared with PBS group.

[0049] Figure 17 Depicted is the inhibition of lung metastatic nodules in a mouse CT26 lung metastasis model one day after 4× Q5D dosing of P-0315, P-0351, P-0313, or PBS control. The first dose began one day after injection of CT26 cells. Unless otherwise noted, all comparisons are compared to the PBS group; ****p<0.0001; **p<0.01; *p<0.05.

[0050] Figures 18A-18B Depicted are A) CD8+ T cell % and B) NK cell % of total blood lymphocytes in CT26-transferred mice. Cell numbers were determined by flow cytometry 4 days after three Q5D i.p. injections of P-0315, P-0351, P-0313, or PBS controls. All comparisons are compared to the PBS group; ****p<0.0001; **p<0.01; *p<0.05.

[0051] Figures 19A-19D depict the antitumor efficacy of Fc IL-15 VitoKine P-0315 compared to the fully active IL-15 Fc fusion P-0313 in an established CT26 murine colorectal cancer tumor model. Shown are growth curves of CT26 sc tumors in individual mice following two Q5D treatments: A) vehicle PBS, B) 0.1 mg / kg P-0315, or C) 0.1 mg / kg P-0313. (D) Mean tumor volume ± SEM for each treatment group over time. All comparisons are compared to vehicle treatment; n = 11 mice / group; ****P < 0.0001.

[0052] Figures 20A-20BDepicted are immunopharmacodynamic profiling of peripheral blood in mice treated with VitoKine P-0315 or the highly active IL-15Fc fusion protein P-0313 in the CT26 murine colorectal cancer tumor model. Following two Q5D treatments, starting 11 days after tumor implantation, the percentage 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.

[0053] Figures 21A-21C Depicted is the immunopharmacodynamic profiling of peripheral blood in mice treated with P-0315 or P-0313 in the CT26 murine colorectal cancer tumor model. Following two Q5D treatments starting 11 days after tumor implantation, increases in the number of circulating (per μl whole blood) A) total leukocytes, B) NK cells, and C) CD8+ T cells were determined by flow cytometry on day 19. ****P < 0.0001 compared to PBS.

[0054] Figures 22A-22C Depicted is the immunopharmacodynamic profiling of spleens following treatment with P-0315 or P-0313 in the CT26 murine colorectal cancer tumor model. Following two Q5D treatments starting 11 days after tumor implantation, increases in the number of A) total leukocytes, B) NK cells, and C) CD8+ T cells in the spleen were determined by flow cytometry on day 19. ****P<0.0001, *P<0.05 compared to PBS.

[0055] Figures 23A-23B Depicted are comparisons of the activity of uncleavable Fc IL-15 VitoKine P-0351 and benchmarks by analyzing the induction of the proliferation marker Ki67 on A) CD56+ NK cells and B) CD8+ T cells.

[0056] Figures 24A-24B Depicted are the protein profile of an 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.

[0057] Figures 25A-25B Depicted is the analysis of A) CD4+Foxp3+ / CD25 in fresh human PBMCs. 高 Treg and B) CD4+Foxp3- / CD25 低pStat5 levels in CD4 conventional T cell subsets were assessed for the activity of two Fc IL-2 VitoKine proteins, P-0320 (IL-2 fused to the C-terminus of Fc) and P-0329 (IL-2 fused to the N-terminus of Fc). The highly active IL-2 Fc fusion protein P-0250 was included as a positive control.

[0058] Figure 26 Depicts the mechanism of VitoKine activation from the expected release of D3 from D2 after protease cleavage and diffusion away. Figure 2 Changes in the diagram.

[0059] Figures 27A-27B Depicted are A) SDS-PAGE analysis of IL-2 VitoKine P-0382 and its activation by MMP-2 digestion followed by Ni-Excel purification. B) Protein profile of MMP-2 activated P-0382 purified by Protein A in bind and elute mode.

[0060] Figures 28A-28B Depicted is the analysis of A) CD4+Foxp3+ / CD25 in fresh human PBMCs. 高 Treg and B) CD4+Foxp3- / CD25 低 pStat5 levels in CD4 conventional T (Tconv) cell subsets were assessed for the activity of protease-activated IL-2 VitoKine P-0382. Both activated samples were purified with Ni-Excel resin to remove proteases (active form 1) or with Protein A to remove proteases and the IL-2Rα Sushi domain produced by proteolysis (active form 2). The highly active IL-2Fc fusion protein P-0250 was included as a positive control.

[0061] Figures 29A-29B Depicted is the analysis of A) CD4+Foxp3+ / CD25 in fresh human PBMCs. 高 Treg and B) CD4+Foxp3- / CD25 低 pStat5 levels in CD4 Tconv cell subsets were assessed for the activity of FcIL-2 VitoKine P-0398 before and after MMP-2 proteolysis. P-0382 differs from P-0398 only in the length of the L2 linker, and the highly active IL-2 Fc fusion protein P-0250 was included for comparison.

[0062] Figures 30A-30BDepicted are the evaluation of the activity of Fc IL-15VitoKine P-0315 versus the antibody IL-15VitoKine P-0485 by determining the induction of the proliferation marker Ki67 on A) CD56+ NK cells and B) CD8+ T cells by flow cytometry.

[0063] Figure 31 Binding of blocking peptides (L01, L02, L03, L04 and L05) to IL-15 was profiled in an ELISA format.

[0064] Figure 32 Depicted are the binding of IL-15 fusion proteins containing IL-2Rβ-based blocking peptides (P-0153, P-0159, P-0160, and P-0161) to plate-coated IL-2Rβ.

[0065] Figures 33A-33E Depicted are size exclusion chromatograms of four IL-2 VitoKine (P-0320, P-0382, P-0362, and P-0379) (BE) and a P-0250 counterpart Fc fusion protein containing a single amino acid substitution S125I in IL-2, compared to the IL-2 Fc fusion protein P-0250 (A).

[0066] Figures 34A-34B Depicted is an SDS-PAGE gel of Fc IL-15 Vitokine P-0389 (A) compared to an SDS-PAGE gel of P-0315 (B).

[0067] Means for carrying out the present disclosure

[0068] The present disclosure provides novel "VitoKine" constructs as a platform technology to reduce systemic on-target toxicity and improve the therapeutic index of cytokines intended for the treatment of cancer, viral infections, autoimmune diseases, or inflammatory diseases. Figure 1 The VitoKine platform consists of Figure 1 The constructs depicted in Figure 2 The proposed activation method is defined as described in . Figure 1 The novel VitoKine constructs of the present invention comprise 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-function moiety domain, 2) a D2 domain ("D2"), which is the "active moiety domain," and 3) a D3 domain ("D3"), which is the "shielding moiety domain." Importantly, the D3 domain is capable of shielding or attenuating the functional activity of D2 until D2 is activated at the intended treatment site.

[0069] These three domains are connected using a linker of variable length and rigidity, coupled to a protease-cleavable sequence that is a peptide substrate for a specific protease subtype whose expression is elevated or dysregulated at the disease site, thereby allowing for the exposure or release of the functional D2 domain at the disease site. The linker length and composition are optimized to promote optimal shielding of the D2 domain from its receptor accessibility, reducing its systemic involvement, while maintaining VitoKine's stability in the bloodstream and allowing for efficient cleavage upon encounter with the specific protease at the intended disease site. The design of "VitoKine" is also based on the rational manipulation of knowledge of the molecular interactions of cytokines with their cognate receptors. Cytokine receptors typically function as oligomeric complexes composed of two to four receptor subunits. Different subunits perform specialized functions, such as ligand binding or signal transduction. The α subunit of the cytokine receptor is the binding receptor that confers ligand specificity, enhancing the interaction of the ligand with the signaling receptor and converting the signaling receptor from low affinity to high affinity. Therefore, the D3 domain of VitoKine is preferably a cognate binding receptor for the D2 domain. After cleavage, the D3 domain can dissociate or reassociate with the D2 domain and fully restore the binding and signaling activity of the D2 domain locally. Therefore, the D3 domain can have a dual role in regulating the function of the D2 domain. When VitoKine is inactive, the D3 domain renders the D2 domain inert, and when VitoKine is cleaved and activated, the D3 domain can participate in D2 function. However, the D3 domain can be any protein, peptide, antibody, antibody fragment, polymer, or nucleotide capable of masking D2 activity.

[0070] On the other hand, the addition of the D3 domain may also lead to a significantly improved developability profile of the VitoKine constructs, increased expression yields, and reduced aggregation propensity.

[0071] The D1 domain can be a half-life extension domain to extend the circulating half-life of VitoKine, and can also serve as an additional domain to mask the functional activity of the D2 domain. The D1 domain can also be a disease or tissue targeting motif, which specifically guides VitoKine to the site of interest and limits VitoKine activation locally to further improve its therapeutic index. Therefore, the "VitoKine" platform allows cytokine selective activation at the intended site, with the benefit of reducing systemic toxicity while increasing the therapeutic effect at the disease site, thereby improving its therapeutic index.

[0072] The D2 domain of the VitoKine construct is the active moiety but remains inert until locally activated by proteases upregulated in diseased tissues. This will limit binding of the active moiety to receptors in the periphery or on the cell surface of non-diseased cells or tissues, preventing overactivation of the pathway and reducing undesirable "off-tissue" "on-target" toxicity. Furthermore, the inertness of the VitoKine active moiety prior to protease activation will significantly reduce potential antigen sinks and thereby extend in vivo half-life, leading to improved biodistribution, bioavailability, and efficacy at the intended treatment site. Furthermore, based on the present invention, the VitoKine platform can enhance the developability profile of proteins, including, but not limited to, increased expression levels and reduced aggregation propensity, such as when using the cognate receptor α as the D3 domain.

[0073] Although cleavable linkers are preferred for most VitoKines to limit systemic activation and release the active domain at the intended site following administration, non-cleavable linkers may also be desirable to provide sustained systemic exposure of the pharmacologically active VitoKine and enhance therapeutic efficacy.

[0074] In an exemplary embodiment, the VitoKine construct comprises an active portion (D2) based on IL-15, based on an IL-15 variant, based on IL-2, or based on an IL-2 variant. For these IL-15 and / or IL-2 based VitoKine constructs, the unique and non-signaling α-subunit of each cytokine receptor is used as one of the shielding domains (D3) via a protease-cleavable linker to reversibly shield the cytokine activity. Depending on the different characteristics of each receptor complex and the different requirements of the different disease indications intended to be treated with the VitoKine molecule, after the linker is cleaved by the protease, the shielding α-subunit can preferably be complexed with the activated cytokine by non-covalent association (e.g., for IL-15) or preferably dissociated (e.g., for IL-2 in selectively expanding Treg cells). Therefore, it may be necessary to perform amino acid modifications on the α-receptor to adjust the binding affinity to its cognate cytokine.

[0075] This concept of coupling cognate receptors, proteins, antibodies, antibody fragments, binding peptides to cytokines via activatable linkers to mask their functional activity until activated at the intended treatment site can be tailored for various cytokines, including but not limited to IL-4, IL-7, IL-9, IL-10, IL-12, IL-22, IL-23 and TGFβ, chemokines such as CXCR3, or various growth factors such as the TNF family, TGFα and TGFβ, and hormones. The same concept can also be applied to other proteins to generate proproteins to achieve enhanced targeting to disease sites and expand therapeutic utility.

[0076] definition

[0077] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. In various embodiments, "peptide", "polypeptide" and "protein" are amino acid chains whose α carbons are connected by peptide bonds. Therefore, the terminal amino acid at one end (amino terminal) of the chain has a free amino group, and the terminal amino acid at the other end (carboxyl terminal) of the chain has a free carboxyl group. As used herein, the term "amino terminal" (abbreviated as N-terminal) refers to the free α-amino group on the amino acid at the amino terminal of a peptide, or refers to the α-amino group (amino group when participating in a peptide bond) of the amino acid at any other position in the peptide. Similarly, the term "carboxyl terminal" (abbreviated as C-terminal) refers to the free carboxyl group on the carboxyl terminal of a peptide, or the carboxyl group of the amino acid at any other position in the peptide. Peptide also includes substantially any polyamino acid, including but not limited to peptide mimetics (peptide mimetic), such as the amino acid connected by ether bonds rather than amide bonds.

[0078] The polypeptides of the present disclosure include polypeptides that have been modified in any manner and for any reason, for example, to: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or alter other physicochemical or functional properties.

[0079] As used herein, an amino acid "substitution" refers to the replacement of an amino acid at a specific position in a parent polypeptide sequence with a different amino acid. Amino acid substitutions can be produced using genetic or chemical methods well known in the art. For example, single amino acid substitutions or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in a naturally occurring sequence (e.g., in portions of a polypeptide outside of the domains forming intermolecular contacts). A "conservative amino acid substitution" refers to the replacement of an amino acid in a polypeptide with a functionally similar amino acid. The following six groups each contain amino acids that are conservative substitutions for each other:

[0080] 1) Alanine (A), serine (S), and threonine (T)

[0081] 2) Aspartic acid (D) and glutamic acid (E)

[0082] 3) Asparagine (N) and glutamine (Q)

[0083] 4) Arginine (R) and Lysine (K)

[0084] 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V)

[0085] 6) Phenylalanine (F), tyrosine (Y) and tryptophan (W)

[0086] A "non-conservative amino acid substitution" refers to a substitution of a member of one of these classes for a member from another class. When making such changes, according to various embodiments, the hydropathic index of the amino acid can be considered. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics. They are: 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); glutamate (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5).

[0087] The importance of the hydropathic amino acid index in conferring interactive biological function on proteins is understood in the art (see, for example, Kyte et al., 1982, J. Mol. Biol. 157: 105-131). It is known that certain amino acids can be substituted with other amino acids having similar hydropathic indices or scores and still retain similar biological activity. When making changes based on the hydropathic index, in various embodiments, substitutions of amino acids whose hydropathic index is within ± 2 are included. In various embodiments, those within ± 1 are included, and in various embodiments, those within ± 0.5 are included.

[0088] It is also understood in the art that substitution of similar amino acids can be effectively made based on hydrophilicity, particularly when the resulting biologically functional protein or peptide is intended for use in immunological embodiments, as disclosed herein. In various embodiments, the greatest local average hydrophilicity of a protein (as determined by the hydrophilicity of its adjacent amino acids) correlates with its immunogenicity and antigenicity, i.e., with the biological properties of the protein.

[0089] The following hydrophilicity 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 making changes based on similar hydrophilicity values, in various embodiments, substitution of amino acids whose hydrophilicity values are within ±2 is included, in various embodiments, those within ±1 are included, and in various embodiments, those within ±0.5 are included.

[0090] Exemplary amino acid substitutions are listed in Table 1.

[0091] Table 1

[0092]

[0093] The skilled artisan will be able to determine suitable polypeptide variants as listed herein using known techniques. In various embodiments, one skilled in the art can identify suitable regions of the molecule that can be altered without destroying activity by targeting regions that are not considered important for activity. In other embodiments, one skilled in the art can identify residues and portions of the molecule that are conserved between similar polypeptides. In additional embodiments, even regions that may be important for biological activity or structure can undergo conservative amino acid substitutions without destroying biological activity or adversely affecting polypeptide structure.

[0094] In addition, one skilled in the art can review structure-function studies that identify residues in similar polypeptides that are important for activity or structure. Based on such comparisons, one can predict the importance of amino acid residues in a polypeptide that correspond to amino acid residues in similar polypeptides that are important for activity or structure. One skilled in the art can choose to substitute chemically similar amino acids for the amino acid residues predicted to be important.

[0095] Those skilled in the art can also analyze the three-dimensional structure and the amino acid sequence relevant to this structure in similar polypeptides.In view of such information, those skilled in the art can predict the arrangement of the amino acid residues of polypeptide in terms of its three-dimensional structure.In various embodiments, those skilled in the art can choose not to carry out radical changes (radical changes) to the amino acid residues predicted on the polypeptide surface, because such residues may participate in important interactions with other molecules.In addition, those skilled in the art can produce test variants comprising single amino acid substitutions at each desired amino acid residue.Then activity assays known to those skilled in the art can be used to screen variants.These variants can be used to collect information about suitable variants.For example, if people find that the change of specific amino acid residues causes destruction, undesirably reduced or inappropriate activity, then variants with such changes can be avoided.In other words, based on the information collected from such routine experiments, those skilled in the art can easily determine to avoid further substituted amino acids therein separately or in combination with other mutations.

[0096] As used herein, the terms "polypeptide fragment" and "truncated polypeptide" refer to polypeptides having an amino-terminal deletion and / or a carboxyl-terminal deletion compared to the corresponding full-length protein. In various embodiments, the length of the fragment can be, for example, at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids. In various embodiments, the length of the fragment can also be, for example, at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 450, at most 400, at most 350, at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 25, at most 10, or at most 5 amino acids. A fragment can also comprise one or more additional amino acids at either or both ends, for example, an amino acid sequence from a different naturally occurring protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence).

[0097] As used herein, the terms "polypeptide variant," "hybrid polypeptide," and "polypeptide mutant" refer to a polypeptide comprising an amino acid sequence in which one or more amino acid residues are inserted into, deleted from, and / or substituted into an amino acid sequence relative to another polypeptide sequence. In various embodiments, the number of amino acid residues to be inserted, deleted, or substituted 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 acids in length. Hybrids of the present disclosure include fusion proteins.

[0098] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, for example, by conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (eg, human serum albumin), phosphorylation, and glycosylation.

[0099] The term "% sequence identity" is used interchangeably with the term "% identity" in this article and refers to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences when using a sequence alignment program. For example, as used herein, 80% identity determined by a defined algorithm means the same as 80% sequence identity, and means that a given sequence is at least 80% identical to another sequence of another 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 greater sequence identity to a given sequence. In various embodiments, % identity 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%.

[0100] The term "% sequence homology" is used interchangeably with the term "% homology" herein and refers to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences when compared using a sequence alignment program. For example, as used herein, 80% homology as determined by a defined algorithm means the same as 80% sequence homology, and thus homologs of a given sequence have a sequence homology greater than 80% relative to the 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 greater sequence homology to a given sequence. In various embodiments, % 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%.

[0101] Exemplary computer programs that can be used to determine the identity between two sequences include, but are not limited to, a set of BLAST programs publicly available on the Internet at the NCBI website, such as BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN. See also Altschul et al., J. Mol. Biol. 215: 403-10, 1990 (with particular reference to the published default settings, i.e., parameters w = 4, t = 17) and Altschul et al., Nucleic Acids Res., 25: 3389-3402, 1997. When evaluating a given amino acid sequence relative to amino acid sequences in GenBank protein sequences and other public databases, the BLASTP program is typically used for sequence searching. The BLASTX program is preferably used to search for nucleic acid sequences that have been translated in all reading frames for amino acid sequences in GenBank protein sequences and other public databases. Both BLASTP and BLASTX are run using the default parameters of an open gap penalty of 11.0 and an extended gap penalty of 1.0 and using the BLOSUM-62 matrix.

[0102] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, a nucleic acid is considered similar to a reference sequence if, in a comparison of a test nucleic acid to a reference nucleic acid, the minimum total probability is, e.g., less than about 0.1, less than about 0.01, or less than about 0.001.

[0103] As used herein, the term "modification" refers to any manipulation of the peptide backbone (eg, amino acid sequence) or post-translational modification of the polypeptide (eg, glycosylation).

[0104] As used herein, the term "knob-into-hole modification" refers to a modification within the interface between the CH3 domains of two immunoglobulin heavy chains. In one embodiment, the "knob-into-hole modification" comprises the amino acid substitution T366W and, optionally, the amino acid substitution S354C in one antibody heavy chain, and the amino acid substitutions T366S, L368A, Y407V, and, optionally, Y349C in the other antibody heavy chain. The knob-into-hole technique is described, for example, in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001).

[0105] As used herein, the term "bioactivatable drug" or "VitoKine" refers to a compound that is a prodrug that, after administration to a subject, undergoes some chemical or physiological processing in vivo to release the drug, converting the bioactivatable drug into a product that is active in the target tissue. A bioactivatable drug is any compound that undergoes bioactivation and then exhibits its pharmacological effect. Therefore, a bioactivatable drug can be considered a drug that contains a specialized non-toxic protective group that is used in a temporary manner to alter or eliminate undesirable properties of the parent molecule.

[0106] As used herein, the term "fusion protein" refers to a fusion polypeptide molecule comprising two or more genes that originally encoded different proteins, wherein the components of the fusion protein are directly linked to each other by peptide bonds or are linked to each other by peptide linkers. As used herein, the term "fusion" refers to components that are directly linked by peptide bonds or components that are linked via one or more peptide linkers.

[0107] "Joint" refers to a molecule that connects two other molecules covalently or by ionic bonds, van der Waals forces or hydrogen bonds, for example, a nucleic acid molecule that hybridizes at the 5' end with a complementary sequence and at the 3' end with another complementary sequence to connect two non-complementary sequences. "Cleaving joint" refers to a joint that can be degraded, digested or otherwise cut off to separate the two components connected by the cleavable joint. Cleaving joints are usually cracked by enzymes, typically peptidases, proteases, nucleases, lipases, etc. Cleaving joints can also be cracked by environmental factors, such as changes in temperature, pH, salt concentration, etc.

[0108] As used herein, the term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 1-30 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides include, for example, (G4S) n 、(SG4) n or G4 (SG4) n Peptide linker. "n" is typically a number between 1 and 10, usually between 2 and 4.

[0109] "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 an agent that effectively produces the expected pharmacological result. "Pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, vehicle, buffer and excipient, such as phosphate buffered saline solution, 5% dextrose aqueous solution, and emulsions, such as oil / water emulsions or water / oil emulsions, and various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21st edition 2005, Mack Publishing Co, Easton. "Pharmaceutically acceptable salts" are salts of compounds that can be formulated for pharmaceutical use, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or salts of organic amines.

[0110] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of the disease in the individual being treated, and the clinical intervention can be performed for prevention or during the course of clinical pathology. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of the symptoms of the disease, disorder, or condition. Additionally, references herein to "treatment" include references to curative, palliative, and prophylactic treatments.

[0111] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease, such as to improve, alleviate, mitigate, and / or delay one or more symptoms thereof. With respect to cancer or other unwanted cell proliferation, an effective amount includes an amount sufficient to: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow, and preferably stop to some extent, the infiltration of cancer cells into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) alleviate to some extent one or more symptoms associated with cancer. An effective amount can be administered in one or more administrations.

[0112] The phrases "administer" or "cause to be administered" refer to actions taken by a medical professional (e.g., a physician) or person controlling a patient's medical care to control and / or permit the administration of the agent / compound in question to a patient. Causing administration can include diagnosing and / or determining an appropriate treatment regimen, and / or prescribing a particular agent / compound to a patient. Such prescribing can include, for example, drafting a prescription form, annotating a medical record, etc. "Causing administration" is also contemplated when describing administration herein.

[0113] The terms "patient," "individual," and "subject" are used interchangeably and refer to mammals, preferably humans or non-human primates, but also domestic mammals (e.g., canines or felines), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., equines, bovines, swine, ovines). In various embodiments, a patient can be a human (e.g., an adult male, an adult female, an adolescent male, an adolescent female, a male child, a female child) under the care of a physician or other health worker in a hospital, psychiatric care facility, such as an outpatient clinic, or other clinical setting. In various embodiments, a patient can be an immunocompromised patient or a patient with a weakened immune system, including but not limited to patients with primary immunodeficiency, AIDS; cancer patients and transplant patients taking certain immunosuppressive drugs; and patients with genetic diseases that affect the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In various embodiments, the patient has an immunogenic cancer, including but not limited to bladder cancer, lung cancer, melanoma, and other cancers reported to have high mutation rates (Lawrence et al., Nature, 499(7457):214-218, 2013).

[0114] The term "immunotherapy" refers to cancer treatments including, but not limited to, treatment with depleting antibodies directed against specific tumor antigens; treatment with antibody-drug conjugates; treatment with agonistic, antagonistic, or blocking antibodies directed against co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, PDL-1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, SIRPa, CD47, GITR, ICOS, CD27, Siglec 7, Siglec 8, Siglec 9, Siglec 15, and VISTA, CD276, CD272, TIM-3, B7-H4; treatment with bispecific T cell-engaging antibodies treatment involving administration of biological response modifiers (such as 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 TGF-β traps); treatment with therapeutic vaccines such as sipuleucel-T; treatment with therapeutic viruses (including but not limited to oncolytic viruses such as T-vec); treatment with dendritic cell vaccines or tumor antigen peptide vaccines or neoantigen vaccines; treatment with NK cells; treatment with chimeric antigen receptors treatment with immunostimulants such as toll-like receptor (TLR)-T cells, CAR-NK cells, DCs or T cells, iPS-induced NK cells, iPS-induced T cells and vaccines such as bacille Calmette-Guérin (BCG), tumor-infiltrating lymphocytes (TILs), adoptively transferred anti-tumor T cells (ex vivo expanded T cells and / or TCR-T cells), TALL-104 cells, and immunostimulants such as toll-like receptor (TLR) agonists CpG, TLR7, TLR8, TLR9, and imiquimod.

