Methods and systems for applications involving antigen-presenting cells - Patents.com

Abstract

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

[0002] The etiology of 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. Treatments for these diseases or conditions are needed. Summary of the Invention [Problem to be solved by the invention]

[0003] The present disclosure generally relates to systems and methods for treating and / or reversing diseases or conditions, such as aging, non-alcoholic steatohepatitis (NASH), neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, obesity, pulmonary fibrosis, congestive heart failure, or cancer, using immunogenic cell death, such as lysosomal induced immunogenic cell death. In certain embodiments, the present application is directed to a composition comprising an antibody and an enzyme capable of producing reactive oxygen species. In some cases, the lysosomes of cells associated with the disease or condition may be targeted by the composition for induced cell death. [Means for solving the problem]

[0004] The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a number of different uses of one or more systems and / or compositions.

[0005] For example, in certain embodiments, by targeting cells likely to contribute to the disease or condition, such as senescent cells, adipocytes, fibroblasts, or cancer cells, the disease or condition may be treated by elimination of the cells, which may, according to some embodiments, be achieved by targeted immunogenic cell death.

[0006] In another aspect, the present disclosure is directed 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 at least a portion of the antibody is configured to bind to a cell associated with the disease or condition, and the enzyme is configured to increase lysosomal membrane permeability of the cell.

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

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

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

[0010] In yet another aspect, the present disclosure is directed to a method comprising administering to a subject a composition comprising an antibody that recognizes a senescent cell, an adipocyte, a cardiac muscle fibroblast, or a pulmonary fibroblast, and an enzyme capable of increasing lysosomal membrane permeability of the recognized senescent cell, an adipocyte, a cardiac muscle fibroblast, or a pulmonary fibroblast.

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

[0012] Several methods of administering a composition to a subject for the treatment of a particular disease or condition are disclosed herein. It should be understood that each of these aspects of the disclosure specifically includes the composition for use in the treatment of a particular disease or condition, as well as the use of the composition for the manufacture of a medicament for the treatment of a particular disease or condition.

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

[0014] Non-limiting embodiments of the present disclosure are described, for example, with reference to the accompanying drawings, which are schematic and are not intended to be drawn to scale unless otherwise indicated. 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 are every component of each embodiment of the present disclosure shown unless illustration is necessary to enable a person skilled in the art to understand the disclosure. The drawings are as follows: [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows an exemplary mechanism for lysosome-directed immunogenic cell death induced by reactive oxygen species, according to some embodiments. [Diagram 2] FIG. 2 shows a schematic perspective diagram of an exemplary fusion protein comprising an exemplary Nanobody bound to an exemplary protein associated with a disease or condition, according to some embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] 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 and Parkinson's disease, obesity, pulmonary fibrosis, congestive heart failure, or cancer) via immunogenic cell death, e.g., lysosome-induced immunogenic cell death.

[0017] The present disclosure includes multiple aspects, which may be used individually or together. For example, in one aspect, the present disclosure is directed to preparing a subject's body and / or brain for treatment, for example, by withdrawal and inhibition of antioxidants, by supplying n-3 with n-6 and other unsaturated fatty acids, and / or by treating a subject with statins.

[0018] In one aspect, the present disclosure is directed to a composition comprising an enzyme. For example, the composition may comprise an enzyme and an antibody. In some embodiments, the enzyme is bound to the antibody. In some cases, the antibody and the enzyme may be expressed as a single fusion protein, as discussed in more detail below.

[0019] The enzyme may be a naturally occurring or synthetically modified enzyme, or may be a fragment thereof. In some embodiments, the enzyme is an oxidoreductase. For example, in various embodiments, the enzyme may be an oxidase or a peroxidase. Other exemplary oxidoreductases that may be used include, but are not limited to, the following:

[0020] According to some embodiments, an oxidase is an enzyme that catalyzes an oxidation-reduction reaction, including reactions involving oxygen (O2) as an electron acceptor. 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, and the like. The enzymes described herein may be recombinant enzymes.

[0021] According to some embodiments, the peroxidase is an enzyme that acts on a substrate such as hydrogen peroxide or lipid peroxide. Non-limiting examples include horseradish peroxidase, cytochrome c peroxidase, ascorbate peroxidase, chloride peroxidase, glutathione peroxidase, haloperoxidase, lactoperoxidase, manganese peroxidase, myeloperoxidase, thyroid peroxidase, vanadium boromoperoxidase, etc.

[0022] In some embodiments, the enzyme is an oxidase or peroxidase suitable for generating the antigens described herein.

[0023] In some embodiments, the composition may be administered to a subject. The subject may also be provided with a substrate for the enzyme. The composition may be used individually or together with other compositions. In some embodiments, the composition is used to treat and / or cure a disease or condition, such as aging, non-alcoholic steatohepatitis (NASH) disease, neurodegenerative diseases such as Alzheimer's and Parkinson's disease, obesity, pulmonary fibrosis, congestive heart failure, or cancer. For example, the enzyme may be bound to an antibody that recognizes a cell associated with the disease or condition (e.g., senescent cells, adipocytes, fibroblasts), as described in further detail below. In some cases, the composition may target the lysosomes of the cell(s). Targeting the lysosomes of the cell may, in some embodiments, induce cell death of a cell associated with the disease or condition (e.g., senescent cells, adipocytes, fibroblasts).

[0024] The enzyme may be an oxidase, peroxidase, or other enzyme capable of generating reactive oxygen species. The composition may be administered to the subject, for example, via injection or other administration techniques, for example, as described herein. The subject may also be provided with a substrate for the enzyme in some cases. Without wishing to be bound by any theory, it is believed that the antibody helps localize the composition to cells associated with the selected disease or condition. Once localized, the substrate for the enzyme may be administered, for example, injected, to the subject, which may trigger a cascade of events that results in attack of the lysosomal membrane, release of lysosomal contents into the cell, and cell death by apoptosis. Thus, in some embodiments, the present disclosure is directed to lysosome-induced immunogenic cell death, LIICD.

