Methods and systems relating to use of antigen presenting cells

Abstract

The present disclosure relates generally to systems and methods for treating and / or reversing a disease or condition using immunogenic cell death, such as lysosomal-induced immunogenic cell death, the disease or condition is, for example, senescence, non-alcoholic steatohepatitis (NASH), neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, obesity, pulmonary fibrosis, congestive heart failure or cancer. In certain embodiments, the present application relates to compositions comprising an antibody and an enzyme capable of producing reactive oxygen species. In some cases, the compositions may target lysosomes of cells associated with the disease or condition to induce cell death.

Description

Technical Field

[0001] The present disclosure generally relates to systems and methods for treating diseases or conditions such as aging, non-alcoholic steatohepatitis (NASH) disease, neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, obesity, pulmonary fibrosis, congestive heart failure, and cancer by targeted apoptosis of specific cells associated with the disease or condition (e.g., senescent cells, adipocytes, fibroblasts, or cancer cells). Background Art

[0002] Diseases and conditions such as aging, nonalcoholic steatohepatitis (NASH) disease, neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, obesity, pulmonary fibrosis, congestive heart failure, and cancer can be multifactorial in their pathogenesis. There is a need to treat these diseases or conditions. Summary of the invention

[0003] The present disclosure generally relates to systems and methods for treating and / or reversing a disease or condition using immunogenic cell death, such as lysosomal-induced immunogenic cell death, such as aging, nonalcoholic steatohepatitis (NASH), neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, obesity, pulmonary fibrosis, congestive heart failure, or cancer. In certain embodiments, the application relates to compositions comprising antibodies and enzymes capable of producing reactive oxygen species. In some cases, the compositions can target the lysosomes of cells associated with the disease or condition to induce cell death.

[0004] In some cases, the presently disclosed subject matter involves related products, alternative solutions to a particular problem, and / or a variety of different uses of one or more systems and / or compositions.

[0005] For example, in certain embodiments, by targeting cells that may contribute to the disease or condition, such as senescent cells, adipocytes, fibroblasts, or cancer cells, a disease or condition may be treated by removing cells. According to some embodiments, such removal may be achieved by targeting immunogenic cell death.

[0006] In another aspect, the disclosure relates to a composition for treating a disease or condition. In some embodiments, the composition comprises: a fusion protein comprising an enzyme and at least a portion of an antibody, wherein the at least a portion of the antibody is configured to bind to a cell associated with the disease or condition, and wherein the enzyme is configured to increase the lysosomal membrane permeability of the cell.

[0007] In one aspect, the disclosure relates to methods for treating a disease or condition. In some embodiments, the method comprises: administering to a subject a composition comprising a fusion protein comprising an enzyme and at least a portion of an antibody, wherein the at least a portion of the antibody recognizes a cell associated with the disease or condition of the subject, and wherein the enzyme is configured to increase the lysosomal membrane permeability of the cell.

[0008] In one aspect, the disclosure relates to a composition. In some embodiments, the composition comprises: an antibody that recognizes a senescent cell, an adipocyte, a cardiac fibroblast, or a lung fibroblast, and an enzyme configured to increase the permeability of the lysosomal membrane of the recognized senescent cell, an adipocyte, a cardiac fibroblast, or a lung fibroblast.

[0009] In another aspect, the present disclosure relates to a composition comprising: an antibody that recognizes senescent cells, adipocytes, cardiac fibroblasts, or lung fibroblasts, and an enzyme capable of generating reactive oxygen species.

[0010] In yet another aspect, the present disclosure relates to a method comprising: administering to a subject a composition comprising an antibody that recognizes senescent cells, adipocytes, cardiac fibroblasts, or lung fibroblasts, and an enzyme that is capable of increasing the permeability of the lysosomal membrane of the identified senescent cells, adipocytes, cardiac fibroblasts, or lung fibroblasts.

[0011] In yet another aspect, the disclosure relates to a method comprising: administering to a subject a composition comprising an antibody that recognizes senescent cells, adipocytes, cardiac fibroblasts, or lung fibroblasts, and an enzyme capable of generating reactive oxygen species.

[0012] Several methods of administering compositions to subjects to treat specific diseases or conditions are disclosed herein. It should be understood that in each such aspect of the present disclosure, compositions for use in treating the specific disease or condition, and the use of compositions for the manufacture of a medicament for treating the specific disease or condition are also specifically included.

[0013] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the present disclosure when considered in conjunction with the accompanying drawings. In the event that the present specification and documents incorporated by reference include conflicting and / or inconsistent disclosures, the present specification shall prevail. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying drawings, which are schematic and are not intended to be drawn to scale unless otherwise specified. In the drawings, each identical or nearly identical component shown is typically represented by a single numeral. For clarity, not every component is labeled in every drawing, nor is every component of every embodiment of the present disclosure shown, where an illustration is not necessary for one of ordinary skill in the art to understand the present disclosure. In the drawings:

[0015] Figure 1 presents an exemplary mechanism of lysosomal-induced immunogenic cell death induced by reactive oxygen species according to some embodiments; and

[0016] Figure 2 Presented are perspective schematic diagrams of exemplary fusion proteins according to some embodiments, comprising exemplary Nanobodies coupled to exemplary proteins associated with a disease or disorder. DETAILED DESCRIPTION

[0017] The present disclosure generally relates to systems and methods for treating or reversing a disease or condition (e.g., aging, non-alcoholic steatohepatitis (NASH) disease, neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, obesity, pulmonary fibrosis, congestive heart failure, or cancer) through immunogenic cell death (e.g., lysosome-induced immunogenic cell death).

[0018] The present disclosure includes multiple aspects that can be used alone or together. For example, in one aspect, the present disclosure relates to preparing the subject's body and / or brain for treatment, such as by withdrawal and suppression of antioxidants, supplying n-3 to n-6 and other unsaturated fatty acids, and / or treating the subject with statins.

[0019] On the one hand, the present disclosure relates to a composition comprising an enzyme. For example, the composition can comprise an enzyme and an antibody. In some embodiments, the enzyme is linked to the antibody. In some cases, the antibody and enzyme can be expressed as a single fusion protein, as discussed in more detail below.

[0020] The enzyme can be a naturally occurring or synthetically modified enzyme or fragment thereof. In some embodiments, the enzyme is an oxidoreductase. For example, in various embodiments, the enzyme can be an oxidase or a peroxidase. Other exemplary oxidoreductases that can be used include, but are not limited to, those described below.

[0021] According to some embodiments, oxidase is an enzyme that catalyzes redox reactions, including reactions involving oxygen (O2) as electron acceptors. Oxygen can be reduced to water (H2O) or hydrogen peroxide (H2O2). Non-limiting examples of oxidases include glucose oxidase, monoamine oxidase, cytochrome P450 oxidase, NADPH oxidase, xanthine oxidase, L-gulonolactone oxidase, laccase, lysyl oxidase, etc. The enzyme described herein can be a recombinant enzyme.

[0022] According to some embodiments, peroxidase is an enzyme that acts on a substrate such as hydrogen peroxide or lipid peroxidase. Non-limiting examples include horseradish peroxidase, cytochrome c peroxidase, ascorbate peroxidase, chloride peroxidase, glutathione peroxidase, haloperoxidase, lactoperoxidase, manganese peroxidase, myeloperoxidase, thyroid peroxidase, vanadium boron peroxidase, etc.

[0023] In some embodiments, the enzyme is an oxidase or peroxidase suitable for producing an antigen as described herein.

[0024] In certain embodiments, the composition can be applied to a subject. It is also possible to provide a substrate for the enzyme to the subject. The composition can be used alone or in combination with other compositions. In certain embodiments, the composition is used to treat and / or cure a disease or illness, such as aging, nonalcoholic steatohepatitis (NASH) disease, neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease), obesity, pulmonary fibrosis, congestive heart failure or cancer. For example, the enzyme can be connected to an antibody that identifies a cell (e.g., senescent cell, adipocyte, fibroblast) associated with a disease or illness, as described in more detail below. In some cases, the composition can target the lysosome of one or more cells. In certain embodiments, the lysosome of the targeted cell can induce cell death of a cell (e.g., senescent cell, adipocyte, fibroblast) associated with a disease or illness.

[0025] The enzyme can be an oxidase, a peroxidase or other enzymes capable of producing reactive oxygen species. The composition can be administered to a subject, for example, by infusion or other administration techniques, such as those described herein. In some cases, a substrate for the enzyme can also be provided to the subject. Without wishing to be bound by any theory, it is believed that antibodies help to locate the composition in cells associated with a selected disease or condition. Once located, the substrate for the enzyme can be administered to the subject, such as by infusion, which can set up a series of events leading to lysosomal membrane onset, lysosomal contents being released into the cell and cell death by apoptosis. Therefore, in some embodiments, the present disclosure relates to lysosomal-induced immunogenic cell death, LIICD.

[0026] In some cases, a substrate for the enzyme may also be administered to the subject. The substrate and enzyme may be administered in any suitable order, for example, sequentially and / or simultaneously. Non-limiting examples of substrates include xanthine for hypoxanthine or xanthine oxidase, or glucose for glucose oxidase. As another example, monoamine oxidase may act on, for monoamine oxidase, serotonin, melatonin, norepinephrine, epinephrine, phenylethylamine, benzylamine, dopamine, tyramine, tryptamine, etc.; for NADPH peroxidase, NADPH; for ascorbic acid peroxidase, ascorbic acid; or similar.

[0027] Without wishing to be bound by any theory, it is believed that certain enzymes (e.g., oxidases and peroxidases) generate reactive oxygen species, such as superoxide, peroxide, hydroxyl radicals, etc., by oxidizing substrates associated with the enzymes. Thus, in some embodiments, substrates for the enzymes can be optionally added (e.g., can be included with the composition, or can be administered to a subject to be treated with the composition) to promote such reactions. In some embodiments, reactive oxygen species are endocytosed by targeted cells. Reactive oxygen species can interact with the membranes of target cells. In some embodiments, oxidation of unsaturated lipids can cause their endocytosis. Figure 1 A cross-sectional schematic diagram of the method is presented, wherein reactive oxygen species 101 reacts with unsaturated lipids 102 to produce oxidized unsaturated lipids 104 that are endocytosed into cells 103. Without wishing to be bound by theory, the interaction between the lysosomes of the targeted cells and the oxidized unsaturated lipids can induce the lysosomal membrane of the cell to become more permeable. For example, in FIG3 , the oxidized unsaturated lipids 104 are endocytosed into cells 103, wherein the lipids 104 are absorbed by lysosomes 105, causing the lysosomal membrane 107 to become a permeable lysosomal membrane 109. Thus, the enzyme can be configured to increase the lysosomal membrane permeability of the cell, for example by producing reactive oxygen species. One of ordinary skill can use galectin spot assays to detect changes in lysosomal membrane permeability.

[0028] Increased lysosomal membrane permeability may result in leakage of degradative enzymes from cells. See again, for example, Figure 1 Once the lysosomal membrane 107 becomes a permeable lysosomal membrane 109, the degradative enzymes 111 can leak through the permeable lysosomal membrane 109. The instability of the cell's lysosomal membrane may disperse the lysosomal enzymes throughout the cell. The cell may then produce cell-specific neoantigens that can be recognized by the immune system, and / or may trigger apoptosis and / or cell death. This is Figure 1, wherein leakage of the degradative enzyme 111 produces degradation byproducts 117 and independently causes the cell 103 to express the neoantigen 113, with the signaling cell 103 for endocytosis by the dendritic cell 115. Thus, reactive oxygen species can be used in some embodiments to cause lysosomal induced immunogenic cell death (LIICD). Thus, it is believed that cells associated with diseases or conditions such as aging, non-alcoholic steatohepatitis (NASH) disease, neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, obesity, pulmonary fibrosis, congestive heart failure, or cancer can be targeted (e.g., with antibodies) for delivery of enzymes that can kill the cells via administration of a composition as described herein.

[0029] Furthermore, it should be understood that in some cases, a drug may be used in the composition instead of and / or in addition to the enzyme that generates the reactive oxygen species.

[0030] In some embodiments, the disclosure relates to a composition comprising an enzyme and an antibody or antibody fragment.Antibodies or antibody fragments can be selected to identify cells (e.g., senescent cells, adipocytes, cancer cells, or fibroblasts) associated with a disease or disorder of a subject.Antibodies or antibody fragments can be, for example, covalently linked to an enzyme by connection, for example.In some embodiments, a composition comprises a fusion protein containing an enzyme and an antibody or antibody fragment, as described in more detail below.For example, in some embodiments, a composition as described herein can, for example, produce antigens by increasing lysosomal membrane permeability and / or leakage and / or by producing reactive oxygen species, etc., as discussed herein.The generation of antigens can allow the immune system of a subject, such as T cells, to identify cells (e.g., senescent cells, adipocytes, cancer cells, or fibroblasts) associated with a disease or disorder of a subject by identifying antigens produced in the cell.In some embodiments, the recognition of T cells can result in lysosomal-induced cell immunogenic cell death.In some embodiments, the immunogenic cell death of cells can train immune responses for target cells (e.g., senescent cells, adipocytes, fibroblasts, or cancer cells), thereby resulting in long-term treatment of diseases or disorders as a result of a sustained immune response.

[0031] In some embodiments, the composition can be used to treat aging disorders. For example, in some embodiments, the enzyme of the composition is configured to induce death of senescent cells. Without wishing to be bound by theory, senescent cells may be unable to undergo cell division, and / or may be associated with aging diseases.

[0032] In the present invention, it has been creatively recognized that by using a composition comprising an enzyme and a senescent cell recognition antibody, in some embodiments, senescent cells can be targeted for immunogenic cell death. The immunogenic cell death of senescent cells can reduce the number or proportion of senescent cells in the body. The reduction in the number or proportion of senescent cells may in turn delay or reverse the aging process.

[0033] According to certain embodiments, the composition for treating senescence comprises at least a portion of an antibody that recognizes senescent cells (e.g., a complete antibody or an antibody fragment). For example, the composition may comprise at least a portion of cankinumab, an anti-DPP4 antibody, an anti-CD9 antibody, or another of various suitable antibodies that recognize senescent cells and are known to those of ordinary skill in the art.

[0034] At least a portion of an antibody (e.g., a whole antibody or antibody fragment) and an enzyme can be linked to each other, as discussed in more detail below. In some embodiments, the enzyme and the antibody form a fusion protein. Fusion proteins may be particularly advantageous for treating aging. In some embodiments, for example, the fusion protein can bind to senescent cells with an affinity higher than the affinity expected for the same enzyme linked to the same antibody or antibody portion by a linker. In some embodiments, the fusion protein also has a longer lifespan in a subject, which can also lead to improved performance of the composition.

[0035] Senescent cells are also associated with diseases such as non-alcoholic steatohepatitis (NASH). In some embodiments, the composition can be used to treat NASH. For example, as described above with respect to senescence, the enzyme of the composition can be configured to induce the death of senescent cells.

[0036] In this article, it has been creatively recognized that by using a composition comprising an enzyme and a senescent cell recognition antibody, in some embodiments, senescent cells can be targeted for immunogenic cell death. The immunogenic cell death of senescent cells can reduce the number or proportion of senescent cells in the body. The reduction in the number or proportion of senescent cells may in turn delay or reverse NASH.

[0037] As discussed above with reference to senescence, according to certain embodiments, a composition useful for treating NASH comprises at least a portion of an antibody (eg, a whole antibody or an antibody fragment) that recognizes a senescent cell.