[0115] "Resistant or refractory cancer" refers to tumor cells or cancers that do not respond to previous anticancer therapies, including, for example, chemotherapy, surgery, radiation therapy, stem cell transplantation, and immunotherapy. Tumor cells can be resistant or refractory at the start of treatment, or they can become resistant or refractory during treatment. Refractory tumor cells include tumors that do not respond at the start of treatment, or tumors that initially respond for a short period of time but ultimately fail to respond to treatment. Refractory tumor cells also include tumors that respond to treatment with anticancer therapy but fail to respond to subsequent rounds of therapy. For the purposes of the present invention, refractory tumor cells also include tumors that appear to be suppressed by treatment with anticancer therapy but recur up to 5 years, sometimes up to 10 years or more, after treatment has stopped. Anticancer therapy can use a single chemotherapeutic agent, radiation alone, targeted therapy alone, surgery alone, or a combination thereof. For ease of description and not limitation, it should be understood that refractory tumor cells are interchangeable with resistant tumors.

[0116] The term "tumor-associated antigen" (TAA) refers to, for example, a cell surface antigen that is selectively expressed by cancer cells or overexpressed in cancer cells relative to most normal cells. The terms "TAA variant" and "TAA mutant" as used herein refer to a TAA comprising an amino acid sequence in which one or more amino acid residues are inserted into, deleted from, and / or substituted into the amino acid sequence relative to another TAA sequence. In various embodiments, the number of amino acid residues to be inserted, deleted, or substituted 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 acids in length.

[0117] The term "neoantigen" refers to a cell surface antigen to which the immune system has not been previously exposed, for example, and in particular, a cell surface antigen generated by alteration of host antigens by radiation, chemotherapy, viral infection, neoplastic transformation / mutation, drug metabolism, etc., which is selectively expressed by or overexpressed in cancer cells relative to most normal cells.

[0118] As used herein, the term "antibody" is used in the broadest sense and encompasses various antibody structures (IgG1, 2, 3 or 4, IgM, IgA, IgE), including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies or bifunctional antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0119] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and 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.

[0120] The term "Fab fragment" as used herein refers to an immunoglobulin fragment comprising the VL domain and constant domain (CL) of the light chain and the VH domain and first constant domain (CH1) of the heavy chain.

[0121] As used herein, the term "variable region" or "variable domain" refers to the domain of an immunoglobulin or antibody heavy or light chain that is typically involved in binding 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, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs).

[0122] As used herein, a "human immunoglobulin" is an immunoglobulin having an amino acid sequence that corresponds to the amino acid sequence of an immunoglobulin produced by a human or human cell or derived from a non-human source using the human immunoglobulin repertoire or other human immunoglobulin encoding sequences. This definition of a human immunoglobulin specifically excludes humanized immunoglobulins that contain non-human antigen-binding residues.

[0123] As used herein, the term "Fc domain" or "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region comprises IgG CH2 and IgG CH3 domains. The CH3 region herein can be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "protuberance" ("knob") in one chain and a corresponding introduced "cavity" ("hole") in another chain; see U.S. Patent No. 5,821,333, which is expressly incorporated herein by reference). Such a variant CH3 domain can be used to promote the heterodimerization of two different immunoglobulin heavy chains as described herein. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system.

[0124] As used herein, the term "effector function" refers to those biological activities attributable to the Fc region of an immunoglobulin, which differs among immunoglobulin subtypes. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0125] As used herein, the term "regulatory T cells" or "Treg cells" refers to a specialized type of CD4+ T cell that can suppress 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 forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)), and play a key role in inducing and maintaining peripheral self-tolerance to antigens, including antigens expressed by tumors.

[0126] As used herein, the term "conventional CD4+ T cells" means CD4+ T cells other than regulatory T cells.

[0127] As used herein, the term "selective activation of Treg cells" means the activation of Treg cells that are substantially not accompanied by other T cell subsets (such as CD4+ helper T cells, CD8+ cytotoxic T cells, NK T cells) or natural killer (NK) cells. Methods for identifying and distinguishing these cell types are described in the embodiments. Activation can include inducing IL-2 receptor signaling (as measured by, for example, detecting phosphorylated STAT5a), inducing proliferation (as measured by, for example, detecting Ki-67) and / or raising the expression of activation markers (such as, for example, CD25).

[0128] As used herein, "specific binding" means that the binding to an antigen is selective and can be distinguished from unwanted or non-specific interactions. The ability of an immunoglobulin to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology.

[0129] As used herein, the term "affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y can generally be characterized by the dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (koff and kon, respectively). A specific method for measuring affinity is surface plasmon resonance (SPR).

[0130] As used herein, the term "reduced binding" refers to a decrease in the affinity of the respective interaction, as measured, for example, by SPR. In contrast, "increased binding" refers to an increase in the binding affinity of the respective interaction.

[0131] As used herein, the term "polymer" generally includes, but is not limited to, homopolymers; copolymers, such as, for example, block, graft, random, and alternating copolymers; and terpolymers; and mixtures and modifications thereof. Furthermore, unless expressly limited otherwise, the term "polymer" shall include all possible geometric configurations of the material. These configurations include, but are not limited to, isotactic, syndiotactic, and random symmetries.

[0132] "Polynucleotide" refers to a polymer comprising nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") and nucleic acid analogs. Nucleic acid analogs include those comprising the following: non-naturally occurring bases, nucleotides that engage with other nucleotides with connections other than naturally occurring phosphodiester bonds, or nucleotides that comprise bases attached by connections other than phosphodiester bonds. Therefore, nucleotide analogs include, for example and without limitation, phosphorothioate, phosphorodithioate, phosphotriester, phosphoramidate, boranophosphate, methylphosphonate, chiral methylphosphonate, 2-O-methyl ribonucleotide, peptide nucleic acid (PNA), etc. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" generally refers to large polynucleotides. The term "oligonucleotide" generally refers to short polynucleotides, generally not more than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (ie, A, T, G, C), this also includes RNA sequences (ie, A, U, G, C) in which "U" replaces "T."

[0133] Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction. The direction of nucleotide addition from 5' to 3' of a nascent RNA transcript is referred to as the direction of transcription. The DNA strand having the same sequence as the mRNA is referred to as the "coding strand"; the sequence on the DNA strand having the same sequence as the mRNA transcribed from that DNA and located 5' to the 5'-end of the RNA transcript is referred to as the "upstream sequence"; the sequence on the DNA strand having the same sequence as the RNA and located 3' to the 3'-end of the coding RNA transcript is referred to as the "downstream sequence."

[0134] "Complementary" refers to the topological compatibility or matching of the interacting surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, and further, the 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 can hybridize with the second polynucleotide under stringent hybridization conditions.

[0135] "Specifically hybridizes to" or "specifically hybridizes" or "selectively hybridizes to" refers to the preferential binding, duplexing or hybridization of a nucleic acid molecule to a particular nucleotide sequence when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA under stringent conditions. The term "stringent conditions" refers to conditions under which a probe will preferentially hybridize to its target subsequence and to other sequences to a lesser extent or not at all. In the context of nucleic acid hybridization experiments such as Southern hybridization and Northern hybridization, "stringent hybridization" and "stringent hybridization wash conditions" are sequence-dependent and are different under different environmental parameters. An extensive guide to 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, NY; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3rd Suppl. ed., NY; and Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

[0136] Typically, highly stringent hybridization and washing conditions are selected to be about 5°C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH conditions) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm of the specific probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids having more than about 100 complementary residues on filters in Southern or Northern blotting is hybridization at 42°C in 50% formalin with 1 mg of heparin overnight. An example of highly stringent washing conditions is 0.15 M NaCl at 72°C for about 15 minutes. An example of stringent washing conditions is 0.2x SSC washing at 65°C for 15 minutes. See Sambrook et al. for a description of SSC buffer. A low stringency wash can be performed to remove background probe signal, followed by a high stringency wash. An exemplary moderate stringency wash for, e.g., a duplex of more than about 100 nucleotides is 1 x SSC at 45° C. for 15 minutes. An exemplary low stringency wash for, e.g., a duplex of more than about 100 nucleotides is 4-6 x SSC at 40° C. for 15 minutes. Typically, a signal-to-noise ratio of 2x (or higher) compared to the signal-to-noise ratio observed for an unrelated probe in a particular hybridization assay indicates that specific hybridization has been detected.

[0137] " Primer " refers to the polynucleotide that can hybridize with the polynucleotide template specificity of appointment and provide the starting point for the synthesis of complementary polynucleotide. When polynucleotide primer is placed under the conditions of induction synthesis (that is, in the presence of nucleotide, complementary polynucleotide template and the agent such as DNA polymerase for polymerization), such synthesis occurs. Primer is generally single-stranded, but can be double-stranded. Primer is generally deoxyribonucleic acid, but many synthetic and naturally occurring primers can be used for many applications. Primer is complementary to template, and primer is designed to hybridize with template as the site of synthesis initiation, but does not need to reflect the accurate sequence of template. In such case, the specific hybridization of primer and template depends on the stringency of hybridization conditions. Primer can be used as detectable part with the partial labeling of for example chromogenicity, radioactivity or fluorescence.

[0138] When used in relation to polynucleotides, "probe" refers to a polynucleotide that can specifically hybridize to a given sequence of another polynucleotide. The probe specifically hybridizes to a target complementary polynucleotide, but does not need to reflect the exact 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. The probe can be labeled with, for example, a colorimetric, radioactive or fluorescent moiety and used as a detectable moiety. In the case where the probe provides a starting point for synthesizing a complementary polynucleotide, the probe can also be a primer.

[0139] " vector " is the polynucleotide that can be used to another nucleic acid being connected thereto and is introduced into the cell.A type of vector is " plasmid ", and it refers to that other nucleic acid segment can be connected to linear or circular double-stranded DNA molecule wherein.Another type of vector is viral vector (for example, replication defective retrovirus, adenovirus and adeno-associated virus), and wherein other DNA segment can be introduced in the viral genome.Some vector can be autonomously replicated (for example, bacterial vector and additional mammalian vector (episomal mammalian vector) comprising bacterial replication origin) in the host cell having introduced them.Other vectors (for example, non-additional mammalian vector) are integrated into the genome of the host cell after being introduced into the host cell, and thereby replicate together with the host genome." expression vector " is a type of vector that can guide the expression of the polynucleotide of selection.

[0140] A "regulatory sequence" is a nucleic acid that affects the expression (e.g., the level, timing, or position of expression) of a nucleic acid to which it is operably linked. A regulatory sequence can, for example, exert its effect directly on the regulated nucleic acid, or exert its effect through the action of one or more other molecules (e.g., a polypeptide bound to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Additional examples of regulatory sequences are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego; Calif. and Baron et al., 1995, Nucleic Acids Res. 23: 3605-06. A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., the level, timing, or position of expression) of the nucleotide sequence.

[0141] "Host cell" is a cell that can be used to express the polynucleotides of the present disclosure. The host cell can be a prokaryotic organism, such as Escherichia coli (E. coli), or the host cell can be a eukaryotic organism, such as a unicellular eukaryotic organism (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., a human cell, monkey cell, hamster cell, rat cell, mouse cell or insect cell) or a hybridoma. Generally, a host cell is a cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, and the nucleic acid can then be expressed in the host cell. The term "recombinant host cell" can be used to represent a host cell that has been transformed or transfected with a nucleic acid to be expressed. The host cell can also be a cell that comprises nucleic acid but does not express the nucleic acid at a desired level, unless a regulatory sequence is introduced into the host cell so that the regulatory sequence becomes operably connected with the nucleic acid. It should be understood that the term host cell refers not only to specific subject's cells, but also to the offspring or potential offspring of such a cell. Because certain modifications may occur in succeeding generations due to, for example, mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0142] The term "isolated molecule" (wherein the molecule is, for example, a polypeptide or polynucleotide) is a molecule that, by virtue of its origin or source of derivation, is (1) not associated with naturally associated components with which it accompanies it in its natural state, (2) is substantially free of other molecules from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cell from which it naturally originates would be "isolated" from its naturally associated components. A molecule may also be rendered substantially free of naturally associated components by separation using purification techniques well known in the art. Molecular purity or homogeneity can be determined by a number of means well known in the art. For example, the purity of a polypeptide sample can be determined using polyacrylamide gel electrophoresis and staining the gel to visualize the polypeptide using techniques well known in the art. For some purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

[0143] A protein or polypeptide is "substantially pure," "substantially homogeneous," or "substantially purified" when at least about 60% to 75% of the sample represents a single species of polypeptide. The polypeptide or protein can be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80%, or 90% w / w of the protein sample, more typically about 95% and preferably will be greater than 99% pure. Protein purity or homogeneity can be indicated by many means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization of individual polypeptide bands by staining the gel with staining agents well known in the art. For certain purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

[0144] As used herein, the term "label" or "labeled" refers to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable marker, such as a polypeptide incorporating a radiolabeled amino acid or attached to a biotinyl moiety that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or an enzyme activity that can be detected by optical methods or calorimetry). In another embodiment, the label or marker can be therapeutic, such as a drug conjugate or a toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: 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, rhodamine, lanthanide fluorophores); enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; a predetermined polypeptide epitope recognized by a secondary reporter (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag); magnetic agents, such as gadolinium chelates; toxins, such as pertussis toxin, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione), mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and analogs or homologs thereof. In various embodiments, the label is attached by spacer arms of various lengths to reduce potential steric hindrance.

[0145] As used herein, the term "heterologous" refers to a non-native or non-natural combination or state, which can be achieved, for example, by replacing existing native components or states with components or states derived from another source. Similarly, expression of a protein in an organism other than the organism in which the protein is naturally expressed constitutes a heterologous expression system and a heterologous protein.

[0146] It should be understood that aspects and embodiments of the present disclosure described herein include "consisting of" and / or "consisting essentially of" these aspects and embodiments.

[0147] Reference herein to "about" a value or parameter includes (and describes) variations with respect to that value or parameter itself. For example, reference to "about X" includes a description of "X."

[0148] As used herein and in the appended claims, the singular forms "a," "an," "or," and "the" include plural referents unless the context clearly dictates otherwise. It should be understood that aspects and variations of the present disclosure described herein include "consisting of" and / or "consisting essentially of" these aspects and variations.

[0149] Description of the VitoKine platform

[0150] The present invention provides a cytokine-based bioactivatable drug ("VitoKine") platform designed to reduce toxicity based on systemic mechanisms and lead to broader therapeutic utility of proteins such as cytokines. Figure 1The novel VitoKine construct of the present invention comprises a D1 domain, an "active part domain" (D2), and a "shielding part domain" (D3), wherein the D1 domain is a targeting domain, a half-life extension domain, or a dual or multifunctional part domain. The proposed method for activating the VitoKine D2 domain is Figure 2 Importantly, because D2 of the VitoKine construct will remain inert or attenuated until locally activated by proteases upregulated in diseased tissue, this will limit binding of the active moiety to receptors in the periphery or on the cell surface of non-diseased cells, preventing overactivation of the pathway and reducing undesirable "out-of-tissue" "on-target" toxicity. Furthermore, the inertness of the VitoKine active moiety prior to protease activation will significantly reduce potential antigen or target sinks, thereby extending in vivo half-life and leading to improved biodistribution and bioavailability at the intended treatment site.

[0151] D1 domain ("targeting domain, half-life extension domain or dual or multifunctional moiety domain")

[0152] In various embodiments, the VitoKine construct of the present invention comprises a D1 domain, which is a targeting portion in the form of an antibody or antibody fragment or protein or peptide directed against a tumor-associated antigen. In various embodiments, the VitoKine construct of the present invention comprises a D1 domain, which is an antibody, antibody fragment, protein or peptide directed against an immune checkpoint regulator. In various embodiments, the VitoKine construct of the present invention comprises a D1 domain, which is an antibody or antibody fragment or protein or peptide as an autoimmune regulator. In various embodiments, the VitoKine construct of the present invention comprises D1, which acts to maintain the D2 domain at a tissue site, such as a tumor microenvironment (TME) or an inflammatory tissue site. In various embodiments, the VitoKine construct of the present invention comprises D1, which is dual-functional, such as tissue targeting and retention. In various embodiments, the VitoKine construct of the present invention comprises a D1 domain, which is a polymer. In various embodiments, the VitoKine construct of the present invention comprises a D1 domain, which is a half-life extending portion. In various embodiments, the VitoKine constructs of the present invention comprise a D1 domain that is an Fc domain.

[0153] Fc domain

[0154] The immunoglobulin of the IgG class is one of the most abundant proteins in human blood. Their circulation half-life can be as long as 21 days. It has been reported that the fusion protein of the Fc region of IgG is combined with the domain of another protein (such as various cytokines and receptors) (see, for example, Capon et al., Nature, 337:525-531, 1989; Chamow et al., Trends Biotechnol., 14:52-60, 1996); U.S. Patent Nos. 5,116,964 and 5,541,087). The prototype fusion protein is a homodimeric protein connected by the cysteine residues in the hinge region of IgG Fc, producing a molecule similar to an IgG molecule, without a heavy chain variable region and CH1 domain and a light chain. The dimer nature of the fusion protein comprising the Fc domain may be advantageous in providing a higher order interaction (i.e., bivalent or bispecific binding) with other molecules. Due to structural homology, Fc fusion proteins exhibit in vivo pharmacokinetic profiles comparable to human IgG of similar isotype.

[0155] The term "Fc" refers to a molecule or sequence comprising the sequence of the non-antigen binding fragment of a complete antibody, whether in monomeric or multimeric form. The original immunoglobulin source of native Fc is preferably human origin, and can be any immunoglobulin, although IgG1 and IgG2 are preferred. Native Fc consists of monomeric polypeptides, which can be linked to dimers or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between the monomer subunits of native Fc molecules varies from 1 to 4 depending on class (e.g., IgG, IgM, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgGA2). An example of native Fc is a disulfide-bonded dimer produced by papain digestion of IgG (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). As used herein, the term "native Fc" is a general term for monomers, dimers, and multimeric forms. The Fc domain contains binding sites for protein A, protein G, various Fc receptors, and complement proteins.

[0156] In various embodiments, the term "Fc variant" refers to a molecule or sequence that is modified from a native Fc but still contains a binding site for the salvage receptor FcRn. International applications WO 97 / 34631 (published September 25, 1997) and WO 96 / 32478 describe exemplary Fc variants and interactions with salvage receptors and are hereby incorporated by reference. In addition, native Fc contains sites that can be removed because they provide structural features or biological activities that are not required for the fusion molecules of the present invention. Therefore, in various embodiments, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues that affect or participate in (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity when expressed in a selected host cell, (4) glycosylation, (5) interaction with complement, such as CDC, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0157] The term "Fc domain" includes natural Fc and Fc variant molecules and sequences as defined above. Like Fc variants and natural Fc, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from a complete antibody or expressed by a recombinant gene or produced by other means. In various embodiments, an "Fc domain" refers to a dimer of two Fc domain monomers (SEQ ID NO: 13), which typically include all or part of the hinge region. In various embodiments, the Fc domain can be mutated to lack effector functions. In various embodiments, each Fc domain monomer in the Fc domain comprises an amino acid substitution in the CH2 antibody constant domain to reduce the interaction or binding between the Fc domain and the Fcγ receptor. In various embodiments, each subunit of the Fc domain comprises two amino acid substitutions that reduce binding and / or effector functions to activating Fc receptors, wherein the amino acid substitutions are L234A and L235A. In various embodiments, each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to an activating Fc receptor and / or effector function, wherein the amino acid substitutions are L234A, L235A, and G237A (SEQ ID NO: 14).

[0158] In various embodiments, the Fc domain can be mutated to further extend the in vivo half-life. In various embodiments, each subunit of the Fc domain comprises three amino acid substitutions that enhance binding to human FcRn, wherein the amino acid substitutions are M252Y, S254T, and T256E (SEQ ID NO: 156), disclosed in U.S. Patent Publication No. 7,658,921. In various embodiments, each subunit of the Fc domain comprises an amino acid substitution that enhances binding to human FcRn, wherein the amino acid substitution is N434A (SEQ ID NO: 166), disclosed in U.S. Patent Publication No. 7,371,826. In various embodiments, each subunit of the Fc domain comprises an amino acid substitution that enhances binding to human FcRn, wherein the amino acid substitutions are M428L and N434S, disclosed in U.S. Patent Publication No. 8,546,543. In various embodiments, half-life extension mutations can be combined with amino acid substitutions that reduce binding to activating Fc receptors and / or effector function.

[0159] In various embodiments, each of the two Fc domain monomers in the Fc domain comprises an amino acid substitution that promotes the heterodimerization of the two monomers. In various other embodiments, the heterodimerization of the Fc domain monomers can be promoted by introducing different but compatible substitutions such as "knob-into-hole" residue pairs in the two Fc domain monomers. "Knob-into-hole" technology is also disclosed in U.S. Patent Publication No. 8,216,805. In another embodiment, an Fc domain monomer comprises a knob mutation T366W, and another Fc domain monomer comprises a hole mutation T366S, L358A, and Y407V. In various embodiments, two Cys residues (S354C on the "knob" side and Y349C on the "hole" side) that form a stabilized disulfide bridge are introduced (SEQ ID NO: 15 and 16). The use of heterodimer Fc can produce a monovalent VitoKine construct.

[0160] In various embodiments, the Fc domain sequence used to prepare the VitoKine construct is the human IgG1-Fc domain sequence set forth in SEQ ID NO: 14:

[0161]

[0162] Wherein SEQ ID NO: 14 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding.

[0163] In various embodiments, the heterodimeric Fc domain sequence used to prepare the VitoKine construct is the Knob-Fc domain sequence set forth in SEQ ID NO: 15:

[0164]

[0165] Wherein SEQ ID NO: 15 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding.

[0166] In various embodiments, the heterodimeric Fc domain sequence used to prepare the VitoKine construct is the Hole-Fc domain sequence set forth in SEQ ID NO: 16:

[0167]

[0168] Wherein SEQ ID NO: 16 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding.

[0169] In various embodiments, the Fc domain sequence used to prepare the VitoKine construct is an IgG1-Fc domain with reduced / eliminated effector function and extended half-life and having the amino acid sequence set forth in SEQ ID NO: 156

[0170]

[0171] SEQ ID NO: 156 includes amino acid substitutions that abolish FcγR and C1q binding (underlined) and amino acid substitutions that extend half-life (bold).

[0172] In various embodiments, the Fc domain sequence used to prepare the VitoKine construct is the human IgG1-Fc domain sequence set forth in SEQ ID NO: 166:

[0173]

[0174] SEQ ID NO: 166 includes amino acid substitutions that abolish FcγR and C1q binding (underlined) and amino acid substitutions that extend half-life (bold).

[0175] In various embodiments, the heterodimeric Fc domain sequence used to prepare the VitoKine construct is the Knob-Fc domain sequence with extended in vivo half-life set forth in SEQ ID NO: 167:

[0176]

[0177] SEQ ID NO: 167 includes amino acid substitutions that abolish FcγR and C1q binding (underlined) and amino acid substitutions that extend half-life (bold).

[0178] In various embodiments, the heterodimeric Fc domain sequence used to prepare the VitoKine construct is the Hole-Fc domain sequence with extended in vivo half-life set forth in SEQ ID NO: 168:

[0179]

[0180] SEQ ID NO: 168 includes amino acid substitutions that abolish FcγR and C1q binding (underlined) and amino acid substitutions that extend half-life (bold).

[0181] Antibodies and protein / peptide conjugates for disease-related targets or tumor-associated antigens

[0182] In various embodiments, D1 can be a targeting moiety in the form of an antibody to a tumor-associated antigen (TAA) or another protein or peptide that exhibits binding affinity to diseased cells or diseased tissue. TAA can be any molecule, macromolecule, combination of molecules, etc. for which an immune response is desired. TAA can be a protein comprising more than one polypeptide subunit. For example, a protein can be a dimer, trimer, or higher polymer. In various embodiments, two or more subunits of a protein can be connected by covalent bonds such as, for example, disulfide bonds. In various embodiments, the subunits of a protein can be held together by non-covalent interactions. Therefore, TAA can be any peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate, or small organic molecule, or any combination thereof, for which a technician wishes to induce an immune response. In various embodiments, a TAA is a peptide comprising 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 subject by injection. In various embodiments, upon administration, a tumor-specific antibody or binding protein is used as a targeting moiety to direct VitoKine to the site of disease, such as a cancer site, where the active domain can be released and interact with its cognate receptor on the diseased cell or tissue.

[0183] Any of the aforementioned markers can be used as disease-associated targets or TAA targets for the VitoKine constructs of the present invention. In various embodiments, one or more disease-associated targets or variants thereof or TAAs, TAA variants, or TAA mutants contemplated for use in the VitoKine constructs and methods of the present disclosure are selected from or derived from the list provided in Table 2.