[0025] In some cases, the substrate for the enzyme can also be administered to the subject.Substrate and enzyme can be administered in any suitable order, for example, sequentially and / or simultaneously.Non-limiting examples of substrate include hypoxanthine or xanthine for xanthine oxidase, or glucose for glucose oxidase.Further examples include monoamine oxidase can act on serotonin, melatonin, norepinephrine, epinephrine, phenethylamine, benzylamine, dopamine, tyramine, tryptamine, etc., NADPH peroxidase can act on NADPH, ascorbic acid peroxidase can act on ascorbic acid, etc.

[0026] 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, and hydroxyl radicals, by oxidizing substrates bound to the enzyme. Thus, in some embodiments, substrates for the enzymes may be added (e.g., included in the composition or administered to a subject treated with the composition) as appropriate to facilitate such reactions. In some embodiments, the reactive oxygen species are endocytosed by the targeted cells. The reactive oxygen species may interact with the membrane of the targeted cells. In some embodiments, oxidation of unsaturated lipids may cause their endocytosis. Figure 1 shows a cross-sectional schematic of this process, where reactive oxygen species 101 react with unsaturated lipids 102 to generate oxidized unsaturated lipids 104, which are endocytosed into the cells 103. Without wishing to be bound by theory, the interaction between the lysosomes of the targeted cells and the oxidized unsaturated lipids may induce the lysosomal membrane of the cells to become more permeable. For example, in Figure 3, oxidized unsaturated lipid 104 is endocytosed into cell 103 where lipid 104 is taken up by lysosome 105, which causes lysosomal membrane 107 to become permeable lysosomal membrane 109. Thus, an enzyme may be configured to increase the lysosomal membrane permeability of a cell, for example, by generating reactive oxygen species. Changes in lysosomal membrane permeability can be detected by one of skill in the art using a galectin puncture assay.

[0027] Increased lysosomal membrane permeability can cause cells to leak degradative enzymes. For example, referring again to FIG. 1, once the lysosomal membrane 107 becomes permeable lysosomal membrane 109, degradative enzymes 111 can leak through the permeable lysosomal membrane 109. When the lysosomal membrane of a cell is destabilized, the lysosomal enzymes can disperse throughout the cell. The cell can then generate cell-specific neo-antigens that can be recognized by the immune system and / or induce apoptosis and / or cell death. This is illustrated in FIG. 1, where leakage of degradative enzymes 111 generates degradation by-products 117, which independently cause the cell 103 to express neo-antigens 113 and signal the cell 103 for endocytosis by dendritic cells 115. Thus, in some embodiments, reactive oxygen species can be useful to trigger lysosome-induced immunogenic cell death (LIICD). Thus, it is contemplated that cells associated with a disease or condition, such as aging, non-alcoholic steatohepatitis (NASH) disease, neurodegenerative diseases such as Alzheimer's and Parkinson's disease, obesity, pulmonary fibrosis, congestive heart failure, or cancer, can be targeted (e.g., with an antibody) for delivery of an enzyme capable of killing the cells by administering the compositions described herein.

[0028] Furthermore, it should be understood that in some cases, agents may be used within the composition in place of and / or in addition to the enzymes that produce reactive oxygen species.

[0029] In some embodiments, the present disclosure is directed to compositions comprising an enzyme and an antibody or antibody fragment. The antibody or antibody fragment may be selected to recognize cells associated with the disease or condition of the subject (e.g., senescent cells, adipocytes, cancer cells, or fibroblasts). The antibody or antibody fragment may be linked to the enzyme via a linkage, such as, for example, a covalent bond. In some embodiments, as described in further detail below, the composition comprises a fusion protein comprising an enzyme and an antibody or antibody fragment. For example, in some embodiments, the compositions described herein can generate antigens, such as by increasing lysosomal membrane permeability and / or leakage and / or by generating reactive oxygen species, as discussed herein. The generation of antigens allows the immune system of the subject, such as T cells, to recognize cells associated with the disease or condition of the subject (e.g., senescent cells, adipocytes, cancer cells, or fibroblasts) by recognizing the antigens generated within the cells. In some embodiments, recognition by T cells can result in lysosome-induced immunogenic cell death of the cells. In some embodiments, immunogenic cell death of a cell trains an immune response against a target cell (e.g., a senescent cell, an adipocyte, a fibroblast, or a cancer cell), potentially resulting in long-term treatment of a disease or condition as a result of a sustained immune response.

[0030] In some embodiments, the composition may be used to treat aging conditions. For example, in some embodiments, the enzyme of the composition is configured to induce the death of senescent cells. Without wishing to be bound by theory, senescent cells may be unable to divide and / or may be associated with diseases of aging.

[0031] It is recognized herein that in some embodiments, senescent cells can be targeted for immunogenic cell death through the use of a composition comprising an enzyme and a senescent cell-recognizing antibody. Immunogenic cell death of senescent cells can reduce the amount or proportion of senescent cells in the body. Reducing the amount or proportion of senescent cells can in turn slow or reverse the aging process.

[0032] According to certain embodiments, the composition useful for treating aging comprises at least a portion of an antibody (e.g., a whole antibody or an antibody fragment) that recognizes senescent cells. For example, the composition may comprise at least a portion of canakinumab, an anti-DPP4 antibody, an anti-CD9 antibody, or another of a variety of suitable antibodies that recognize senescent cells and are known to those skilled in the art.

[0033] At least a portion of the antibody (e.g., a whole antibody or an antibody fragment) and the enzyme may be conjugated to each other, as discussed in more detail below. In some embodiments, the enzyme and the antibody form a fusion protein. This fusion protein may be particularly advantageous for treating aging. In some embodiments, for example, the fusion protein may bind to senescent cells with higher affinity than would be expected from the same enzyme linked to the same antibody or antibody portion by a linker. In some embodiments, the fusion protein may also have a longer life span in a subject, which may also result in improved performance of the composition.

[0034] Senescent cells are also involved in diseases such as non-alcoholic steatohepatitis (NASH). In some embodiments, the composition can be used to treat NASH. For example, as described above with reference to aging, the enzyme of the composition can be configured to induce the death of senescent cells.