[0038] At least a portion of the antibody (e.g., a whole antibody or antibody fragment) and the enzyme can be linked to each other, as discussed in more detail below. In some embodiments, the enzyme and the antibody form a fusion protein. The fusion protein may be particularly advantageous for treating NASH. In some embodiments, for example, the fusion protein can bind to senescent cells with an affinity higher than the affinity expected for the same enzyme linked to the same antibody or antibody portion by a linker. In some embodiments, the fusion protein also has a longer lifespan in the subject, which can also lead to improvements in the performance of the composition.

[0039] Senescent cells can also lead to neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In some embodiments, the composition can be used to treat neurodegenerative diseases. For example, as described above with respect to senescence, the enzyme of the composition can be configured to induce the death of senescent cells.

[0040] In this article, it has been creatively recognized that by using a composition comprising an enzyme and a senescent cell recognition antibody, in some embodiments, senescent cells can be targeted for immunogenic cell death. The immunogenic cell death of senescent cells can reduce the number or proportion of senescent cells in the body. The reduction in the number or proportion of senescent cells may in turn delay or reverse neurodegenerative diseases.

[0041] As discussed above with reference to senescence, according to certain embodiments, a composition useful for treating a neurodegenerative disease comprises at least a portion of an antibody (eg, a whole antibody or an antibody fragment) that recognizes a senescent cell.

[0042] At least a portion of an antibody (e.g., a whole antibody or antibody fragment) and an enzyme can be linked to each other, as discussed in more detail below. In some embodiments, the enzyme and the antibody form a fusion protein. Fusion proteins may be particularly advantageous for treating neurodegenerative diseases. In some embodiments, for example, the fusion protein can bind to senescent cells with an affinity higher than the affinity expected for the same enzyme linked to the same antibody or antibody portion by a linker. In some embodiments, the fusion protein also has a longer lifespan in a subject, which can also lead to improvements in the performance of the composition.

[0043] In certain embodiments, the composition can be used to treat obesity diseases. For example, the enzyme of the composition can be configured to be connected to adipocytes (also referred to as lipocytes). The abundance of adipocytes that are too many and / or too large may be relevant to obesity and many related conditions, such as insulin resistance / diabetes.

[0044] In this article, it has been creatively recognized that, by using a composition comprising an enzyme and an adipocyte recognition antibody, in some embodiments, adipocytes can be targeted for immunogenic cell death. The immunogenic cell death of adipocytes can reduce the number or ratio of adipocytes in the body. The reduction of the number or ratio of adipocytes may in turn delay or reverse obesity.

[0045] According to certain embodiments, the composition for treating obesity comprises at least a portion of an antibody that recognizes adipocytes (e.g., a complete antibody or an antibody fragment). For example, the composition may comprise at least a portion of an adipose-specific IgG antibody, an anti-ASC-1 antibody, an anti-PAT2 antibody, an anti-P2RX5 antibody, an adipocyte-type fatty acid binding protein (A-FABP), or another of various suitable antibodies that recognize adipocytes and are known to those of ordinary skill in the art.

[0046] In some embodiments, the fusion protein of the present invention can be used to bind to the adipocyte with the affinity higher than the affinity expected by the same enzyme of the same antibody or antibody part connected to the same antibody or antibody part.In some embodiments, the fusion protein of the present invention can be used to bind to the adipocyte with the affinity higher than the affinity expected by the same enzyme of the same antibody or antibody part connected to the same antibody or antibody part.In some embodiments, the fusion protein of the present invention can be used to bind to the adipocyte with the affinity higher than the affinity expected by the same enzyme of the same antibody or antibody part connected to the same antibody or antibody part.

[0047] In some embodiments, the composition can be used to treat pulmonary fibrosis. For example, the enzyme of the composition can be configured to be connected to fibroblasts (e.g., lung fibroblasts). Too many fibroblasts in lung tissue (e.g., caused by fibroblast proliferation) may be related to pulmonary fibrosis.

[0048] In this article, it has been creatively recognized that by using a composition comprising an enzyme and a fibroblast recognition antibody (e.g., a lung fibroblast recognition antibody), in some embodiments, fibroblasts (e.g., lung fibroblasts) can be targeted for immunogenic cell death. The immunogenic cell death of fibroblasts can reduce the number or proportion of fibroblasts in the lungs of a subject. The reduction in the number or proportion of fibroblasts may in turn delay or reverse pulmonary fibrosis.

[0049] According to certain embodiments, the composition for treating pulmonary fibrosis comprises at least a portion of an antibody that recognizes fibroblasts (e.g., a complete antibody or an antibody fragment). For example, the composition may comprise at least a portion of another of the various suitable antibodies that recognize fibroblasts and are known to those of ordinary skill in the art, such as TE-7, ER-TR7, or 1B10, anti-CD82, anti-a-SMA, anti-ITG-P1, or other antibodies that recognize fibroblasts.

[0050] At least a portion of an antibody (e.g., a whole antibody or antibody fragment) and an enzyme can be connected to each other, as discussed in more detail below. In certain embodiments, enzyme and antibody form a fusion protein. Fusion protein may be particularly advantageous for treating pulmonary fibrosis. In certain embodiments, for example, the fusion protein can be bound to fibroblasts (e.g., lung fibroblasts) with an affinity higher than the affinity expected by the same enzyme connected to the same antibody or antibody portion by a joint. In certain embodiments, the fusion protein also has a longer lifespan in a subject, which can also result in an improvement in composition performance.

[0051] In some embodiments, the composition can be used to treat congestive heart failure. For example, the enzyme of the composition can be configured to be attached to fibroblasts (e.g., cardiac fibroblasts). Excessive fibroblasts in myocardial tissue (e.g., caused by fibroblast proliferation) may be associated with congestive heart failure.

[0052] In this article, it has been creatively recognized that by using a composition comprising an enzyme and a fibroblast recognition antibody (e.g., a cardiac fibroblast recognition antibody), in some embodiments, fibroblasts (e.g., cardiac fibroblasts) can be targeted for immunogenic cell death. The immunogenic cell death of fibroblasts can reduce the number or proportion of fibroblasts in the lungs of a subject. The reduction in the number or proportion of fibroblasts may in turn delay or reverse congestive heart failure.

[0053] According to certain embodiments, a composition useful for treating congestive heart failure comprises at least a portion of an antibody (eg, a whole antibody or an antibody fragment) that recognizes fibroblasts, as described above.

[0054] At least a portion of an antibody (e.g., a whole antibody or antibody fragment) and an enzyme can be connected to each other, as discussed in more detail below. In certain embodiments, the enzyme and the antibody form a fusion protein. The fusion protein may be particularly advantageous for treating congestive heart failure. In certain embodiments, for example, the fusion protein may be bound to fibroblasts (e.g., cardiac fibroblasts) with an affinity higher than the affinity expected by the same enzyme connected to the same antibody or antibody portion by a joint. In certain embodiments, the fusion protein also has a longer lifespan in a subject, which may also result in an improvement in the performance of the composition.

[0055] In some embodiments, the composition can be used to treat cancer diseases. For example, the enzyme of the composition can be configured to be attached to the cancer cell. In this article, it has been creatively recognized that by using a composition comprising an enzyme and a cancer cell recognition antibody, in some embodiments, the cancer cell can be targeted to perform immunogenic cell death. The immunogenic cell death of the cancer cell can treat cancer.

[0056] According to certain embodiments, the composition for treating cancer comprises at least a portion of an antibody that recognizes cancer cells (e.g., a complete antibody or an antibody fragment). For example, the antibody can be an antibody to epidermal growth factor receptor (EGFR), which can be overexpressed in cancer cells, or can be an antibody to any of a variety of other antigens or other markers that are uniquely expressed or overexpressed in cancer cells. As a specific example, the antibody can be (SEQ ID NO 1).

[0057] At least a portion of an antibody (e.g., a whole antibody or antibody fragment) and an enzyme can be connected to each other, as discussed in more detail below. In certain embodiments, enzyme and antibody form a fusion protein. Fusion protein may be particularly advantageous for treating cancer. In certain embodiments, for example, the fusion protein can be bound to cancer cells with an affinity higher than the affinity expected by the same enzyme connected to the same antibody or antibody portion by a joint. In certain embodiments, the fusion protein also has a longer lifespan in a subject, which can also result in an improvement in composition performance.

[0058] Enzyme can be covalently linked to another part of the composition (e.g., antibody or antibody portion, etc.). In certain embodiments, enzyme and other parts of the composition (e.g., antibody or antibody portion) are directly connected so that enzyme and other parts form a single polypeptide chain. For example, the composition can be expressed as a single fusion protein containing enzyme (e.g., oxidase, such as xanthine oxidase) and another part of the composition (e.g., antibody or antibody portion). However, in other embodiments, other methods can be used to connect the enzyme to the composition, for example, directly combined with each other, or combined by one or more cross-linking agents. Non-limiting examples include glutaraldehyde, NHS-ester (N-hydroxysuccinimide) (e.g., dithiobis (succinimidyl propionate), dithiobis (sulfosuccinimidyl propionate) etc.), PEG groups, amide esters (e.g., dimethyl adipamide, dimethyl suberimide, dimethyl pimelimide, etc.), maleimide, pyridyl, carbodiimide, isocyanate, etc. The antibody and enzyme can be coupled by any suitable system, such as amine to amine, sulfhydryl to sulfhydryl, amine to sulfhydryl, carboxyl to amine, sulfhydryl to carbohydrate, hydroxyl to sulfhydryl, or the like. One of ordinary skill in the art will be familiar with methods for cross-linking or conjugating proteins to each other.

[0059] "Fusion protein" generally refers to a hybrid polypeptide comprising protein domains from at least two different proteins. One protein can be located at the amino terminal (N-terminal) portion of the fusion protein or the carboxyl terminal (C-terminal) protein, thereby forming an "amino terminal fusion protein" or "carboxyl terminal fusion protein", respectively. Fusion proteins can contain different domains, such as an enzyme and an antibody; or an enzyme and an antibody fragment. In some embodiments, the enzyme is fused to the N-terminus of the antibody or antibody fragment. In some embodiments, the enzyme is fused to the C-terminus of the antibody or antibody fragment.

[0060] In some embodiments, the enzyme is separated from the antibody or antibody fragment by a linker region. The linker region can be configured to spatially separate the antibody from the enzyme. In some embodiments, the linker region can have an amino acid sequence comprising a repeating subsequence. For example, in some embodiments, the linker region has an amino acid sequence comprising (GGGGS) n(SEQ ID NO 6), wherein is an integer greater than or equal to 1 (e.g., 1, 2, 3, 4, 5, 6, 8, 10 or greater). In some embodiments, the linker region is GGGGSGGGGS (SEQ ID NO 3). In other embodiments, the composition does not include a linker region.

[0061] In some embodiments, the fusion protein of the present disclosure comprises an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs: 4-5. For example, the fusion protein may comprise an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or at least 99.5% identical to any one of SEQ ID NOs: 4-5. In some embodiments, the fusion protein comprises an amino acid sequence that is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to any one of SEQ ID NOs: 4-5. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or at least 99.5% identical to any one of SEQ ID NOs: 4-5. In some embodiments, the fusion protein consists of the amino acid sequence of any one of SEQ ID NOs: 4-5.

[0062] Further provided herein are enzyme variants and fusion proteins comprising such enzyme variants.In certain embodiments, fusion proteins described herein include modifications.When fusion proteins are mentioned herein, all variants and derivatives thereof are encompassed.Polypeptides comprising modifications have additional features except amino acid content.As used herein, "modification" or "derivatives" of proteins or polypeptides (e.g., fusion proteins as described herein) produce modified or derived polypeptides, which are forms of given peptides chemically modified relative to reference peptides, wherein the modifications include but are not limited to oligomerization or polymerization, modification of amino acid residues or peptide backbones, cross-linking, cyclization, conjugation, pegylation, glycosylation, acetylation, phosphorylation, acylation, carboxylation, lipidation, thioglycolic acid amidation, alkylation, methylation, polyglycosylation, glycosylation, polysialylation, adenylation, pegylation, fusion with other heterologous amino acid sequences, or substantially change the stability, solubility or other properties of the peptides while substantially maintaining other modifications of the activity of the polypeptides described herein. It should be understood that the fusion protein comprising such modifications is cross-linked, cyclized, conjugated, acylated, carboxylated, lipidated, acetylated, thioglycolic acid amidated, alkylated, methylated, polyglycosylated, glycosylated, polysialylated, phosphorylated, adenylated, pegylated or a combination thereof. In some embodiments, the modified fusion protein of the present disclosure may contain non-amino acid elements, such as polyethylene glycol, lipids, polysaccharides or monosaccharides and phosphates. The fusion protein of the present disclosure may include modifications disclosed herein at the C-terminus (e.g., C-terminal amidation), N-terminus (e.g., N-terminal acetylation). Terminal modifications are useful and well known to reduce sensitivity to protease digestion, and are therefore used to extend the half-life of polypeptides in biological fluids where proteases may be present. In some embodiments, the fusion protein described herein is further modified within the sequence, such as by terminal-NH2 acylation, such as acetylation or thioglycolic acid amidation, by terminal-carboxyl amidation, such as modification with ammonia, methylamine and similar terminal modifications.

[0063] Terminal modification is useful to reduce the sensitivity of protease digestion, and therefore can be used to extend the half-life of polypeptides in solution, particularly in biological fluids where proteases may be present. Amino terminal modification includes methylation (e.g., --NHCH3 or --N(CH3)2), acetylation (e.g., with acetic acid or its halogenated derivatives, such as α-chloroacetic acid, α-bromoacetic acid or α-iodoacetic acid), addition of benzyloxycarbonyl (Cbz) group, or blocking the amino terminal with any end-capping group containing a carboxylate functional group defined by RCOO-- or a sulfonyl functional group defined by R--SO2--, wherein R is selected from the group consisting of: alkyl, aryl, heteroaryl, alkylaryl, etc., and similar groups. Deamino acid (deamino acid) can also be incorporated into the N-terminus (so that there is no N-terminal amino group) to reduce the sensitivity to proteases or to limit the conformation of the polypeptide. In certain embodiments, the N-terminus is acetylated by acetic acid or acetic anhydride.

[0064] The carboxyl terminal modification includes replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxyl terminal to introduce structural constraints. It is also possible to cyclize the peptides described herein, or to incorporate deamino or decarboxylation residues at the peptide termini so that there are no terminal amino or carboxyl groups to reduce sensitivity to proteases or to restrict the conformation of the peptide. Methods for the synthesis of cyclic peptides are known in the art. The C-terminal functional groups of the peptides described herein include amides, amide lower alkyl groups, amide di(lower alkyl groups), lower alkoxy groups, hydroxyl groups and carboxyl groups, and lower ester derivatives thereof, and pharmaceutically acceptable salts thereof.

[0065] In some embodiments, the fusion proteins described herein are phosphorylated. Peptides can also be easily modified by phosphorylation and other methods. In some embodiments, other side chains can also be used, such as alkyl, lower (C 1-6 ) alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl amide di(lower alkyl), lower alkoxy, hydroxy, carboxyl and lower ester derivatives thereof, as well as substitution of naturally occurring genetically encoded amino acids (or stereoisomeric D amino acids) side chains with 4-, 5-, 6- to 7-membered heterocyclic rings. For example, proline analogs in which the ring size of the proline residue is changed from 5-membered to 4, 6 or 7-membered can be used.