[0184] Table 2

[0185]

[0186]

[0187]

[0188]

[0189] Additional 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, CA 19-9, and CA 23H1. 72-4, CAM17.1, Numa, K-ras, β-catenin, CDK4, Muni-1, p16, TAGE, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, β-HCG, BCA225, BTAA, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, 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\cyclophilin C-related protein), TAAL6, TAG72, TLP and TPS.

[0190] Immune checkpoint modulators

[0191] Many immune checkpoint protein antigens expressed on various immune cells have been reported, including, for example, CD152 (expressed by activated CD8+T cells, CD4+T cells and regulatory T cells), CD279 (expressed on tumor infiltrating lymphocytes, by activated T cells (both CD4 and CD8), regulatory T cells, activated B cells, activated NK cells, anergic T cells, monocytes, dendritic cells), CD274 (expressed on T cells, B cells, dendritic cells, macrophages, vascular endothelial cells, islet cells) and CD223 (expressed by activated T cells, regulatory T cells, anergic T cells, NK cells, NKT cells and plasmacytoid dendritic cells) (see, e.g., Pardoll, D., Nature Reviews Cancer, 12: 252-264, 2012). Antibodies that bind to the antigens identified as immune checkpoint proteins are known to those skilled in the art. For example, various anti-CD276 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20120294796 (Johnson et al.) and references cited therein); various anti-CD272 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20140017255 (Mataraza et al.) and references cited therein); various anti-CD152 / CTLA-4 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20130136749 (Korman et al.) and references cited therein); various anti-LAG-3 / CD223 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20110150892 (Thudium et al.) and references cited therein); Various anti-CD279 / PD-1 antibodies have been described in the art (see, e.g., U.S. Patent No. 7,488,802 (Collins et al.) and references cited therein); various anti-PD-L1 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20130122014 (Korman et al.) and references cited therein); various anti-TIM-3 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20140044728 (Takayanagi et al.) and references cited therein); and various anti-B7-H4 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20110085970 (Terrett et al.) and references cited therein). Each of these references is hereby incorporated by reference in its entirety with respect to the specific antibodies and sequences taught therein.

[0192] In various embodiments, D1 may comprise an antibody, antibody fragment, protein, or peptide that exhibits binding to an immune checkpoint protein antigen present on the surface of an immune cell. In various embodiments, the immune checkpoint protein antigen is selected from the group consisting of, but not limited to, CD276, CD272, CD152, CD223, CD279, CD274, CD40, SIRPa, CD47, OX-40, GITR, ICOS, CD27, 4-1BB, TIM-3, B7-H4, Siglec 7, Siglec 8, Siglec 9, Siglec15, and VISTA.

[0193] In various embodiments, D1 may comprise antibodies against immune checkpoint protein antigens present on the surface of tumor cells, selected from the group consisting of, but not limited to, PD-L1, B7-H3, and B7-H4.

[0194] Regulators of autoimmune and inflammatory disorders

[0195] Any of the aforementioned proteins that are highly expressed on various inflammatory tissues or immune cells can be used as autoimmune / inflammatory disease targets for the VitoKine constructs of the present invention. In various embodiments, one or more autoimmune / inflammatory disease targets, variants thereof, or mutants / isoforms thereof contemplated for use in the VitoKine constructs and methods of the present disclosure are selected from or derived from the list provided in Table 3. These targets may also be used as cancer targeting targets.

[0196] Table 3 Targets of autoimmune and inflammatory disorders or cancer

[0197]

[0198]

[0199] In various embodiments, the D1 targeting moiety can be an inflammatory tissue specific antibody, antibody fragment, another protein or peptide that exhibits binding to diseased cells or the disease microenvironment, such as TNF, TNFR, integrin A4β7, IL-6Rα, BLYS, TSLP.

[0200] polymer

[0201] In various embodiments, D1 can be a polymer, such as polyethylene glycol (PEG). In various embodiments, a polymer, such as PEG, can be covalently attached to the N-terminus or C-terminus or an internal position using conventional chemical methods, such as chemical conjugation. In various embodiments, a polymer, such as PEG, can be covalently attached to the N-terminus of the D2 domain via site-specific conjugation or substitution of other amino acids or engineered specific amino acids of a cytokine.

[0202] Half-life extension

[0203] In various embodiments, other half-life extending moieties can be used as the D1 domain in the present invention to increase the serum half-life of VitoKine. Half-life extending moieties include, but are not limited to, Fc domains, Fc variants, antibodies, antibody fragments (Fab, ScFv), and EXTEN (Schellenberger et al., Nat. Biotechnol. 27: 1 186-1 192, 2009) and human serum albumin protein.

[0204] D2 domain ("active portion domain")

[0205] D2 is the active portion of the VitoKine construct, whose activity is reversibly shielded within the construct and restored upon protease cleavage at the disease site. The active portion can be any protein, including but not limited to any native or variant interleukin or cytokine polypeptide. Importantly, because the "active portion" of the VitoKine construct will remain inert or attenuated until locally activated by proteases upregulated in diseased tissue, this will limit binding of the active portion to receptors in the periphery or on the cell surface of non-diseased cells, preventing overactivation of the pathway and reducing undesirable "off-tissue" or "on-target" toxicity. Furthermore, the inertness of the VitoKine active portion prior to protease activation will significantly reduce potential antigenic or target silencing, thereby extending the in vivo half-life and resulting in improved biodistribution and exposure at the intended treatment site.

[0206] IL-15

[0207] Interleukin-15 (IL-15) is a cytokine that was identified by two independent groups based on its ability to stimulate the proliferation of IL-2-dependent CTLL-2 T cell lines in the presence of neutralizing anti-IL-2 antibodies (Steel et al., Trends in Pharmacological Sciences, 33(1):35-41, 2012). IL-15 and interleukin-2 (IL-2) have similar biological properties in vitro and share a common receptor (R) signaling component (IL-2 / 15Rβγc ) are consistent. However, the specificity of IL-15 and IL-2 is provided by unique private α chain receptors, which complete the IL-15Rαβγ and IL-2Rαβγ heterotrimeric high-affinity receptor complex and thereby allow different responsiveness depending on the expressed ligand and high-affinity receptor. Interestingly, both IL-15 transcripts and IL-15Rα transcripts have a much wider tissue distribution than IL-2 / IL-2Rα. In addition, various complex post-transcriptional regulatory mechanisms strictly control IL-15 expression. Therefore, based on complex regulation and the different modes of expression of IL-15 and IL-15Rα, the key in vivo function of this receptor / ligand pair may be different from the in vivo function of IL-2 and IL-2Rα. To date, studies examining the biology of IL-15 have identified several key non-redundant roles, such as the importance and function of IL-15 in the development of natural killer (NK) cells, NK-T cells and intestinal intraepithelial lymphocytes. The role of IL-15 during autoimmune processes, such as rheumatoid arthritis, and malignancies, such as adult T-cell leukemia, suggests that dysregulation of IL-15 may lead to deleterious effects on the host (Fehniger et al., Blood, 97:14-32, 2001).

[0208] As used herein, the terms "native IL-15" and "native interleukin-15" in the context of a protein or polypeptide refer to any naturally occurring mammalian interleukin-15 amino acid sequence, including immature or precursor forms and mature forms. Non-limiting examples of GenBank accession numbers for amino acid sequences of native mammalian interleukin-15 from various species include NP_032383 (Mus musculus, immature form), AAB60398 (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 naturally occurring mammalian IL-15. In other embodiments, native IL-15 is a mature form of naturally occurring mammalian IL-15. In various embodiments, native IL-15 is a precursor form of naturally occurring human IL-15. In various embodiments, native IL-15 is a mature form of naturally occurring 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 set forth in SEQ ID NO: 1:

[0209]

[0210] In various embodiments, the IL-15-based domain D2 comprises the amino acid sequence of the mature form of human IL-15 set forth in SEQ ID NO: 2:

[0211]

[0212] In various embodiments, the IL-15-based domain D2 is an IL-15 variant (or mutant) comprising a sequence derived from the mature human IL-15 polypeptide sequence as set forth in SEQ ID NO: 2. Natural amino acids, the positions of natural amino acids in the mature sequence, and variant amino acids are used herein to refer to variants (or mutants) of IL-15. For example, "huIL-15S58D" refers to a human IL-15 comprising an S to D substitution at position 58 of SEQ ID NO: 2. In various embodiments, the D2 domain of the present invention comprises an IL-15 domain that is an IL-15 variant (also referred to herein as an IL-15 mutant domain). In various embodiments, the IL-15 variant comprises an amino acid sequence that differs from the native (or wild-type) IL-15 protein. In various embodiments, the IL-15 variant binds to the IL-15Rα polypeptide and acts as an IL-15 agonist or antagonist. In various embodiments, the IL-15 variant having agonist activity has superagonist activity. In various embodiments, IL-15 variants can act as IL-15 agonists or antagonists, independent of their association with IL-15Rα. IL-15 agonists are exemplified by comparable or increased biological activity compared to wild-type IL-15. IL-15 antagonists are exemplified by reduced biological activity compared to wild-type IL-15 or by the 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, such as a substitution or deletion, compared to the native IL-15 sequence, such a change resulting in IL-15 agonist or antagonist activity. In various embodiments, the amino acid substitution / deletion is located at a position adjacent to the IL-15Rβ and / or γ CIn various embodiments, the amino acid substitution / deletion does not affect the binding to the IL-15Rα polypeptide or the ability to produce IL-15 variants. Based on the known IL-15 structure, IL-15 and homologous molecules such as IL-2 with a known structure, suitable amino acid substitutions / deletions for producing IL-15 variants can be identified by rational or random mutagenesis and functional assays or other empirical methods as provided herein. In addition, suitable amino acid substitutions can be conservative or non-conservative changes and insertions of additional amino acids. In various embodiments, the IL-15 variants of the present invention comprise one or more amino acid substitutions or deletions at positions 30, 31, 32, 58, 62, 63, 67, 68 or 108 of the mature human IL-15 sequence listed in SEQ ID NO: 2. In various embodiments, a D30T ("D30" refers to the amino acid and residue position in the native mature human IL-15 sequence, and "T" refers to the amino acid residue substituted at that position in the IL-15 variant), V31Y, H32E, T62D, I68F, or Q108M substitution results in an IL-15 variant with antagonist activity, and an S58D substitution results in an IL-15 variant with agonist activity. In various embodiments, the IL-15 variant comprises the amino acid sequence set forth in SEQ ID NO: 3:

[0213]

[0214] Exemplary Fc IL-15 VitoKine constructs are provided in Table 4:

[0215] Table 4

[0216]

[0217]

[0218] In various embodiments, the antibody IL-15 VitoKine or IL-15 Fc fusion molecule comprises two or more heterodimeric chains, as listed in Table 5:

[0219] Table 5

[0220]

[0221] In various embodiments, the IL-15 based D2 domain comprises an IL-15 construct comprising an IL-2Rβ based blocking peptide selected from the constructs having the amino acid sequence set forth in SEQ ID NOs: 66-70.

[0222] In various embodiments, the IL-15 based D2 domain comprises an IL-15 construct comprising an IL-2Rβ based blocking peptide and having two or more heterodimeric chains as listed in Table 6:

[0223] Table 6

[0224]

[0225] IL-2

[0226] Interleukin-2 (IL-2) is a classic Th1 cytokine produced by T cells following activation via the T cell antigen receptor and the co-stimulatory molecule CD28. Regulation of IL-2 occurs through the activation of signaling pathways and transcription factors that act on the IL-2 promoter to produce new gene transcription, but also involves regulation of the stability of IL-2 mRNA. IL-2 binds to multi-chain receptors, including a highly regulated α chain and β and γ chains that mediate signaling via the Jak-STAT pathway. IL-2 delivers activation, growth, and differentiation signals to T cells, B cells, and NK cells. IL-2 is also important in mediating activation-induced cell death of T cells, a function that provides a key mechanism for terminating the immune response. A commercially available unglycosylated human recombinant IL-2 product, aldesleukin (available from Prometheus Laboratories Inc., San Diego Calif. under the trademark IL-2 is a cytokine derived from human interleukin-2 (derived from des-alanyl-1, serine-125) and has been approved for administration to patients suffering from metastatic renal cell carcinoma and metastatic melanoma. IL-2 has also been suggested for administration to patients suffering from or infected with hepatitis C virus (HCV), human immunodeficiency virus (HIV), acute myeloid leukemia, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, juvenile rheumatoid arthritis, atopic dermatitis, breast cancer, and bladder cancer. Unfortunately, the short half-life and severe toxicity limit the optimal administration of IL-2.

[0227] As used herein, the terms "natural IL-2" and "natural interleukin-2" in the context of a protein or polypeptide refer to any naturally occurring mammalian interleukin-2 amino acid sequence, including immature or precursor forms and mature forms. Non-limiting examples of GenBank accession numbers for amino acid sequences of natural mammalian interleukin-2 of various species include NP_032392.1 (Mus musculus, immature form), NP_001040595.1 (Macaca mulatta, immature form), NP_000577.2 (Human, precursor form), CAA01199,1 (Human, immature form), AAD48509.1 (Human, immature form) and AAB20900.1 (Human). In various embodiments of the present invention, natural IL-2 is an immature or precursor form of naturally occurring mammalian IL-2. In other embodiments, natural IL-2 is a mature form of naturally occurring mammalian IL-2. In various embodiments, natural IL-2 is a precursor form of naturally occurring human IL-2. In various embodiments, the native IL-2 is a mature form of naturally occurring 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 set forth in SEQ ID NO: 6:

[0228]

[0229] In various embodiments, the IL-2-based domain D2 comprises the amino acid sequence of the wild-type sequence of the mature form of human IL-2 set forth in SEQ ID NO:8 comprising a cysteine to serine substitution at position 125, but does not alter IL-2 receptor binding compared to naturally occurring IL-2:

[0230]

[0231] In various embodiments, the IL-2-based domain D2 is an IL-2 variant (or mutant) comprising a sequence derived from a mature human IL-2 polypeptide sequence as set forth in SEQ ID NO: 8. In various embodiments, the IL-2 variant comprises an amino acid sequence different from that of the natural (or wild-type) IL-2 protein. In various embodiments, the IL-2 variant binds to the IL-2Rα polypeptide and acts as an IL-2 agonist or antagonist. In various embodiments, the IL-2 variant having agonist activity has superagonist activity. In various embodiments, the IL-2 variant can act as an IL-2 agonist or antagonist, regardless of its association with IL-2Rα. IL-2 agonists are exemplified by having comparable or increased biological activity compared to wild-type IL-2. IL-2 antagonists are exemplified by having reduced biological activity compared to wild-type IL-2 or by the ability to inhibit IL-2-mediated responses. In various embodiments, the sequence of the IL-2 variant has at least one amino acid change, such as a substitution or deletion, compared to the native IL-2 sequence, such a change resulting in IL-2 agonist or antagonist activity. In various embodiments, the IL-2 variant has an amino acid sequence derived from SEQ ID NO: 8, has reduced / eliminated binding to IL-2Rα, so as to selectively activate and proliferate effector T cells (Teff) for treating cancer; exemplary amino acid substitutions are listed in Table 7. In various embodiments, the IL-2 variant has an amino acid sequence derived from SEQ ID NO: 8, has reduced binding to IL-2Rβ and / or γc, and has enhanced selectivity in activating and proliferating regulatory T cells (Treg) for treating autoimmune diseases; exemplary amino acid substitutions are listed in Table 7. As will be appreciated by those skilled in the art, all mutations can be optionally and independently combined in any manner to achieve optimal affinity and activity modulation.

[0232] Table 7

[0233]

[0234] Exemplary IL-2 based VitoKine constructs are provided in Table 8:

[0235] Table 8

[0236]

[0237] In various embodiments, the active portion is an amino acid sequence selected from the group consisting of, but not limited to, 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). In various embodiments, the active portion is a heterodimeric human IL-12 cytokine comprising SEQ ID NO: 21 as chain 1 and SEQ ID NO: 22 as chain 2. In various embodiments, the active portion is a heterodimeric human IL-23 cytokine comprising as chain 1 SEQ ID NO: 23 and as chain 2 SEQ ID NO: 22.

[0238] D3 domain ("masked portion domain")

[0239] The D3 domain is a "shielding domain" and is primarily used to reversibly shield the activity of the D2 domain in specific VitoKine constructs. The D3 domain is capable of shielding the functional activity of D2 until activated at the intended treatment site. In various embodiments, the VitoKine constructs of the present invention comprise a "shielding domain" (D3) that is a cognate receptor / binding partner of the D2 protein or cytokine. In various embodiments, the D3 domain is a variant or specific binder of the cognate receptor / binding partner of the D2 domain, such as a peptide or antibody fragment. In various embodiments, the D3 domain has enhanced binding to the D2 domain compared to the wild-type cognate receptor / binding partner. In various embodiments, the D3 domain has reduced or eliminated binding to the D2 domain compared to the wild-type cognate receptor / binding partner. In various embodiments, the D3 domain is a protein, or peptide, or antibody, or antibody fragment that is capable of shielding the activity of D2. In various embodiments, the D3 domain is a DNA, RNA fragment or a polymer such as PEG connected by a 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-15RαSushi 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-15RαSushi+ domain containing 1-30 additional IL-15Rα residues at the C-terminus of the Sushi domain (e.g., SEQ ID NO: 5). 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-2RαSushi domain. In various embodiments, the D3 domain is capable of masking the functional activity of D2 until D2 is activated at the intended treatment site.

[0240] IL-15 receptor α

[0241] IL-15 receptor is a type I cytokine receptor, composed of beta (β) and gamma (γ) subunits shared with IL-2 receptors and alpha (α) subunits that bind IL-15 with high affinity. Full-length human IL-15Rα is a type 1 transmembrane protein with a signal peptide of 32 AA, an extracellular domain of 173 AA, a transmembrane domain of 21 AA, a cytoplasmic tail of 37 AA, and multiple N-connections or O-connected glycosylation sites (Anderson et al., J.Biol Chem, 270: 29862-29869, 1995). It has been previously demonstrated that the natural soluble form of the IL-15Rα chain corresponding to the complete extracellular domain of IL-15Rα acts as a high-affinity IL-15 antagonist. However, in stark contrast to this finding, it has been shown that a recombinant soluble sushi domain of IL-15Rα, which has a large binding affinity for IL-15, acts as a potent IL-15 agonist by enhancing its binding and biological effects (proliferation and prevention of apoptosis) through the IL-15Rβ / γ heterodimer, while the sushi domain does not affect IL-15 binding and the function of the IL-15Rα / β / γ tripartite membrane receptor. These results suggest that such a soluble sushi domain, if naturally produced, may be involved in the trans-presentation mechanism of IL-15 (Mortier et al., J. Biol Chem, 281(3):1612-1619, 2006).

[0242] As used herein, the terms "native IL-15Rα" and "native interleukin-15 receptor α" in the context of a protein or polypeptide refer to any naturally occurring mammalian interleukin-15 receptor α ("IL-15Rα") amino acid sequence, including immature or precursor forms and mature forms and naturally occurring isoforms. Non-limiting examples of GenBank accession numbers for the amino acid sequences of various naturally occurring mammalian IL-15Rα include NP_002180 (human), ABK41438 (cynomolgus), NP_032384 (musculus), Q60819 (musculus), CA141082 (human). In various embodiments, the naturally occurring IL-15Rα is an immature form of a naturally occurring mammalian IL-15Rα polypeptide. In various embodiments, the naturally occurring IL-15Rα is a mature form of a naturally occurring mammalian IL-15Rα polypeptide. In various embodiments, the naturally occurring IL-15Rα is a form of a naturally occurring mammalian IL-15Rα polypeptide. In various embodiments, the native IL-15Rα is a full-length form of a naturally occurring mammalian IL-15Rα polypeptide. In various embodiments, the native IL-15Rα is an immature form of a naturally occurring human IL-15Rα polypeptide. In various embodiments, the native IL-15Rα is a mature form of a naturally occurring human IL-15Rα polypeptide. In various embodiments, the native IL-15Rα is a full-length form of a naturally occurring 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 set forth in SEQ ID NO: 4:

[0243]

[0244] In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is an IL-15Rα Sushi+ domain comprising the amino acid sequence of a mature human IL-15Rα polypeptide, as set forth in SEQ ID NO: 5:

[0245]

[0246] In various embodiments, a truncated co-receptor of IL-15, IL-15RαSushi+ (SEQ ID NO: 5), which reproduces most of the binding affinity of full-length IL-15Rα (SEQ ID NO: 4), is used as the D3 domain to mask the activity of IL-15 by adjusting the cleavable or non-cleavable linker connecting IL-15 and IL-15RαSushi+ to prepare an IL-15VitoKine. As will be appreciated by those skilled in the art, the length of the D3 domain can vary from the sequence listed in SEQ ID NO: 5, provided that the D3 domain can reproduce most of the binding activity of full-length IL-15α (SEQ ID NO: 4), i.e., the D3 domain is a functional fragment. The unique design of IL-15VitoKine is that it fully utilizes the unique characteristics of the IL-15 pathway, including the exceptionally high affinity between IL-15 and IL-15α (30 pM), and the fact that complexing with IL-15α enhances the activity of IL-15 in vivo. After cleavage of the linker connecting IL-15 and IL-15αSushi+ by proteases upregulated at disease sites, IL-15αShushi+ or any functional fragment derived from IL-15α ECD is expected to remain non-covalently associated with IL-15 and enhance IL-15 activity.

[0247] IL-2 receptor

[0248] The IL-2 receptor (IL-2R) is a heterotrimeric protein expressed on the surface of certain immune cells, such as lymphocytes, that binds and responds to a cytokine called IL-2. IL-2R has three subunits: α (CD25), β (CD122), and γ c (CD132, which is a chain shared by 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 Tac antigen or p55) is encoded by the gene IL-2RA on chromosome 10p14-15. The gene for the β chain of the human receptor (IL-2RB, CD122) is located on chromosome 22q11.2-12, while the shared IL-2Rγ CThe gene for the IL-2 chain (IL-2RG) is located on chromosome Xq13. The assembly of all three subunits of the receptor is important for signal transduction into B cells and T cells. IL-2R is present (temporarily or permanently) on the cell surface of almost all hematopoietic cells (including lymphoid lineage T, B and NK cells, as well as myeloid cells such as macrophages, monocytes and neutrophils). The signal is transmitted to the cell through the Janus kinases Jak1 and Jak3. Phosphorylation of the cytoplasmic portion of the receptor's β chain enables the STAT-3 and STAT-5 factors to form homodimers. Homodimers of STAT-3 and STAT-5 show increased affinity for the cell nucleus, where they bind to specific DNA elements and enhance the transcription of IL-2-dependent genes.

[0249] As used herein, the terms "native IL-2Rα" and "native interleukin-2 receptor α" in the context of a protein or polypeptide refer to any naturally occurring mammalian interleukin-2 receptor α ("IL-2Rα") amino acid sequence, including immature or precursor forms and mature forms and naturally occurring isoforms. Non-limiting examples of GenBank accession numbers for the amino acid sequences of various naturally occurring mammalian IL-2Rα include NP_032393.3 (Mus musculus), CAK26553.1 (Human), and NP_000408.1 (Human). In various embodiments, the naturally occurring IL-2Rα is an immature form of a naturally occurring mammalian IL-2Rα polypeptide. In various embodiments, the naturally occurring IL-2Rα is a mature form of a naturally occurring mammalian IL-2Rα polypeptide. In various embodiments, the naturally occurring IL-2Rα is a form of a naturally occurring mammalian IL-2Rα polypeptide. In various embodiments, the naturally occurring IL-2Rα is a full-length form of a naturally occurring mammalian IL-2Rα polypeptide. In various embodiments, the native IL-2Rα is an immature form of a naturally occurring human IL-2Rα polypeptide. In various embodiments, the native IL-2Rα is a mature form of a naturally occurring human IL-2Rα polypeptide. In various embodiments, the native IL-2Rα is a full-length form of a naturally occurring 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 set forth in SEQ ID NO:9:

[0250]

[0251] In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is an IL-2Rα Sushi domain comprising the amino acid sequence of a mature human IL-2Rα polypeptide, as set forth in SEQ ID NO: 10:

[0252]

[0253] In various embodiments, IL-2RαSushi (SEQ ID NO: 10) is used to mask IL-2 activity to prepare IL-2VitoKine. Unlike IL-15Rα, which contains a single sushi domain, IL-2Rα contains two sushi domains separated by a linker. In various embodiments, IL-2VitoKine comprises an IL-2RαSushi variant that comprises amino acid substitutions to disrupt the specific non-covalent interaction between IL-2Rα and IL-2, thereby reducing the binding affinity of IL-2Rα to IL-2. Although native IL-2Rα binds to IL-2 with a moderate affinity of 30nM, IL-2Rα may still not dissociate after cleavage of the linker. The association of IL-2Rα with IL-2 can reduce the activity of IL-2 and / or tilt the balance of T cell subsets in an undesirable direction. As affinity-reducing mutations (such as K38E or Y43A or a combination of these two substitutions) are introduced into IL-2RαSushi, the IL-2RαSushi domain may dissociate from IL-2 after protease cleavage of the linker.