[0035] In some embodiments, the present invention recognizes that senescent cells can be targeted for immunogenic cell death through the use of a composition comprising an enzyme and a senescent cell-recognizing antibody.The immunogenic cell death of senescent cells can reduce the amount or proportion of senescent cells in the body.The reduction in the amount or proportion of senescent cells can then delay or reverse NASH.

[0036] As discussed above with reference to aging, according to certain embodiments, a composition useful for treating NASH comprises at least a portion of an antibody (e.g., a whole antibody or an antibody fragment) that recognizes senescent cells.

[0037] At least a portion of the antibody (e.g., whole antibody or antibody fragment) and the enzyme may be linked to each other, as will be discussed in more detail below. In some embodiments, the enzyme and the antibody form a fusion protein. This fusion protein may be particularly advantageous for treating NASH. In some embodiments, for example, the fusion protein may bind to senescent cells with higher affinity than would be expected from the same enzyme linked to the same antibody or antibody portion by a linker. In some embodiments, the fusion protein may also have a longer life span in a subject, which may result in improved performance of the composition.

[0038] Senescent cells may also contribute to neurodegenerative diseases, such as Alzheimer's and Parkinson's. In some embodiments, the composition may be used to treat neurodegenerative diseases. For example, as described above with reference to aging, the enzyme of the composition may be configured to induce the death of senescent cells.

[0039] It is recognized herein that in some embodiments, senescent cells can be targeted for immunogenic cell death through the use of a composition comprising an enzyme and a senescent cell-recognizing antibody. Immunogenic cell death of senescent cells can reduce the amount or proportion of senescent cells in the body. The reduction in the amount or proportion of senescent cells can in turn delay or reverse neurodegenerative diseases.

[0040] As discussed above with reference to aging, according to certain embodiments, a composition useful for treating a neurodegenerative disease comprises at least a portion of an antibody (e.g., a whole antibody or an antibody fragment) that recognizes senescent cells.

[0041] At least a portion of the antibody (e.g., a whole antibody or an antibody fragment) and the enzyme may be conjugated 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 may bind to senescent cells with higher affinity than would be expected from the same enzyme linked to the same antibody or antibody portion by a linker. In some embodiments, the fusion protein may also have a longer life span in a subject, which may also result in improved performance of the composition.

[0042] In some embodiments, the composition can be used to treat the disease of obesity. For example, the enzyme of the composition can be configured to bind to fat cells (also known as adipocytes). An excess of fat cells and / or an excess of fat cells of excessive size can be associated with obesity and many related diseases, such as insulin resistance / diabetes.

[0043] In some embodiments, the present invention recognizes that adipocytes can be targeted for immunogenic cell death through the use of a composition comprising an enzyme and an adipocyte-recognizing antibody. Immunogenic cell death of adipocytes can reduce the amount or proportion of adipocytes in the body. Reduction in the amount or proportion of adipocytes can then slow or reverse obesity.

[0044] According to certain embodiments, the composition useful for treating obesity comprises at least a portion of an antibody (e.g., a whole antibody or an antibody fragment) that recognizes adipocytes. For example, the composition may comprise at least a portion of an adipocyte-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 a variety of suitable antibodies that recognize adipocytes and are known to those skilled in the art.

[0045] At least a portion of the antibody (e.g., a whole antibody or an antibody fragment) and the enzyme may be conjugated 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 the treatment of obesity. In some embodiments, for example, the fusion protein may bind to adipocytes with higher affinity than would be expected from the same enzyme linked to the same antibody or antibody portion by a linker. In some embodiments, the fusion protein may also have a longer life in the subject, which may also result in improved performance of the composition.

[0046] In some embodiments, the composition can be used to treat pulmonary fibrosis disease.For example, the enzyme of the composition can be configured to bind to fibroblasts (e.g., pulmonary fibroblasts).Excess of fibroblasts (e.g., due to fibroblast proliferation) in lung tissue can be associated with pulmonary fibrosis.

[0047] In some embodiments, the present invention recognizes that fibroblasts (e.g., lung fibroblasts) can be targeted for immunogenic cell death through the use of a composition comprising enzyme and fibroblast-recognizing antibody (e.g., lung fibroblast-recognizing antibody).The immunogenic cell death of fibroblasts can reduce the amount or proportion of fibroblasts in the lung of a subject.The reduction in the amount or proportion of fibroblasts can then slow or reverse pulmonary fibrosis.

[0048] According to certain embodiments, the composition useful for treating pulmonary fibrosis comprises at least a portion of an antibody (e.g., whole antibody or antibody fragment) that recognizes fibroblasts.For example, the composition may comprise at least a portion of fibroblast antibody TE-7, fibroblast antibody ER-TR7, or monoclonal antibody fibroblast 1B10, anti-CD82 antibody, anti-a-SMA antibody, anti-ITG-P1 antibody, or another of a variety of suitable antibodies that recognize fibroblasts and are known to those skilled in the art.

[0049] At least a portion of the antibody (e.g., a whole antibody or an antibody fragment) and the enzyme may be conjugated 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 pulmonary fibrosis. In some embodiments, for example, the fusion protein may bind to fibroblasts (e.g., lung fibroblasts) with higher affinity than would be expected from the same enzyme linked to the same antibody or antibody portion by a linker. In some embodiments, the fusion protein may also have a longer life span in a subject, which may result in improved performance of the composition.

[0050] In some embodiments, the composition can be used to treat congestive heart failure.For example, the enzyme of the composition can be configured to bind to fibroblasts (e.g., myocardial fibroblasts).Excess of fibroblasts (e.g., due to fibroblast proliferation) in myocardial tissue can be associated with congestive heart failure.

[0051] In some embodiments, the present invention recognizes that fibroblasts (e.g., cardiac fibroblasts) can be targeted for immunogenic cell death through the use of a composition comprising enzyme and fibroblast-recognizing antibody (e.g., cardiac fibroblast-recognizing antibody).The immunogenic cell death of fibroblasts can reduce the amount or proportion of fibroblasts in the lung of a subject.The reduction in the amount or proportion of fibroblasts can then delay or reverse congestive heart failure.