[0066] Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic.Heterocyclic groups preferably contain one or more nitrogen, oxygen and / or sulfur heteroatoms.The example of such groups includes furazanyl, furanyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g. 1-piperazinyl), piperidinyl (e.g. 1-piperidinyl, piperido), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g. 1-pyrrolidinyl), pyrrolinyl, pyrrole, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g. thiomorpholino) and triazolyl groups.These heterocyclic groups can be substituted or unsubstituted.When the group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen or substituted or unsubstituted phenyl.

[0067] In some embodiments, the fusion protein described herein can be connected to one or more polymer parts. In some embodiments, these polymers are covalently connected to the fusion protein of the present disclosure. In some embodiments, for the therapeutic use of the final product preparation, the polymer is pharmaceutically acceptable. Those skilled in the art will be able to select the desired polymer based on considerations such as whether the polymer-peptide conjugate is used for treatment, and if so, consider the desired dosage, circulation time, resistance to proteolysis and other considerations.

[0068] All combinations of different modifications and derivatizations are contemplated for the fusion proteins described herein.

[0069] Other aspects of the present disclosure provide methods for producing fusion proteins. Fusion proteins can be produced and separated by expressing recombinant nucleic acids in appropriate cells (e.g., bacterial cells or eukaryotic cells). In order to produce fusion proteins, nucleic acids encoding fusion proteins can be introduced into cells (e.g., bacterial cells or eukaryotic cells, such as yeast cells or insect cells). Cells can be cultured under conditions that allow fusion proteins to be expressed from nucleic acids encoding fusion proteins. Fusion proteins comprising signal peptides can be secreted, for example, into culture medium and can be recovered subsequently. Fusion proteins can be separated using any method for purifying proteins known in the art.

[0070] The nucleic acid encoding the fusion protein described herein can be obtained by any method known in the art and the nucleotide sequence of the nucleic acid can be determined. Those skilled in the art can identify the nucleotide sequence encoding the fusion protein from the amino acid sequence of the fusion protein. The nucleic acid encoding the fusion protein disclosed herein can be double-stranded or single-stranded DNA or RNA. In certain embodiments, the nucleotide sequence encoding the fusion protein can be codon optimized to adapt to different expression systems (for example, for mammalian expression).

[0071] In some embodiments, the nucleic acid is contained in a vector, such as an expression vector. In some embodiments, the vector comprises a promoter operably linked to the nucleic acid.

[0072] A variety of promoters can be used to express the fusion proteins described herein, including but not limited to the cytomegalovirus (CMV) intermediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, simian virus 40 (SV40) early promoter, Escherichia coli lac UV5 promoter, and herpes simplex virus promoter.

[0073] Regulatable promoters may also be used. Such regulatable promoters include promoters that use the lac repressor of E. coli as a transcriptional regulator to regulate transcription from mammalian cell promoters with the lac operator, promoters that use the tetracycline repressor (tetR), and the like. Other systems include the use of estradiol, RU486, diphenolsterone, or FK506 dimers of rapamycin, VP16, or p65.

[0074] A regulatable promoter including a repressor with an operator can be used. In one embodiment, the lac repressor from E. coli can be used as a transcriptional regulator to regulate transcription from a mammalian cell promoter with a lac operator. In one embodiment, a tetracycline inducible switch is used.

[0075] In addition, the vector may include, for example, some or all of the following: a selectable marker gene, such as a neomycin gene for selecting stable or transient transfectants in mammalian cells; an enhancer / promoter sequence from the immediate early gene of human CMV for high-level transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma replication origin and ColE1 for correct episomal replication; an internal ribosome binding site (IRES), a multifunctional multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known in the art and are available.

[0076] The expression vector comprising the nucleic acid can be transferred to a host cell by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the fusion proteins described herein. In some embodiments, the expression of the fusion proteins described herein is regulated by a constitutive, inducible, or tissue-specific promoter.

[0077] Host cells used to express the fusion proteins described herein may include bacterial cells (eg, E. coli), eukaryotic cells, etc. In one embodiment, mammalian cells (eg, Chinese hamster ovary cells (CHO)) may be used.

[0078] A variety of host-expression vector systems can be utilized to express the fusion proteins described herein. Such host-expression systems represent vehicles by which the coding sequences of the isolated fusion proteins described herein can be produced and subsequently purified, but also represent cells that in situ express the fusion proteins described herein when transformed or transfected with the appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors comprising the coding sequence of the fusion protein described herein, such as bacteria (e.g., Escherichia coli and Bacillus subtilis); yeast (e.g., Pichia pastoris) transformed with recombinant yeast expression vectors containing sequences encoding the fusion protein described herein; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing sequences encoding the fusion protein described herein; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing sequences encoding the fusion protein described herein; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphoid cells, Per C.6 cells carrying recombinant expression constructs containing promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or promoters of mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0079] In bacterial systems, a variety of expression vectors can be advantageously selected according to the purpose of the expressed fusion protein. For example, when a large amount of such proteins are to be produced, in order to generate the pharmaceutical composition of the fusion protein described herein, it may be desirable to instruct the expression of a high-level fusion protein product that is easily purified. Such vectors include, but are not limited to, the E. coli expression vector pUR278, in which the coding sequence can be individually connected to a vector in a frame with a lac Z coding region to produce a fusion protein; pIN vectors, etc. pGEX vectors can also be used to express exogenous polypeptides as fusion proteins with glutathione S-transferase (GST). Typically, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads, followed by elution in the presence of free glutathione. pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST portion.

[0080] In mammalian host cells, many virus-based expression systems can be utilized. In the case where adenovirus is used as an expression vector, the target coding sequence can be connected to an adenovirus transcription / translation control composition, such as a late promoter and a three-part leader sequence. The chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion of the non-essential regions of the viral genome (e.g., region E1 or E3) will result in recombinant viruses being feasible and capable of expressing immunoglobulin molecules in infected hosts. The effective translation of the inserted antibody coding sequence may also require specific start signals. These signals include an ATG start codon and adjacent sequences. In addition, the start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and start codons can be natural and synthetic in a variety of sources. Expression efficiency can be enhanced by including appropriate transcription enhancer elements, transcription terminators, etc.

[0081] In addition, host cell strains that regulate the expression of the inserted sequence or modify and process the gene product in a desired ad hoc manner can be selected. Such modifications (e.g., glycosylation) and processing (e.g., cutting) of the protein product may be important for the function of the protein. The purification and modification of recombinant proteins are well known in the art, so that the design of polyprotein precursors can include multiple embodiments that are easily understood by the technician. Any known protease or peptidase known in the art can be used for the described modification of precursor molecules, such as thrombin or factor Xa, enterokinase, furin and AcTEV, and foot-and-mouth disease virus protease C3, etc.

[0082] Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure the correct modification and processing of the expressed foreign proteins. For this reason, eukaryotic host cells with appropriate processing of primary transcripts, gene product glycosylation and phosphorylation cellular mechanisms can be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.

[0083] For the long-term high-yield production of recombinant proteins, stable expression is preferred. For example, the cell line of the fusion protein described herein can be engineered to stably express. Instead of using an expression vector containing a viral origin of replication, the host cell can be transformed with DNA and selectable markers controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). After introducing foreign DNA, the engineered cells can be grown in enriched medium for 1-2 days and then switched to selective medium. The selectable markers in the recombinant plasmid confer resistance to selection, and allow cells to stably integrate the plasmid into their chromosomes and grow to form cloning sites, which can be cloned and expanded into the cell line. The method can be advantageously used for engineering the cell line expressing the fusion protein described herein. Such engineered cell lines can be particularly useful for screening and evaluating the fusion protein that directly or indirectly interacts with the fusion protein described herein.

[0084] The expression level of the fusion described herein can be increased by vector amplification. When the marker in the vector system expressing the fusion protein described herein is amplifiable, the increase of the inhibitor level present in the culture of the host cell will increase the copy number of the marker gene. Because the amplified region is related to the nucleotide sequence of the fusion protein described herein or the fusion protein described herein, the output of the fusion protein also can increase.

[0085] Once a fusion described herein has been recombinantly expressed, it can be purified by any method known in the art for purifying a polypeptide, polyprotein or antibody (e.g., similar to antibody purification schemes based on antigen selectivity), for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for a specific antigen (optionally following protein A selection where the polypeptide comprises an Fc domain (or portion thereof)) and size exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying polypeptides or antibodies.

[0086] In some embodiments, fusion proteins can advantageously resist degradation relative to other compositions comprising linked antibodies and enzymes. Fusion proteins can also result in unexpectedly high performance of the molecule. For example. For example, relative to the activity of a composition in which an enzyme is cross-linked with an antibody or antibody fragment, an enzyme, antibody or antibody fragment can have unexpectedly high activity when comprised by the fusion protein of the composition. This can advantageously improve the effectiveness of the composition comprising the fusion protein.

[0087] However, the enzyme can be connected to another part of the composition (e.g., an antibody or antibody portion) by cross-linking. The part and the enzyme can be cross-linked using a cross-linking agent. Cross-linking can include a synthetic cross-linking agent, or can include a direct coupling between the amino acid side chains of the antibody and the enzyme. In certain embodiments, one or more cross-linking agents are used to cross-link the antibody and the enzyme. Non-limiting examples of cross-linking agents include glutaraldehyde, NHS-esters (N-hydroxysuccinimide) (e.g., dithiobis (succinimidyl propionate), dithiobis (sulfosuccinimidyl propionate), etc.), PEG groups, amide esters (e.g., dimethyl adipamide, dimethyl suberimide, dimethyl pimelimide, etc.), maleimide, pyridyl, carbodiimide, isocyanate, etc.

[0088] The antibody and enzyme can be coupled by any suitable cross-linking system, such as amine to amine, sulfhydryl to sulfhydryl, amine to sulfhydryl, carboxyl to amine, sulfhydryl to carbohydrate, hydroxyl to sulfhydryl, or the like. One of ordinary skill in the art will be familiar with methods for cross-linking or conjugating proteins to each other.

[0089] In certain embodiments, the composition may include a treatment agent, or may be used with a treatment agent. Treatment agents may be selected to increase the sensitivity of cells to the composition. In certain embodiments, the subject is pretreated with a treatment agent. In certain embodiments, the treatment agent is used as a part of a composition as described herein or is applied to the subject simultaneously with the application of the composition as described herein. The subject may also receive the treatment agent after the application of the composition and the disclosure is not limited thereto. Treating the subject with a treatment agent may prepare the subject for the successful attack on the lysosomal membrane that can be used to treat the disease. Treatment using a treatment agent may provide a more effective attack on the lysosomal membrane. However, it should also be understood that it is not necessary to use a treatment agent, and in some cases, the composition may be applied without the use of a treatment agent.

[0090] In some embodiments, the treating agent is configured to increase the concentration of reactive oxygen species near the cells targeted by the composition. Without wishing to be bound by theory, it is believed that, as described above, oxidation processes (e.g., those caused by the action of oxidases or peroxidases) can be used to treat diseases or conditions. Thus, the treating agent can increase the rate of oxidation by increasing the concentration of reactive oxygen species near the cells, or can increase the total amount of oxidation.

[0091] In some embodiments, the increase in reactive oxygen species concentration is associated with the level of antioxidants in the subject, as antioxidants can reduce or inhibit such tumor treatment. In some embodiments, the treating agent reduces the level of antioxidants near the target cells. For example, the treating agent can remove or inhibit antioxidants in the subject, as discussed in more detail below.

[0092] It should be understood that, according to some embodiments, one or more treatment agents can be used separately or together. For example, a statin, a fatty acid, or both can be administered to a subject, as discussed in more detail below. Treatment agents can be administered separately and / or together. If administered separately, the treatment agents can be administered simultaneously and / or sequentially in any suitable order. Any suitable method of administration and dosing regimen can be used, including those discussed herein. In addition, one or more treatment agents can be present in a suitable pharmaceutically acceptable carrier. Whether or not a treatment agent is used, antioxidants can be removed or inhibited from the subject.

[0093] In some embodiments, the treatment agent comprises fatty acids. The fatty acids may include unsaturated fatty acids. For example, the treatment agent may include n-3 to n-6 fatty acids. In some embodiments, the treatment agent is a fatty acid. In some embodiments, the administration of fatty acids results in a decrease in the antioxidant status of the subject. Unsaturated fatty acids, including n-3 to n-6 fatty acids, may be fed to the subject, or may be administered by any of a variety of other appropriate methods. In the context of the present disclosure, it has been creatively recognized that treating a subject with unsaturated fatty acids may increase the local concentration of unsaturated fatty acids that are oxidizable by reactive oxygen species. This may accelerate lysosomal-induced cell death by accelerating the degradation of lysosomal membranes.

[0094] One or more fatty acids can be applied to the subject. One or more fatty acids can be applied simultaneously and / or sequentially. Unsaturated fatty acids can contain one or more double bonds and / or triple bonds in the fatty acid chain. Fatty acids can be relatively short-chain fatty acids. Non-limiting examples of such unsaturated fatty acids include n-3 to n-8 (e.g., n-3, n-4, n-5, n-6, n-7 or n-8) fatty acids. Unsaturated fatty acids can be monounsaturated and / or polyunsaturated fatty acids. Non-limiting examples of unsaturated fatty acids include, but are not limited to, CH2=CHCOOH, CH3CH=CHCOOH, CH3CH2CH=CHCOOH, CH3CH2CH2CH=CHCOOH, CH2=CHCH2COOH, CH2=CH CH2CH2COOH, CH2=CHCH2CH2CH2COOH, CH2=CHCH2CH=CHCOOH, CH2=CHCH=CHCOOH, CH2=CHCH=CHCOOH, etc. Furthermore, in some cases, the fatty acids can be derived from naturally occurring sources, such as krill oil, fish oil, safflower oil, soybean oil, flaxseed oil, canola oil, algae oil, and the like.

[0095] A wide range of fatty acid dosages can be used. In some embodiments, the fatty acid can be administered to a subject, such as a human, at a dosage of at least 0.1 g, at least 0.2 g, at least 0.3 g, at least 0.4 g, at least 0.5 g, at least 0.6 g, at least 0.7 g, at least 0.8 g, at least 0.9 g, at least 1 g, at least 2 g, at least 3 g, at least 4 g, at least 5 g, at least 6 g, at least 7 g, at least 8 g, at least 9 g, at least 10 g, at least 11 g, at least 12 g, at least 13 g, at least 14 g, or at least 15 g. In certain embodiments, fatty acid may be administered at a dosage of no more than 15 g, no more than 14 g, no more than 13 g, no more than 12 g, no more than 11 g, no more than 10 g, no more than 9 g, no more than 8 g, no more than 7 g, no more than 6 g, no more than 5 g, no more than 4 g, no more than 3 g, no more than 2 g, no more than 1 g, no more than 0.9 g, no more than 0.8 g, no more than 0.7 g, no more than 0.6 g, no more than 0.5 g, no more than 0.4 g, no more than 0.3 g, no more than 0.2 g, or no more than 0.1 g. In certain embodiments, any combination of these is also possible, for example, the dosage may be between 1 g and 15 g, between 1 g and 10 g, between 5 g and 10 g, between 0.5 g and 1 g, etc.