[0254] L1 connector and L2 connector

[0255] Cleavable linker

[0256] Cleavable joints or joints sensitive to disease-related enzymes can include parts, such as protein substrates, which can be specifically cleaved by proteases present at elevated levels compared to non-disease tissues at the disease site. There are reports in the literature that in various types of cancers such as solid tumors, the level of enzymes with known substrates increases. See, for example, La Rocca et al., Brit. J. Cancer 90: 1414-1421 and Ducry et al., Bioconjug. Chem. 21: 5-13, 2010, wherein each document is incorporated herein by reference in its entirety. In various embodiments, the protease capable of cleaving protease cleavable joints is selected from the group consisting of: metalloproteinases such as matrix metalloproteinases (MMP) 1-28, and serine proteases such as urokinase-type plasminogen activator (uPA) and protein cleavage enzymes (Matriptase), cysteine proteases such as legumin, aspartic proteases and cathepsin proteases. Exemplary protease substrate peptide sequences are provided in Table 9:

[0257] Table 9

[0258]

[0259] Exemplary protease substrate peptide sequences that can be used as protease cleavable linkers, with or without peptide spacers of varying lengths at the C-terminus or N-terminus, or both, of the D2 domain are provided in Table 10:

[0260] Table 10

[0261] Protease Substrate peptide SEQ ID NO: MMP-2, 7, 9, 14 SPLGLAGS 71 MMP-2, 7, 9, 14, protein cleavage enzymes EPLELRAG 72 Proteinase, uPA, legumin LSGRSDNH 73 MMP-2 GPLGIAGQ 74 MMP-2,14 GTAHLMGG 75 MMP-14 RIGSLRTA 76 MMP-14 SGRSENIRTA 157 MMP-2,9 GPLGMLSQ 77 MMP-9,uPA RPSASRSA 78 MMP PLGLAG 79 uPA LGGSGRSANAlLE 80 uPA GGSGRSANAI 81 uPA SGRSA 82 legume protein AANL 83 legume protein GPTNKVR 158 Cathepsin C GFFY 84 Cathepsin D GPICFRLG 85 Cathepsin E RQAGFSL 86 Proteinase RQARAVGG 159 Prostate-specific antigen HSSKLQ 87

[0262] In various embodiments, the protease is MMP-9 or MMP-2. In further specific embodiments, the protease is uPA. In further specific embodiments, the protease is MMP-14. In further specific embodiments, the protease is legumin. In various embodiments, a VitoKine molecule comprises two different protease-cleavable linkers. In various embodiments, the protease-cleavable linker comprises the protease recognition sequence 'GPLGMLSQ' (SEQ ID NO: 77). In various embodiments, the protease-cleavable linker comprises the protease recognition sequence 'LGGSGRSANAILE' (SEQ ID NO: 80). In various embodiments, the protease-cleavable linker comprises the protease recognition sequence 'SGRSENIRTA' (SEQ ID NO: 157). In various embodiments, the protease-cleavable linker comprises the protease recognition sequence 'GPTNKVR' (SEQ ID NO: 158). In various embodiments, the linker (e.g., a cleavable linker) can be cleaved by a tumor-associated protease. In various embodiments, the cleavable linker can be cleaved by other disease-specific proteases in diseases other than cancer, such as inflammatory diseases.

[0263] In various embodiments, a peptide spacer can be incorporated into either side of the protease cleavable sequence or into both sides of the protease cleavable sequence, or as a non-cleavable linker without a protease substrate site. The peptide spacer is used to position the cleavable linker to be more accessible to the enzyme responsible for cleavage. The length of the spacer can be varied or optimized to balance accessibility for enzymatic cleavage and the spatial constraints required to reversibly shield the D2 domain from exerting its biological activity. The spacer can comprise 1-100 amino acids. Suitable peptide spacers are known in the art and include, but are not limited to, peptide linkers containing flexible amino acid residues such as glycine and serine. In various embodiments, the spacer can comprise the motif GS, GGS, GGGGS, GGSG, or SGGG. In various embodiments, the spacer can comprise 1 to 12 amino acids including the motif G, S, GS (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 GSGSGSGSGSGSGS (SEQ ID NO: 125). In other embodiments, the spacer can comprise the motif (GGGGS) n , wherein n is an integer from 1 to 10. In other embodiments, the spacer may further comprise amino acids other than glycine and serine.

[0264] Exemplary protease-cleavable linkers having spacer peptides flanking the protease substrate peptide (underlined) are provided in Table 11:

[0265] Table 11

[0266]

[0267] In various embodiments, the cleavable linker can be activated by mechanisms other than proteolysis, including but not limited to hydrolysis, such as a releasable PEGylated polymer that can be shed via a controlled release mechanism under varying pH conditions.

[0268] Non-cleavable linker

[0269] Non-cleavable linkers provide covalent attachment and additional structural and / or spatial flexibility between protein domains. As known in the art, peptide linkers containing flexible amino acid residues such as glycine and serine can be used as non-cleavable linkers. In various embodiments, non-cleavable linkers can include 1-100 amino acids. In various embodiments, the spacer can include the motif GS (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 can include the motif (GGGGS) n, where n is an integer from 1 to 10. In other embodiments, the linker can also include amino acids other than glycine and serine. In another embodiment, non-cleavable linkers can be simple chemical bonds, such as amide bonds (e.g., by chemical conjugation of PEG). Non-cleavable linkers are stable under physiological conditions and at lesion sites such as cancer sites or inflammatory disease sites.

[0270] Exemplary non-cleavable linkers are provided in Table 12:

[0271] Table 12

[0272]

[0273]

[0274] Combinations of cleavable and non-cleavable linkers

[0275] In various embodiments, the L1 linker and the L2 linker can both be cleavable linkers or both be non-cleavable linkers or a combination of cleavable and non-cleavable linkers to generate different forms of the active portion of the D2 domain to achieve different therapeutic intent or balance risk / benefit ratios or comply with different properties of cytokines. Exemplary active forms released by linker cleavage are Figure 2 Depicted in Figure 2. Active forms 1 and 3, derived from cleavage of L1 and cleavage of both L1 and L2, respectively, are short-acting cytokines with varying degrees of functional activity depending on the D3 conformation. Cleavage and release of D1 from the half-life extension or disease tissue targeting moiety increases the local concentration of the activated D2 domain. Following local action, the short-acting active form can be rapidly eliminated from the systemic circulation to reduce toxicity. In contrast, active form 2, derived from L2 cleavage, is a fully functional, long-acting, and tissue-targeted conserved cytokine that persists at the disease site for a longer period of time and with enhanced efficacy.

[0276] polynucleotides

[0277] In another aspect, the present disclosure provides an isolated nucleic acid molecule comprising a polynucleotide encoding an IL-15, an IL-15 variant, an IL-15Rα, an IL-15Rα variant, an Fc, an Fc variant, an IL-15-Fc fusion protein, an IL-15RαSushi-Fc fusion protein, or a VitoKine construct of the present disclosure. The subject nucleic acid may be single-stranded or double-stranded. Such a nucleic acid may be a DNA or RNA molecule. DNA includes, for example, cDNA, genomic DNA, synthetic DNA, DNA amplified by PCR, and combinations thereof. Genomic DNA encoding the VitoKine construct is obtained from a genomic library, which is available for many species. Synthetic DNA can be obtained by chemically synthesizing overlapping oligonucleotide fragments and then assembling the fragments to reconstruct part or all of the coding region and flanking sequences. RNA can be obtained from a prokaryotic expression vector that directs high-level synthesis of mRNA, such as a vector using a T7 promoter and RNA polymerase. DNA molecules of the present disclosure include full-length genes as well as polynucleotides and fragments thereof. The full-length gene may also comprise a sequence encoding an N-terminal signal sequence. Such nucleic acids may be used, for example, in methods for preparing novel VitoKine constructs.

[0278] 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 joint described herein.

[0279] In various embodiments, the recombinant nucleic acids of the present disclosure can be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory sequences are known in the art and are selected to direct the expression of the VitoKine construct. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, a promoter sequence, a leader or signal sequence, a ribosome binding site, transcription start and stop sequences, translation start and stop sequences, and an enhancer or activation sequence. The present disclosure contemplates constitutive or inducible promoters known in the art. The promoter may be a naturally occurring promoter or a hybrid promoter combining elements of more than one promoter. The expression construct may be present in an episome in the cell, such as a plasmid, or the expression construct may be inserted into a chromosome. In various embodiments, the expression vector comprises a selective marker gene to allow selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

[0280] In another aspect of the present disclosure, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding a VitoKine construct and operably linked to at least one regulatory sequence. The term "expression vector" refers to a plasmid, phage, virus, or vector used to express a polypeptide from a polynucleotide sequence. Vectors suitable for expression in host cells are readily available and utilize standard recombinant DNA techniques to insert nucleic acid molecules into the vector. Such vectors can include a variety of expression control sequences that, when operably linked to a DNA sequence, control the expression of that DNA sequence and can be used in these vectors to express a DNA sequence encoding a VitoKine construct. Such useful expression control sequences include, for example, the early and late promoters of SV40, the tet promoter, the early promoter mediated by adenovirus or cytomegalovirus, the RSV promoter, the lac system, the trp system, the TAC or TRC system, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter region of lambda phage, the control region of the fd coat protein, the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter of acid phosphatases such as PhoS, the promoter of yeast α-mating factor, the polyhedron promoter of the baculovirus system, and other sequences known to control gene expression in prokaryotes or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed and / or the type of protein desired to be expressed. In addition, the copy number of the vector, the ability to control the copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. Exemplary expression vectors suitable for expression of VitoKine are pDSRa and its derivatives comprising the VitoKine polynucleotide, as well as any additional suitable vectors known in the art or described below.

[0281] The recombinant nucleic acids of the present disclosure can be produced by ligating the cloned gene or a portion thereof into a vector suitable for expression in prokaryotes, eukaryotic cells (yeast, birds, insects, or mammals), or both. Expression vehicles for producing recombinant VitoKine constructs include plasmids and other vectors. For example, suitable vectors include the following types of plasmids: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotes such as E. coli.

[0282] Some mammalian expression vectors include prokaryotic sequences that are beneficial to vector propagation in bacteria and one or more eukaryotic transcription units expressed in eukaryotic cells. PcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfecting eukaryotic cells. Some of these vectors are modified by sequences from bacterial plasmids such as pBR322 to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, viruses such as derivatives of bovine papillomavirus (BPV-1) or derivatives of Epstein-Barr virus (pHEBo, pREP derived and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems are found in the description of gene therapy delivery systems below. In some cases, it may be desirable to express recombinant polypeptides using a baculovirus expression system. 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 pBlueBac III containing B-gal).

[0283] In various embodiments, vectors will be designed for producing the subject VitoKine constructs in CHO cells, such as the pcmv-Script vector (Stratagene, La Jolla, Calif.), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), and the pCI-neo vector (Promega, Madison, Wis.). It will be apparent that the subject gene constructs can be used to cause expression of the subject VitoKine constructs in cells proliferating in culture, for example, to produce proteins, including fusion proteins or variant proteins, for purification.

[0284] The present disclosure also relates to host cells transfected with recombinant genes comprising nucleotide sequences encoding the amino acid sequences of one or more subject VitoKine constructs. The host cells can be prokaryotic or eukaryotic. For example, the VitoKine constructs of the present disclosure can 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 are known to those skilled in the art, such as Chinese hamster ovary (CHO) cells or human embryonic kidney 293 (HEK293) cells.

[0285] Accordingly, the present disclosure also relates to methods for producing the subject VitoKine constructs. For example, host cells transfected with an expression vector encoding the VitoKine construct can be cultured under appropriate conditions that allow expression of the VitoKine construct to occur. The VitoKine construct can be secreted from cells containing the VitoKine construct and separated from a mixture of cells and culture medium containing the VitoKine construct. Alternatively, the VitoKine construct can be retained in the cytoplasm or in a membrane fraction, and the cells can be harvested, lysed, and the protein isolated. The cell culture comprises host cells, culture medium, and other by-products. Suitable culture media for cell culture are well known in the art.

[0286] The polypeptides and proteins of the present disclosure can be purified according to protein purification techniques well known to those skilled in the art. These techniques, at one level, involve the crude fractionation of protein fractions and non-protein fractions. After the peptide or polypeptide is separated from other proteins, the peptide or polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). As used herein, the terms "isolated polypeptide" or "purified polypeptide" are intended to refer to a composition that can be separated from other components, wherein the polypeptide is purified to any degree relative to its naturally available state. Thus, a purified polypeptide also refers to a polypeptide that has been separated from the environment in which it may naturally occur. Generally, "purified" will refer to a polypeptide composition that has undergone fractionation to remove various other components, and the polypeptide composition substantially retains its expressed biological activity. When the term "substantially purified" is used, the designation will refer to a peptide or polypeptide composition in which the polypeptide or peptide forms a majority component of the composition, such as about 50%, about 60%, about 70%, about 80%, about 85%, or about 90% or more of the protein in the composition.

[0287] Various techniques suitable for purification will be well known to those skilled in the art. These techniques include, for example, precipitation with ammonium sulfate, PEG, antibodies (immunoprecipitation), or by heat denaturation followed by centrifugation; chromatography, such as affinity chromatography (Protein A column), ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, hydroxyapatite chromatography, hydrophobic interaction chromatography; isoelectric focusing; gel electrophoresis; and combinations of these techniques. As is generally known in the art, it is believed that the order in which the various purification steps are performed can be changed, or that certain steps can be omitted, and still result in a suitable method for preparing a substantially purified polypeptide.

[0288] Pharmaceutical composition

[0289] In another aspect, the present disclosure provides pharmaceutical compositions comprising a VitoKine construct mixed with a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are well known and understood by those of ordinary skill in the art and have been extensively described (see, for example, Remington's Pharmaceutical Sciences, 18th edition, compiled by A.R. Gennaro, Mack Publishing Company, 1990). A pharmaceutically acceptable carrier can be included to alter, maintain, or preserve, for example, pH, osmotic pressure concentration, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or penetration of the composition. Such pharmaceutical compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the polypeptide. Suitable pharmaceutically acceptable carriers include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as PEG-100). agents) (such as caffeine, polyvinyl pyrrolidone, 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 diluents; emulsifiers; hydrophilic polymers (such as polyvinyl pyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronic, PEG, sorbitan esters) esters), polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (sucrose or sorbitol); tonicity enhancers (such as alkali metal halides (preferably sodium chloride or potassium chloride), mannitol and sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.

[0290] The main vehicle or carrier in the pharmaceutical composition can be aqueous or non-aqueous in nature. For example, suitable vehicles or carriers can be water for injection, physiological saline solution or artificial cerebrospinal fluid that may be supplemented with other materials common in the composition for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are other exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5, which can also include sorbitol or a suitable substitute for sorbitol. In one embodiment of the present disclosure, the composition with the desired degree of purity can be prepared by mixing with an optional formulation agent (formulation agent) (Remington's Pharmaceutical Sciences, the same) to store in the form of a lyophilized block or aqueous solution. In addition, the therapeutic composition can be formulated as a lyophilized product using suitable excipients such as sucrose. The best pharmaceutical composition will be determined by a person of ordinary skill in the art depending on, for example, the intended route of administration, delivery form and desired dosage.

[0291] When parenteral administration is envisioned, the therapeutic pharmaceutical composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired VitoKine construct in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, wherein the polypeptide is formulated as a sterile, isotonic solution suitable for storage. In various embodiments, pharmaceutical formulations suitable for injectable administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks solution, Ringer solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. In addition, suspensions of the active compound can be prepared as suitable oily injection suspensions. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compound and allow the preparation of highly concentrated solutions.

[0292] In various embodiments, the therapeutic pharmaceutical composition can be formulated as for targeted delivery using a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, pearls, and lipid-based systems, and lipid-based systems include oil-in-water emulsions, micelles, mixed micelles, and liposomes. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Exemplary phospholipids include lecithin, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. The targeting of liposomes can also be based on, for example, organ-specificity, cell-specificity, and organelle-specificity and known in the art.

[0293] In various embodiments, oral administration of the pharmaceutical composition is contemplated. Pharmaceutical compositions administered in this form may be formulated with or without carriers commonly used in the formulation of solid dosage forms such as tablets and capsules. In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), one or more therapeutic compounds of the present disclosure may be combined with one or more pharmaceutically acceptable carriers such as sodium citrate or dibasic calcium phosphate. phosphate) and / or any of the following: (1) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and / or gum arabic; (3) humectants such as glycerol; (4) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retarding agents such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as, for example, cetyl alcohol and glyceryl monostearate; (8) absorbents such as kaolin and bentonite; (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 also contain a buffer. The solid composition of similar type can also be used as the filler in the soft-filled and hard-filled gelatin capsule using excipients such as lactose (lactose) or lactose (milk sugar) and high molecular weight polyethylene glycol etc.. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to active ingredients, liquid dosage forms can include inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifying agents such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (especially, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0294] In various embodiments, it is envisioned that the pharmaceutical composition is topically applied to the skin or to a mucous membrane. Topical preparations can also include one or more of the various agents known as skin or stratum corneum penetration enhancers. Examples of these agents are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methanol or isopropyl alcohol, dimethyl sulfoxide (DMSO) and azone. Other agents can also be included to make the preparation cosmetically acceptable. Examples of these agents are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers and surfactants. Keratin softeners (keratolytic agents) such as those known in the art can also be included. Examples are salicylic acid and sulphur. Formulations 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 with any preservatives, buffers, or propellants that may be required. In addition to the subject compound of the present disclosure (e.g., a VitoKine construct), ointments, pastes, creams, and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0295] Other pharmaceutical compositions contemplated for use herein include formulations comprising the polypeptide in sustained or controlled delivery formulations. The techniques for formulating various other sustained or controlled delivery vehicles such as liposome vehicles, bioerodible microparticles or porous beads, and depot injections are also known to those skilled in the art.

[0296] The effective amount of the pharmaceutical composition to be used for treatment will depend on, for example, treatment background and treatment goal. It will be understood by those skilled in the art that the appropriate dosage level for treatment will therefore depend in part on the indication, route of administration, and patient's size (body weight, body surface or organ size) and condition (age and overall health) of the molecule being delivered, the polypeptide being used for and change. Accordingly, clinicians can adjust dosage and change route of administration to obtain optimal therapeutic effect. Typical dosage can depend on the factors mentioned above and in the range of from about 0.0001mg / kg to about 100mg / kg or more. Peptide compositions can preferably be injected or administered intravenously. Long-acting pharmaceutical compositions can be administered once every three to four days, once a week, or once every two weeks depending on the half-life and clearance rate of a 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 the dosage for achieving the desired effect is reached. Therefore, the composition can be administered as a single dose or as multiple doses (with the same or different concentrations / dosages) over time or as a continuous infusion. Further improvements of appropriate dosage are routinely carried out. Appropriate dosages can be determined through use of appropriate dose-response data.

[0297] The route of administration of the pharmaceutical composition is according to known methods, such as oral; by intravenous, intraperitoneal, intratumoral, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal vein, intralesional approach, intramedullary, intrathecal, intraventricular, intravesicular, transdermal, subcutaneous or intraperitoneal injection; and intranasal, intestinal, local, sublingual, urethra, vaginal or rectal mode; by sustained release system or by implant device. When desired, composition can be administered by bolus injection, or continuously administered by infusion, or administered by implant device. Alternatively or additionally, composition can be administered locally via the implantation of the film, sponge or another suitable material that the desired molecule has been adsorbed or encapsulated thereon. When using implant device, the device can be implanted in any suitable tissue or organ, and the delivery of the desired molecule can be administered via diffusion, sustained release bolus or continuously administered.

[0298] Therapeutic uses

[0299] The present disclosure provides a method for treating cancer cells in a subject, comprising administering to the subject a therapeutically effective amount (as a monotherapy or in a combination therapy regimen) of a VitoKine construct of the present disclosure in a pharmaceutically acceptable carrier, wherein such administration inhibits the growth and / or proliferation of the cancer cells. In particular, the VitoKine constructs of the present disclosure are useful in treating disorders characterized by cancer. Such disorders include, but are not limited to, solid tumors such as breast cancer, respiratory tract cancer, brain cancer, cancer of the reproductive organs, cancer of the digestive tract, cancer of the urinary tract, eye cancer, liver cancer, skin cancer, head and neck cancer, thyroid cancer, parathyroid cancer and their distant metastases, lymphomas, sarcomas, multiple myeloma, and leukemias. Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ. Examples of cancers of the respiratory tract include, but are not limited to, small cell lung cancer and non-small cell lung cancer, as well as bronchial adenomas and pleuropulmonary blastomas. Examples of brain cancer include, but are not limited to, brainstem and hypothalamic gliomas, cerebellar and cerebral astrocytomas, neuroblastomas, medulloblastomas, ependymomas, and neuroectodermal and pineal tumors. Tumors of male / male reproductive organs include, but are not limited to, prostate cancer and testicular cancer. Tumors of female / female reproductive organs include, but are not limited to, endometrial cancer, cervical cancer, ovarian cancer, vaginal cancer, vulvar cancer, and uterine sarcomas. Tumors of the digestive tract include, but are not limited to, anal cancer, colon cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, liver cancer, breast cancer, pancreatic cancer, rectal cancer, small intestine cancer, and salivary gland cancer. Tumors of the urethra include, but are not limited to, bladder cancer, penile cancer, kidney cancer, renal pelvis cancer, ureteral cancer, and urethral cancer. Eye cancers 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 fibrolamellar variant), bile duct cancer (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma. Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer. Head and neck cancers include, but are not limited to, nasopharyngeal carcinoma and lip and oral cancer. Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and central nervous system lymphoma. Sarcomas include, but are not limited to, soft tissue sarcomas, osteosarcomas, malignant fibrous histiocytomas, lymphosarcoma, and rhabdomyosarcomas. Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia.In various embodiments, the cancer will be one with high expression of TGF-β family members, such as activin A, myostatin, TGF-β, and GDF15, 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.

[0300] In various embodiments, the VitoKine constructs can be used as a single agent to treat 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.

[0301] In another aspect, the present disclosure provides a method for treating an autoimmune disease in a subject, the method comprising administering to the subject a therapeutically effective amount (as a monotherapy or in a combination therapy regimen) of a VitoKine construct of the present disclosure in a pharmaceutically acceptable carrier. An "autoimmune disease" refers to a non-malignant disease or disorder that arises from and is directed against an individual's own tissues. Examples of autoimmune diseases or disorders include, but are not limited to, inflammatory reactions, such as inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); reactions associated with inflammatory bowel disease (such as 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, cutaneous lupus); diabetes (e.g., type 1 diabetes or insulin-dependent diabetes); multiple sclerosis and juvenile-onset diabetes.

[0302] In another aspect, the present disclosure provides a method for treating an inflammatory disease in a subject, comprising administering to the subject a therapeutically effective amount of a VitoKine construct of the present disclosure in a pharmaceutically acceptable carrier (as a monotherapy or in a combination therapy regimen). "Inflammatory diseases" include all diseases associated with acute or chronic inflammation. Acute inflammation is the body's initial response to a noxious stimulus and is caused by an increased movement of plasma and white blood cells (such as, for example, granulocytes) from the blood into damaged tissue. A number of biochemical events propagate and mature the inflammatory response, which involves the local vasculature, the immune system, and various cells within the damaged tissue. Long-term inflammation is referred to as chronic inflammation, which results in progressive changes in the cell types present at the site of inflammation and is characterized by the simultaneous destruction and healing of tissues from the inflammatory process. Examples of inflammatory diseases are well known in the art. In various embodiments, the inflammatory disease is 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, diversion colitis, Behcet's syndrome, and indeterminate colitis.

[0303] In another aspect, the present disclosure provides a method for treating a viral infection in a subject, comprising administering to the subject a therapeutically effective amount (as a monotherapy or in a combination therapy regimen) of a VitoKine construct of the present disclosure in a pharmaceutically acceptable carrier. In various embodiments, the viral infection to be treated can be caused by an infectious agent 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, those caused by hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza virus, varicella virus, adenovirus, herpes simplex type 1 (HSY-I) virus, herpes simplex type 2 (HSY-II) virus, rinderpest virus, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papillomavirus, papovavirus, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsackievirus, mumps virus, measles virus, rubella virus, poliovirus, smallpox virus, Epstein Barr virus, human immunodeficiency virus type 1 (HIV-I), human immunodeficiency virus type II (HIV-II), and the causative agent of viral diseases such as viral meningitis, encephalitis, dengue fever, or smallpox.