[0052] 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.

[0053] At least a portion of the antibody (e.g., a whole antibody or an antibody fragment) and the enzyme may be conjugated 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 congestive heart failure. In some embodiments, for example, the fusion protein may bind to fibroblasts (e.g., cardiac fibroblasts) with higher affinity than would be expected from the same enzyme linked to the same antibody or antibody portion by a linker. In some embodiments, the fusion protein may also have a longer life in a subject, which may result in improved performance of the composition.

[0054] In some embodiments, the composition can be used to treat cancer disease. For example, the enzyme of the composition can be configured to bind to cancer cells. In some embodiments, the present invention recognizes that cancer cells can be targeted for immunogenic cell death through the use of a composition comprising an enzyme and a cancer cell recognition antibody. The immunogenic cell death of cancer cells can treat cancer.

[0055] According to certain embodiments, the composition useful for treating cancer comprises at least a portion of an antibody (e.g., a whole antibody or an antibody fragment) that recognizes cancer cells. For example, the antibody may be an antibody against epidermal growth factor receptor (EGFR), which may be overexpressed on cancer cells, or an antibody against any of a variety of other antigens or other markers that are uniquely expressed or overexpressed on cancer cells. As a specific example, the antibody may be (SEQ ID NO: 1).

[0056] At least a portion of the antibody (e.g., a whole antibody or an antibody fragment) and the enzyme may be conjugated 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 cancer. In some embodiments, for example, the fusion protein may bind to cancer cells with higher affinity than would be expected from the same enzyme attached to the same antibody or antibody portion by a linker. In some embodiments, the fusion protein may also have a longer life in a subject, which may result in improved performance of the composition.

[0057] The enzyme may be covalently linked to another portion of the composition (e.g., an antibody or antibody portion). In some embodiments, the enzyme and the other portion of the composition (e.g., an antibody or antibody portion) are directly linked such that the enzyme and the other portion form a single polypeptide chain. For example, the composition may be expressed as a single fusion protein that includes both the enzyme (e.g., an oxidase such as xanthine oxidase) and the other portion of the composition (e.g., an antibody or antibody portion). However, in other embodiments, other methods may be used to link the enzyme to the composition, for example, directly linked to each other or linked via one or more crosslinkers. Non-limiting examples include glutaraldehyde, NHS-esters (N-hydroxysuccinimide) (e.g., dithiobis(succinimidyl propionate), dithiobis(sulfosuccinimidyl propionate), etc.), PEG groups, imidoesters (e.g., dimethyl adipimidate, dimethyl suberimidate, dimethyl pimelimidate, etc.), maleimides, pyridyls, carbodiimides, isocyanates, etc. The antibody and enzyme can be attached via any suitable system, for example, amine-amine, sulfhydryl-sulfhydryl, amine-sulfhydryl, carboxyl-amine, sulfhydryl-carbohydrate, hydroxyl-sulfhydryl, etc. One of skill in the art will be familiar with methods for crosslinking or conjugating proteins to one another.

[0058] A "fusion protein" generally refers to a hybrid polypeptide that contains protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein, or at the carboxy-terminal (C-terminal) portion of the fusion protein, thereby forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein," respectively. A fusion protein may contain different domains, for example, an enzyme and an antibody, or an enzyme and an antibody fragment. In some embodiments, an enzyme is fused at the N-terminus of an antibody or antibody fragment. In some embodiments, an enzyme is fused at the C-terminus of an antibody or antibody fragment.

[0059] In some embodiments, the enzyme is separated from the antibody or antibody fragment by a linker region. The linker region may be configured to spatially separate the antibody from the enzyme. In some embodiments, the linker region may have an amino acid sequence that includes a repeating subsequence. For example, in some embodiments, the linker region may have an amino acid sequence that includes a repeating subsequence, such as (GGGGS) n (SEQ ID NO:6), where n is an integer equal to or greater than 1 (e.g., 1, 2, 3, 4, 5, 6, 8, 10, or more). In some embodiments, the linker region is GGGGSGGGGS (SEQ ID NO:3). In other embodiments, the composition does not include a linker region.

[0060] 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 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 of 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.

[0061] Further provided herein are enzyme variants and fusion proteins comprising such enzyme variants. In some embodiments, the fusion proteins described herein comprise modifications. When fusion proteins are referred to herein, it encompasses all variants and derivatives thereof. Polypeptides comprising modifications have additional characteristics other than amino acid content. As used herein, a "modification" or "derivative" of a protein or polypeptide (e.g., a fusion protein described herein) refers to a form of a given peptide that is chemically modified relative to a reference peptide, including, but not limited to, oligomerization or polymerization, modification of amino acid residues or peptide backbone, crosslinking, cyclization, conjugation, PEGylation, glycosylation, acetylation, phosphorylation, acylation, carboxylation, lipidation, thioglycolamidation, alkylation, methylation, polyglycylation, glycosylation, polysialylation, adenylation, PEGylation, fusion to additional heterologous amino acid sequences, or other modifications that substantially alter the stability, solubility or other properties of the peptide while substantially retaining the activity of the polypeptide described herein. It should be understood that the fusion protein containing such modifications is crosslinked, cyclized, conjugated, acylated, carboxylated, lipidated, acetylated, thioglycolamidated, alkylated, methylated, polyglycylated, 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 contain the 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 susceptibility to proteinase digestion, and thus serve to extend the half-life of the polypeptide in solution, especially in biological fluids where proteases may be present.In some embodiments, the fusion proteins described herein are further modified in sequence by terminal NH3 acylation, e.g., acetylation, or thioglycolic acid amidation, terminal carboxylamidation, e.g., modification with ammonia, methylamine, and similar terminal modifications.