[0096] In some embodiments, the fatty acid can be administered to a subject, such as a human, at a dose of at least 1 mg / kg, at least 2 mg / kg, at least 3 mg / kg, at least 5 mg / kg, at least 10 mg / kg, at least 20 mg / kg, at least 30 mg / kg, at least 50 mg / kg, at least 100 mg / kg, at least 200 mg / kg, at least 300 mg / kg, at least 500 mg / kg, at least 1 g / kg, or at least 2 mg / kg. In some cases, the fatty acid can be administered at a dose of no more than 2 g / kg, no more than 1 g / kg, no more than 500 mg / kg, no more than 300 mg / kg, no more than 200 mg / kg, no more than 100 mg / kg, no more than 50 mg / kg, no more than 30 mg / kg, no more than 20 mg / kg, no more than 10 mg / kg, no more than 5 mg / kg, no more than 3 mg / kg, no more than 2 mg / kg, or no more than 1 mg / kg. In some embodiments, any combination of these is also possible, for example, the dose can be 1 to 2 g / kg, 500 mg / kg to 1 g / kg, 400 to 800 mg / kg, etc. Appropriate doses, dose rates, and treatments using fatty acids can be determined using the methods described herein.

[0097] In some embodiments, the treatment agent comprises a statin. Statins can be co-administered with fatty acids, or can be administered without the administration of fatty acids. Statins, also known as HMG-CoA reductase inhibitors, can be used to reduce blood lipid levels. Non-limiting examples of statins include atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, cerivastatin, mevastatin and simvastatin. One or more statins can be administered to a subject, for example, simultaneously and / or sequentially.

[0098] A wide range of statin dosages can be used. For example, statins can be administered to a subject, such as a human, at a dosage of at least 1 mg, at least 2 mg, at least 3 mg, at least 5 mg, at least 10 mg, at least 20 mg, at least 30 mg, at least 40 mg, at least 50 mg, at least 60 mg, at least 70 mg, at least 80 mg, at least 90 mg or at least 100 mg. In some cases, statins can be administered at a dosage of no more than 100 mg, no more than 90 mg, no more than 80 mg, no more than 70 mg, no more than 60 mg, no more than 50 mg, no more than 40 mg, no more than 30 mg, no more than 20 mg, no more than 10 mg, no more than 5 mg, no more than 3 mg, no more than 2 mg or no more than 1 mg. In certain embodiments, any combination of these is also possible, for example, dosage can be 10 mg to 20 mg, 20 mg to 40 mg, 40 mg to 80 mg, 5 mg to 10 mg, etc.

[0099] In addition, in some embodiments, the statin can be administered to a subject, such as a human, at a dose of at least 1 mg / kg, at least 2 mg / kg, at least 3 mg / kg, at least 4 mg / kg, at least 5 mg / kg, at least 6 mg / kg, at least 7 mg / kg, at least 8 mg / kg, at least 9 mg / kg, at least 10 mg / kg, at least 11 mg / kg, at least 12 mg / kg, at least 13 mg / kg, at least 14 mg / kg, or at least 15 mg / kg. In some embodiments, the statin can be administered at a dose of no more than 15 mg / kg, no more than 14 mg / kg, no more than 13 mg / kg, no more than 12 mg / kg, no more than 11 mg / kg, no more than 10 mg / kg, no more than 9 mg / kg, no more than 8 mg / kg, no more than 7 mg / kg, no more than 6 mg / kg, no more than 5 mg / kg, no more than 4 mg / kg, no more than 3 mg / kg, no more than 2 mg / kg, or no more than 1 mg / kg. In some embodiments, combinations of any of these are also possible, for example, the dose may be between 1 mg / kg and 15 mg / kg, between 1 mg / kg and 10 mg / kg, between 5 mg / kg and 10 mg / kg, etc. Appropriate doses, dose rates, and treatments using fatty acids can be determined using the methods described herein.

[0100] According to certain embodiments, if the subject is taking an antioxidant, the antioxidant can be removed or inhibited from the subject. For example, the use of the antioxidant can be stopped completely, or the dosage of the antioxidant can be reduced, such as by reducing the amount and / or frequency, as part of preparing to treat the tumor. Non-limiting examples of antioxidants include beta-carotene, vitamin A, and vitamin E. Other examples include vitamin C, glutathione, lipoic acid, uric acid, and panthenol.

[0101] As described above, some embodiments of the present disclosure relate to compositions comprising enzymes such as oxidoreductases. For example, the enzyme can be an oxidase or a peroxidase. Other exemplary oxidoreductases that can be used include, but are not limited to, transglutaminase 3 (e.g., human transglutaminase 3); hydroxyacid oxidase 1 (e.g., human hydroxyacid oxidase 1); nitrate reductase (NAD[P]H) (e.g., from Pichia pastoris); native Aspergillus niger catalase; NADH peroxidase (e.g., from Pseudomonas fluorescens); L-malate dehydrogenase (e.g., from Escherichia coli); L-lactate dehydrogenase (e.g., porcine L-lactate dehydrogenase) ; D-lactate dehydrogenase (e.g., from Leuconostoc mesenteroides); isocitrate dehydrogenase (e.g., isocitrate dehydrogenase from Bacillus subtilis); inositol dehydrogenase (e.g., from Bacillus subtilis); prokaryotic 3-hydroxybutyrate dehydrogenase; galactose dehydrogenase (e.g., from Escherichia coli); galactose mutarotase (e.g., from Escherichia coli); formate dehydrogenase (e.g., from Candida boidinii); manganese peroxidase (e.g., from natural white rot fungus (Nematoloma frowardii) manganese peroxidase); cholesterol oxidase (e.g., from Nocardia spp.); 15-lipoxygenase-2 (e.g., human 15-lipoxygenase-2); 3-acetylpyridine-adenine dinucleotide phosphate; 3-oxo-5-β-steroid 4-dehydrogenase (e.g., human 3-oxo-5-β-steroid 4-dehydrogenase); 3α-hydroxysteroid dehydrogenase; 5-lipoxygenase (e.g., human 5-lipoxygenase); Acyl-CoA oxidase; acyl-CoA dehydrogenase 8 (e.g., human acyl-CoA dehydrogenase 8); human aflatoxin B1 aldehyde reductase member 2 (e.g., human aflatoxin B1 aldehyde reductase member 2); alanine dehydrogenase (e.g., from Bacillus cereus); alcohol dehydrogenase (e.g., human alcohol dehydrogenase or alcohol dehydrogenase from Escherichia coli); aldehyde dehydrogenase 2 (e.g., human aldehyde dehydrogenase 2); aldose reductase (e.g., human aldose reductase); bilirubin oxidation enzyme; biotinylated luciferase (e.g., from Escherichia coli); bovine catalase-polyethylene glycol; bovine superoxide dismutase-polyethylene glycol; catalase (e.g., from psychrotolerant bacteria); cholesterol oxidase (e.g., from Escherichia coli); copper / zinc superoxide dismutase; cyclohexanone monooxygenase (e.g., from Acinetobacter species); cyclooxygenase (e.g., human cyclooxygenase 1 or 2 or sheep cyclooxygenase 2); cytochrome p450 reductase (e.g., human cytochrome p450 reductase); D-2-hydroxyacetateglutarate dehydrogenase (e.g., from Acidaminococcus fermentans); D-2-hydroxyisocaproate dehydrogenase; D-3-hydroxybutyrate dehydrogenase; D-amino acid dehydrogenase; D-amino acid oxidase (e.g., human D-amino acid oxidase); diaphorase (e.g., from Escherichia coli); dihydrofolate reductase (e.g., human dihydrofolate reductase); dimethylglycine oxidase (e.g., from Arthrobacter sphaeroides);disulfide oxidoreductase; D-lactate dehydrogenase (e.g., from E. coli); DT diaphorase (e.g., human DT diaphorase or rat DT diaphorase); FMN reductase (e.g., from E. coli); formate dehydrogenase (e.g., from Candida boidinii or recombinant E. coli); free methionine-(R)-sulfoxide reductase (e.g., from E. coli); fructosyl-amino acid oxidase (e.g., from Corynebacterium spp. or E. coli); fructosyl amino acid oxidase; fructosyl-peptide oxidase (e.g., from E. coli); galactose dehydrogenase (e.g., from recombinant E. coli); galactose oxidase (e.g., from Dactylospora dendrogensis); glucose dehydrogenase (e.g., from E. coli); glucose-6 -phosphate dehydrogenase (e.g., from Escherichia coli); glutamate dehydrogenase (e.g., from Escherichia coli or thermophilic bacteria); glutathione reductase (e.g., human glutathione reductase from Escherichia coli); glyceraldehyde-3-phosphate dehydrogenase (e.g., human or mouse glyceraldehyde-3-phosphate dehydrogenase); glycerol-3-phosphate dehydrogenase (e.g., from Escherichia coli); glycerol-3-phosphate oxidase (e.g., from Escherichia coli); glycine oxidase H244K (e.g., from Bacillus subtilis); histamine dehydrogenase (e.g., from Escherichia coli); inosine monophosphate dehydrogenase (e.g., from Staphylococcus aureus); type II inosine monophosphate dehydrogenase (e.g., type II human inosine monophosphate dehydrogenase); isocitrate dehydrogenase (NA D+) (e.g., from bacteria or yeast); ketoamine oxidase; ketoreductase; L-2-hydroxyisocaproate dehydrogenase; laccase (e.g., from Bacillus subtilis or Escherichia coli); lactaldehyde dehydrogenase (e.g., from Escherichia coli); lactate dehydrogenase (e.g., native human lactate dehydrogenase 1, 2, 3, or 5, or chicken heart lactate dehydrogenase); lactate oxidase (e.g., from Aerococcus viride); lactate dehydrogenase; leucine dehydrogenase; Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase; L-glutamate dehydrogenase; L-glutamate oxidase (e.g., from Streptomyces species); L-lactate dehydrogenase; L-lactate dehydrogenase (e.g., from Bacillus stearothermophilus); L-leucine dehydrogenase; L-phenylalanine dehydrogenase; luciferase (e.g., from E. coli or from Pseudomonas firefly); lytic cellulose monooxygenase (e.g., from Thermospora fusca); lytic chitin monooxygenase (e.g., from Bacillus licheniformis or from Lactococcus lactis); malate dehydrogenase; mannitol dehydrogenase; Mn-superoxide dismutase; monoamine oxidase (e.g., human monoamine oxidase A or human monoamine oxidase B); isocitrate dehydrogenase 1R132H (e.g., human isocitrate dehydrogenase 1R132H or isocitrate dehydrogenase 1R132H from E. coli); N-acylmannosamine 1-dehydrogenase (e.g., from Pseudomonas species); native 6-phospho-D-gluconate dehydrogenase (e.g., from E. coli); native Acremonium species ascorbate oxidase;Natural green aerosol glycerol 3-phosphate oxidase; natural green aerosol pyruvate oxidase; natural Agaricus bisporus laccase; natural Alcaligenes faecalis 3-hydroxybutyrate dehydrogenase; natural Alcaligenes species choline oxidase; natural alcohol dehydrogenase; natural Arthrobacter sphaeroides choline oxidase; natural Arthrobacter sphaeroides uricase; natural Arthrobacter species acyl-CoA oxidase; natural Arthrobacter species tyramine oxidase; natural Aspergillus niger glucose oxidase; natural Aspergillus niger nitrate reductase (NAD[P]H); natural Aspergillus species catalase; natural Aspergillus species glucose oxidase; natural Bacillus cereus L-leucine dehydrogenase; natural Bacillus fastidious uricase; natural Bacillus licheniformis NADH oxidase; natural Bacillus megaterium diaphorase (NADH); natural Bacillus pumilus bilirubin oxidase / laccase; natural Bacillus species glucose-6-phosphate dehydrogenase; natural Bacillus species leucine dehydrogenase; natural Bacillus species sarcosine oxidase; natural Bacillus species uricase; natural Bacillus species Bacillus species 12α-hydroxysteroid dehydrogenase; natural Bacillus stearothermophilus alanine dehydrogenase; natural Bacillus stearothermophilus diaphorase 1; natural Bacillus stearothermophilus leucine dehydrogenase; natural Bacillus stearothermophilus polynucleotide phosphorylase; natural Bacillus stearothermophilus superoxide dismutase; natural Bacillus subtilis bilirubin oxidase; natural Bacillus subtilis L-alanine dehydrogenase; natural baker's yeast (Saccharomyces cerevisiae) glucose-6-phosphate dehydrogenase; natural baker's yeast (Saccharomyces cerevisiae) glutathione GSH reductase; natural baker's yeast (Saccharomyces cerevisiae) glyceraldehyde-3-phosphate dehydrogenase; natural tobacco pipe peroxidase; natural bovine catalase; natural bovine glutamate dehydrogenase; natural bovine glutathione peroxidase; natural bovine lactoperoxidase; natural bovine L-glutamate dehydrogenase; natural bovine L-lactate dehydrogenase; natural bovine L-lactate dehydrogenase; natural bovine malate dehydrogenase; natural bovine plasma amine oxidase; natural bovine superoxide dismutase; natural bovine xanthine oxidase; natural Caldariomyces fumago chloroperoxidase; natural Candida boidinii formate dehydrogenase; natural Candida species alcohol oxidase; natural Candida species uricase; natural Candida utilis L-glutamate dehydrogenase (NADP); natural canine superoxide dismutase; natural Cellulomonas species glycerol dehydrogenase; natural chicken glyceraldehyde-3-phosphate dehydrogenase; natural chicken L-lactate dehydrogenase; natural chicken malate dehydrogenase (oxaloacetate decarboxylation); natural chicken sulfite oxidase; natural Clostridium kluyveri diaphorase; natural Clostridium species diaphorase; natural coral algae bromoperoxidase; natural versicolor laccase; natural corn nitrate reductase; natural Corynebacterium glutamicum catalase; natural eastern diamondback rattlesnake L-amino acid oxidase; natural black rattlesnake L-amino acid oxidase; natural South American rattlesnake venom L-amino acid oxidase; natural Cucurbita species ascorbate oxidase;Natural Cucurbita species L-ascorbate oxidase; natural Dactylospora dendriticus galactose oxidase; natural diaphorase (NADPH) (e.g., from Bacillus megaterium); natural Escherichia coli 1-5-anhydroglucitol-6-phosphate dehydrogenase; natural Escherichia coli galactose 1-dehydrogenase; natural Escherichia coli sarcosine oxidase; natural Enterobacter aerogenes glycerol dehydrogenase; natural Enterobacter aerogenes myo-inositol dehydrogenase; natural horse spleen apoferritin; natural Escherichia coli nitrate reductase (cytochrome); natural Escherichia coli superoxide dismutase; natural Escherichia coli thioredoxin reductase; natural industrial Gluconobacter D-fructose dehydrogenase; natural Gluconobacter species D-fructose dehydrogenase; natural glutamate dehydrogenase (NAD P+); natural soy bean (soybean) lipoxygenase; natural Hansenula species alcohol oxidase; natural horseradish peroxidase; natural horseradish peroxidase; natural horseradish polyperoxidase; natural horseradish superoxide dismutase; natural human catalase; natural human eosinophil peroxidase; natural human glutathione peroxidase; natural human glyceraldehyde-3-phosphate dehydrogenase; natural human lactate dehydrogenase; natural human lactoperoxidase; natural human myeloperoxidase (e.g., isoform A, B, or C); natural human superoxide dismutase; natural laccase (e.g., from Trichoderma unicolor or from white rot fungi); natural lactate dehydrogenase (e.g., from thermophilic bacteria); natural Lactobacillus delbrueckii D-lactate dehydrogenase; natural Lactobacillus religiosa D- Lactate dehydrogenase; natural Leuconostoc mesenteroides D-lactate dehydrogenase; natural Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase; natural Leuconostoc mesenteroides mannitol dehydrogenase; natural lignin peroxidase (e.g., from Phanerochaete chrysosporium); natural malate dehydrogenase (e.g., from thermophilic bacteria); natural Micrococcus lysodeikticus catalase; natural microbial cholesterol oxidase; natural microbial D-lactate dehydrogenase; natural microbial glucose dehydrogenase (FAD-dependent); natural microbial glucose dehydrogenase (NAD(P)-dependent); natural microbial glucose dehydrogenase (PQQ-dependent); natural microbial glucose-6-phosphate dehydrogenase; natural microbial glutamate dehydrogenase (NAD-dependent); natural microbial L- α-Glycerol phosphate oxidase; natural microbial malate dehydrogenase; natural microbial p-hydroxybenzoate hydroxylase; natural microbial pyruvate oxidase; natural microbial sarcosine oxidase; natural microbial sorbitol dehydrogenase; natural microbial xanthine oxidase; natural microbial pyruvate oxidase; natural mouse catalase; natural mushroom tyrosinase; natural mushroom polyphenol oxidase; natural Myrocera verrucosum bilirubin oxidase; natural nitrate reductase (e.g., from Aspergillus species); natural Nocardia erythropolis cholesterol oxidase; natural Nocardia species cholesterol dehydrogenase; natural Pediococcus species glycerol 3-phosphate oxidase; natural Pediococcus species glycerol-3-phosphate oxidase; natural Pediococcus species lactate oxidase;Natural Pediococcus species L-α-glycerophosphate oxidase; Natural Photinus pyralis (firefly) luciferase; Natural Photorhabditis elegans (Lux) bacterial luciferase; Natural Pichia pastoris alcohol oxidase; Natural plant-derived diamine oxidase; Natural Pleurotus ostreatus laccase; Natural porcine apo D-amino acid oxidase; Natural porcine cytochrome C reductase; Natural porcine D-amino acid oxidase; Natural porcine diamine oxidase; Natural porcine heart lactate dehydrogenase; Natural porcine isocitrate dehydrogenase (NADP); Natural porcine lactate dehydrogenase; Natural porcine fatty amide dehydrogenase; Natural porcine L-lactate dehydrogenase; Natural porcine L-lactate dehydrogenase; Natural porcine malate dehydrogenase, IFCC quality porcine heart; Natural porcine malate dehydrogenase; Natural porcine NAD(P)H dehydrogenase (quinone); Natural porcine superoxide dismutase; Natural porcine α- Ketoglutarate dehydrogenase; native Proteus species glutamate dehydrogenase (NADP-dependent); native Proteus species L-glutamate dehydrogenase (NADP); native Pseudomonas fluorescens galactose 1-dehydrogenase; native Pseudomonas leucoderma β-hydroxybutyrate dehydrogenase; native Pseudomonas species cholesterol oxidase; native Pseudomonas species D-3-hydroxybutyrate dehydrogenase; native Pseudomonas species formaldehyde dehydrogenase; natural Pseudomonas species glucose dehydrogenase; natural Pseudomonas species N-acylhexosamine oxidase hydroxylase from Pseudomonas species; p-hydroxybenzoate hydroxylase from Pseudomonas species; protocatechuate 3,4-dioxygenase from Pseudomonas species; protocatechuate 3,4-dioxygenase from Pseudomonas species; sarcosine dehydrogenase from Pseudomonas species; 3α-hydroxysteroid dehydrogenase from Pseudomonas testosteroni; cytochrome P450 reductase from rabbit; glyceraldehyde-3-phosphate dehydrogenase from rabbit; glycerol-3-phosphate dehydrogenase from rabbit; lactate dehydrogenase from rabbit; lactate dehydrogenase from rabbit; pyruvate kinase / lactate Acid dehydrogenase rabbit muscle; natural rabbit α-glycerol phosphate dehydrogenase; natural rabbit α-glycerol phosphate dehydrogenase-triose phosphate isomerase; natural rat sorbitol dehydrogenase; natural rat thioredoxin reductase; natural Rhodopseudomonas sphericalus β-hydroxybutyrate dehydrogenase; natural Rhus verniciflua laccase; natural Rosa roxburghii superoxide dismutase; natural Saccharomyces cerevisiae alcohol dehydrogenase; natural sheep 6-phosphoglucose dehydrogenase; natural sheep cyclooxygenase; natural sheep sorbitol dehydrogenase; natural spinach glutathione reductase; natural spinach (Spinacia oleracea)(spinach) ferredoxin-NADP+ reductase; native Sporosarcina species L-phenylalanine dehydrogenase; native Staphylococcus epidermidis D-lactate dehydrogenase; native Streptococcus thermophilus glycerol 3-phosphate oxidase; native Streptomyces species cholesterol oxidase; natural Thermoactinomycetes intermedia phenylalanine dehydrogenase; natural Thermoanaerobacterium brunneri alcohol dehydrogenase, NADP+ dependent; natural Thermoanaerobacterium species aromatic alcohol dehydrogenase, NADP+ dependent;Natural heat-stable fungal catalase for semiconductor technology; Natural heat-stable fungal catalase for textile technology; Natural yellow fever malate dehydrogenase; Natural toruloides glucose-6-phosphate dehydrogenase; Natural versicolor laccase; Natural Trichoderma viride lysine oxidase; Natural multifunctional peroxidase (e.g. from tobacco pipe fungus); Natural Vibrio fischeri (luminescent bacterial luciferase); Natural wheat germ glutathione reductase; Natural white rot fungus (Phanerochaete chrysosporium) manganese peroxidase; Natural xanthine dehydrogenase (e.g. from cow's milk); Natural xanthine dehydrogenase; Natural yeast 6-phosphoglucose dehydrogenase; Natural yeast alcohol dehydrogenase; Natural yeast aldehyde dehydrogenase; Natural yeast formate dehydrogenase; Natural yeast malate dehydrogenase; Natural zucchini ascorbate oxidase; Natural Zymomonas mobilis alcohol dehydrogenase; Natural Zymomonas mobilis glucokinase; Natural Zymomonas mobilis glucose-6-phosphate dehydrogenase; NiFe-type cytoplasmic hydrogenase (e.g., from Pyrococcus furiosus); Nitrate reductase (e.g., from Arabidopsis thaliana); Nitric oxide synthase (e.g., mouse nitric oxide synthase); Nitroreductase (e.g., from Escherichia coli); Oxalate oxidase (e.g., from Bacillus subtilis); Phosphite dehydrogenase; Phosphate coli); prokaryotic galactose dehydrogenase; prostaglandin F synthase (e.g., human prostaglandin F synthase); protocatechuate 3,4-dioxygenase; pyranose oxidase (e.g., from Coriolus species or Escherichia coli); pyruvate oxidase (e.g., from Escherichia coli); R-carbonyl reductase; recombinant keto-acid reductoisomerase (e.g., from Mycobacterium tuberculosis); sarcosine oxidase (e.g., from Escherichia coli); S-carbonyl reductase; secondary alcohol dehydrogenase; sulfite oxidase (e.g., human sulfite oxidase); oxidase); taurine dioxygenase (e.g., from Escherichia coli); thioredoxin reductase (NADPH) (e.g., from yeast); thyroid peroxidase (e.g., human thyroid peroxidase); UDP-Glc dehydrogenase (e.g., from Streptococcus pyogenes); uricase (e.g., from Candida utilis or Escherichia coli); uronate dehydrogenase (e.g., from Agrobacterium tumefaciens); valine dehydrogenase; xanthine oxidase (e.g., from Arthrobacter); α-hydroxysteroid dehydrogenase (e.g., from Bacillus pumilus); or β-galactose dehydrogenase S (e.g., from Pseudomonas fluorescens). ;