[0304] Bacterial diseases caused by bacteria (e.g., Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Candida albicans, Proteus vulgaris, Staphylococcus viridans, and Pseudomonas aeruginosa) that can be prevented, treated, and / or managed according to the methods described herein include, but are not limited to, mycobacteria, rickettsiae, mycoplasmas, Neisseria, S. pneumoniae, Borrelia burgdorferi (Lyme disease), Bacillus anthracis, and leukemia. anthracis), tetanus, streptococci, staphylococci, mycobacteria, pertussis, cholera, plague, diphtheria, chlamydia, Staphylococcus aureus, and Legionella.

[0305] Protozoal diseases caused by protozoa that can be prevented, treated, and / or managed according to the methods described herein include, but are not limited to, Leishmania, kokzidioa, trypanosoma, or malaria.

[0306] Parasitic diseases caused by parasites that can be prevented, treated, and / or managed according to the methods described herein include, but are not limited to, Chlamydia and Rickettsia.

[0307] "Therapeutically effective amount" or "therapeutically effective dose" refers to that amount of the administered therapeutic agent that will relieve to some extent one or more symptoms of the disorder being treated.

[0308] The therapeutically effective dose can be determined by IC 50 Initially estimated from cell culture assays. A dose can then be formulated in animal models to achieve an IC including the IC as determined in cell culture. 50 The circulating plasma concentration range of the drug can be used to more accurately determine the dosage that is useful in humans. The levels in plasma can be measured, for example, by HPLC. The exact composition, route of administration, and dosage can be selected by the individual physician in view of the subject's condition.

[0309] The dosage regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic response or a preventive response). For example, a single bolus injection can be administered, several divided doses (multiple or repeated or maintenance) can be administered over time, and the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic scenario. For ease of administration and consistency of dosage, it is particularly beneficial to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically dispersed units suitable as single doses for a mammalian subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. The specifications of the dosage unit form of the present disclosure will be primarily determined by the unique characteristics of the antibody and the specific therapeutic or preventive effect to be achieved.

[0310] Therefore, it will be understood by those skilled in the art that, based on the disclosure provided herein, dosage and administration regimens are adjusted according to methods well known in the therapeutic field. That is, the maximum tolerated dose can be easily determined, and the effective amount that provides a detectable therapeutic benefit to the subject can also be determined, and the time requirement for applying each agent to provide a detectable therapeutic benefit to the subject can also be determined. Therefore, although certain dosages and administration regimens are exemplified herein, these examples are by no means limiting the dosages and administration regimens that can be provided to the subject when practicing this disclosure.

[0311] It should be noted that dosage values may vary with the type and severity of the condition to be alleviated and may include a single dose or more than one dose. It should also be understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges listed herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. In addition, the dosage regimen of the compositions of the present disclosure may be based on a number of factors, including the type of disease, the age, weight, sex, medical condition, severity of the condition, route of administration, and the specific antibody used. Thus, the dosage regimen may vary widely but can be routinely determined using standard methods. For example, the dosage may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or experimental values. Thus, the present disclosure includes intra-subject dose-escalation as determined by a skilled person. Determining appropriate dosages and regimens is well known in the relevant art and will be understood to be within the skill of the art once the teachings disclosed herein are provided.

[0312] Exemplary, non-limiting daily dosing ranges for a therapeutically or prophylactically effective amount of VitoKine or a VitoKine variant of the present disclosure can be 0.0001 to 100 mg / kg body weight, 0.0001 to 90 mg / kg body weight, 0.0001 to 80 mg / kg body weight, 0.0001 to 70 mg / kg body weight, 0.0001 to 60 mg / kg body weight, 0.0001 to 50 mg / kg body weight, 0.0001 to 40 mg / kg body weight, 0.0001 to 30 mg / kg body weight, 0.0001 to 20 mg / kg body weight, 0.0001 to 30 mg / kg body weight, 0.0001 to 40 mg / kg body weight, 0.0001 to 50 mg / kg body weight, 0.0001 to 60 mg / kg body weight, 0.0001 to 70 mg / kg body weight, 0.0001 to 80 mg / kg body weight, 0.0001 to 90 mg / kg body weight, 0.0001 to 100 mg / kg body weight, 0.0001 to 150 mg / kg body weight, 0.0001 to 150 mg / kg body weight, 0.0001 to 160 mg / kg body weight, 0.0001 to 170 mg / kg body weight, 0.0001 to 180 mg / kg body weight, 0.0001 to 190 mg / kg body weight, 0.0001 to 200 mg / kg body weight, 0.0001 to 250 mg / kg body weight, 0.0001 to 250 mg / kg body weight, 0.0001 to 0.01 to 10 mg / kg body weight, 0.0001 to 5 mg / kg body weight, 0.0001 to 4 mg / kg body weight, 0.0001 to 3 mg / kg body weight, 0.0001 to 2 mg / kg body weight, 0.0001 to 1 mg / kg body weight, 0.001 to 50 mg / kg body weight, 0.001 to 40 mg / kg body weight, 0.001 to 30 mg / kg body weight, 0.001 to 20 mg / kg body weight, 0.001 to 10 mg / kg body weight, 0.001 to 5 mg / kg body weight, 0.001 to 4 mg / kg body weight, 0.001 to 3 mg / kg body weight, 0.001 to 2 mg / kg body weight, 0.001 to 1 mg / kg body weight, 0.010 to 50 mg / kg body weight, 0.010 to 40 mg / kg body weight, 0.010 to 30 mg / kg body weight, 0.010 to 20 mg / kg body weight, 0.010 to 10 mg / kg body weight, 0.010 to 5 mg / kg body weight, 0.010 to 4 mg / kg body weight, 0.010 to 3 mg / kg body weight, 0.010 to 2 mg / kg body weight, 0.010 to 1 mg / kg body weight, 0.1 to 50 mg / kg body weight, 0.1 to 40 mg / kg body weight, 0.1 to The present invention provides the present invention relates to an agent that can be used in combination with other pharmaceutical compositions and / or pharmaceutical products. The present invention provides an agent that can be used in combination with other pharmaceutical compositions and / or pharmaceutical products. The present invention provides an agent that can be used in combination with other pharmaceutical compositions and / or pharmaceutical products. The present invention provides an agent that can be used in combination with other pharmaceutical compositions and / or pharmaceutical products. The present invention provides an agent that can be used in combination with other pharmaceutical compositions and / or pharmaceutical products. The present invention provides an agent that can be used in combination with other pharmaceutical compositions and / or pharmaceutical products.

[0313] Toxicity and therapeutic index of the pharmaceutical compositions of the present disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD 50 (a dose lethal to 50% of the population) and ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between the toxic dose and the therapeutically effective dose is the therapeutic index, and the therapeutic index can be expressed as the ratio LD 50 / ED 50 Compositions that exhibit large therapeutic indices are generally preferred.

[0314] The dosing frequency of administration of the VitoKine construct pharmaceutical composition depends on the nature of the therapy and the specific disease being treated. Subjects can be treated at regular intervals, such as once a week or once a month, until the desired therapeutic outcome is achieved. Exemplary dosing frequencies include, but are not limited to: once a week without interruption; once a week every other week; once every 2 weeks; once every 3 weeks; once a week without interruption for 2 weeks, then once a month; once a week without interruption for 3 weeks, then once a month; once a month; once every two months; once every 3 months; once every 4 months; once every 5 months; or once every 6 months, or once a year.

[0315] Combination therapy

[0316] As used herein, the terms "co-administration," "co-administered," and "incombination with," in reference to a VitoKine construct of the present disclosure and one or more other therapeutic agents, are intended to mean, and do mean and include, the following: simultaneous administration of such combination of a VitoKine construct of the present disclosure and one or more therapeutic agents to a subject in need of treatment, wherein such components are formulated together into a single dosage form that releases said components to said subject at substantially the same time; substantially simultaneous administration of such combination of a VitoKine construct of the present disclosure and one or more therapeutic agents to a subject in need of treatment, wherein such components are formulated separately from one another into separate dosage forms that are taken by said subject at substantially the same time, whereupon said components are released to said subject at substantially the same time; and one or more therapeutic agents are administered sequentially to a subject in need of treatment, wherein such components are formulated separately from one another into separate dosage forms that are taken by the subject at successive times with significant time intervals between each administration, whereupon the components are released to the subject at substantially different times; and, such combinations of a VitoKine construct of the present disclosure and one or more therapeutic agents are administered sequentially to a subject in need of treatment, wherein such components are formulated together into a single dosage form that releases the components in a controlled manner, whereupon the components are released to the subject simultaneously, sequentially, and / or overlappingly at the same and / or different times, where each part can be administered by the same or different routes.

[0317] On the other hand, the present disclosure provides a method for treating a subject's cancer or cancer metastasis, the method comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention in combination with a second therapy, wherein the second therapy 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 for preventive cancer prevention, prevention of postoperative cancer recurrence and metastasis, and as an adjunct to other conventional cancer therapies. The present disclosure recognizes that the effectiveness of conventional cancer therapies (e.g., chemotherapy, radiotherapy, phototherapy, immunotherapy, and surgery) can be enhanced by the use of the combined methods described herein.

[0318] A large amount of conventional compounds have been shown to have anti-neoplastic activity. These compounds have been used as the medicament in chemotherapy to reduce solid tumors, prevent metastasis and further grow or reduce the number of the malignant T cells in leukemia or myeloid malignancies. Although chemotherapy is effective in treating various types of malignant tumors, many anti-neoplastic compounds induce undesirable side effects. Shown, when two or more different treatment combinations, treatment can work collaboratively, and allows to reduce the dosage of every kind of treatment, thereby reducing the harmful side effects of every kind of compound produced at higher dosage. In other cases, for treatment, it is that the malignant tumor of refractory nature can respond to the combination therapy of two or more different treatments.

[0319] In various embodiments, a second anticancer agent, such as a chemotherapeutic agent, will be administered to the patient. A list of exemplary chemotherapeutic agents includes, but is not limited to, daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, mechlorethamine, 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, dapoxetine ... Platinum, oxaliplatin, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fludarabine, ifosfamide, hydroxyureataxanes (such as paclitaxel and docetaxel) and / or anthracycline antibiotics, and combinations of agents such as, but not limited to, DA-EPOCH, CHOP, CVP or FOLFOX. In various embodiments, the dose of such chemotherapeutic agents includes, but is not limited to, about 10 mg / m 2 , 20mg / m 2 , 30mg / m 2 , 40mg / m 2 , 50mg / m 2 , 60mg / m 2 , 75mg / m 2 , 80mg / m 2 , 90mg / m 2 , 100mg / m 2 , 120mg / m 2 , 150mg / m 2 , 175mg / m 2 , 200mg / m 2 , 210mg / m 2 , 220mg / m 2 , 230mg / m 2, 240mg / m 2 , 250mg / m 2 , 260mg / m 2 and 300 mg / m 2 Any of .

[0320] In various embodiments, the combination treatment methods of the present disclosure may also include administering to the subject a therapeutically effective amount of immunotherapy, including, but not limited to, treatment with depleting antibodies directed against specific tumor antigens; treatment with antibody-drug conjugates; treatment with agonistic antibodies, antagonistic antibodies, or blocking antibodies directed against co-stimulatory or co-inhibitory molecules (immune checkpoints), such as, but not limited to, antibodies against CTLA-4, PD-1, PDL-1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, SIRPa, CD47, GITR, ICOS, CD27, Siglec7, Siglec 8, Siglec 9, Siglec 15, and VISTA, CD276, CD272, TIM-3, B7-H4; treatment with bispecific T cell-engaging antibodies Treatments such as blinatumomab; treatments involving 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); treatments using therapeutic vaccines (including but not limited to oncolytic viruses such as T-vec) or using therapeutic vaccines such as sipuleucel-T; treatments using dendritic cell vaccines or tumor antigen peptides or neoantigen vaccines; treatments using chimeric antigen receptor (CAR)-T cells; treatments using CAR-NK cells; treatments using NK cells; treatments using iPS-induced NK cells treatment with iPS-induced T cells; treatment with iPS-induced CAR-T cells or iPS-induced CAR-NK cells; treatment with tumor infiltrating lymphocytes (TILs); treatment with adoptively transferred anti-tumor T cells (ex vivo expanded T cells and / or TCR-T cells); treatment with TALL-104 cells; and treatment with immunostimulants such as Toll-like receptor (TLR) agonists CpG, TLR7, TLR8, TLR9 and vaccines such as Bacillus Calmette-Guérin (BCG) and imiquimod; wherein the combination therapy provides increased effector cell killing of tumor cells, i.e., there is a synergistic effect between the VitoKine construct and the immunotherapy when co-administered.

[0321] In various embodiments, the combination treatment methods of the present disclosure may also include administering to the subject a therapeutically effective amount of an anti-inflammatory agent for autoimmune diseases, inflammatory diseases, and other immune disorders, including but not limited to treatment with depleting antibodies directed against specific immune cells; treatment with regulatory antibodies (agonistic, antagonistic, or blocking) as target modulators of the immune response to targets (ligands or their receptors), including but not limited to 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-21R, 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 its related receptors, integrin family (e.g., α4β7), TSLP, complement 5 (C5) or C5a, IgE, APRIL, TACI, BCMA, CD20, CD22, CD40 / CD40L, B7H1, B7H2, ICOS, BAFF, BCR, BLys, B7RP1, T LR7, TLR8, TLR8, TLR9; treatment using regulatory small molecules (agonistic or antagonistic) as target modulators of the immune response to targets including but not limited to NFkB, Jak1, Jak2, Jak3, Tyk2, Syk, BTK, PIK3, cyclooxygenase 2 and NMDA receptor; wherein the combination treatment provides increased efficacy in modulating the immune response, i.e., there is a synergistic effect between the VitoKine construct and the anti-inflammatory therapy when co-administered.

[0322] In various embodiments, the combination therapy comprises administering a VitoKine construct and a second composition simultaneously, either in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, the VitoKine construct composition and the second composition are administered sequentially, i.e., the VitoKine construct composition is administered before or after the second composition is administered. In various embodiments, the administration of the VitoKine construct composition and the second composition is simultaneous, i.e., the administration periods of the VitoKine construct composition and the second composition overlap. In various embodiments, the administration of the VitoKine construct composition and the second composition is non-simultaneous. For example, in various embodiments, the administration of the VitoKine construct composition is terminated and the second composition is then administered. In various embodiments, the administration of the second composition is terminated and the VitoKine construct composition is then administered.

[0323] The following examples are provided to more fully illustrate the present disclosure but should not be construed as limiting the scope of the present disclosure.

[0324] Example 1

[0325] Construction and production of IL-15VitoKine constructs

[0326] The goal is to design an IL-15VitoKine construct that will remain inert until locally activated by a protease that is upregulated in cancer or diseased tissue. This article describes VitoKine with wild-type IL-15 (SEQ ID NO: 2) or IL-15 mutein (e.g., SEQ ID NO: 3) as the active portion that is reversibly shielded between the Fc domain and IL-15RαSushi+ (SEQ ID NO: 5). These constructs include one or two cleavable linkers that are recognized by tumor-specific proteases. In the presence of tumor cells expressing proteases, the linker connecting the Fc and IL-15 mutein and / or the linker connecting IL-15 and IL-15αSushi+ will be cleaved, thereby restoring IL-15 activity. Notably, due to the unusually high affinity between IL-15 and IL-15α (30 pM), the IL-15αSushi+ released after proteolysis is expected to remain non-covalently associated with IL-15. Produced and Figure 1 Fc IL-15 VitoKine constructs with various linker and peptide spacer compositions are schematically depicted in , and their respective sequences are listed as SEQ ID NOs: 25-43, 162-165, and 169-174.

[0327] All genes were codon-optimized for expression in mammalian cells, synthesized, and subcloned into a recipient mammalian expression vector (GenScript). Protein expression was driven by a CMV promoter, and a synthetic SV40 polyadenylation (polyA) signal sequence was present at the 3' end of the CDS. A leader sequence was engineered at the N-terminus of the construct to ensure proper signaling and processing for secretion.

[0328] The constructs were generated by co-transfecting HEK293-F cells grown in suspension with mammalian expression vectors using polyethyleneimine (PEI, 25,000 MW linear, Polysciences). If two or more expression vectors were used, the vectors were transfected at a 1:1 ratio. For transfection, HEK293 cells were grown in serum-free FreeStyle TM HEK293 cells were cultured in 1000 ml shake flasks (working volume 330 mL) at 0.8 × 10 6 The density of individual cells / ml was inoculated and transfected after 24 hours. The expression vector that is 330 μ g DNA is mixed with 16.7 ml Opti-mem culture medium (ThermoFisher) by a total amount. After adding 0.33 mg PEI diluted in 16.7 ml Opti-mem culture medium, the mixture was vortexed for 15 sec, and then incubated at room temperature for 10 min. The DNA / PEI solution was then added to the cells and incubated at 37 ℃ in an incubator with 8% CO2 atmosphere. On the 4th day, sodium butyrate (Millipore Sigma) was added to the cell culture at a final concentration of 2 mg / L to help maintain protein expression. After cultivating for 6 days, the supernatant was collected and used for purification by centrifuging at 2200 rpm for 20 min. The solution was sterile filtered (0.22 μ m filter, Corning). Protein A affinity chromatography was used to purify the secreted protein from the cell culture supernatant.

[0329] For affinity chromatography, the supernatant was loaded onto a HiTrap MabSelectSure protein AFF column (CV = 5 mL, GE Healthcare) equilibrated with 25 ml of phosphate buffered saline pH 7.2 (ThermoFisher). 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, 25 mM sodium chloride, pH 3.2. The protein solution was neutralized by adding 3% 1 M Tris pH 10.2. The target protein was washed with The samples were concentrated using an Ultra-15 concentrator 10 kDa NMWC (Merck Millipore Ltd.).

[0330] The samples were analyzed by SDS-PAGE and staining with Coomassie (Imperial R The purity and molecular weight of the purified constructs were analyzed by staining with ELISA kit according to the manufacturer's instructions. Precast gel system (4-12% or 8-16% Bis-Tris, ThermoFisher). The protein concentration of the purified protein samples was determined by measuring the UV absorbance at 280 nm (Nanodrop spectrophotometer, ThermoFisher) divided by the molar extinction coefficient calculated based on the amino acid sequence. The aggregate content of the constructs was analyzed on an Agilent 1200 high performance liquid chromatography (HPLC) system. The samples were injected onto an AdvanceBio size exclusion column ( 4.6 x 150 mm, 2.7 μm, LC column, Agilent).

[0331] P-0315 is a dimeric C-terminal Fc-fused IL-15 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 spectrum of the Fc IL-15 VitoKine, an SDS-PAGE analysis of P-0315 (SEQ ID NO: 33) is shown in Figure 3A. The size exclusion chromatogram is in Figure 3B.

[0332] Example 2

[0333] The in vitro activity of IL-15 is effectively masked in the VitoKine format

[0334] IL-15VitoKine P-0172 (SEQ ID NO: 27) comprises an IL-15 / IL-15RαSushi+ fusion polypeptide connected by a short GS (SEQ ID NO: 116) peptide linker, which in the homodimeric fusion format is connected to the C-terminus of the homodimeric Fc domain via a uPA cleavable linker. P-0198 is a dimeric C-terminal Fc-IL-15 fusion protein with non-covalently complexed IL-15αSushi. These two molecules have similar configurations between Fc and IL-15 fusions, with significant differences in IL-15αSushi incorporation. One is fused via a short GS linker (P-0172), while the other is free via non-covalent bonding (P-0198). The binding activity of P-0172 to IL-2Rβ was determined by enzyme-linked immunosorbent assay (ELISA) and compared with P-0198 (comprising SEQ ID NOs: 45, 44, and 5), a highly active IL-15 / IL-15Rα-Fc fusion protein.

[0335] Briefly, IL-2Rβ-ECD (SEQ ID NO: 12) was coated onto the wells of a Nunc Maxisorp 96-well microplate at 1 μg / well. After incubation overnight at 4°C and blocking with superblock (ThermoFisher), a 3-fold serial dilution of IL-15 compound starting from 100 nM was added to each well at 100 μl / well. After incubation at room temperature for 1 hour, 100 μl / well of goat anti-human IgG Fc-HRP (1:5000 dilution in diluent) was added to each well and incubated 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 was added to each well, the plate was developed at room temperature in the dark 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 illustrated in FIG4A , VitoKine P-0172 binds to IL-2Rβ with significantly reduced potency compared to P-0198 (12.2 nM vs. 0.21 nM), which may be due to the spatial restriction caused by the short covalent connection between IL-15 and IL-15αSushi, indicating that the IL-15αSushi in the VitoKine platform effectively shields the IL-15 domain from binding to its receptor.

[0336] The functional activity of IL-15VitoKine P-0172 was further evaluated compared to P-0198 by examining IL-15-mediated induction of CD69 expression on human NK cells and CD8+ T cells from fresh human peripheral blood mononuclear cells (PBMCs) by FACS analysis. CD69 is a cell surface glycoprotein that is induced early during lymphoid activation involving NK cells and T cells.

[0337] In brief, human PBMCs were separated from buffy coats purchased from Oklahoma Blood Institute by Ficoll-Hypaque centrifugation. 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 x g and resuspended in FACS buffer. After blocking Fc receptors by adding human TruStain FcX (1:50 dilution), cells were stained with anti-human CD56-FITC antibodies, anti-human CD69-PE antibodies, and anti-human CD8-APC antibodies (1:50 dilution). After incubation with antibodies for 30 minutes at room temperature, cells were collected and washed, resuspended in FACS buffer, and analyzed by flow cytometry. CD69 expression was determined by gating CD56+NK and CD8+T cells, and data were expressed as the % of CD69 positive cells in the gated population.

[0338] As shown in Figures 4B and 4C, VitoKine P-0172 significantly reduced CD69 activation of CD8+ T cells and NK cells and was only measurable at the highest concentration tested, with potency at least 2-3 logs lower than that of P-0198. This indicates that IL-15 activity is effectively masked in the VitoKine format. The masking effect was more pronounced in the PBMC CD69 activation assay than in the IL-2Rβ ELISA binding assay, indicating that the severe impairment of IL-15 activity under physiological conditions is more pronounced than under in vitro reconstitution conditions of the ELISA. Due to the presence of spatial constraints, VitoKine severely disrupts the binding of IL-15 to IL-2Rβ and γ expressed on lymphocytes. C complex, and thus leads to inefficient pathway activation and severely impaired activity.

[0339] The biological activity of monomeric IL-15 VitoKine was also examined. P-0170 (SEQ ID NO: 26 and 15) is the monomeric counterpart of P-0172, with the same linker and fusion configuration. Compared to the highly active IL-15Fc fusion protein P-0166, P-0170 showed a significantly reduced ability to activate CD69 on CD8+ T cells ( Figure 5), indicating that the monomeric VitoKine platform effectively masks the biological activity of IL-15 in the D2 domain.

[0340] Example 3

[0341] Comparison of FcIL-15VitoKine shielding efficiency of different linker lengths and compositions between IL-15 and IL-15αSushi+ and between Fc and IL15

[0342] like Figure 1 As depicted in [1], IL-15 VitoKine was constructed by fusing human IL-15 between two different domains, such as a half-life-extending Fc domain and a cognate high-affinity co-receptor α domain of IL-15, via peptide linkers L1 and L2. The two linkers, connecting Fc to IL-15 versus connecting IL-15 to the IL-15α Sushi domain, as well as the length and composition of the linkers, were examined for their differential effects on the bioactivity of IL-15 to achieve the desired activity impairment.

[0343] FACS analysis of the activation marker CD69 of immune cell subsets of fresh human PBMCs was performed to evaluate IL-15 and IL-15αSushi+IL-15VitoKine with different lengths of the non-cleavable linker (L2). The same protocol as in Example 2 was followed.

[0344] P-0204 (SEQ ID NO:30), P-0205 (SEQ ID NO:31), and P-0206 (SEQ ID NO:32) are Fc IL-15 vitokines that share the same uPA-cleavable linker sequence (L1) between Fc and IL-15, but the linker (L2) connecting IL-15 and IL-15α Sushi+ domains varies in length in the three vitokines and is (GGGGS)3 (SEQ ID NO:127), (GGGGS)2 (SEQ ID NO:126), and GGGGS (SEQ ID NO:118), respectively.

[0345] like Figures 6A-6BAs shown in Figure 6, Fc IL-15VitoKine with different linker lengths between IL-15 and IL-15αSushi+ ranging from 5 to 15 amino acids all resulted in a significant reduction in the potency of activated CD8+ T cells (Figure 6A) or NK cells (Figure 6B). Comparing the potency of P-0206, P-0205, and P-0204, it is clear that the shorter the linker length connecting IL-15 and IL-15αSushi+ domains, the less active VitoKine becomes; indicating that the degree of reduced activity can be further regulated by the length of the L2 linker; in summary, by adjusting the length of the linker (L2) between IL-15 and IL-15α to produce an appropriate level of spatial restriction, we shielded IL-15 between the Fc domain and the cognate high-affinity co-receptor α of IL-15, thereby almost completely losing IL-15 activity.