[0062] Terminal modifications are useful for reducing susceptibility to proteinase digestion and therefore may play a role in extending the half-life of a polypeptide in solution, particularly in biological fluids where proteases may be present. Amino-terminal modifications include methylation (e.g., --NHCH3 or --N(CH3)2), acetylation (e.g., with acetic acid or its halogenated derivatives, such as a-chloroacetic acid, a-bromoacetic acid, or a-iodoacetic acid), addition of a benzyloxycarbonyl (Cbz) group, or blocking of the amino terminus with any blocking group, including a carboxylate functional group defined by RCOO-- or a sulfonyl functional group defined by R--SO2--, where R is selected from the group consisting of alkyl, aryl, heteroaryl, alkylaryl, etc. Also, des amino acids can be incorporated at the N-terminus (so that the N-terminal amino group is absent) to reduce susceptibility to proteases or restrict the conformation of the polypeptide. In certain embodiments, the N-terminus is acetylated with acetic acid or acetic anhydride.

[0063] Modifications of the carboxy terminus include the introduction of structural constraints by replacing free acids with carboxamide groups or forming cyclic lactams at the carboxy terminus. The peptides described herein can also be cyclized or incorporate des amino or des carboxy residues at the terminus of the peptide to reduce susceptibility to proteases or restrict peptide conformation, eliminating the presence of amino or carboxyl groups at the terminus. Methods for cyclic peptide synthesis are known in the art. C-terminal functional groups of the peptides described herein include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and lower ester derivatives thereof, as well as pharmaceutically acceptable salts thereof.

[0064] In some embodiments, the fusion proteins described herein are phosphorylated. Peptides can also be easily modified by phosphorylation and other methods. In some embodiments, the naturally occurring side chains of the genetically encoded amino acids (or stereoisomeric D amino acids) can also be replaced with other side chains, such as alkyl, lower (C 1-6 ) alkyl, cyclic 4-, 5-, 6- to 7-membered alkyl, amido, amido lower alkyl amido di(lower alkyl), lower alkoxy, hydroxy, carboxy and their lower ester derivatives, and 4-, 5-, 6- to 7-membered heterocycles. For example, proline analogs in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members can be employed.

[0065] The cyclic group may be saturated or unsaturated, and if unsaturated, may be aromatic or non-aromatic. Heterocyclic groups preferably contain one or more nitrogen, oxygen, and / or sulfur heteroatoms. Examples of such groups include furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g., morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl groups. These heterocyclic groups may be substituted or unsubstituted. When a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.

[0066] In some embodiments, the fusion proteins described herein may be linked to one or more polymer moieties. In some embodiments, these polymers are covalently attached to the fusion proteins of the present disclosure. In some embodiments, the polymer is pharma- ceutically acceptable for therapeutic use of the final product preparation. Those skilled in the art will be able to select the desired polymer based on such considerations as whether the polymer-peptide conjugate will be used therapeutically, if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations.

[0067] All combinations of the different modifications and derivatizations are envisioned for the fusion proteins described herein.

[0068] Another aspect of the present disclosure provides a method for producing a fusion protein. The fusion protein can be produced and isolated by expression form recombinant nucleic acid in a suitable cell (e.g., bacterial cell or eukaryotic cell). To produce the fusion protein, the nucleic acid encoding the fusion protein can be introduced into a cell (e.g., bacterial cell or eukaryotic cell such as yeast cell or insect cell). The cell can be cultured under conditions that allow the fusion protein to be expressed from the nucleic acid encoding the fusion protein. The fusion protein including the signal peptide can be secreted, for example, into the culture medium and then recovered. The fusion protein can be isolated using any method of purifying proteins known in the art.

[0069] 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 of the present disclosure can be DNA or RNA, double-stranded or single-stranded. In some embodiments, the nucleotide sequence encoding the fusion protein can be codon-optimized to accommodate different expression systems (e.g., for mammalian expression).

[0070] In some embodiments, the nucleic acid is contained within a vector, such as an expression vector. In some embodiments, the vector comprises a promoter operably linked to the nucleic acid.

[0071] A variety of promoters can be used for expression of 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, E. coli lac UV5 promoter, and herpes simplex tk virus promoter.

[0072] Controllable promoters can also be used, including those that use the lac repressor from E. coli as a transcriptional modulator to control transcription from mammalian cell promoters with the lac operator, and those that use the tetracycline repressor (tetR). Other systems include FK506 dimers, VP16 or p65 with astroradiol, RU486, diphenol murislerone, or rapamycin.

[0073] A controllable promoter containing a repressor may be used in conjunction with an operon. In one embodiment, the lac repressor from E. coli may function as a transcriptional modulator to control transcription from a mammalian cell promoter with a lac operator. In one embodiment, a tetracycline-inducible switch is used.

[0074] In addition, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene, for the selection of stable or transient transfectants in mammalian cells; an enhancer / promoter sequence from the immediate early gene of human CMV for high-level transcription; a transcription termination signal and an RNA processing signal from SV40 for mRNA stability; an SV40 polyoma origin of replication and ColE1 for proper episomal replication; an internal ribosome binding site (IRES), a versatile multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Methods for making suitable vectors and vectors containing transgenes are well known and available in the art.

[0075] An expression vector containing the nucleic acid can be transfected into a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation), and the transfected cells can then be cultured by conventional techniques to produce the fusion proteins described herein. In some embodiments, expression of the fusion proteins described herein is controlled by a constitutive, inducible, or tissue-specific promoter.

[0076] Host cells used to express the fusion proteins described herein may include bacterial cells such as E. coli, eukaryotic cells, etc. In one embodiment, mammalian cells such as Chinese hamster ovary cells (CHO) may be used.

[0077] A variety of host-expression vector systems may be utilized to express the fusion proteins described herein. Such host-expression systems represent vehicles in which the coding sequences of the isolated fusion proteins described herein may be produced and subsequently purified, but also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequences, may express the fusion proteins described herein in situ. These include microorganisms such as bacteria [e.g., Escherichia coli and B. subtilis] transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the coding sequences of the fusion proteins described herein; yeast [e.g., Saccharomyces pichia] transformed with recombinant yeast expression vectors containing the coding sequences of the fusion proteins described herein; pichia)]; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing sequences encoding the fusion proteins described herein; plant cell systems infected with recombinant viral 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 proteins described herein; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphocytic cells, Per C.6 cells) harboring recombinant expression constructs containing promoters derived from the genome of a mammalian cell (e.g., metallothionein promoter) or from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0078] In bacterial systems, a number of expression vectors may be conveniently selected depending on the intended use of the expressed fusion protein. For example, when producing large quantities of such proteins, a vector that induces expression of a high level of a fusion protein product that can be easily purified may be desirable for the production of pharmaceutical compositions of the fusion proteins described herein. Such vectors include, but are not limited to, the E. coli expression vector pUR278; pIN vectors, in which coding sequences can be individually ligated into the vector in frame with the lac Z coding region to produce a fusion protein. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). Generally, 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 moiety.