[0102] The compositions described herein can be administered to a subject (e.g., a human) using any of a variety of suitable techniques, as discussed in more detail below. The compositions can be administered to a subject at any suitable dose. For example, the compositions can be administered to a subject at a dose of greater than or equal to 10 mg, greater than or equal to 15 mg, greater than or equal to 20 mg, greater than or equal to 25 mg, greater than or equal to 30 mg, greater than or equal to 40 mg, greater than or equal to 50 mg, greater than or equal to 60 mg, greater than or equal to 70 mg, greater than or equal to 80 mg, greater than or equal to 90 mg, greater than or equal to 100 mg, greater than or equal to 200 mg, greater than or equal to 300 mg, greater than or equal to 500 mg, greater than or equal to 1000 mg, greater than or equal to 1500 mg, greater than or equal to 2000 mg, greater than or equal to 2500 mg, greater than or equal to 3000 mg, greater than or equal to 5000 mg, etc. In some embodiments, the composition can be applied at a dosage of less than or equal to 5000 mg, less than or equal to 3000 mg, less than or equal to 2500 mg, less than or equal to 2000 mg, less than or equal to 1500 mg, less than or equal to 1000 mg, less than or equal to 500 mg, less than or equal to 300 mg, less than or equal to 200 mg, less than or equal to 100 mg, less than or equal to 90 mg, less than or equal to 80 mg, less than or equal to 70 mg, less than or equal to 60 mg, less than or equal to 50 mg, less than or equal to 40 mg, less than or equal to 30 mg, less than or equal to 25 mg, less than or equal to 20 mg, less than or equal to 15 mg, less than or equal to 10 mg, etc. Combinations of these ranges are also possible. For example, the dosage can be greater than or equal to 10 mg and less than or equal to 20 mg, greater than or equal to 50 mg and less than or equal to 100 mg, or greater than or equal to 100 mg and less than or equal to 200 mg.

[0103] In some embodiments, the composition can be administered to a subject (e.g., a human) at a dose of greater than or equal to 1 mg / kg, greater than or equal to 2 mg / kg, greater than or equal to 3 mg / kg, greater than or equal to 4 mg / kg, greater than or equal to 5 mg / kg, greater than or equal to 6 mg / kg, greater than or equal to 7 mg / kg, greater than or equal to 8 mg / kg, greater than or equal to 9 mg / kg, greater than or equal to 10 mg / kg, greater than or equal to 11 mg / kg, greater than or equal to 12 mg / kg, greater than or equal to 13 mg / kg, greater than or equal to 14 mg / kg, or greater than or equal to 15 mg / kg. In some embodiments, statins can be administered at a dose of less than or equal to 15 mg / kg, less than or equal to 14 mg / kg, less than or equal to 13 mg / kg, less than or equal to 12 mg / kg, less than or equal to 11 mg / kg, less than or equal to 10 mg / kg, less than or equal to 9 mg / kg, less than or equal to 8 mg / kg, less than or equal to 7 mg / kg, less than or equal to 6 mg / kg, less than or equal to 5 mg / kg, less than or equal to 4 mg / kg, less than or equal to 3 mg / kg, less than or equal to 2 mg / kg, or less than or equal to 1 mg / kg. Combinations of these ranges are also possible. For example, in some embodiments, the dose is greater than or equal to 1 mg / kg and less than or equal to 15 mg / kg, greater than or equal to 1 mg / kg and less than or equal to 10 mg / kg, greater than or equal to 5 mg / kg, or less than or equal to 10 mg / kg.

[0104] On the other hand, the composition described herein can be administered to a subject. The composition can be administered alone. In some embodiments, the composition is administered in combination with cofactors, other therapeutic agents (e.g., treatment agents), etc. For example, the composition can be administered alone, or in combination with substrates such as hypoxanthine, xanthine, glucose, for example, as discussed herein, and / or in combination with fatty acids, statins, etc. For example, see International Patent Application Publication No. WO 2019 / 099687, incorporated herein by reference.

[0105] The composition can be applied as a pharmaceutically acceptable formulation (e.g., a pharmaceutically acceptable carrier, such as those described below) in a therapeutically effective, pharmaceutically acceptable amount. The term "effective amount" of a composition (e.g., a composition described herein) refers to an amount necessary or sufficient to achieve a desired biological effect. For example, an effective amount of a composition for treating a tumor can be an amount sufficient to reduce the size of the tumor. In conjunction with the teachings provided herein, by selecting among various active compositions and weighting factors (such as efficacy, relative bioavailability, subject weight, severity of adverse side effects, and mode of administration), an effective preventive or therapeutic treatment regimen can be planned that does not cause significant toxicity and is also fully effective in treating a specific subject. The effective amount of any particular application can vary according to factors such as the disease or condition being treated, the specific composition being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of the composition without excessive experimentation.

[0106] As used herein, the terms "treat," "therapeutic," "treating," and the like refer to administering a composition to a subject that can increase the subject's resistance to a disease, or resist further progression of a disease, control the progression of a disease, and / or slow the progression of symptoms of a disease or reduce the severity of symptoms of a disease. The effective amount can depend on the specific disease being treated and the desired outcome.

[0107] In some embodiments, the therapeutically effective dose of the composition can be initially determined from an animal model. The administered dose can be adjusted based on the relative bioavailability and efficacy of the administered compound. It is within the capabilities of ordinary technicians to adjust the dose to achieve maximum efficacy based on the above methods and other methods well known in the art.

[0108] When administering the composition to a subject, the dosage, dosing regimen, route of administration, etc. can be selected to affect the known activity of these compositions. The dosage can be estimated based on the results of the experimental model, optionally in combination with the results of the assay of the composition described herein. Depending on the mode of administration, the dosage can be appropriately adjusted to achieve the desired local or systemic drug level. The dosage can be administered once or multiple times per day. Multiple doses per day can be used to achieve appropriate systemic levels of the composition in the subject or in the cells targeted by the composition.

[0109] The dose can be selected to provide a therapeutically effective amount of the composition to the cells targeted by the composition. The dose can be given at the maximum safe dose. The maximum safe dose can be selected so that a high therapeutically effective amount of the composition is delivered to the subject, but the subject is at minimal risk for potential harmful side effects.

[0110] The dose of the composition can be selected to have a desired concentration at the targeted cells, to have a desired efficacy, to have a desired longevity within the subject, to have a desired administration rate, to have a desired frequency of administration, to function in an appropriate manner when administered concurrently with other therapies (e.g., as in an admixture), or for any of a variety of other purposes known to those of ordinary skill in the art.

[0111] The dosage can be selected at least in part based on the condition associated with the subject. For example, the dosage can be selected at least in part based on the species, age, sex, body weight, size, environment, metabolism, physical condition or current health status of the subject. In some cases, a subject-specific maximum dose can be used. In some cases, the dosage can be applied in a manner that limits the harmful effects on the subject (for example, the dosage can be delivered orally, nasally, intravenously, etc. according to the needs of the subject).

[0112] Doses of the compositions may be administered daily, weekly, or monthly, and any other amount of time therebetween.

[0113] The dosage may include any appropriate amount of the composition. In some embodiments, the dosage of the composition is greater than or equal to 0.1 micrograms, greater than or equal to 0.5 micrograms, greater than or equal to 10 micrograms, greater than or equal to 50 micrograms, greater than or equal to 100 micrograms, greater than or equal to 500 micrograms, greater than or equal to 1000 micrograms or more. In some embodiments, the dosage of the composition is less than or equal to 10,000 micrograms, less than or equal to 5,000 micrograms, less than or equal to 1000 micrograms, less than or equal to 500 micrograms, less than or equal to 100 micrograms, less than or equal to 50 micrograms or less. Combinations of these ranges are also possible. For example, in some embodiments, the dosage of the composition is greater than or equal to 0.5 micrograms and less than or equal to 10,000 micrograms.