[0346] exist Figure 7 The effect of the linker (L1) connecting Fc and IL-15 on the bioactivity of VitoKine was also examined. P-0204 and P-0203 (SEQ ID NO: 29) share the same 15-amino acid flexible (G4S)3 linker (L2) between IL-15 and IL-15Rα, but the length of the L1 linker is different, and P-0203 contains a peptide spacer that is 7 GS residues longer than P-0204 on the flanks of the uPA substrate peptide connecting Fc and IL-15. Despite the different lengths of the L1 linker connecting Fc and IL-15, the bioactivity of P-0204 and P-0203 is similar ( Figure 7 ), indicating that the L1 linker connecting Fc and IL-15 has the least damaging effect on IL-15 activity when it spans 13 to 35 amino acid residues. However, L1 linkers shorter or longer than 13 to 35 amino acid residues or in the context of different cytokines may affect the masking activity of the D2 domain. In the same study, P-0202 was included. P-0202 and P-0203 share the same L1 linker connecting Fc and IL-15, but the L2 linker connecting IL-15 and the IL-15α Sushi domain of P-0202 is 13 amino acids shorter than that of P-0203. P-0202 showed lower biological activity than P-0203, confirming that the linker L2 is more important than the linker L1 in terms of VitoKine's masking activity.

[0347] The effect of linker composition or linker peptide sequence on VitoKine activity was studied by measuring Ki67 expression in the nuclei of NK cells and CD8 T cells after IL-15VitoKine treatment. Ki67 is a marker of cell proliferation, and an in vitro human PBMC assay was established. Briefly, purified human PBMCs were treated with serial dilutions of IL-15VitoKine compounds and incubated at 37°C for 5 days. On day 5, the cells were washed once with FACS buffer (1% FBS / PBS) and first stained with Fc blocking agent 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 fully resuspended with 200 μl / well of 1X Foxp3 fixation and permeabilization working solution and incubated in the dark at room temperature for 30 minutes. After centrifugation, 200 μl of 1X permeabilization buffer was added to each well for another wash. The cell pellet was resuspended in permeabilization buffer with anti-human Ki67-PE (1:25 dilution). After incubation at room temperature for 30 minutes, the cells were collected and washed, resuspended in FACS buffer, and analyzed by flow cytometry. Data are expressed as the percentage of Ki67-positive cells in the gated population.

[0348] Since the L2 linker exerts a significant effect on IL-15VitoKine activity compared to the L1 linker, we examined the effect of different sequence compositions of the L2 linker on the biological activity of IL-15VitoKine. 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) connecting Fc and IL-15. The linkers connecting IL-15 and IL-15Rα are both 10 amino acids long but have different sequences. The linker is (G4S) 2 in P-0351, an MMP-14 substrate peptide (SEQ ID NO: 157) in P-0488, or a legumin substrate peptide (SEQ ID NO: 160) in P-0489.

[0349] like Figures 8A-8BAs shown in Figure 8 , all three IL-15 VitoKines had severely impaired potency in proliferating NK cells (Figure 8A) or CD8+ T cells (Figure 8B) compared to the highly active IL-15 / IL-15RαFc fusion protein P-0156 (SEQ ID NO: 175+176). Different peptide linker sequences had subtle effects 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, the more structural constraints it imposes on the VitoKine molecule, which can lead to more severe activity impairment. However, the effect of the L2 linker sequence composition on the activity of VitoKine is minimal, and the data support that different cleavable linkers can be incorporated as L2 linkers to effectively shield the activity of the D2 domain, thereby expanding the design and application range of VitoKine.

[0350] Together, these data demonstrate that the L2 linker, which connects the IL-15 (D2) and IL-15Rα Sushi+ (D3) domains, plays an important role in masking D2 activity to produce an inert VitoKine. The level of active inertness can be further tuned by adjusting the L2 linker length and varying the linker sequence / flexibility. The choice of the cleavable L2 linker length and sequence should balance the presence of a specific protease at the intended disease indication site, the accessibility of the substrate peptide to the protease, and the desired rate of proteolysis.

[0351] Example 4

[0352] Determine appropriate reaction conditions for complete protease cleavage

[0353] Initial in vitro protease cleavage experiments were performed using the IL-15 VitoKine constructs P-0315 and P-0203 to determine the protease cleavage and optimal cleavage conditions of MMP-2 and uPA, respectively. P-0315 (SEQ ID NO: 33) contains a uPA-cleavable linker connecting the Fc and IL-15 domains and an MMP-2 / 9-cleavable linker connecting the IL-15 and IL-15RαSushi+ domains. P-0203 (SEQ ID NO: 29) contains a single protease-cleavable linker (uPA) connecting the Fc and IL-15 domains. The linker between the IL-15 and IL-15RαSushi+ domains in P-0203 is a flexible (G4S)3 linker. Recombinant human uPA and MMP-2 were purchased from BioLegend. MMP-2 was provided in latent form and activated with para-aminophenylmercuric acetate (APMA, Millipore Sigma) according to the manufacturer's instructions.

[0354] For proteolytic cleavage by MMP-2, 4 μg of P-0315 were incubated with 30 ng, 100 ng or 300 ng of APMA-activated MMP-2 at 37°C in the manufacturer's recommended assay buffer (100 mM Tris, 20 mM CaCl2, 300 mM NaCl, 0.1% (w / v) Brij 35, pH 7.5) for 3 hours. To stop the reaction, SDS-PAGE loading dye was added to the reaction and the mixture was heated at 95°C for 5 minutes. To assess 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 treatment with all tested concentrations of MMP-2. This was indicated by a size change and the appearance of a clear band of ~9 KDa in the SDS page gel ( Figure 9 ), the sharp band of ~9 KDa is the IL-15RαSushi+ domain cleaved from P-0315.

[0355] The cleavage of uPA was assessed using P-0203. First, different amounts of uPA were added to 2 μg of P-0203 in 20 μl PBS pH 7.2 buffer, and the reaction mixture was incubated at 37°C for 2 hours. The cleavage performed with 0, 25 ng, 50 ng, 100 ng and 300 ng of uPA is illustrated in Figure 10A. The three arrows in Figure 10A represent non-reduced (NR) samples and indicate the changes in the Fc chain as uPA proteolysis occurs. In "Partial cut", the IL-15 / IL-15RαSushi+ fusion polypeptide is cleaved from only one of the two Fc chains, while in "Full cut", the IL-15 / IL-15RαSushi+ fusion polypeptide is cleaved from both Fc chains. The fuzzy bands circled in Figure 10A are the IL-15 / IL-15RαSushi+ fusion polypeptides cleaved from the Fc, and this fuzzy appearance is likely due to glycosylation. In the reduced (R) sample, the upper band is the Fc chain attached to the IL-15 / IL-15RαSushi+ fusion polypeptide, and the lower clear band is the Fc chain from which the IL-15 / IL-15RαSushi+ fusion polypeptide has been cleaved.

[0356] The SDS-PAGE gel clearly shows that, along with the increase of uPA amount, the amount of protein that is fully cut in non-reduced sample increases gradually. Similarly, the amount of the Fc chain of cracking in the reduced sample increases, indicating that the level of cracking increases. However, there is no condition to cause complete cracking. In order to achieve complete digestion, similar uPA digestion reactions are hatched for a longer time. Figure 10 B shows 2 μ g P-0203 with 50ng, 100ng and 300ng of uPA cracking for 24 hours at 37 ℃. Data show that 100ng uPA and 24 hours hatching cause almost complete cracking.

[0357] Example 5

[0358] Protease cleavage of Fc IL-15 VitoKine P-0203 to obtain activated IL-15 product

[0359] VitoKine P-0203 (SEQ ID NO: 29) contains a uPA substrate peptide linker (SEQ ID NO: 90) flanked by spacer peptides at both ends that connects Fc and IL-15, and a second 15-amino acid flexible linker (GGGGS) 3 (SEQ ID NO: 127) connects IL-15 and IL-15RαSushi+ domains. 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 PBS pH 7.2 buffer at 37°C for 24 hours. To stop the reaction, 25 μl of Ni-Excel resin (50% slurry balanced in PBS, GE Healthcare) was added to remove the 6-His-tagged uPA from the solution. At the same time, 50 μl of MabSelectSure protein A resin (50% slurry balanced in PBS, GE Healthcare) was added to the reaction to remove the cleaved Fc fraction and uncut or incompletely digested P-0203. After incubation with the two affinity resins at room temperature for 15 min, the resin was removed by centrifugation and the protein containing protease-activated P-0203, i.e., IL-15 / IL-15RαSushi+ fusion polypeptide (in Figure 2 As can be seen in Figures 11A and 11B, the activated P-0203 fragment migrates together with a fuzzy band that is likely due to glycosylation.

[0360] Example 6

[0361] Protease cleavage of Fc IL-15 VitoKine P-0315 to obtain activated IL-15 product

[0362] VitoKine P-0315 (SEQ ID NO: 33) contains a uPA substrate peptide linker (SEQ ID NO: 92) connecting the Fc and IL-15, and a second 10-amino acid MMP-2 / 9 cleavable linker (SEQ ID NO: 95) between the IL-15 and IL-15Rα Sushi+ domains. The IL-15 domain in P-0315 contains an S58D substitution to enhance binding to the receptor β subunit. Two activated forms of P-0315 are generated by protease digestion.

[0363] An activated form of P-0315 was obtained by in vitro protease cleavage using MMP-2 ( Figure 2 Schematically illustrated as active form 2 in FIG. 1 ). 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 added to P-0315 (80 μg) in 0.4 ml of the manufacturer's recommended assay buffer (100 mM Tris, 20 mM CaCl 2 , 300 mM NaCl, 0.1% (w / v) Brij 35, pH 7.5). After incubation at 37° C. for 3 hours, 50 μl of MabSelectSure Protein A resin (50% slurry balanced in PBS, GE Healthcare) was added to the reaction. The desired activated form 1 was eluted with 25 mM sodium citrate, 25 mM sodium chloride, pH 3.2. The protein was neutralized by adding 3% 1 M Tris, pH 10.2. To assess cleavage, samples were separated on 4%-12% Tris-Bis SDS-PAGE gels (Figure 12A). Lane 1 shows P-0315 before MMP-2 digestion in the presence of a reducing agent, and lanes 2 and 3 are non-reduced and reduced P-0315 after MMP-2 proteolysis but before protein A purification. The IL-15Rα-sushi+ domain appears as a sharp band at 9 KDa on the gel, confirming efficient cleavage of MMP-2 at the MMP-2 / 9 substrate peptide linker. After protein A purification, samples (lanes 4 and 5) showed the same migration pattern. This data indicates that the IL-15RαSushi+ domain released from the covalent linkage remains non-covalently associated with IL-15, which is fused to Fc, as shown in Figure 12A. Figure 2 This association is strong enough to withstand the low pH conditions during elution of Protein A. Figure 12B further illustrates the two non-covalently associated components of this activated form.

[0364] Another activated form of P-0315 was obtained by proteolytic cleavage of P-0315 by both uPA and MMP-2 ( Figure 2 Schematically illustrated as active form 3 in Figure 3). Briefly, 100 μg of P-0315 was incubated with 5 μg of ... Ni-Excel resin (50 μl of a 50% slurry equilibrated in PBS, GE Healthcare) was added to remove His-tagged MMP-2 and uPA from the solution. Simultaneously, 100 μl of MabSelectSure Protein A resin (50% slurry equilibrated in PBS, GE Healthcare) was added to the reaction to remove the cleaved Fc fraction and the remaining uncleaved or incompletely digested P-0315. After incubation with both affinity resins for 15 min at room temperature, the resin was removed by centrifugation and the slurry containing the His-tagged MMP-2 and uPA was recovered. Figure 2 12C , active form 3 of P-0315 contained a non-covalent complex of IL-15 / IL-15RαSushi+ as expected from a dual proteolytic reaction; IL-15 migrated as a fuzzy band, while IL-15RαSushi+ was a sharp band at ˜9 kDa, as observed in active form 2 ( FIG12B ).

[0365] Example 7

[0366] Activity Assessment of Protease-Activated Fc IL-15 VitoKine by Human PBMC Assay

[0367] As detailed in Example 2, FACS analysis of the activation marker CD69 of immune cell subsets from fresh human PBMCs was performed to assess the activity of protease-activated IL-15 VitoKine. P-0203 and its corresponding activated form produced by uPA digestion (P-0203 activated form; in Figure 2 Schematically illustrated as active form 1) in comparison, Figures 13A-13BThe activity of VitoKine before protease activation was approximately 3 logs lower than that of the highly active IL-15 / IL-15RαFc fusion protein P-0165, which is consistent with the activity of VitoKine described in Example 3. uPA digestion significantly restored the potency of activating both CD56+ NK cells (Figure 13A) and CD8+ T cells (Figure 13B), but was still significantly lower than that of P-0165, which may be due to the covalent linkage of the IL-15 and IL-15α domains. Increasing the length of the flexible linker connecting IL-15 and IL-15α is expected to enhance the potency of the activated form. Paradoxically, extending the linker length may also reduce the activity shielding efficiency of the D3 domain and thus lead to VitoKine constructs with higher basal activity.

[0368] The bioactivity of another IL-15Fc, VitoKine P-0315, and its two activated forms were evaluated by measuring CD69 activation in immune cell subsets derived from fresh human PBMCs. Figures 14A-14B As observed in the , the activity of uncleaved P-0315 was barely measurable, confirming the effective shielding of the active moiety in the VitoKine format. Figure 2 As described in, active form 2 of P-0315 comprises Fc-fused IL-15 non-covalently complexed with the IL-15RαSushi+ domain released by MMP-2 cleavage; active form 2 of P-0315 is structurally similar to the positive control P-0313, a highly potent IL-15IL-15RαFc fusion protein. Figure 2 As depicted in Figure 14 , active form 3 of P-0315 comprises a free IL-15 domain cleaved from the Fc domain by uPA and an IL-15RαSushi+ domain released by MMP-2 cleavage, the two forming a non-covalent complex. Both activated forms of P-0315 showed complete or near-complete potency restoration in activating both CD56+ NK cells (Figure 14A) and CCD8+ T cells (Figure 14B); active form 3 was moderately more active than active form 2. The lack of the Fc domain in active form 3 can be beneficial when transient activation of the intended pathway in the tumor microenvironment is desired.

[0369] The activity of P-0315 before and after MMP-2 proteolysis was also studied by measuring the Ki67 expression in the nuclei of NK cells (Figure 15 A) and CD8+T cells (Figure 15 B) after treatment. P-0351, which comprises two non-cleavable flexible joints, was included for comparison. These data further demonstrate the activity inertness of VitoKine and the recovery of about 3 logarithms of efficacy in both NK cells and CD8+T cells after in vitro proteolytic activation. The observation that P-0351 and P-0315 have the same activity shows that the two cleavable joints in P-0315 remain intact during production, expression and storage, and are specific for their respective proteases.

[0370] In conclusion, cleavage of IL-15VitoKine P-0315 by MMP-2 / 9 and / or uPA results in activation of the molecule, and cytokine activity is restored to levels similar to that of the highly active IL-15 compound P-0313, with an EC in the subnanomolar range. 50 .

[0371] Example 8

[0372] Fc IL-15VitoKine has minimal systemic cytokine effects in healthy mice

[0373] The goal of the VitoKine platform technology is to reduce systemic on-target toxicity and enhance the therapeutic window. VitoKine masks active cytokines in an inert state and prevents active cytokines from binding to receptors in the periphery or on the cell surface of non-lesional cells. Therefore, the VitoKine platform limits the overactivation of cytokine pathways and reduces undesirable "out-of-tissue" "on-target" toxicity. It is intended that VitoKine be locally activated by proteases that are upregulated in diseased tissues. To evaluate this hypothesis, protease-cleavable and non-cleavable VitoKine were administered to healthy mice and compared with the highly active IL-15Fc fusion protein to evaluate their systemic cytokine effects.

[0374] P-0313 (SEQ ID NOs: 47 and 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 Fc IL-15 VitoKine containing two protease-cleavable linkers. P-0351 (SEQ ID NO: 25) is an Fc IL-15 VitoKine containing two non-cleavable linkers. Vehicle (PBS) was included as a negative control. The compound was administered as a single ip injection to healthy BALB / c mice (8-10 weeks old, n=6 / group) at doses of 0.1 mg / kg and 0.3 mg / kg. Blood samples were collected before dosing (day -1) or on days 3, 5, and 7 after dosing for immunophenotyping.

[0375] After lysing red blood cells with BD pharm lysis buffer, total viable mononuclear blood cells were counted by trypan blue dead cell exclusion. After blocking Fc receptors with purified anti-mouse CD16 / CD32 (1:50 dilution), cells were stained with anti-mouse CD3-FITC, anti-mouse CD49b-APC, and anti-mouse CD8-Percpcy5.5 (1:50 dilution). After incubation for 30 minutes, cells were collected and washed, resuspended in FACS buffer, and analyzed by flow cytometry.

[0376] like Figures 16A-16C As shown in Figure 16A, the fully active IL-15Fc fusion protein P-0313 significantly expanded peripheral blood cytotoxic CD8+ T cells (Figure 16A), NK cells (Figure 16B) and total leukocytes (Figure 16C) in a dose-dependent manner at two test doses. Cell expansion was observed on the 3rd day, peaked on the 5th day, and returned to near baseline on the 7th day. In contrast, neither cleavable (P-0315) nor non-cleavable (P-0351) VitoKine showed an increase in CD8 T cells throughout the 7-day study. A slight and delayed increase in NK cell expansion was observed in mice treated with high-dose cleavable VitoKine P-0315. P-0351 and low-dose P-0315 did not show signs of increase in any target cell population tested. Overall, both tested VitoKines showed minimal systemic activation and expansion of targeted lymphocyte populations compared to the active molecule P-0313, and demonstrated successful masking and delay of IL-15 activity in the periphery.

[0377] Example 9

[0378] Inhibition of colon cancer cell lung metastasis by Fc IL-15 VitoKine in mice

[0379] The anti-metastatic efficacy and immune response of the IL-15Fc VitoKine molecule were investigated in the mouse CT26 lung metastasis model. Briefly, 1x10 5 Mouse colon cancer cells CT26-WT (ATCC CRL-2638) were injected intravenously into female Balb / C mice (9-11 weeks old). Four Q5D treatments were started via intraperitoneal injection the next day (day 1). The treatment group (6 in total, n=7 / group) included 0.3 mg / kg P-0315, 0.3 mg / kg P-0351, and 0.1 mg / kg P-0313. P-0315 (SEQ ID NO: 33) is an Fc IL-15 VitoKine containing two protease-cleavable linkers. P-0351 (SEQ ID NO: 25) is an uncleaved Fc IL-15 VitoKine. P-0313 (SEQ ID NO: 47 and 5) is a fully active IL-15 / IL-15RαFc fusion molecule. Vehicle (PBS) was included as a negative control. On day 17, all mice were sacrificed for tissue harvest. Lungs were inflated with 15% India ink and destained in Fekete solution (10% formaldehyde, 5% glacial acetic acid and 60% ethanol).Lung tumor nodules were counted and the anti-metastatic effect was expressed as the difference in the number of tumor nodules between the treatment groups and the vehicle control.

[0380] like Figure 17 As described in, P-0313 has a significant effect in inhibiting the formation and growth of lung metastases. At 0.1 mg / kg, P-0313 treatment resulted in near-complete inhibition of lung metastases. The cleavable VitoKine P-0315 demonstrated 70% inhibition of lung nodule development; the anti-metastatic efficacy of all three doses (0.3 mg / kg, 1 mg / kg, or 3 mg / kg) was comparable. The non-cleavable VitoKine P-0351 demonstrated a relatively weaker but still significant effect in reducing metastatic development, suggesting some inherent basal activity at high doses. However, P-0315 demonstrated significantly superior anti-metastatic efficacy to P-0351 (p<0.05; Figure 17 ), suggesting that proteolytic cleavage of one or both linkers in P-0315 and subsequent release of the active form of IL-15 may contribute to the superior in vivo efficacy of P-0315 over P-0351. The development of tumor metastasis can lead to increased proteolytic activity near the tumor microenvironment.

[0381] On day 15 (4 days after the third treatment), the immune response after IL-15 compound treatment was investigated by flow cytometric analysis of mouse peripheral blood. Compared to controls, an expansion of CD8+ T cells was observed in mice treated with active IL-15Fc fusion protein P-0313, but not in mice treated with cleavable VitoKine P-0315 or non-cleavable VitoKine P-0351, indicating that the anti-colon cancer metastasis efficacy of VitoKine was observed without causing an increase in systemic CD8+ T cells. Figure 17 and Figure 18A). However, after repeated administration, peripheral blood NK cells increased in all three IL-15 compound-treated groups, with the most significant increase in the non-cleavable VitoKine group (Figure 18B). In the VitoKine-treated group, the increase in the systemic expansion of NK cells rather than CD8+T cells indicated that NK cells were more sensitive to IL-15 treatment than CD8+T cells, and that the inherent basal activity of VitoKine may lead to NK cell expansion. Therefore, it is crucial to adjust the dosage concentration of IL-15VitoKine to reduce residual systemic effects. The significant increase in NK cells in the P-0351 group also indicates that the low-efficacy non-cleavable VitoKine can weakly but continuously activate this pathway and lead to a long-term immune response.

[0382] Example 10

[0383] Fc IL-15VitoKine P-0315 inhibits established CT26 tumor growth in mice with minimal systemic cytokine activation

[0384] The antitumor efficacy and immune response of Fc IL-15VitoKine P-0315 compared to the fully active IL-15 / IL-15Rα-Fc fusion protein P-0313 were investigated in the CT26 murine colorectal cancer tumor model. Briefly, female Balb / C mice (10-12 weeks old) were injected subcutaneously in the right flank with 1×10 5 On day 11, when the average tumor volume was 70 mm 3 At 4 hr, mice were randomly divided into three groups (n=11 / group) and received an intraperitoneal injection of 0.1 mg / kg of vehicle (PBS) or P-0315 or P-0313 on the same day of randomization. An additional intraperitoneal injection of each test agent was performed on day 16 (2×Q5D). Tumors were measured three times a week using a caliper, and tumor volume was calculated as follows: volume=0.5×(width) 2x (length). To study immune responses, non-terminal peripheral blood was collected in heparin-treated tubes on day 19. On day 21, all mice were sacrificed for tissue harvest.

[0385] As shown in Figure 19A, PBS-treated mice rapidly developed large subcutaneous tumors, while mice treated with P-0315 or P-0313 were roughly equivalent in delaying tumor growth (Figures 19B and 19C). On day 21 after tumor inoculation, the average tumor volume in control-treated mice was ∼1000 mm 3 , compared to an average tumor volume of ∼450 mm in mice treated with P-0315 or P-0313. 3 (****, P < 0.0001; one-way ANOVA with Tukey post hoc test) Figure 19D Notably, P-0313 initially showed a greater reduction in tumor burden than P-0315, but the difference tapered off as treatment progressed. The delayed antitumor effect of P-0315 may be due to the time it takes for the appropriate amount of protease to develop access to and cleave the substrate peptide linker and activate VitoKine.

[0386] Next, the effect of P-0315 on the proliferation of CD8+ T cells and NK cells in peripheral blood was studied by flow cytometry compared to P-0313 and vehicle. Similar assessments were performed on the effect of P-0315 on the total WBC population and lymphocyte subsets (CD8+ T cells and NK cells) in the periphery and spleen.

[0387] Injection of fully active IL-15 / IL-15RαFc fusion protein P-0313 into tumor-bearing mice induced significant lymphocyte proliferation and expansion in both peripheral blood and spleen ( Figures 20A-20B 、 Figures 21A-21C 、 Figures 22A-22C). Compared with the PBS group, after P-0313 treatment, Ki67 proliferation in peripheral NK cells increased to 4 times (61% vs. 15%; Figure 20A), and Ki67 proliferation in CD8+ cells increased to 5.3 times (46% vs. 8.6%; Figure 20B). Similarly, P-0313 treatment resulted in significant cell expansion of total leukocytes, NK cells, and CD8+T cells in peripheral blood (Figures 21A-21C) and spleen (Figures 22A-22C). For example, it was observed that the number of total peripheral WCB cells expanded to 6 times, and the number of CD8+T cells expanded to 5 times; the number of NK cells increased significantly to 85 times. In the spleen, the most obvious cell expansion of NK cells was also observed (10 times), followed by CD8+T cells, which expanded to 2.9 times. Total spleen WBC expanded moderately to 1.7 times. The robust activation of cytotoxic CD8+ T cells and NK cells is consistent with the overall immunomodulatory properties of IL-15, and an effective immune response may be the main contributor to the antitumor activity of P-0313 in vivo. However, significantly altered lymphocyte subsets in the blood may cause toxicity and reduce the therapeutic index.