[0079] In mammalian host cells, a number of viral-based expression systems are available. When adenovirus is used as an expression vector, the coding sequence of interest may be ligated to the adenovirus transcription / translation control compositions, such as the late promoter and tripartite leader sequence. This chimeric gene may then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., regions E1 or E3) results in recombinant viruses that are viable and capable of expressing immunoglobulin molecules in infected hosts. Specific initiation signals may also be required for efficient translation of the inserted antibody coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, to ensure translation of the entire insert, the initiation codon must be in phase with the reading frame of the desired coding sequence. These exogenous translational control signals and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, and the like.

[0080] In addition, a host cell line can be selected that modulates the expression of the inserted sequence or modifies and processes the gene product in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Purification and modification of recombinant proteins are well known in the art, so that the design of polyprotein precursors can include a number of embodiments that are readily understood by those skilled in the art. Any known protease or peptidase known in the art can be used for the described modifications of precursor molecules, such as thrombin or factor Xa, enterokinase, furin, and AcTEV, and foot and mouth disease virus protease C3.

[0081] Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. To ensure the correct modification and processing of expressed foreign proteins, appropriate cell lines or host systems can be selected. For this purpose, eukaryotic host cells that have the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product 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.

[0082] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines can be engineered that stably express the fusion proteins described herein. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. After introduction of the foreign DNA, engineered cells can be grown in enriched medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express the fusion proteins described herein. Such engineered cell lines can be particularly useful for screening and evaluating fusion proteins that interact directly or indirectly with the fusion proteins described herein.

[0083] The expression level of the fusion described herein can be increased by vector amplification.If the marker in the vector system expressing the fusion protein described herein is amplifiable, the increase in the level of inhibitor present in the culture of the host cell increases the copy number of the marker gene.Since the amplified region is associated with the fusion protein described herein or the nucleotide sequence of the fusion protein described herein, the production of the fusion protein also increases.

[0084] Once a fusion described herein is recombinantly expressed, it may be purified by any method known in the art for the purification of polypeptides, polyproteins or antibodies (e.g., similar to antibody purification schemes based on antigen selectivity), such as, for example, chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen (optionally after Protein A selection if the polypeptide contains an Fc domain (or portion thereof)), and sizing column chromatography), centrifugation, differential lysis, or any other standard technique for the purification of polypeptides or antibodies.

[0085] In some embodiments, fusion proteins can be advantageously resistant to degradation compared to other compositions that contain linked antibodies and enzymes. Fusion proteins can also provide unexpectedly high molecular performance. For example, an enzyme, antibody or antibody fragment may have unexpectedly high activity when included in the fusion protein of a composition compared to the activity of a composition in which the enzyme is crosslinked with the antibody or antibody fragment. This can advantageously improve the efficacy of the composition that contains the fusion protein.

[0086] However, the enzyme may be linked to another moiety of the composition (e.g., an antibody or antibody moiety) via a crosslinker. The moiety and the enzyme may be crosslinked using a crosslinker. The crosslinker may include synthetic crosslinkers or may include direct bonds between amino acid side chains of the antibody and the enzyme. In some embodiments, the antibody and the enzyme are crosslinked using one or more crosslinkers. Non-limiting examples of crosslinkers include glutaraldehyde, NHS-esters (N-hydroxysuccinimide) (e.g., dithiobis(succinimidyl propionate), dithiobis(sulfosuccinimidyl propionate), etc.), PEG groups, imidoesters (e.g., dimethyl adipimidate, dimethyl suberimidate, dimethyl pimelimidate, etc.), maleimides, pyridyls, carbodiimides, isocyanates, etc.

[0087] The antibody and enzyme can be attached via any suitable coupling system, e.g., amine-amine, sulfhydryl-sulfhydryl, amine-sulfhydryl, carboxyl-amine, sulfhydryl-carbohydrate, hydroxyl-sulfhydryl, etc. One of skill in the art will be familiar with methods for crosslinking or conjugating proteins to one another.

[0088] The composition may, in certain embodiments, include or be administered with a therapeutic agent. The therapeutic agent may be selected to enhance the sensitivity of the cells to the composition. In some embodiments, the subject is pre-treated with a therapeutic agent. In some embodiments, the subject is administered a therapeutic agent as part of the composition described herein or simultaneously with administration of the composition. The subject may also be administered a therapeutic agent after administration of the composition, and the disclosure is not so limited. Treatment of the subject with a therapeutic agent may prepare the subject for successful attack on the lysosomal membrane, which may be useful in treating the disease. Treatment with a therapeutic agent may provide a more efficient attack on the lysosomal membrane. However, it should also be understood that the use of a therapeutic agent is not required, and in some cases, the composition may be administered without the use of a therapeutic agent.

[0089] In some embodiments, the therapeutic agent is configured to increase the concentration of reactive oxygen species in the vicinity of the cells targeted by the composition.Without wishing to be bound by theory, it is believed that, as discussed above, oxidation processes (e.g., those caused by the action of oxidase or peroxidase) can be useful in the treatment of diseases or conditions.Therefore, the therapeutic agent can increase the concentration of reactive oxygen species in the vicinity of cells, thereby increasing the rate of oxidation or increasing the total amount of oxidation.

[0090] In some embodiments, the increase in the concentration of reactive oxygen species is related to the level of antioxidants in the subject, as antioxidants can reduce or inhibit such tumor treatment. In some embodiments, the therapeutic agent reduces the level of antioxidants in the vicinity of the targeted cells. For example, as discussed in more detail below, the therapeutic agent may remove or inhibit antioxidants in the subject.