[0114] The dose of the composition can be administered at any of a variety of average rates. In some embodiments, the dose is administered at a rate of greater than or equal to 0.01 μg / hour, greater than or equal to 0.05 μg / hour, greater than or equal to 0.1 μg / hour, greater than or equal to 0.5 μg / hour, greater than or equal to 1 μg / hour, greater than or equal to 5 μg / hour, greater than or equal to 10 μg / hour, greater than or equal to 50 μg / hour, greater than or equal to 100 μg / hour, greater than or equal to 500 μg / hour, greater than or equal to 1000 μg / hour, or greater. In some embodiments, the dose is administered at a rate of less than or equal to 10,000 μg / hour, less than or equal to 5,000 μg / hour, less than or equal to 1,000 μg / hour, less than or equal to 500 μg / hour, less than or equal to 100 μg / hour, less than or equal to 50 μg / hour, less than or equal to 10 μg / hour, less than or equal to 10 μg / hour, less than or equal to 1 μg / hour, less than or equal to 0.5 μg / hour, less than or equal to 0.1 μg / hour, or less. Combinations of these ranges are possible. For example, in some embodiments, the dose is administered at a rate greater than or equal to 0.01 μg / hour and less than or equal to 10,000 μg / hour.

[0115] The composition can be used in an extended period of time. In certain embodiments, the dosage is applied in a time period greater than or equal to 1h, greater than or equal to 2h, greater than or equal to 4h, greater than or equal to 6h, greater than or equal to 12h, greater than or equal to 24h, greater than or equal to 3 days, greater than or equal to 7 days, greater than or equal to 10 days, greater than or equal to 14 days, greater than or equal to 21 days, greater than or equal to 30 days, greater than or equal to 45 days, greater than or equal to 60 days, greater than or equal to 90 days or longer. In certain embodiments, the dosage is applied in a time period less than or equal to 365 days, less than or equal to 200 days, less than or equal to 90 days, less than or equal to 60 days, less than or equal to 45 days, less than or equal to 30 days, less than or equal to 21 days, less than or equal to 14 days, less than or equal to 10 days, less than or equal to 7 days, less than or equal to 3 days, less than or equal to 24h, less than or equal to 12h or less. The combination of these ranges is possible. For example, in some embodiments, the dose is administered over a period of time greater than or equal to 1 hour and less than or equal to 365 days.

[0116] The subject can be any suitable subject. For example, the subject can be a mammal, such as a human or a non-human animal, such as a dog, a cat, a horse, a rabbit, a cow, a pig, a sheep, a goat, a rat (e.g., Rattus norvegicus), a mouse (e.g., Rattus rattus), a guinea pig, a non-human primate (e.g., a monkey, a chimpanzee, a baboon, an ape, a gorilla, etc.), etc.

[0117] Administration of the compositions disclosed herein can be accomplished by any of a variety of medically acceptable methods that allow the compositions to reach their target cells. The particular mode selected can of course depend on factors such as those previously described, such as the particular composition, the severity of the condition of the subject being treated, the dose desired for therapeutic efficacy, and the like. As used herein, a "medically acceptable" mode of treatment is one that is capable of producing effective levels of the composition in the subject without causing clinically unacceptable side effects. In the case of neurodegenerative diseases, the blood-brain barrier may prevent the drug from reaching senescent cells. In this case, a medically acceptable mode can be administered through the cerebrospinal fluid, i.e., intrathecal administration.

[0118] The composition can be administered to a subject using any medically acceptable variety of methods. Administration can be local (e.g., to a specific region, physiological system, tissue, organ, or cell type) or systemic, depending on the condition to be treated. For example, the composition can be administered orally, vaginally, rectally, buccally, pulmonary, topically, nasally, transdermally, by parenteral injection or implantation, via surgical administration, or any other administration method that achieves access to the tumor. In some cases, more than one administration method can be used, for example, if two or more compositions are to be administered.

[0119] Examples of parenteral administration methods that can be used include intravenous, intradermal, subcutaneous, intracavitary, intramuscular, intraperitoneal, epidural or intrathecal administration. Examples of implantation administration methods include implantation or injection of any implantable or injectable drug delivery system. Oral administration can be used in some embodiments. Oral administration may be advantageous because it is convenient for the subject and the dosing regimen is also convenient. Compositions suitable for oral administration can be administered using discrete units, such as hard or soft capsules, pills, cachets, tablets, lozenges, dissolving films or lozenges. Other oral compositions suitable for use include solutions or suspensions, such as syrups, elixirs or emulsions. Solutions or suspensions may contain aqueous or non-aqueous liquids. In some embodiments, the composition can be used to fortify food or beverages.

[0120] When systemic delivery of the composition is desired, it can be formulated for parenteral administration by injection, for example by push injection or continuous infusion. The formulation for injection can be present in unit dosage form, for example in an ampoule with a preservative added or in a multidose container. The composition can take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and can contain preparatants (e.g., suspending agents, stabilizers and / or dispersants).

[0121] Pharmaceutical formulations for parenteral administration may include aqueous solutions of the active compound in water-soluble form. In some cases, the suspension of the active compound may be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles may include, but are not limited to, fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. The suspension may contain a suitable stabilizer. In certain embodiments, the suspension includes an agent that increases the solubility of the compound. Stabilizers or solubility increasers may allow the preparation of highly concentrated solutions.

[0122] In some embodiments, the composition can be formulated as a depot preparation. In some cases, such long-acting formulations can be formulated with suitable polymers or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

[0123] The composition can be prepared in the form of a liquid pharmaceutical preparation. In certain embodiments, the liquid pharmaceutical preparation comprises a solution, a suspension or an emulsion. The composition can be included in a liquid pharmaceutical preparation in any of a variety of forms. For example, the composition can be composed of an aqueous solution or a saline solution (e.g., suitable for inhalation), can be microencapsulated, can be encapsulated, can be coated on microscopic gold particles, can be contained in liposomes, can be atomized, can be aerosolized, or can be dried onto a sharp object to be scraped into the skin.

[0124] The composition can be prepared in the form of a solid pharmaceutical preparation. For example, the composition can be contained in particles; powders; tablets; coated tablets; (micro) capsules; suppositories; syrups; emulsions; suspensions; creams; or drops or preparations with extended release of active compounds. In some embodiments, the composition is prepared in the form of a solid pharmaceutical preparation, which is configured to dissolve or otherwise be converted into a pharmaceutical liquid preparation. For example, the composition can be prepared in the form of a dry powder, which can be dissolved in water to form an aqueous solution. The liquid or solid composition may include additives such as excipients. In some embodiments, the additive includes a disintegrant, a binder, a coating agent, a swelling agent, a lubricant, a flavoring agent, a sweetener, or a solubilizer, but any of a variety of additives can be used, and the present disclosure is not limited thereto.

[0125] In some embodiments, the composition can be administered to provide continuous exposure to the composition over a specific period of time (eg, hours, days, weeks, months, or years).

[0126] For example, the composition can be administered by repeated administration or by controlled release. For example, the composition can be controlled release within the time period so that it maintains the dosage of the composition during the time period. Controlled release can be achieved, for example, by oral dosage forms, bolus injections, transdermal patches, subcutaneous implants or other methods such as those described herein. In some cases, it may be necessary to maintain a substantially constant concentration of the composition.

[0127] Other delivery systems suitable for use in certain embodiments may include delayed release, delayed release, sustained release or controlled release delivery systems. Such systems can advantageously avoid the need for repeated administration, increasing the convenience of the subject. Many types of delivery systems are available and known to those of ordinary skill in the art. The composition can be administered, for example, using a delivery system, which is, for example, a polymer-based system such as polylactic acid and / or polyglycolic acid, polyanhydride, polycaprolactone and / or a combination of these; a lipid-based non-polymer system, including sterols, such as cholesterol, cholesterol esters and fatty acids or neutral fats, such as monoglycerides, diglycerides and triglycerides; a hydrogel release system; a liposome-based system; a phospholipid-based system; a silicone rubber system; a peptide-based system; a wax coating; tablets compressed using conventional adhesives and excipients; or partially fused implants. Specific examples include, but are not limited to, an erosion system in which the composition is contained in a matrix in a form, or a diffusion system in which the active component controls the release rate. The formulation may exist as, for example, microspheres, hydrogels, polymer reservoirs, cholesterol matrices or polymer systems. In some embodiments, the system may allow for sustained or controlled release of the composition, for example by controlling the diffusion or erosion / degradation rate of the formulation. Additionally, in some embodiments, a pump-based hardware delivery system may be used.

[0128] In some embodiments, implant delivery composition is used.Implant can be configured for short-term or long-term release composition.In some embodiments, delivery system is long-term release implant.In some embodiments, the use of long-term release implant may be particularly suitable.As used herein, "long-term release" refers to that the implant comprising the composition as described herein is constructed and arranged to deliver therapeutically effective levels.In some embodiments, the composition is delivered with therapeutically effective levels in a time period greater than or equal to 1h, greater than or equal to 2h, greater than or equal to 4h, greater than or equal to 6h, greater than or equal to 12h, greater than or equal to 24h, greater than or equal to 3 days, greater than or equal to 7 days, greater than or equal to 10 days, greater than or equal to 14 days, greater than or equal to 21 days, greater than or equal to 30 days, greater than or equal to 45 days, greater than or equal to 60 days, greater than or equal to 90 days or greater. In some embodiments, the composition is delivered at a therapeutically effective level in a time period of less than or equal to 365 days, less than or equal to 200 days, less than or equal to 90 days, less than or equal to 60 days, less than or equal to 45 days, less than or equal to 30 days, less than or equal to 21 days, less than or equal to 14 days, less than or equal to 10 days, less than or equal to 7 days, less than or equal to 3 days, less than or equal to 24h, less than or equal to 12h or less. Combinations of these ranges are possible. For example, in some embodiments, in some embodiments, the composition is delivered at a therapeutically effective level in a time period of greater than or equal to 1h and less than or equal to 365 days. Long-term release implants are well known to those of ordinary skill in the art and include some release systems described herein.

[0129] In certain embodiments, the composition can be combined with a suitable pharmaceutically acceptable carrier. In general, pharmaceutically acceptable carriers suitable for use are well known to those of ordinary skill in the art. As used herein, "pharmaceutically acceptable carriers" refer to non-toxic materials that do not significantly interfere with the effectiveness of the biological activity of one or more active compounds to be administered, but are used as formulation ingredients, such as to stabilize or protect one or more active compounds in the composition before use. For example, the composition can be incorporated into liposomes, incorporated into polymer release systems, suspended in liquids (e.g., in dissolved form or colloidal form), or other methods (such as those described herein). The carrier may include one or more compatible solid or liquid fillers, diluents or encapsulated materials suitable for application to humans or other vertebrates. The term "carrier" means an organic or inorganic component, which may be natural or synthetic, combined with one or more active compounds of the present disclosure to facilitate application. The carrier may be mixed together or otherwise mixed with one or more compositions as described herein, and / or mixed with each other in a manner such that there is no interaction that will significantly impair the desired drug efficacy. The carrier may be soluble or insoluble, depending on the application. Examples of well-known carriers include, but are not limited to, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier may be soluble or insoluble.

[0130] In some embodiments, the formulations described herein can be administered in a pharmaceutically acceptable solution. Pharmaceutically acceptable solutions can routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, emulsifiers, diluents, excipients, chelating agents, fillers, desiccants, antioxidants, antimicrobials, preservatives, adhesives, fillers, silicon dioxide, solubilizers, stabilizers, and other optional therapeutic ingredients that can be used with the composition. For example, if the formulation is a liquid, the carrier can be a solvent, a partial solvent, or a non-solvent, and can be water-based or organic-based. Non-limiting examples of suitable formulation ingredients include diluents, such as calcium carbonate, sodium carbonate, lactose, kaolin, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch or alginic acid; binders, such as starch, gelatin or acacia; lubricants, such as magnesium stearate, stearic acid or talc; time delay materials, such as glyceryl monostearate or glyceryl distearate; suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone; dispersing or wetting agents, such as lecithin or other naturally occurring phospholipids; thickening agents, such as cetyl alcohol or beeswax; buffers, such as acetic acid and its salts, citric acid and its salts, boric acid and its salts, or phosphoric acid and its salts; or preservatives, such as benzalkonium chloride, chlorobutanol, parabens or thimerosal. The compositions of the present disclosure can be formulated into solid, semisolid, liquid or gaseous preparations, such as tablets, capsules, elixirs, powders, granules, ointments, solutions, depots, inhalants or injections, etc.

[0131] Suitable buffers include, but are not limited to, acetic acid and salt (1%-2% w / v); citric acid and salt (1%-3% w / v); boric acid and salt (0.5%-2.5% w / v); and phosphoric acid and salt (0.8%-2% w / v). Suitable preservatives include, but are not limited to, benzalkonium chloride (0.003%-0.03% w / v); chlorobutanol (0.3%-0.9% w / v); parabens (0.01%-0.25% w / v) and thimerosal (0.004%-0.02% w / v).

[0132] Preparations may include solutions, suspensions or emulsions as described above. Solutions, suspensions or emulsions may be sterile. In certain embodiments, solutions, suspensions or emulsions are aqueous. In certain embodiments, solutions, suspensions or emulsions are non-aqueous. In certain embodiments, sterile aqueous or non-aqueous solutions, suspensions or emulsions may be isotonic with the blood of the subject. Non-limiting examples of non-aqueous solvents are polypropylene glycol, polyethylene glycol, vegetable oils such as olive oil, sesame oil, coconut oil, groundnut oil, peanut oil, mineral oil, injectable organic esters such as ethyl oleate or fixed oils, including synthetic monoglycerides or diglycerides. Solutions, suspensions or emulsions may include aqueous carriers. Exemplary aqueous carriers that may be used include, but are not limited to, water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. The preparation may also include parenteral vehicles, which are configured to improve the suitability of solutions, suspensions or emulsions for parenteral delivery. Parenteral vehicles include sodium chloride solution, 1,3-butanediol, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements (such as those based on Ringer's dextrose), etc. In some embodiments, preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, etc.

[0133] In some embodiments, the compositions described herein may be associated or contacted with a suitable carrier. A suitable carrier may include one or more auxiliary components. The preparation may be prepared by any of a variety of suitable techniques. For example, the preparation may be prepared by uniformly and closely combining the composition with a liquid carrier, a finely divided solid carrier, or both. In some embodiments, the resulting preparation may be shaped (e.g., formed into tablets, pills, etc.).

[0134] The compositions discussed herein can be administered per se (pure) or in the form of pharmaceutically acceptable salts. Although non-pharmaceutically acceptable salts can be used to prepare the compositions, when used in medicines, the salts should be pharmaceutically acceptable. The term "pharmaceutically acceptable salts" includes salts of the compositions described herein prepared in combination with, for example, an acid or a base. Pharmaceutically acceptable salts can be prepared as alkali metal salts, such as lithium salts, sodium salts or potassium salts; or as alkaline earth metal salts, such as magnesium salts or calcium salts. Examples of suitable bases that can be used to form salts include ammonium or mineral bases, such as sodium hydroxide, lithium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and the like. Examples of suitable acids that can be used to form salts include inorganic or mineral acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, phosphorous acid, and the like. Other suitable acids include organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, glucuronic acid, galacturonic acid, salicylic acid, formic acid, naphthalene-2-sulfonic acid, etc. Other suitable acids also include amino acids such as arginine, aspartic acid, glutamic acid, etc. Moreover, such salts can be prepared as alkali metal salts or alkaline earth metal salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

[0135] On the other hand, the present disclosure also provides any of the above-mentioned compositions in the kit, optionally including instructions for treating a disease or condition (e.g., by targeting senescent cells, adipocytes, cancer cells, or fibroblasts). In some cases, the kit may include a description of the use of the composition discussed herein. The kit may also include instructions for use of a combination of two or more compositions. Instructions for administering the composition by any suitable technique as previously described may also be provided, for example, orally, intravenously, by a pump or implantable delivery device, or via another known drug delivery route.