[0388] In stark contrast to P-0313, treatment with FcIL-15VitoKine P-0315 resulted in minimal alterations in the homeostasis of lymphocyte subsets in the blood. Figures 20A-20B The results of Ki67 proliferation observation of peripheral NK cells and CD8+ T cells were shown in Figures 21A-21C The results of the observation of cell expansion of total leukocytes, NK cells and CD8+ T cells in peripheral blood are shown in Figure 22. The only notable immunopharmacodynamic effect after P-0315 treatment was a 4-fold increase in the number of NK cells in the spleen (Figure 22B). Since P-0315 was roughly equivalent to P-0313 in delaying the growth of established CT26 tumors (Figure 19A- Figure 19D ), therefore, the in vivo anti-tumor activity of P-0315 is likely due to the proteolysis and subsequent activation of the cleavable linker of VitoKine near the tumor microenvironment. Because activated VitoKine is only present near the tumor, the response of peripheral lymphocytes to the administration of the inert VitoKine molecule is far less pronounced than that of the fully active P-0313.

[0389] In summary, IL-15Fc VitoKine, exemplified by P-0315, effectively delayed tumor growth without significantly altering the proliferation and expansion of lymphocyte subsets in the blood and spleen. Therefore, the overactivation of pathways, undesirable off-tissue and on-target toxicities, and undesirable target silencing typically associated with fully active cytokines can be prevented or reduced by the VitoKine format without compromising anti-tumor effects.

[0390] Example 11

[0391] Uncleavable VitoKine as a reduced-potency form of cytokine

[0392] It is known in the art that cytokines that are effective in vitro may not elicit the strongest lymphocyte response in vivo. Highly effective cytokines are often associated with stronger receptor stimulation, internalization and desensitization, reduced signaling, proliferation and function, and increased cell death or clonal depletion. Therefore, there may be a great need for cytokines with reduced potency to prevent overly potent lymphocyte activation and achieve sustained and enhanced pharmacodynamic effects and anti-tumor efficacy in vivo.

[0393] In vitro, the non-cleavable Fc IL-15 VitoKine P-0351 exhibited significantly reduced potency compared to the fully active IL-15 compound, but P-0351 showed anti-metastatic efficacy and significant NK cell responses in the mouse CT26 lung metastasis model (Example 8). Therefore, the non-cleavable VitoKine construct can be used as a reduced potency cytokine with sustained activity to optimize in vivo pharmacodynamics.

[0394] In terms of inducing Ki67 proliferation in both NK cells and CD8+ T cells (Figures 23A and 23B), P-0351 showed the same potency as the benchmark molecule (SEQ ID NOs: 177 and 178), which is equivalent to XENP024306 in patent application WO2018071919A1. XENP024306 is an IL-15 / IL-15RαFc fusion molecule containing amino acid substitutions (D30N / E64Q / N65D) in IL-15 and half-life extension mutations in the Fc. The triple mutation in the IL-15 chain of XENP024306 reportedly resulted in a 200-fold reduction in potency in vitro, but XENP024306 was shown to be more active in vivo, likely due to optimized in vivo pharmacodynamics.

[0395] Likewise, by avoiding or reducing the overactivation and undesirable target silencing typically associated with fully active cytokines, the attenuation of the potency of P-0351 is expected to result in more sustained exposure to improve pharmacodynamics (PD). Thus, P-0651 (SEQ ID NO: 170), the half-life-extended counterpart of P-0351, could promote a longer half-life and further extend PD in vivo.

[0396] Example 12

[0397] Construction and production of Fc IL-2 VitoKine (Treg IL-2 VitoKine) for selective expansion of regulatory T cells for the treatment of autoimmune diseases, inflammatory disorders, transplantation, and other disorders

[0398] The goal is to design IL-2VitoKine constructs that will remain inert until locally activated by proteases upregulated at sites of inflammation. Low-dose wild-type IL-2 that preferentially stimulates Tregs compared to effector T cells and IL-2 muteins with reduced binding affinity to IL-2Rβ have been reported to expand the selectivity window. These molecules could be developed as therapeutic agents for the prevention of autoimmune diseases. Interference with IL-2Rβ and / orγ C Additional mutations that combine but do not affect the interaction with IL-2Rα could also expand the selectivity window for Treg activation over Teff.

[0399] IL-2Fc VitoKine comprises wild-type IL-2 or an IL-2 mutant with increased selectivity for stimulating Tregs over effector T cells as the active moiety, which is reversibly shielded between the Fc domain and IL-2RαSushi (SEQ ID NO: 10). IL-2Rα (SEQ ID NO: 9) comprises two sushi domains separated by a native peptide linker region. The IL-2VitoKine construct comprises one or two cleavable linkers recognized by proteases that are reported to be upregulated at sites of inflammatory disorders. While the linker connecting the Fc and IL-2 / mutant can be either cleavable or non-cleavable, the linker connecting IL-2 and IL-2RαSushi is preferably capable of being specifically cleaved by a protease.

[0400] The activity of IL-2 mutant proteins that selectively stimulate Tregs is expected to be restored after protease cleavage, release of IL-2Rα from IL-2, and diffusion away. Due to the nM binding affinity between IL-2Rα and IL-2, it is possible that IL-2RαSushi remains non-covalently associated with IL-2 after linker cleavage; therefore, the interaction of IL-2 with IL-2Rα on Treg cells remains blocked. To address this potential problem, IL-2Rα mutant proteins with amino acid substitutions at the interface with IL-2 were designed to weaken their binding to IL-2. Therefore, after protease cleavage of the linker, the IL-2RαSushi mutant can dissociate and then diffuse away from IL-2, which is a similar phenomenon to that of IL-2RαSushi. Figure 2 A slightly different activation mechanism is described in Figure 26 Schematically illustrated in ).

[0401] Representative amino acid substitutions were made at position 38 (i.e., K38E) and position 43 (i.e., Y43A) of the IL-2Rα domain. Other IL-2Rα variants with substitutions on residues that interact with IL-2 are expected to disrupt the interaction between IL-2 and IL-2Rα and may also be incorporated. As will be appreciated by those skilled in the art, all mutations can be optionally and independently combined in any manner to achieve optimal affinity regulation. IL-2VitoKine molecules comprising different linker compositions, wild-type or variant IL-2 and wild-type or variant IL-2RαSushi were generated, and their respective sequences are listed in SEQ ID NOs: 49-65.

[0402] Gene synthesis, expression vector construction, and protein production, purification, and characterization followed the same procedures detailed in Example 1. As an example of the protein profile of IL-2 VitoKine, an SDS-PAGE analysis of P-0320 is shown in FIG24A. The size exclusion chromatogram in FIG24B shows that after the initial protein A capture step without a polishing step, there is <5% aggregation. The low aggregation tendency indicates that IL-2 VitoKine has a favorable developability profile.

[0403] Example 13

[0404] Fc IL-2 VitoKine for the selective expansion of effector T cells for the treatment of cancer and other disorders

[0405] Construction and production of (Teff IL-2VitoKine)

[0406] The goal is to design an IL-2VitoKine construct that will remain inert until locally activated by a protease that is present or upregulated only at the tumor site. The preferential amplification of IL-2 to Treg represents the undesirable effect of IL-2 on cancer immunotherapy because Treg can suppress effector T cell responses. In order to overcome these limitations, IL-2 is designed to have amino acid substitutions at the interface bound to IL-2Rα, including F42A and R38E (PNAS, 1991.88: 4636-4640), to reduce / eliminate binding to IL-2Rα. Other mutations that only interfere with binding to IL-2Rα without affecting interaction with IL-2Rβγ, such as R38A, T41A, T41G, T41V, Y107G, Y107H, Y107L or Y107V, can also be incorporated. As will be understood by those skilled in the art, all mutations can be optionally and independently combined in any way to achieve optimal affinity regulation.

[0407] The Fc IL-2VitoKine construct comprises wild-type IL-2 or an IL-2 variant with reduced / eliminated binding to IL-2Rα as the active portion, which is reversibly shielded between the Fc domain and IL-2RαSushi (SEQ ID NO: 10). These constructs contain one or two cleavable linkers recognized by proteases that are reported to be upregulated in various types of cancers, such as solid tumors. Although the linker connecting the Fc and IL-2 / mutant protein can be either cleavable or non-cleavable, the linker connecting IL-2 and IL-2RαSushi is preferably capable of being specifically cleaved by the protease. After cleavage, IL-2Rα can preferably associate with IL-2 to increase selectivity for Teff function. After protease cleavage, IL-2Rα is released from IL-2 and diffuses away, and the activity of the IL-2 mutant protein is restored. Figure 1 IL-2 VitoKine molecules incorporating different IL-2 muteins as active moieties are schematically depicted in FIG. Exemplary Fc IL-2 VitoKine molecules for selective expansion of Teff cells were constructed and produced, and their respective sequences are listed in SEQ ID NOs: 59-61.

[0408] Gene synthesis, expression vector construction, and protein production, purification, and characterization followed the same procedures as detailed in Example 1.

[0409] Example 14

[0410] In vitro activity evaluation of Fc IL-2 VitoKine

[0411] The biological activity of IL-2VitoKine on T cells was determined by measuring the levels of phosphorylated STAT5 (pStat5) in specific T cell subsets in fresh human PBMCs. Stat5 is known to be involved in the binding of IL-2 to the transmembrane IL-2Rβγ. C The downstream intracellular signaling induced by the complex was measured by flow cytometry using an antibody against the pStat5 peptide in fixed and permeabilized cells. Briefly, human PBMCs were isolated from buffy coats of healthy donors purchased from the Oklahoma Blood Institute by Ficoll-Hypaque centrifugation. 2 × 10 5Each PBMC was treated with a serial dilution of the test compound for 30 minutes. The cells were then treated with Foxp3 / transcription factor staining buffer (EBIO) according to the manufacturer's instructions. The cells were then fixed with Cytofix buffer and permeabilized with Perm buffer III (BD Biosciences) and then washed. After blocking Fc receptors 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, anti-FOXP3-APC, anti-pSTAT5-FITC and anti-CD4-PerCP-Cy5.5 antibodies at a concentration recommended by the manufacturer. The cells were then collected and washed, resuspended in FACS buffer, and analyzed by flow cytometry. The flow cytometry data of Treg and CD4 effector T cell subsets were gated to Foxp3+ / CD25 respectively. 高 and Foxp3- / CD25 低 Data are expressed as the percentage of pStat5-positive cells in the gated population.

[0412] The pStat5 activation of IL-2 VitoKine 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 the wild-type IL-2 domain fused to the Fc domain at its N-terminus via a uPA-cleavable linker and connected to the IL-2RαSushi domain at its C-terminus via a flexible (GGGGS)3 (SEQ ID NO: 127) linker. P-0329 contains the wild-type IL-2 domain fused to the Fc domain at its C-terminus via a uPA-cleavable linker and connected to the IL-2RαSushi domain at its N-terminus via a flexible (GGGGS)3 linker. P-0250 is a highly active IL-2 Fc fusion protein. For the compounds tested, the percentage of pStat5-positive cells in Treg and CD4+ conventional T cell (Tconv) subsets was Figures 25A-25B It was clearly observed that pStat5 activation in Tregs was significantly reduced for both IL-2 VitoKines compared to the fully active IL-2 fusion protein, and pStat5 activation in CD4+ Tconv cells was barely measurable. These data clearly demonstrate the effective masking of IL-2 activity in the VitoKine format.

[0413] Example 15

[0414] Protease Activation and In Vitro Activity Assessment of IL-2VitoKine

[0415] IL-2VitoKine P-0382 (SEQ ID NO: 51) contains a flexible GGGSGGGS linker (SEQ ID NO: 115) connecting the Fc and IL-2, and a 10-amino acid MMP-2 / 9 cleavable linker (SEQ ID NO: 77) between IL-2 and the IL-2RαSushi domain. The IL-2RαSushi domain in P-0382 contains an amino acid substitution (K38E) designed to reduce the binding affinity of the IL-2RαSushi domain to IL-2, ensuring that the IL-2RαSushi domain dissociates after protease cleavage of the linker and subsequently diffuses away from IL-2.

[0416] 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 buffer exchanged and added to 120 μg of P-0382 in 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 20 hrs, half of the reaction was purified with MabSelectSure Protein A resin and the activated VitoKine was eluted with 25 mM sodium citrate, 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 activated VitoKine was collected by removing the Ni resin via centrifugation. Protein A purification was performed to confirm that the IL-2Rα Sushi domain was not non-covalently associated with IL-2 after cleavage from the polypeptide chain, as shown in Figure 2. Figure 26 Schematically illustrated. Figures 27A-27B The samples were evaluated on a 4%-12% Tris-Bis SDS-PAGE as shown in Figure 27 . Despite the increased amount of protease and the prolonged reaction time compared to the structurally similar IL-15 VitoKine (e.g., P-0315), the reaction did not result in complete cleavage. Comparison of MMP-2 treated samples with and without Protein A purification ( Figures 27A and 27B ) indeed confirmed that the IL-2Rα Sushi domain was released from the covalent linkage and did not co-purify with the Fc-IL-2 fusion polypeptide.

[0417] Although cleavage was incomplete, two MMP-2 activated samples, one Ni-Excel flow-through (active form 1) and the other Protein A eluate (active form 2), were evaluated in the pStat5 activation assay described in Example 13, and the data are presented in Table 1. Figures 28A-28B P-0382 showed very low activity in Treg cells and was barely measurable against CD4+ Tconv cells, again demonstrating effective masking of the active moiety in the IL-2 VitoKine format. Both activated samples showed nearly complete restoration of activity. Its potency was slightly lower than that of P-0250, likely due to incomplete proteolysis.

[0418] The presence of the MMP-2-cleaved IL-2RαSushi domain in the active form 1 sample did not appear to alter the activity of the activated IL-2 VitoKine, as active forms 1 and 2 were comparable in inducing pStat5 phosphorylation in both Treg and Tconv cells (Figures 28A and 28B). The data indicate that the IL-2RαSushi domain generated by MMP-2 cleavage does not associate with IL-2 and should not interfere with the binding of IL-2 to the receptor complex expressed on lymphocytes.

[0419] MMP-2 proteolysis of P-0382 did not result in complete cleavage, and it was hypothesized that the extension of the cleavable linker could make the substrate peptide more accessible to the protease responsible for cleavage. The 10-amino acid linker in P-0382 (SEQ ID NO: 95) was replaced with a 15-amino acid MMP-2 / 9-cleavable linker (SEQ ID NO: 94) containing additional flanking residues, and a new VitoKine construct, P-0398 (SEQ ID NO: 52), was generated. P-0398 was activated by in vitro protease cleavage using MMP-2, following the same protocol detailed above. Threefold lower amounts of MMP-2 (1.5 μg MMP-2 for 180 μg P-0398 and 3.3 μg MMP-2 for 120 μg P-0382) resulted in complete digestion of P-0398, as evidenced by the presence of only a "completely cleaved" band on an SDS-PAGE gel (data not shown).

[0420] The biological activity of the activated P-0398 of IL-2RαSushi domain removed by protein A purification was determined in pStat5 assay (Figure 29 A and Figure 29 B). Activated P-0398 is similar to IL-2Fc fusion molecule P-0250 in sequence and structure, and they have almost the same effect in inducing Stat5 phosphorylation in both Treg cells and Tconv cells. Although two kinds of VitoKine (P-0382 and P-0398) have significantly impaired biological activity (4 logarithms) due to the covalent connection with IL-2RαSushi domain, there seems to be a trend that the P-0398 comprising a longer L2 joint is more active. Similar to the observations of IL-15Fc VitoKine, the active inert level of IL-2VitoKine can be further adjusted by adjusting the L2 joint length. Likewise, the choice of cleavable L2 linker length and sequence should be balanced between the presence of a specific protease at the site of intended disease indication, the accessibility of the substrate peptide to the protease, and the desired rate of proteolysis.

[0421] In summary, compared to IL-15 VitoKine, IL-2 VitoKine requires a longer L2 linker for optimal enzymatic accessibility to achieve complete proteolysis. MMP-2 cleavage of exemplary IL-2 VitoKine constructs P-0382 and P-0398 results in full activation of the molecule. Activated IL-2 VitoKine achieves similar biological activity to the highly active IL-2 Fc fusion compound P-0250.

[0422] Example 16

[0423] Construction of antibody VitoKine

[0424] The use of recombinant antibody-cytokine fusion proteins (immunocytokines) is expected to improve the therapeutic index of cytokines by targeting cytokines to disease sites. However, fusing fully active cytokines to antibodies can lead to peripheral activation and lack of tumor targeting. The inertness of VitoKine's activity before activation at the intended treatment site makes the antibody VitoKine a new and innovative form of immunocytokine. In addition to tumor-targeted antibodies, immune checkpoint blocking antibodies that bypass the immunosuppressive effects in the tumor microenvironment or immunostimulatory antibodies that enhance existing responses can also be used to construct the antibody VitoKine, which can lead to further enhancement of the anti-tumor activity of the immune system. In addition, antibody VitoKine targeting inflammatory problem sites can be used to treat anti-autoimmune and chronic inflammatory disorders.

[0425] Following this concept, an antibody VitoKine protein containing IL-15 or IL-2 as the D2 domain was constructed. Exemplary antibodies include PD-1 blocking antibody JS-001, PD-L1 blocking antibody Tecentriq, anti-CTLA4 antibody ipilimumab (ipilimumab), agonist CD40 antibody RO7009789, tumor antigen targeting antibodies (including L19 for the extracellular domain of fibronectin, rituximab for CD20, Herceptin for Her-2, cetuximab for EGFR) and anti-inflammatory antibodies (Vedolizumab for integrin α4β7 and Humira for TNFα). The sequence of the exemplary antibody VitoKine is listed in SEQ ID NO: 128-143.

[0426] Gene synthesis, expression vector construction, and protein production, purification, and characterization followed the same procedures detailed in Example 1. The bioactivity of the exemplary anti-PDL1 antibody IL-15 VitoKine P-0485 (SEQ ID NOs: 180 and 181) was tested by measuring Ki67 expression in NK cells ( FIG. 30A ) and CD8+ T cells ( FIG. 30B ) after treatment of human PBMCs with the IL-15 VitoKine compound. P-0485 shares the same L1 and L2 linkers and D2 and D3 domains with its Fc VitoKine counterpart, P-0315. Figures 29A-29B The data show that Figures 15A-15B Both VitoKines had comparable and severely impaired bioactivity compared to activated P-0315 as shown in Figure 2. P-0485 appeared to have slightly higher potency, which may be due to lymphocyte activation caused by PD-L1 blockade.

[0427] Example 17

[0428] Generation of protease-activatable inert IL-15 or IL-2 fusion proteins based on IL-15Rβ blocking peptides

[0429] A different approach to producing protease-activatable inert IL-15 or IL-2 fusion proteins is to genetically fuse a blocking peptide (e.g., one based on IL-2Rβ) to IL-15 or IL-2 via a cleavable linker. The blocking peptides explored are based on two IL-2Rβ loops (SEQ ID NOs: 97 and 98) containing key residues that make direct contact with IL-15. The peptides listed in Table 13 are based on the sequences of these two loops.

[0430] Table 13

[0431]

[0432] The five peptides L01 to L05 (SEQ ID NO: 97-101) in Table 13 were synthesized and their binding to IL-15 was evaluated in an ELISA format. Briefly, IL-15 / IL-15RαSushi+Fc fusion protein P-0153 (SEQ ID NO: 44 and 46) was coated on the wells of a Nunc Maxisorp 96-well microplate at 1 μg / well, and a 3-fold serial dilution of biotinylated peptide starting from 100 μM was added to each well. Streptavidin-HRP complex was added at the concentration recommended by the manufacturer, and the signal was developed by TMB substrate. Figure 31 As depicted in , specific binding was observed for L03 (SEQ ID NO: 99), which is the cyclized loop 2 (SEQ ID NO: 98).

[0433] A loop 2-based sequence was used as a blocking peptide and incorporated into an IL-15 fusion protein. Exemplary sequences of fusion proteins comprising an IL-2Rβ-based blocking peptide fused to IL-15 via a cleavable linker and a peptide spacer (SEQ NO ID: 102-106) are shown in Table 13, where bold represents an IL-15Rβ-based blocking peptide, a wavy underline represents a cleavable linker, and a straight underline represents a spacer peptide. IL-15αSushi+ (SEQ ID NO: 5) was co-expressed with an IL-15 fusion protein containing a blocking peptide and formed a non-covalent complex.

[0434] Gene synthesis, expression vector construction, and protein production, purification, and characterization followed the same procedures detailed in Example 1. These IL-15 fusion proteins containing blocking peptides were first tested in an ELISA assay to assess their ability to bind to IL-2Rβ. Figure 32 As described in , there was a moderate decrease in binding affinity compared to P-0153 due to the attachment of different blocking peptides. However, PBMC assays evaluating the activation of immune cells (including CD56+ NK cells or CD8+ T cells) by these IL-15 fusion proteins containing blocking peptides did not show a significant decrease in activity (data not shown), indicating that the shielding efficiency of the tested blocking peptides was insufficient. Blocking peptides of different lengths can be explored, including the entire extracellular domain of IL-2Rβ, to observe the efficiency of activity shielding. The same approach can be applied to IL-2 in a similar manner.

[0435] Example 18

[0436] VitoKine format improves developability of fusion proteins

[0437] As is known in the art, naturally occurring IL-2 proteins tend to be less stable and prone to aggregation. This was demonstrated in our experiments, where wild-type IL-2 Fc fusion protein (P-0250) was expressed at low levels (transiently expressed in HEK-293F cells at approximately 3 mg / L) and had a high tendency to aggregate, as illustrated by the SEC chromatogram depicted in Figure 33A. Four IL-2 VitoKine molecules, P-0320, P-0382, P-0362, and P-0379, were compared to P-0250. P-0320 (SEQ ID NO: 49) comprises a wild-type IL-2 domain, the N-terminus of which is fused to the Fc domain, and the C-terminus of which is connected to the IL-2RαSushi domain. The L1 linker connecting Fc and IL-2 is a cleavable linker containing the uPA substrate peptide and flanking spacer peptides (SEQ ID NO: 92), and the L2 linker between IL-2 and IL-2RαSushi is a flexible (GGGGS)3 linker (SEQ ID NO: 127). P-0382 (SEQ ID NO: 51) differs from P-0320 only in the linker sequence; the L1 linker of P-0382 is a flexible (G3S)2 linker (SEQ ID NO: 115), and the L2 linker is an MMP-2 / 9 cleavable linker (SEQ ID NO: 95). P-0362 (SEQ ID NO: 53) and P-0379 (SEQ ID NO: 59) differ from P-382 by a single point mutation. P-0362 contains a K38E mutation in the IL-2RαSushi domain, while P-0379 contains an F42A substitution in the IL-2 domain. P-0250 (SEQ ID NO: 48) is an IL-2Fc fusion protein in which IL-2 is fused to the C-terminus of Fc using a flexible (G3S)2 (SEQ ID NO: 115) linker.

[0438] The size exclusion plots for these five molecules are shown in Figures 33A-33E. It is very apparent from the chromatograms that all four IL-2 VitoKine constructs have significantly improved purity characteristics over the IL-2 Fc fusion protein. P-0250 contains over 25% of undesirable high molecular weight species. In contrast, all four VitoKine molecules show a sharp monomer peak with a monomer content of over 96%. There is no apparent effect of linker variation, mutations in IL-2 or IL-2RαSushi on quality. This significant increase in protein quality is clearly attributed to the fusion of the IL-2RαSushi domain in VitoKine.

[0439] In addition to protein quality, the expression level of IL-2 VitoKine was also improved, especially the VitoKine format with a GS linker between Fc and IL-2 and a 10-amino acid MMP-2 / 9 activatable linker between IL-2 and IL-2RαSushi. Although protein expression levels may vary between batches due to cell growth conditions, it is clear that VitoKine expression levels are consistently several times higher than IL-2Fc fusion protein. Table 14 lists protein expression titers (mg / L) and protein monomer percentages.

[0440] Table 14

[0441] Protein ID Expression titer (mg / L) Monomer detected by SEC P-0250 3.1 74.3％ P-0320 9.3 96.2％ P-0382 23.3 97.8％ P-0362 18.1 100％ P-0379 16.6 99.1％

[0442] In addition, the through engineering approaches work that the inventor carries out to IL-2 has also identified that the single amino acid replacement of serine is substituted with isoleucine at position 125, and this replacement has resulted in the general improvement of the developability of the IL-2Fc fusion construct with the biological activity retained completely.The Ile at position 125 of IL-2 variants with different mutation backgrounds in wild-type IL-2 and Fc fusion forms replaces and all results in 4 to 11 times of expression level improvement, and aggregation tendency is uniformly low.In table 15, the expression level (mg / L) and the purity of the material of the protein A purification assessed with the aggregation percentage of exemplary molecules by SEC chromatography are summarized.Two kinds of molecules in the same row of table 15 have identical other amino acid replacements, and difference is only that residue 125 is serine or isoleucine.