[0091] It should be understood that, according to some embodiments, one or more therapeutic agents may be used individually or together. For example, the subject may be administered a statin, a fatty acid, or both, as discussed in more detail below. The therapeutic agents may be administered individually and / or together. When administered individually, the therapeutic agents may be administered simultaneously and / or sequentially in any suitable order. Any suitable administration method and administration schedule may be used, including those discussed herein. Furthermore, the one or more therapeutic agents may be in a suitable, pharma- ceutically acceptable carrier. Antioxidants may be removed or suppressed from the subject with or without the use of a therapeutic agent.

[0092] In some embodiments, the therapeutic agent comprises a fatty acid. The fatty acid may comprise an unsaturated fatty acid. For example, the therapeutic agent may comprise an n-3 to n-6 fatty acid. In some embodiments, the therapeutic agent is a fatty acid. In some embodiments, administration of the fatty acid reduces the antioxidant status of the subject. The unsaturated fatty acid, including the n-3 to n-6 fatty acid, may be given to the subject or may be administered by any of a variety of other suitable methods. In the context of the present disclosure, it is recognized by the present invention that treating a subject with an unsaturated fatty acid may increase the local concentration of unsaturated fatty acid that reactive oxygen species can oxidize. This may promote lysosome-induced cell death by promoting the degradation of lysosomal membranes.

[0093] One or more fatty acids may be administered to the subject. One or more fatty acids may be administered simultaneously and / or sequentially. The unsaturated fatty acid may contain one or more double and / or triple bonds in the fatty acid chain. The fatty acid may be a relatively small chain fatty acid. 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. The unsaturated fatty acid may be a monounsaturated fatty acid and / or a polyunsaturated fatty acid. Non-limiting examples of unsaturated fatty acids include, but are not limited to, CH2=CHCOOH, CH3CH=CHCOOH, CH3CH2CH=CHCOOH, CH3CH2CH2CH=CHCOOH, CH2=CHCH2COOH, CH2=CHCH2CH2COOH, CH2=CHCH2CH2COOH, CH2=CHCH2CH=CHCOOH, CH2=CHCH=CHCOOH, CH2=CHCH=CHCOOH, and the like. Additionally, in some cases, the fatty acids may be derived from naturally occurring sources such as, for example, krill oil, fish oil, safflower oil, soybean oil, linseed oil, canola oil, algae oil, and the like.

[0094] A wide range of dosages of fatty acids may be used. In some embodiments, fatty acids may be administered to a subject, such as a human, in 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 some embodiments, the fatty acid may be administered in a dosage of 15 g or less, 14 g or less, 13 g or less, 12 g or less, 11 g or less, 10 g or less, 9 g or less, 8 g or less, 7 g or less, 6 g or less, 5 g or less, 4 g or less, 3 g or less, 2 g or less, 1 g or less, 0.9 g or less, 0.8 g or less, 0.7 g or less, 0.6 g or less, 0.5 g or less, 0.4 g or less, 0.3 g or less, 0.2 g or less, or 0.1 g or less. Also, in some embodiments, any combination of these is possible, for example, the dosage may be between 1-15 g, between 1-10 g, between 5-10 g, between 0.5-1 g, etc.

[0095] In some embodiments, the fatty acid may be administered to a subject, such as a human, at a dosage 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 may be administered at a dosage of 2 g / kg or less, 1 g / kg or less, 500 mg / kg or less, 300 mg / kg or less, 200 mg / kg or less, 100 mg / kg or less, 50 mg / kg or less, 30 mg / kg or less, 20 mg / kg or less, 10 mg / kg or less, 5 mg / kg or less, 3 mg / kg or less, 2 mg / kg or less, or 1 mg / kg or less. Also, in some embodiments, any combination of these is possible, for example, dosages can be 1-2 g / kg, 500 mg / kg-1 g / kg, 400-800 mg / kg, etc. Appropriate dosages, administration rates, and treatments with fatty acids can be determined using the methods described herein.

[0096] In some embodiments, the therapeutic agent comprises a statin. The statin may be co-administered with fatty acid or may be administered without fatty acid. Statins are also known as HMG-CoA reductase inhibitors and can be used to lower lipid levels. Non-limiting examples of statins include atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, cerivastatin, mevastatin, and simvastatin. One or more statins may be administered to a subject, for example, simultaneously and / or sequentially.

[0097] A wide range of doses of statins may be used. For example, statins may 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 may be administered at a dosage of 100 mg or less, 90 mg or less, 80 mg or less, 70 mg or less, 60 mg or less, 50 mg or less, 40 mg or less, 30 mg or less, 20 mg or less, 10 mg or less, 5 mg or less, 3 mg or less, 2 mg or less, or 1 mg or less. Also, in some embodiments, any combination of these is possible, for example, the dosage may be 10-20 mg, 20-40 mg, 40-80 mg, 5-10 mg, etc.

[0098] Further, in some embodiments, the statin may be administered to a subject, such as a human, at a dosage 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 may be administered at a dosage of 15 mg / kg or less, 14 mg / kg or less, 13 mg / kg or less, 12 mg / kg or less, 11 mg / kg or less, 10 mg / kg or less, 9 mg / kg or less, 8 mg / kg or less, 7 mg / kg or less, 6 mg / kg or less, 5 mg / kg or less, 4 mg / kg or less, 3 mg / kg or less, 2 mg / kg or less, or 1 mg / kg or less. Also, in some embodiments, any combination of these is possible, for example, the dosage may be between 1-15 mg / kg, between 1-10 mg / kg, between 5-10 mg / kg, etc. Appropriate dosages, administration rates, and treatments with fatty acids can be determined using the methods described herein.

[0099] According to certain embodiments, if the subject is taking antioxidants, the antioxidants may be removed or inhibited from the subject.For example, as part of tumor treatment preparation, antioxidants may be withdrawn completely or the dosage, e.g., amount and / or frequency, of antioxidants may be reduced.Non-limiting examples of antioxidants include beta-carotene, vitamin A, and vitamin E.Other examples include vitamin C, glutathione, lipoic acid, uric acid, and ubiquinol.