[0136] The kit described herein may also include one or more containers. The container may include compositions and other ingredients as described above. In some cases, the kit may also include instructions for mixing, diluting and / or administering the compositions disclosed herein. The kit may also include other containers with one or more solvents, surfactants, preservatives and / or diluents (e.g., saline (0.9% NaCl) or 5% dextrose) and containers for mixing, diluting components in a sample or administering to a subject in need of such treatment.

[0137] The composition of the kit can be provided in any suitable form, for example as a liquid pharmaceutical preparation or as a solid pharmaceutical preparation. When the composition provided is a dry powder, the composition can be reconstituted by adding a suitable solvent which can also be provided in some cases. In embodiments where a composition in liquid form is used, the liquid form can be concentrated or ready-to-use. The solvent will depend on the composition and the mode of use or administration. Suitable solvents for pharmaceutical compositions are well known, for example as described above, and are available from the literature. The solvent will depend on the composition and the mode of use or administration.

[0138] In yet another aspect, the disclosure includes promoting one or more of the above embodiments, eg, promoting treatment or prevention of a tumor in vitro or in vivo, eg, by administering to a subject a composition, such as those described herein.

[0139] The treatments disclosed herein can be administered to any subject, e.g., a human or non-human animal, e.g., dogs, cats, horses, rabbits, cows, pigs, sheep, goats, rats (e.g., Rattus norvegicus), mice (e.g., House mouse), guinea pigs, non-human primates (e.g., monkeys, chimpanzees, baboons, apes, gorillas, etc.), etc.

[0140] Antibodies as described herein may comprise biopolymers, such as polypeptides. For example, antibodies may comprise proteins. In some embodiments, antibodies are glycoproteins. However, in some embodiments, antibodies comprising proteins are glycoproteins. Antibodies may be substantially encoded by immunoglobulin genes or immunoglobulin gene fragments. Any of a variety of immunoglobulin genes or fragments thereof are known to those of ordinary skill in the art. For example, antibodies may be substantially encoded by immunoglobulin genes, including κ, λ, α, γ, δ, ε and μ constant region genes, as well as numerous immunoglobulin variable region genes and fragments thereof. Antibodies may be encoded by light chain immunoglobulin genes or fragments thereof. Light chain immunoglobulins may be divided into κ type or λ type.

[0141] Antibodies can be encoded by heavy chain immunoglobulin genes or fragments thereof. Heavy chain immunoglobulins can be classified as gamma, mu, alpha, delta, or epsilon (which in turn define the immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, respectively).

[0142] Immunoglobulin structural units may comprise tetramers. Each tetramer may be composed of two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain.

[0143] In some embodiments, the light chain has a molecular weight greater than or equal to 15 kDa, greater than or equal to 20 kDa, greater than or equal to 25 kDa or greater. In some embodiments, the light chain has a molecular weight less than or equal to 35 kDa, less than or equal to 30 kDa, less than or equal to 25 kDa or less. Combinations of these ranges are possible. For example, in some embodiments, the light chain has a molecular weight greater than or equal to 15 kDa and less than or equal to 35 kDa.

[0144] In some embodiments, the heavy chain has a molecular weight greater than or equal to 50 kDa, greater than or equal to 55 kDa, greater than or equal to 60 kDa or greater. In some embodiments, the heavy chain has a molecular weight less than or equal to 70 kDa, less than or equal to 65 kDa, less than or equal to 60 kDa or less. Combinations of these ranges are possible. For example, in some embodiments, the heavy chain has a molecular weight greater than or equal to 50 kDa and less than or equal to 70 kDa.

[0145] The N-terminus of each polypeptide chain can define the variable region of the immunoglobulin structural unit. The variable region may be primarily responsible for antigen recognition. In some embodiments, the variable region comprises greater than or equal to 95, greater than or equal to 98, greater than or equal to 100, greater than or equal to 103, greater than or equal to 105 or more amino acids. In some embodiments, the variable region comprises less than or equal to 115, less than or equal to 113, less than or equal to 110, less than or equal to 108, less than or equal to 105 or less amino acids. Combinations of these ranges are possible. For example, in some embodiments, the variable region comprises greater than or equal to 95 and less than or equal to 115 amino acids.

[0146] The antibodies may exist as intact immunoglobulins. However, in some embodiments, the antibodies exist in the form of any of a variety of immunoglobulin fragments.

[0147] Immunoglobulin fragments can be produced by digestion with any of a variety of peptidases (e.g., pepsin). For example, in some embodiments, immunoglobulin fragments can be formed by digesting antibodies with pepsin. In some exemplary embodiments, pepsin is used to digest the Fc domain of an antibody, for example, by degrading the disulfide bonds in the hinge region of the Fc domain to produce F(ab)'2. According to certain embodiments, F(ab)'2 is a dimer of Fab, which itself is a light chain connected to VH-CH1 by a disulfide bond. F(ab)'2 can be reduced to destroy the disulfide bonds in the hinge region, thereby converting the (Fab')2 dimer into a Fab' monomer. According to some embodiments, the Fab' monomer comprises a portion of the hinge region of the Fab and Fc domains.

[0148] In some embodiments, the immunoglobulin fragments are synthesized de novo.Immunoglobulin fragments can be produced by any of a variety of methods known to those of ordinary skill in the art, such as by chemical synthesis, by utilizing recombinant DNA methods, or by "phage display" methods.

[0149] Examples of antibodies include single chain antibodies, such as single chain Fv (scFv) antibodies, in which the variable heavy chain and variable light chain are linked together (directly or through a peptide linker) to form a continuous polypeptide. In one embodiment, the antibody is a monoclonal antibody.

[0150] In some embodiments, the composition comprising the antibody or antibody fragment is configured such that the antibody or antibody fragment retains a relatively high affinity for the target molecule. Without wishing to be bound by theory, the affinity of the antibody or antibody fragment for the target may be related to the dissociation constant (K) between the antibody or antibody fragment and the target. D ) is inversely proportional to the D A value of 0.05 corresponds to a higher affinity. In some embodiments, the K of an antibody or antibody fragment is D Values ​​less than or equal to 10 under physiological conditions -6 M, less than or equal to 10 -7 M, less than or equal to 10 -8 M, less than or equal to 10 -9 M, less than or equal to 10 -10 M or less. In some embodiments, the K of the antibody or antibody fragment is D Value greater than or equal to 10 -13 M, greater than or equal to 10 -12 M, greater than or equal to 10 -11 M or greater. Combinations of these ranges are also possible. For example, in some embodiments, the K of an antibody or antibody fragment is D Value greater than or equal to 10 -12 M and less than or equal to 10 -6 M. Composition of K D Values ​​can be determined by tests known to those of ordinary skill in the art.

[0151] In some embodiments, the K of the composition D The K of a pure antibody or antibody fragment similar to the antibody or antibody fragment of the composition may be relatively close to D This relatively high affinity of the composition for its target may be related to the fact that the composition is a fusion protein.

[0152] As used herein, the term "antibody fragment" may refer to at least a portion of an antibody that retains the ability to specifically interact with an antigen's epitope (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, any antibody fragment described elsewhere herein, including Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (VL or VH), Camelidae VHH domains, multispecific antibodies formed by antibody fragments, such as bivalent fragments including two Fab fragments linked by disulfide bonds at the hinge region, and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments may also be incorporated into single domain antibodies, large antibodies, mini antibodies, nano antibodies, intrabodies, double antibodies, three antibodies, four antibodies, v-NARs, and double-scFvs.

[0153] In some embodiments, the antibody may be a minibody or a nanobody. In the context of the present disclosure, it has been creatively recognized that the use of a minibody or a nanobody may advantageously increase the binding affinity of the antibody relative to a full-sized antibody.

[0154] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the present disclosure.

[0155] Example 1

[0156] In the prophetic example, an 80-year-old human subject with muscle weakness is provided with a composition comprising an antibody that recognizes senescent cells and an enzyme. In the example, the antibody is a commercially available antibody, such as a senescent cell recognition antibody or a monoclonal antibody.

[0157] The enzyme can cause lysosomal leakage, thereby leading to the production of antigens. In the example, the enzyme is an oxidase that produces reactive oxygen species, thereby damaging or destroying lysosomes and causing them to leak. The oxidase produces an exemplary reactive oxygen species, i.e., superoxide. In the example, the oxidase is xanthine oxidase.

[0158] The xanthine oxidase is covalently linked to an antibody and administered intravenously or intramuscularly to a subject. Upon reaching the target cells of the composition, the enzyme reacts with the lysosomes, causing damage or destruction, thereby producing antigens, thereby enhancing the subject's immune response. In this way, the subject's immune system is able to more effectively target the subject's senescent cells.

[0159] Example 2

[0160] In this prophetic example, a composition comprising an antibody that recognizes senescent cells and an enzyme is provided to a human subject having NASH. In some cases, the antibody is a commercially available antibody, such as a senescent cell recognition antibody or a monoclonal antibody.

[0161] This enzyme can cause lysosomal leakiness, which can lead to the production of antigens. In the example described, the enzyme is an oxidase that produces reactive oxygen species, which damages or destroys lysosomes and causes them to leak. The oxidase produces an exemplary reactive oxygen species, namely superoxide. In the example described, the oxidase is xanthine oxidase.

[0162] The xanthine oxidase is covalently linked to an antibody and administered intravenously or intramuscularly to a subject. Upon reaching the target cells of the composition, the enzyme reacts with the lysosomes, causing damage or destruction, thereby producing antigens, thereby enhancing the subject's immune response. In this way, the subject's immune system is able to more effectively target the subject's senescent cells.

[0163] Example 3

[0164] In this prophetic example, a composition comprising an antibody that recognizes senescent cells and an enzyme is provided to a human subject with Alzheimer's disease. In some cases, the antibody is a commercially available antibody, such as a senescent cell recognition antibody or a monoclonal antibody.

[0165] This enzyme can cause lysosomal leakiness, which can lead to the production of antigens. In the example described, the enzyme is an oxidase that can produce reactive oxygen species, thereby damaging or destroying the lysosome and causing it to leak. The oxidase may be capable of producing reactive oxygen species, such as superoxide. In the example described, the oxidase is xanthine oxidase.

[0166] The xanthine oxidase is covalently linked to an antibody and administered intravenously or intramuscularly to a subject. Upon reaching the target cells of the composition, the enzyme reacts with the lysosomes, causing damage or destruction, thereby producing antigens, thereby enhancing the subject's immune response. In this way, the subject's immune system is able to more effectively target the subject's senescent cells.

[0167] Example 4

[0168] In the prophetic example, a human subject is treated as in Example 1, but in addition, a treatment agent is provided to the subject to extract and / or inhibit the antioxidant that affects the effect of the composition. In one example, an unsaturated fatty acid, such as CH2=CHCOOH, is provided to the subject. The effect of the fatty acid is to inhibit the effect of the antioxidant, resulting in a greater response to the composition.

[0169] Example 5

[0170] In the prophetic example, a human subject is treated as in Example 1, but in addition, the subject is exposed to a statin, such as atorvastatin, which can be used to inhibit the effects of the antioxidant, resulting in a greater response to the composition.

[0171] Example 6

[0172] In the indicative example, an amino to amino cross-linking agent is used to prepare a composition comprising a senescent cell recognition antibody and a xanthine oxidase. Senescent cells recognized by the antibody are cultured in a 96-well plate and treated with a conjugate at an appropriate concentration of 10 micrograms / ml to 100 micrograms / mL. Hypoxanthine (an exemplary enzyme substrate of xanthine oxidase) is provided to the cell culture medium at a concentration of 1mM. Immunogenic cell death (ICD) markers are measured at 1h, 2h, 4h, 6h, 12h and 24h after hypoxanthine is provided, including calreticulin exposure, extracellular ATP formation and HMGB1 formation. The result diagram is shown in Figure 3. Appropriately, calreticulin forms earlier, followed by ATP formation, followed by HMGB1 formation. This confirms that cells produce markers of ICD, and in the process of death, it is expected to form new antigens that activate T cells and guide them to senescent cells.

[0173] Example 7

[0174] Once senescent cells are killed during the process of inducing ICD, the subject with the disease becomes “immune” to such cells. Thus, ICD protects the subject from future effects of senescent cells.

[0175] To demonstrate this, in this prophetic example, the senescent cell fragments killed in Example 2 were collected and injected into the left flank of BALB / c mice. Eight days after the injection, live senescent cells were injected into the right flank of each mouse. The senescent cells in the right flank of the mice were monitored for 120 days. The mice treated with ICD dead cells were compared to mice injected with saline. At 120 days, 92% of the mice injected with ICD dead cells had no senescent cells, while only 7% of the mice injected with saline had no senescent cells.

[0176] Example 8

[0177] In the prophetic example, a composition comprising an antibody that recognizes excessively large adipocytes and an enzyme is provided for an 80-year-old human subject having a BMI of 55. In some cases, the antibody is a commercially available antibody, such as an adipocyte recognition antibody or a monoclonal antibody.

[0178] This enzyme can cause lysosomal leakiness, which can lead to the production of antigens. In the example described, the enzyme is an oxidase that produces reactive oxygen species, which damages or destroys lysosomes and causes them to leak. The oxidase produces an exemplary reactive oxygen species, namely superoxide. In the example described, the oxidase is xanthine oxidase.

[0179] Xanthine oxidase is covalently linked to an antibody and administered intravenously or intramuscularly to a subject. Upon reaching the target cells of the composition, the enzyme reacts with the lysosomes, causing damage or destruction, thereby producing antigens, thereby enhancing the subject's immune response. In this way, the subject's immune system is able to more effectively target the oversized fat cells in the subject's body. After 3 months of treatment, the subject's BMI dropped to 37.

[0180] Example 9

[0181] In the indicative example, a conjugate between an oversized adipocyte recognition antibody and xanthine oxidase was prepared using an amino to amino crosslinker. The oversized adipocytes recognized by the antibody were cultured in a 96-well plate and treated with a conjugate at an appropriate concentration of 10 micrograms / ml to 100 micrograms / mL. Hypoxanthine was provided to the cell culture medium at a concentration of 1mM. The markers of immunogenic cell death (ICD) were determined at 1h, 2h, 4h, 6h, 12h and 24h, including calreticulin exposure, extracellular ATP formation and HMGB1. The result is shown in Figure 3. Appropriately, calreticulin forms earlier, followed by ATP formation, followed by HMGB1 formation. This confirms that cells produce markers of ICD, and in the process of death, it is expected to form new antigens that activate T cells and guide them to oversized adipocytes.

[0182] Example 10

[0183] In the prophetic example, for an 80-year-old human subject with dyspnea, a composition comprising an antibody that recognizes lung fibroblasts and an enzyme is provided. In some cases, the antibody is a commercially available antibody, such as a fibroblast recognition antibody or a monoclonal antibody.

[0184] This enzyme can cause lysosomal leakiness, which can lead to the production of antigens. In the example described, the enzyme is an oxidase that produces reactive oxygen species, which damages or destroys lysosomes and causes them to leak. The oxidase produces an exemplary reactive oxygen species, namely superoxide. In the example described, the oxidase is xanthine oxidase.

[0185] Xanthine oxidase is covalently linked to an antibody and administered intravenously or intramuscularly to a subject. Upon reaching the target cells of the composition, the enzyme reacts with the lysosomes, causing damage or destruction, thereby producing antigens, thereby enhancing the subject's immune response. In this way, the subject's immune system is able to more effectively target the subject's fibroblasts. After treatment, the subject's ability to breathe is enhanced.

[0186] Example 11

[0187] Once the fibroblasts were killed during the process of inducing ICD, the animals with fibrotic lungs became "immune" to these cells. Thus, ICD protected the animals from future lung fibrosis.