[0443] Table 15 S125I substitution improves the developability profile of various IL-2Fc fusion proteins

[0444]

[0445] In summary, the VitoKine platform significantly improves the developability profile of proteins, as demonstrated by the increased protein expression and greatly reduced aggregation tendency of the Fc IL-2 VitoKine construct. In addition, IL-2 (wild-type or variant) VitoKine constructs incorporating the beneficial IL-2 S125I amino acid may have a further enhanced developability profile.

[0446] Example 19

[0447] VitoKine D3 domain selection can significantly impact protein expression

[0448] The D3 domain of the VitoKine platform has also been explored, and the D3 domain is a variant or unrelated protein domain of the homologous receptor of the D2 domain. Based on crystal structure analysis (Wang et al., Science 310: 1159-1163, 2005), IL-2Rαsushi domains 1 and 2 participate in chain exchange events, and the result is that residues 1-19 of IL-2Rα are part of sushi domain 2, and residues 102-122 are part of sushi domain 1. Such a structural arrangement is reflected in IL-2RαSushi variants (SEQ ID NO: 147), which contain IL-2Rα (SEQ ID NO: 10) residues 102-122 at the N-terminus and IL-2Rα residues 20-68 at the C-terminus. Such IL-2RαSushi variants contain most of the residues that interact with IL-2, and are presumed to have reproduced most of the activity with assumed structural integrity. IL-2 VitoKine P-0321 (SEQ ID NO: 179) was generated by replacing the IL-2RαSushi domain in P-0320 (SEQ ID NO: 49) with an IL-2RαSushi variant. Unexpectedly, P-0321, which contains an IL-2RαSushi variant as the D3 domain, was not expressed at all or was expressed at such a low level that no material could be captured and purified.

[0449] Similarly, the IL-15αSushi+ domain in VitoKine P-0315 (SEQ ID NO: 33) was replaced with IL-2RαSushi (SEQ ID NO: 10) and the resulting protein was P-0389 (SEQ ID NO: 42). P-0389 was expressed at significantly lower levels than P-0315. More notably, purified P-0389 was primarily high molecular weight aggregates, as shown in the SDS-PAGE gel image depicted in Figure 34A. For comparison purposes, an SDS-PAGE gel image of the corresponding molecule P-0315 is shown in Figure 34B. In addition, despite the presence of the MMP-2 / 9 substrate peptide in the sequence, purified P-0389 was resistant to MMP-2 digestion, suggesting that the molecule was not properly folded or that aggregation restricted access to the protease.

[0450] In summary, D3 is a key component of the VitoKine construct. In addition to functioning as a shielding moiety, D3 can significantly impact the developability profile of the protein, both positively and negatively.

[0451] All articles and methods disclosed and claimed herein can be prepared and executed without undue experimentation in view of the present disclosure. Although the articles and methods of the present disclosure have been described according to preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the articles and methods without departing from the spirit and scope of the present disclosure. It will be apparent to those skilled in the art that all such variations and equivalents, whether existing or developed later, are considered to be within the spirit and scope of the present disclosure as defined by the appended claims. All patents, patent applications, and publications mentioned in the specification indicate the level of ordinary technicians in the field to which the present disclosure belongs. For all purposes, all patents, patent applications, and publications are incorporated herein by reference in their entirety, and to the extent that each individual publication is individually and specifically indicated to be incorporated herein by reference in its entirety for any and all purposes. The disclosure exemplarily stated herein can be suitably practiced under the condition that any one or more elements not specifically disclosed herein do not exist. It will be understood, therefore, that although the present disclosure has been particularly disclosed by preferred embodiments and optional features, those skilled in the art may seek modifications and variations of the concepts disclosed herein, and that such modifications and variations are considered to be within the scope of the present disclosure as defined by the appended claims.

[0452] Sequence Listing

[0453] The nucleic acid and amino acid sequences listed in the accompanying Sequence Listing are shown using the standard letter abbreviations for nucleotide bases and the single-letter code for amino acids as set forth in 37 CFR 1.822.

[0454] SEQ ID NO: 1 is the amino acid sequence of human IL-15 precursor.

[0455] SEQ ID NO: 2 is the amino acid sequence of the mature form of human IL-15.

[0456] SEQ ID NO: 3 is the amino acid sequence of an IL-15 variant polypeptide.

[0457] SEQ ID NO: 4 is the amino acid sequence of human IL-15Rα.

[0458] SEQ ID NO: 5 is the human IL-15Rα, sushi domain + amino acid sequence.

[0459] SEQ ID NO: 6 is the amino acid sequence of human IL-2 precursor.

[0460] SEQ ID NO: 7 is the naturally occurring amino acid sequence of the mature form of human IL-2.

[0461] SEQ ID NO: 8 is the wild-type amino acid sequence of the mature form of human IL-2.

[0462] SEQ ID NO: 9 is the amino acid sequence of human IL-2Rα (CD25) precursor.

[0463] SEQ ID NO: 10 is the amino acid sequence of human IL-2Rα, sushi domain.

[0464] SEQ ID NO: 11 is the amino acid sequence of human IL-2Rβ precursor.

[0465] SEQ ID NO: 12 is the amino acid sequence of the extracellular domain of human IL-2Rβ.

[0466] SEQ ID NO: 13 is the human IgG1-Fc amino acid sequence.

[0467] SEQ ID NO: 14 is the sequence of human IgG1-Fc with reduced / ablated effector function.

[0468] SEQ ID NO: 15 is the Knob-Fc amino acid sequence.

[0469] SEQ ID NO: 16 is the Hole-Fc amino acid sequence.

[0470] SEQ ID NO: 17 is the amino acid sequence of the mature form of human IL-4.

[0471] SEQ ID NO: 18 is the amino acid sequence of the mature form of human IL-7.

[0472] SEQ ID NO: 19 is the amino acid sequence of the mature form of human IL-9.

[0473] SEQ ID NO: 20 is the amino acid sequence of the mature form of human IL-10.

[0474] SEQ ID NO: 21 is the sequence of the mature form of human IL-12 subunit α.

[0475] SEQ ID NO: 22 is the sequence of the mature form of human IL-12 subunit β.

[0476] SEQ ID NO: 23 is the sequence of the mature form of human IL-23 subunit α.

[0477] SEQ ID NO: 24 is the sequence of the mature form of human IL-27 subunit β.

[0478] SEQ ID NOs: 25-43 are the amino acid sequences of various Fc IL-15 VitoKine constructs.

[0479] SEQ ID NO: 44 is the amino acid sequence of Hole-Fc-IL-15 fusion protein.

[0480] SEQ ID NO: 45 is the amino acid sequence of the Knob-Fc-IL-15 fusion protein.

[0481] SEQ ID NO: 46 is the amino acid sequence of the Knob-Fc-IL-15Rα-Sushi+ fusion protein.

[0482] SEQ ID NO: 47 is the amino acid sequence of the Fc-IL-15S58D fusion protein.

[0483] SEQ ID NO: 48 is the amino acid sequence of the IL-2 fusion protein.

[0484] SEQ ID NOs: 49-65 are the amino acid sequences of various Fc IL-2 VitoKine constructs.

[0485] SEQ ID NOs: 66-70 are the amino acid sequences of various IL-15 constructs comprising a blocking peptide.

[0486] SEQ ID NOs: 71-87 and 157-159 are the amino acid sequences of various protease substrate peptides.

[0487] SEQ ID NOs: 88-96 and 160-161 are the amino acid sequences of various protease-cleavable linkers comprising various spacer peptides flanking the protease substrate peptide.

[0488] SEQ ID NOs: 97-106 are the amino acid sequences of various blocking peptide sequences.

[0489] SEQ ID NOs: 107-127 are the amino acid sequences of various non-cleavable linker sequences.

[0490] SEQ ID NOs: 128-146 are the amino acid sequences of various antibody VitoKine constructs.

[0491] SEQ ID NO: 147 is the human IL-2Rα variant sequence.

[0492] SEQ ID NOs: 148-149 are the amino acid sequences of Hole-Fc-IL-15 fusion constructs.

[0493] SEQ ID NOs: 150-155 are the amino acid sequences of various Fc IL-2 VitoKine constructs.

[0494] SEQ ID NO: 156 is a human IgG1-Fc sequence with reduced / eliminated effector function and extended half-life.

[0495] SEQ ID NOs: 162-165 are the amino acid sequences of various Fc IL-15 VitoKine constructs.

[0496] SEQ ID NO: 166 is the sequence of human IgG1-Fc with reduced / eliminated effector function and extended half-life.

[0497] SEQ ID NO: 167 is the amino acid sequence of Knob-Fc with extended half-life.

[0498] SEQ ID NO: 168 is the amino acid sequence of Hole-Fc with extended half-life.

[0499] SEQ ID NOs: 169-174 are the amino acid sequences of various Fc IL-15 VitoKine constructs.

[0500] SEQ ID NOs: 175-178 are the amino acid sequences of various IL-15Fc fusion constructs.

[0501] SEQ ID NO: 179 is the amino acid sequence of the Fc IL-2 VitoKine construct.

[0502] SEQ ID NOs: 180-181 are the amino acid sequences of the antibody IL-15 VitoKine constructs. SEQ ID NOs: 182-192 are the nucleotide sequences of various Fc IL-15 VitoKine constructs.

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[0535] The present invention also provides the following items:

[0536] 1. A bioactivatable polypeptide drug construct comprising, in the N-terminal to C-terminal direction (D1-D2-D3): 1) a tissue or disease site targeting portion D1 domain ("D1"), 2) a bioactivatable portion D2 domain ("D2"), and 3) a shielding portion D3 domain ("D3"); wherein D1 functions to target the bioactivatable portion to an intended treatment site; and wherein D3 is capable of shielding the functional activity of D2 until D2 is activated at the intended treatment site.

[0537] 2. A bioactivatable polypeptide drug construct comprising, in the N-terminal to C-terminal direction (D1-D2-D3): 1) a half-life extending moiety D1 domain ("D1"), 2) a bioactivatable moiety D2 domain ("D2"), and 3) a shielding moiety D3 domain ("D3"); wherein D1 functions to target the bioactivatable moiety to an intended therapeutic site and extend the half-life of D2; and wherein D3 is capable of shielding the functional activity of D2 until D2 is activated at the intended therapeutic site.

[0538] 3. A bioactivatable polypeptide drug construct comprising, in the N-terminal to C-terminal direction (D1-D2-D3): 1) a dual-functional portion D1 domain ("D1"), 2) a bioactivatable portion D2 domain ("D2"), and 3) a shielding portion D3 domain ("D3"); wherein D1 functions to target and maintain the bioactivatable portion at an intended therapeutic site; and wherein D3 is capable of shielding the functional activity of D2 until D2 is activated at the intended therapeutic site.

[0539] 4. A bioactivatable polypeptide drug construct comprising, in the N-terminal to C-terminal direction (D3-D2-D1), 1) a shielding portion, a D3 domain ("D3"), 2) a bioactivatable portion, a D2 domain ("D2"), and 3) a tissue or disease site targeting portion, a D1 domain ("D1"); wherein D1 functions to target the bioactivatable portion to an intended treatment site; and wherein D3 is capable of shielding the functional activity of D2 until D2 is activated at the intended treatment site.

[0540] 5. A bioactivatable polypeptide drug construct comprising, in the N-terminal to C-terminal direction (D3-D2-D1), 1) a shielding portion, a D3 domain ("D3"), 2) a bioactivatable portion, a D2 domain ("D2"), and 3) a half-life extending portion, a D1 domain ("D1"); wherein D1 functions to target the bioactivatable portion to an intended therapeutic site and extend the half-life of D2; and wherein D3 is capable of shielding the functional activity of D2 until D2 is activated at the intended therapeutic site.

[0541] 6. A bioactivatable polypeptide drug construct comprising, in the N-terminal to C-terminal direction (D3-D2-D1), 1) a shielding portion, D3 domain ("D3"), 2) a bioactivatable portion, D2 domain ("D2"), and 3) a dual-functional portion, D1 domain ("D1"); wherein D1 functions to target and maintain the bioactivatable portion at an intended therapeutic site; and wherein D3 is capable of shielding the functional activity of D2 until D2 is activated at the intended therapeutic site.

[0542] 7. The construct according to any one of items 1 to 6, wherein the D1 domain, D2 domain and D3 domain of the construct are each in the form of a monomer, each in the form of a dimer, or together in the form of a combination of a dimer and a monomer.

[0543] 8. A construct according to any one of items 1 to 7, wherein the D1 domain is selected from the group consisting of: an antibody, or an antibody fragment, or a ligand or a variant thereof, or a receptor or a variant thereof that is capable of binding to a tumor-associated antigen (TAA) or a tissue-specific antigen or target; a cell surface molecule or an extracellular matrix protein; a protease and any post-translationally modified residue.

[0544] 9. The construct of claim 8, wherein the D1 domain is an antibody or antibody fragment directed against an immune checkpoint regulator.

[0545] 10. The construct according to any one of items 1 to 7, wherein the D1 domain is an Fc domain selected from the group consisting of a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, an IgA Fc domain, an IgD Fc domain, an IgE Fc domain, an IgG Fc domain, and an IgM Fc domain.

[0546] 11. The construct according to item 10, wherein the Fc domain is an Fc domain having a silent effector function and / or having a half-life extension function.

[0547] 12. The construct according to any one of items 10 to 11, wherein the Fc domain is an Fc domain having an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 156, and SEQ ID NOs: 166-168.

[0548] 13. The construct according to any one of items 1 to 12, wherein the D2 domain is a cytokine selected from the group consisting of interleukin-2 (IL-2) (SEQ ID NO: 8), 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 (12α) (SEQ ID NO: 21), interleukin-12β (IL-12β) (SEQ ID NO: 22), interleukin-15 (IL-15) (SEQ ID NO: 2), interleukin-23α (IL-23α) (SEQ ID NO: 23) and transforming growth factor β (TGFβ) (SEQ ID NO: 24) or a variant thereof.

[0549] 14. The construct according to item 13, wherein the D2 domain is IL-15 (SEQ ID NO: 2).

[0550] 15. The construct according to item 14, wherein the D2 domain is an IL-15 variant polypeptide comprising one or more amino acid substitutions or deletions at positions 30, 31, 32, 58, 62, 63, 67, 68 or 108 of SEQ ID NO: 2.

[0551] 16. The construct according to item 15, wherein the D2 domain is an IL-15 variant polypeptide having the amino acid sequence set forth in SEQ ID NO: 3.

[0552] 17. The construct according to item 13, wherein the D2 domain is IL-2 (SEQ ID NO: 8).

[0553] 18. The construct according to item 13, wherein the D2 domain is an IL-2 variant polypeptide comprising one or more amino acid substitutions or deletions at positions 19, 20, 38, 41, 42, 44, 88, 107, 125 or 126 of SEQ ID NO:8.

[0554] 19. The construct according to any one of items 1 to 18, wherein D2 is attached to D1 via a peptide linker ("L1") selected from the group consisting of a protease-cleavable peptide linker and a non-cleavable peptide linker.

[0555] 20. The construct according to item 19, wherein the protease-cleavable peptide linker is selected from the group of sequences listed in SEQ ID NOs: 71-96 and 157-161.

[0556] 21. The construct according to item 19, wherein the non-cleavable peptide linker is selected from the group of sequences listed in SEQ ID NOs: 107-127.

[0557] 22. The construct according to any one of items 1 to 21, wherein the D3 domain is selected from the group consisting of a cognate receptor / binding partner (or a variant thereof) and any binding partner identified for D2 and capable of masking the activity of D2.

[0558] 23. The construct according to any one of items 1 to 21, wherein the D3 domain is selected from the group consisting of proteins, peptides, DNA fragments, RNA fragments, polymers, antibodies and antibody fragments that are capable of masking the activity of D2.

[0559] 24. The construct according to item 22, wherein the D3 domain is a cognate receptor / binding partner of IL-15 (or a variant thereof) and comprises the amino acid sequence set forth in SEQ ID NO: 4 or any functional fragment thereof.

[0560] 25. The construct according to item 22, wherein the D3 domain is a cognate receptor / binding partner of IL-15 (or a variant thereof) and comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0561] 26. The construct according to item 22, wherein the D3 domain is a cognate receptor / binding partner of IL-2 (or a variant thereof) and comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0562] 27. The construct according to any one of items 1 to 26, wherein D2 is attached to D3 via a peptide linker ("L2") selected from the group consisting of a protease-cleavable peptide linker and a non-cleavable peptide linker.

[0563] 28. The construct according to item 27, wherein the protease-cleavable peptide linker is selected from the group of sequences listed in SEQ ID NOs: 71-96 and 157-161.

[0564] 29. The construct according to item 27, wherein the non-cleavable peptide linker is selected from the group of sequences listed in SEQ ID NO: 107-127.

[0565] 30. The construct according to any one of items 1 to 29, wherein both L1 and L2 are protease-cleavable peptide linkers.

[0566] 31. The construct according to any one of items 1 to 29, wherein both L1 and L2 are non-cleavable peptide linkers.

[0567] 32. The construct according to any one of items 1 to 29, wherein L1 is a protease-cleavable peptide linker and L2 is a non-cleavable peptide linker.

[0568] 33. The construct according to any one of items 1 to 29, wherein L1 is a non-cleavable peptide linker and L2 is a protease-cleavable peptide linker.

[0569] 34. A pharmaceutical composition comprising the construct according to any one of items 1 to 33 in admixture with a pharmaceutically acceptable carrier.

[0570] 35. A method of treating cancer or cancer metastasis in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to item 34.

[0571] 36. The method of claim 35, wherein the method further comprises a second therapeutic agent or therapy capable of treating cancer or cancer metastasis in the subject.

[0572] 37. A method of treating an autoimmune disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to item 34.

[0573] 38. The method of claim 37, wherein the method further comprises a second therapeutic agent or therapy capable of treating the subject's autoimmune disease.

[0574] 39. A method of treating an inflammatory disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to item 34.

[0575] 40. The method of claim 39, wherein the method further comprises a second therapeutic agent or therapy capable of treating the inflammatory disease in the subject.

[0576] 41. A method of treating a viral infection in a subject, the method comprising administering a therapeutically effective amount of the pharmaceutical composition according to item 34.

[0577] 42. The method of claim 41, wherein the method further comprises a second therapeutic agent or therapy capable of treating the viral infection in the subject.

[0578] 43. A nucleic acid molecule encoding the construct according to any one of items 1 to 33.

[0579] 44. An expression vector comprising the nucleic acid molecule according to item 43.

[0580] 45. A host cell comprising the expression vector according to item 44 or the nucleic acid molecule according to item 43.

[0581] 46. A method for producing a bioactivatable polypeptide drug construct according to any one of items 1 to 33, the method comprising culturing the host cell according to item 45 under conditions that promote expression of the bioactivatable polypeptide drug construct, and recovering the bioactivatable polypeptide drug construct protein.

[0582] 47. An isolated bioactivatable polypeptide drug construct protein produced by the method according to item 46.

Claims

1. A bioactivatable polypeptide drug construct, said construct being D1-D2-D3 in the N-terminal to C-terminal direction, comprising: 1) a functional portion D1 domain, 2) a bioactivatable portion D2 domain, and 3) a shielding portion D3 domain; wherein said functional portion D1 domain is selected from the group consisting of: a D1 domain that functions to target the bioactivatable portion to an intended therapeutic site, a D1 domain that functions to target the bioactivatable portion to an intended therapeutic site and to extend the half-life of D2, and a D1 domain that functions to target the bioactivatable portion to an intended therapeutic site and to retain the bioactivatable portion at the intended therapeutic site; wherein D3 is capable of shielding the functional activity of D2 until D2 is activated at the intended therapeutic site; wherein D1 is an Fc domain consisting of the amino acid sequence set forth in SEQ ID NO: 14; wherein D2 is an IL-2 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 7 or 8; and wherein D3 is a cognate receptor / binding partner for IL-2 and ... The amino acid sequence listed in NO:10; wherein D2 is attached to D1 via a peptide linker L1 selected from the group consisting of a protease-cleavable peptide linker and a non-cleavable peptide linker; and wherein D2 is attached to D3 via a peptide linker L2 selected from the group consisting of a protease-cleavable peptide linker and a non-cleavable peptide linker.

2. The construct according to claim 1, wherein the protease-cleavable peptide linker is selected from the group of sequences set forth in SEQ ID NOs: 71-96 and 157-161.

3. The construct according to claim 1, wherein the non-cleavable peptide linker is selected from the group of sequences set forth in SEQ ID NOs: 107-127.

4. The construct of any one of claims 1 to 3, wherein both L1 and L2 are protease-cleavable peptide linkers; or wherein both L1 and L2 are non-cleavable peptide linkers; or wherein L1 is a protease-cleavable peptide linker and L2 is a non-cleavable peptide linker; or wherein L1 is a non-cleavable peptide linker and L2 is a protease-cleavable peptide linker.

5. The construct according to claim 1, wherein the construct consists of the amino acid sequence set forth in SEQ ID NOs: 49-65, 150-155 and 179.

6. construct according to claim 1, wherein the construct consists of the amino acid sequence set forth in SEQ ID NO:49, 51 or 52.

7. A pharmaceutical composition comprising the construct according to any one of claims 1 to 6 in admixture with a pharmaceutically acceptable carrier.

8. Use of the construct according to any one of claims 1 to 6 in the preparation of a pharmaceutical composition for the treatment of colorectal cancer or colon cancer lung metastasis.

9. The use of claim 8, wherein the treatment further comprises a second therapeutic agent or therapy capable of treating cancer or cancer metastasis in the subject.

10. A nucleic acid molecule encoding the bioactivatable polypeptide drug construct according to any one of claims 1 to 6. An expression vector comprising the nucleic acid molecule according to claim 10 . A host cell comprising the expression vector according to claim 11 .

13. A method for producing a bioactivatable polypeptide drug construct according to any one of claims 1 to 6, the method comprising culturing the host cell according to claim 12 under conditions that promote expression of the bioactivatable polypeptide drug construct, and recovering the bioactivatable polypeptide drug construct protein.

14. An isolated bioactivatable polypeptide drug construct protein produced by the method according to claim 13.

15. A bioactivatable polypeptide drug construct comprising, in the N-terminal to C-terminal direction (D1-D2-D3): 1) a tissue or disease site targeting portion, D1 domain ("D1"), 2) a bioactivatable portion, D2 domain ("D2"), and 3) a shielding portion, D3 domain ("D3"); wherein D1 functions to target the bioactivatable portion to an intended treatment site; and wherein D3 is capable of shielding the functional activity of D2 until D2 is activated at the intended treatment site.

16. A bioactivatable polypeptide drug construct comprising, in the N-terminal to C-terminal direction (D1-D2-D3): 1) a half-life extending moiety D1 domain ("D1"), 2) a bioactivatable moiety D2 domain ("D2"), and 3) a shielding moiety D3 domain ("D3"); wherein D1 functions to target the bioactivatable moiety to an intended therapeutic site and extend the half-life of D2; and wherein D3 is capable of shielding the functional activity of D2 until D2 is activated at the intended therapeutic site.

17. A bioactivatable polypeptide drug construct comprising, in the N-terminal to C-terminal direction (D1-D2-D3): 1) a dual-functional portion, D1 domain ("D1"), 2) a bioactivatable portion, D2 domain ("D2"), and 3) a shielding portion, D3 domain ("D3"); wherein D1 functions to target and maintain the bioactivatable portion at an intended therapeutic site; and wherein D3 is capable of shielding the functional activity of D2 until D2 is activated at the intended therapeutic site.

18. A bioactivatable polypeptide drug construct comprising, in the N-terminal to C-terminal direction (D3-D2-D1), 1) a shielding portion, a D3 domain ("D3"), 2) a bioactivatable portion, a D2 domain ("D2"), and 3) a tissue or disease site targeting portion, a D1 domain ("D1"); wherein D1 functions to target the bioactivatable portion to an intended treatment site; and wherein D3 is capable of shielding the functional activity of D2 until D2 is activated at the intended treatment site.

19. A bioactivatable polypeptide drug construct comprising, in the N-terminal to C-terminal direction (D3-D2-D1), 1) a shielding portion, a D3 domain ("D3"), 2) a bioactivatable portion, a D2 domain ("D2"), and 3) a half-life extending portion, a D1 domain ("D1"); wherein D1 functions to target the bioactivatable portion to an intended therapeutic site and extend the half-life of D2; and wherein D3 is capable of shielding the functional activity of D2 until D2 is activated at the intended therapeutic site.

20. A bioactivatable polypeptide drug construct comprising, in the N-terminal to C-terminal direction (D3-D2-D1), 1) a shielding portion, D3 domain ("D3"), 2) a bioactivatable portion, D2 domain ("D2"), and 3) a dual-functional portion, D1 domain ("D1"); wherein D1 functions to target and maintain the bioactivatable portion at an intended therapeutic site; and wherein D3 is capable of shielding the functional activity of D2 until D2 is activated at the intended therapeutic site.

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