[0188] To demonstrate this, in this prophetic example, the fibroblast fragments killed in Example 2 were collected and injected into the left flank of BALB / c mice. Eight days after the injection, live fibroblasts were injected into the right flank of each mouse. The mice were monitored for 120 days for lung fibrosis. The mice treated with ICD dead cells were compared to mice injected with saline. At 120 days, 92% of the mice injected with ICD dead cells had no lung fibroblasts, while only 7% of the mice injected with saline had no lung fibroblasts.

[0189] Example 12

[0190] In the prophetic example, for an 80-year-old human subject with congestive heart failure, a composition comprising an antibody that recognizes cardiac fibroblasts and an enzyme is provided. In some cases, the antibody is a commercially available antibody, such as a fibroblast recognition antibody or a monoclonal antibody.

[0191] This enzyme can cause lysosomal leakiness, which can lead to the production of antigens. In the example described, the enzyme is an oxidase that produces reactive oxygen species, which damages or destroys lysosomes and causes them to leak. The oxidase produces an exemplary reactive oxygen species, namely superoxide. In the example described, the oxidase is xanthine oxidase.

[0192] Xanthine oxidase is covalently linked to an antibody and administered intravenously or intramuscularly to a subject. Upon reaching the target cells of the composition, the enzyme reacts with the lysosomes, causing damage or destruction, thereby producing antigens, thereby enhancing the subject's immune response. In this way, the subject's immune system is able to more effectively target the subject's fibroblasts. After treatment, the subject's ejection volume increased significantly.

[0193] Example 13

[0194] In the indicative example, a conjugate between a fibroblast recognition antibody and xanthine oxidase was prepared using an amino to amino crosslinker. Fibroblasts recognized by the antibody were cultured in 96-well plates and treated with a conjugate at an appropriate concentration of 10 micrograms / ml to 100 micrograms / mL. Hypoxanthine was provided to the cell culture medium at a concentration of 1mM. Markers of immunogenic cell death (ICD) were measured at 1h, 2h, 4h, 6h, 12h and 24h, including calreticulin exposure, extracellular ATP formation and HMGB1. Appropriately, calreticulin formation was earlier, followed by ATP formation, followed by HMGB1 formation. This confirmed that cells produced markers of ICD, and in the process of death, it was expected to form new antigens that activated T cells and directed them to fibroblasts.

[0195] Example 14

[0196] This example describes the production and analysis of two exemplary compositions comprising a xanthine oxidase enzyme fragment having a C-terminal truncation ("XO"; SEQ ID NO 2) connected to an epidermal growth factor receptor nanobody suitable for targeting cancer cells ("VHH 9G8"; SEQ ID NO 1). These compositions are connected in the form of fusion proteins. The fusion protein also includes a linker region between XO and VHH 9G8, which has the amino acid sequence GGGGSGGGGS (SEQ ID NO 3). In one of the exemplary compositions, the xanthine oxidase is located at the N-terminus of the nanobody ("XO-VHH 9G8"; SEQ ID NO 5). In another exemplary composition, the xanthine oxidase is located at the C-terminus of the nanobody ("VHH 9G8-XO"; SEQ ID NO 4). Appropriate controls - isolated VHH 9G8 and isolated XO were also included in the experiment. Table 1 lists the amino acid sequences associated with these substances.

[0197] Table 1. Amino acid sequences associated with various compositions described herein.

[0198]

[0199]

[0200]

[0201]

[0202] Each fusion protein was prepared by cloning into the pCZN4b expression vector and amplifying in TOP 10 E. coli. HEK293 cells were cultured and harvested, and their supernatants were collected - each fusion protein was purified from the supernatant by column purification (Ni-NTA column) using 20mM imidazole, 50mM Tris, 300mM NaCl, pH 8 wash buffer at 1mL / min. The fusion protein was then eluted from the column using 200mM imidazole, 50mM tris, 300mM NaCl, pH 8 elution buffer at 1mL / min. The fusion protein was dialyzed overnight with PBS and the protein concentration was determined using the BCA assay. Each fusion protein and control were His-tagged.

[0203] Figure 2 The binding of fusion protein 202 to epidermal growth factor receptor 205 ("EGFR"). Figure 2 In the embodiment, the fusion protein 202 is VHH 9G8-XO and comprises the Nanobody 203 (VHH 9G8) and the enzyme fragment 201 (XO) connected by a linker 207 (GGGGSGGGGS).

[0204] Table 1 reports the dissociation constants (K D ), and the association rate constants (k ) associated with the binding of each fusion protein and the VHH 9G8 control to EGFR a ) and the dissociation rate constant (k d ), as determined by surface plasmon resonance (SPR). Surface plasmon resonance was performed using a Biacore 300 injector with a contact time of 120 seconds, a dissociation time of 240 seconds, and a flow rate of 30 microliters / minute. The results show that the binding of the exemplary fusion proteins (XO-VHH 9G8 and VHH 9G8-XO) is almost as good as the nanobody control (VHH 9G8). These results demonstrate the usefulness of the compositions as described herein, and demonstrate the unexpected result that the compositions comprising nanobody-containing fusion proteins such as XO-VHH 9G8 and VHH 9G8-XO can have unexpectedly high affinities, comparable to the affinities of pure antibodies.

[0205] Table 1: Dissociation constants of compositions comprising EGFR antibodies.

[0206] <![CDATA[K D (M)]]> <![CDATA[k a (M -1 s -1 )]]> <![CDATA[k d (s -1 )]]> VHH 9G8 <![CDATA[2.85x10 -7 ]]> <![CDATA[1.97x10 4 ]]> <![CDATA[5.6x10 -3 ]]> VHH 9G8-XO <![CDATA[1.34x10 -7 ]]> <![CDATA[1.77x10 4 ]]> <![CDATA[2.37x10 -3 ]]> XO-VHH 9G8 <![CDATA[4.51x10 -8 ]]> <![CDATA[9.54x10 4 ]]> <![CDATA[4.3x10 -3 ]]>

[0207] Although several embodiments of the present disclosure have been described and shown herein, a person of ordinary skill in the art will easily envision a variety of other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantages described herein, and such variations and / or modifications are each considered to be within the scope of the present disclosure. More generally, it will be readily understood by those skilled in the art that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications for which the teachings of the present disclosure are intended. Those skilled in the art will recognize that, or will be able to determine many equivalent examples of the specific embodiments of the present disclosure described herein without using excessive routine experiments. Therefore, it should be understood that the aforementioned embodiments are presented only as examples, and within the scope of the attached claims and their equivalents, the present disclosure can be practiced in a manner different from that specifically described and claimed. The present disclosure relates to each individual feature, system, product, material, and / or method described herein. In addition, if such features, systems, products, materials, and / or methods are not mutually contradictory, any combination of two or more such features, systems, products, materials, and / or methods is included within the scope of the present disclosure.

[0208] As used herein in the specification and claims, the indefinite article "a" or "an" should be understood to mean "at least one", unless explicitly stated to the contrary.

[0209] As used herein in the specification and claims, the phrase "and / or" should be understood to mean "either or both" of the elements so combined, i.e., elements that are present together in some cases and separately in other cases. In addition to the elements specifically indicated by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically indicated elements, unless clearly indicated to the contrary. Thus, as a non-limiting example, when used in conjunction with open language such as "comprising," a reference to "A and / or B" may refer to A and B (optionally including elements other than B) in one embodiment; to B and not A (optionally including elements other than A) in another embodiment; to both A and B (optionally including other elements) in yet another embodiment; etc.

[0210] As used herein in the specification and claims, "or" shall be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be understood to be inclusive, i.e., including at least one, but also including more than one of a plurality of elements or a list of elements, and optionally other unlisted items. Only explicitly contrary terms such as "only one" or "exactly one" or when used in the claims, "consisting of..." will refer to the inclusion of exactly one element of a plurality of elements or a list of elements. Generally speaking, the term "or" as used herein shall only be understood to mean an exclusive alternative (e.g., "the other of one or two") preceded by an exclusive term such as "any one", "one", "only one" or "exactly one". When used in the claims, "consisting essentially of..." shall have its ordinary meaning as used in the field of patent law.

[0211] As used herein in the specification and claims, with respect to a list of one or more elements, the phrase "at least one" should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. The definition also allows for the optional presence of elements in addition to the elements specifically indicated within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those specifically indicated. Thus, as a non-limiting example, "at least one of A and B" (or equivalently "at least one of A or B", or equivalently "at least one of A and / or B") may refer in one embodiment to at least one A, optionally including more than one A, without the presence of B (and optionally including elements other than B); in another embodiment to at least one B, optionally including more than one B, without the presence of A (and optionally including elements other than A); in yet another embodiment to at least one A, optionally including more than one A, and at least one B, optionally including more than one B (and optionally including other elements); etc.

[0212] As used herein, "wt%" is an abbreviation for weight percent. As used herein, "at%" is an abbreviation for atomic percent.

[0213] Some embodiments may be embodied as a method, various examples of which have been described. The actions performed as part of the method may be ordered in any suitable manner. Thus, embodiments may be constructed in which the actions are performed in an order different from the order shown, the order may include different (e.g., more or fewer) actions than described, and / or may involve performing some actions simultaneously, even though the actions are shown as being performed sequentially in the embodiments specifically described above.

[0214] The use of ordinal numbers such as "first", "second", "third", etc. in the claims to modify claim elements does not in itself mean any priority, precedence or order of one claim element over another, nor does it mean the temporal order of performing method actions. It only serves as a label to distinguish one claim element with a certain name from another element with the same name (but using ordinal numbers) to distinguish these claim elements.

[0215] In the claims and in the foregoing specification, all conjunctions such as "comprising," "including," "carrying," "having," "containing," "involving," "having," and the like shall be construed as open ended, i.e., meaning including but not limited to. Only the conjunctions "consisting of" and "consisting essentially of" shall be closed or semi-closed conjunctions, respectively, as provided in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedures.

Claims

1. A composition for treating a disease or condition, comprising: a fusion protein comprising an enzyme and at least a portion of an antibody, wherein said at least a portion of said antibody is configured to bind to a cell associated with a disease or condition, and wherein the enzyme is configured to increase lysosomal membrane permeability of the cell.

2. A method for treating a disease or condition, the method comprising: administering to a subject a composition comprising a fusion protein comprising an enzyme and at least a portion of an antibody, wherein said at least a portion of said antibodies recognizes a cell associated with said disease or condition in said subject, and wherein the enzyme is configured to increase lysosomal membrane permeability of the cell.

3. The composition or method of any preceding claim, wherein the enzyme is configured to generate reactive oxygen species.

4. A composition or method as claimed in any preceding claim, wherein the enzyme is an oxidase or a peroxidase.

5. A composition or method as claimed in any preceding claim, wherein the enzyme is an oxidase.

6. A composition or method as claimed in any preceding claim, wherein the oxidase is glucose oxidase.

7. A composition or method as claimed in any preceding claim, wherein the oxidase is xanthine oxidase.

8. A composition or method as claimed in any preceding claim, wherein the enzyme is a peroxidase.

9. The composition or method of any preceding claim, wherein the at least a portion of the antibody is a whole antibody.

10. The composition or method of any preceding claim, wherein the at least a portion of the antibody is an antibody fragment.

11. The composition or method of any preceding claim, wherein the antibody recognizes the cell.

12. The composition or method of any preceding claim, wherein the antibody recognizes a virus.

13. A composition or method as claimed in any preceding claim, wherein the antibody recognises extracellular material.

14. The composition or method of any preceding claim, wherein the disease or condition is a disease, and wherein the disease is atherosclerosis or obesity.

15. The composition or method of any preceding claim, wherein the disease or disorder is a disorder of aging.

16. The composition or method of any preceding claim, wherein the antibody recognizes a cell, and wherein the cell is a senescent cell or an adipocyte.

17. The composition or method of any preceding claim, wherein the antibody recognizes extracellular material, and wherein the extracellular material is plaque.

18. The composition or method of any preceding claim, wherein the antibody recognizes a virus, and wherein the virus is a coronavirus or HIV.

19. A composition or method as claimed in any preceding claim, wherein the antibody is a Nanobody.

20. A composition or method as claimed in any preceding claim, wherein the antibody of the composition is present at a concentration of less than 10 -6 M of K D The value identifies the cell, virus or extracellular material.

21. A composition or method as claimed in any preceding claim, wherein the antibody of the composition is present at a concentration of less than 10 -7 M of K D The value identifies the cell, virus or extracellular material.

22. A composition or method as claimed in any preceding claim, wherein the antibody of the composition is present at a concentration less than the K of the pure antibody. D Value K D The value identifies the cell, virus or extracellular material.

23. A composition comprising: Antibodies that recognize senescent cells, adipocytes, cardiac fibroblasts, or lung fibroblasts, and The enzyme is configured to increase the permeability of the lysosomal membrane and is targeted to identified senescent cells, adipocytes, cardiac fibroblasts or lung fibroblasts.

24. A composition comprising: Antibodies that recognize senescent cells, adipocytes, cardiac fibroblasts, or lung fibroblasts, and enzymes that produce reactive oxygen species.

25. The composition of any one of claims 23 or 24, wherein the enzyme is an oxidase.

26. The composition of claim 25, wherein the oxidase is glucose oxidase.

27. The composition of claim 25, wherein the oxidase is xanthine oxidase.

28. The composition of any one of claims 23 or 24, wherein the enzyme is a peroxidase.

29. The composition of any one of claims 23-28, wherein the antibody and the enzyme are directly covalently linked.

30. The composition of any one of claims 23-29, wherein the antibody and the enzyme are linked via a linker.

31. A method comprising: A composition is administered to a subject, wherein the composition comprises an antibody that recognizes senescent cells, adipocytes, cardiac fibroblasts or lung fibroblasts, and an enzyme that can increase the permeability of lysosomal membranes, and the target of the composition is the recognized senescent cells, adipocytes, cardiac fibroblasts or lung fibroblasts.

32. A method comprising: A composition comprising an antibody that recognizes senescent cells, adipocytes, cardiac fibroblasts, or lung fibroblasts and an enzyme capable of generating reactive oxygen species is administered to a subject.

33. The method of any one of claims 31-32, wherein the enzyme is an oxidase.

34. The method of claim 33, wherein the oxidase is glucose oxidase.

35. The method of claim 33, wherein the oxidase is xanthine oxidase.

36. The method of any one of claims 31-35, wherein the enzyme is a peroxidase.

37. The method of any one of claims 31-36, wherein the antibody and the enzyme are directly covalently linked in a fusion protein.

38. The method of any one of claims 31-37, wherein the antibody and the enzyme are linked via a linker.

39. The method of any one of claims 31-38, further comprising administering a fatty acid to the subject.

40. The method of claim 39, wherein the fatty acid comprises an unsaturated n-3 to n-6 fatty acid.

41. The method of any one of claims 39 or 40, wherein the composition comprises the fatty acid.

42. The method of any one of claims 39-41, wherein administering the composition and administering the fatty acid occur simultaneously.

43. The method of any one of claims 39-42, wherein administering the composition and administering the fatty acid occur sequentially.

44. The method of any one of claims 39-43, further comprising administering a statin to the subject.

45. The method of claim 44, wherein the composition comprises the statin.

46. ​​The method of any one of claims 44 or 45, wherein administering the composition and administering the statin occur simultaneously.

47. The method of any one of claims 45-46, wherein administering the composition and administering the statin occur sequentially.

48. The method of claims 39-47, wherein administration of the composition is followed by administration of a substrate for the enzyme.

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