[0100] As mentioned above, some embodiments of the present disclosure are directed to compositions that include an enzyme, such as an oxidoreductase. For example, the enzyme can be an oxidase or a peroxidase. Other exemplary oxidoreductases that may 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 Pseudomona flurescens]; 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., from Bacillus subtilis). subtilis); myo-inositol dehydrogenase (e.g., from Bacillus subtilis); prokaryotic 3-hydroxybutyrate dehydrogenase; galactose dehydrogenase (e.g., from Escherichia coli); galactose mutarotase (e.g., from E. coli); formate dehydrogenase [e.g., from Candida boidinii]; manganese peroxidase (e.g., native Nematoloma frowardii manganese peroxidase); cholesterol oxidase [e.g., from Nocardia sp.) ];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);aflatoxin B1 aldehyde reductase member 2 (e.g., human aflatoxin B1 aldehyde reductase member 2);alanine dehydrogenase [e.g., Bacillus cereus ( cereus)]; alcohol dehydrogenase (e.g., human alcohol dehydrogenase or alcohol dehydrogenase from E. coli); aldehyde dehydrogenase 2 (e.g., human aldehyde dehydrogenase 2); aldose reductase (e.g., human aldose reductase); bilirubin oxidase; biotinylated luciferase (e.g., from E. coli); bovine catalase-polyethylene glycol; bovine superoxide dismutase-polyethylene glycol; catalase (e.g., from psychrotolerant bacteria); cholesterol oxidase (e.g., from E. coli); Cu / Zn superoxide dismutase; cyclohexanone monooxygenase [e.g., from Acinetobacter sp.), cyclooxygenase (e.g., human cyclooxygenase 1 or 2, or ovine cyclooxygenase 2); cytochrome p450 reductase (e.g., human cytochrome p450 reductase); D-2-hydroxyglutarate dehydrogenase (e.g., from Acidaminococcus fermentans); D-2-hydroxyisocaproic aid 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); phymethylglycine oxidase (e.g., from Arthrobacter globiformis). globifomis); disulfide oxidoreductase; D-lactate dehydrogenase (e.g., from Escherichia coli); DT diaphorase (e.g., human DT diaphorase or rat DT diaphorase); FMN reductase (e.g., from Escherichia 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 sp.) or from 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 Dactylium dendroides ( dendroides)]; glucose dehydrogenase (e.g., from E. coli); glucose-6-phosphate dehydrogenase (e.g., from E. coli); glutamate dehydrogenase (e.g., from E. coli or from thermophilic bacteria); glutathione reductase (e.g., human glutathione reductase from E. coli); glyceraldehyde-3-phosphate dehydrogenase (e.g., human or mouse glyceraldehyde-3-phosphate dehydrogenase); glycerol-3-phosphate dehydrogenase (e.g., from E. coli); glycine oxidase H244K (e.g., from Bacillus subtilis); histamine dehydrogenase (e.g., from E. coli); inosine monophosphate dehydrogenase [e.g., Staphylococcus aureus aureus]; inosine monophosphate dehydrogenase type II (e.g., human inosine monophosphate dehydrogenase type II); isocitrate dehydrogenase (NAD+) (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 viridans]; lactate dehydrogenase; leucine dehydrogenase; Leuconostoc mesenteroides mesenteroides glucose-6-phosphate dehydrogenase; L-glutamate dehydrogenase; L-glutamate oxidase [e.g., Streptomyces sp.)]; L-lactate dehydrogenase; L-lactate dehydrogenase [e.g., from Bacillus stearothermophilus]; L-leucine dehydrogenase; L-phenylalanine dehydrogenase; luciferase [e.g., from Escherichia coli or from Photinus pyralis]; soluble cellulose monooxygenase [e.g., from Thermobifida fusca]; soluble 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 1 R132H (e.g., human isocitrate dehydrogenase 1 R132H or isocitrate dehydrogenase 1 R132H from Escherichia coli); N-acylmannosamine 1-dehydrogenase [e.g., from Pseudomonas sp.]; native 6-phospho-D-gluconate dehydrogenase (e.g., from Escherichia coli); native Acremonium sp. ascorbate oxidase; native Aerococcus viridans glycerol 3-phosphate oxidase; native Aerococcus viridans pyruvate oxidase; native Agaricus bisporus laccase; native Alcaligenes faecalis 3-hydroxybutyrate dehydrogenase; native Alcaligenes sp. sp.)choline oxidase;native alcohol dehydrogenase;native Arthrobacter globiformischoline oxidase;native Arthrobacter globiformisuricase;native Arthrobacter sp.) acyl-CoA oxidase;native Arthrobacter sp. tyramine oxidase;native Aspergillus niger glucose oxidase;native Aspergillus niger nitrate reductase (NAD[P]H);native Aspergillus sp. catalase;native Aspergillus sp. glucose oxidase;native Bacillus cereus L-leucine dehydrogenase;native Bacillus fastidiosus uricase;native Bacillus licheniformis NADH oxidase;native Bacillus megaterium diaphorase (NADH);native Bacillus pumilus bilirubin oxidase / laccase;native Bacillus sp. sp.) glucose-6-phosphate dehydrogenase;native Bacillus leucine dehydrogenase;native Bacillus sarcosine oxidase;native Bacillus uricase;native Bacillus 12α-hydroxysteroid dehydrogenase;native Bacillus stearothermophilus alanine dehydrogenase;native Bacillus stearothermophilus diaphorase 1;native Bacillus stearothermophilus leucine dehydrogenase;native Bacillus stearothermophilus polynucleotide phosphorylase;native Bacillus stearothermophilus superoxide dismutase;native Bacillus subtilis bilirubin oxidase;native Bacillus subtilis L-alanine dehydrogenase;native baker's yeast [(S. cerevisiae)] glucose-6-phosphate dehydrogenase;native baker's yeast (S. cerevisiae) glutathione reductase;native baker's yeast (S.cerevisiae) glyceraldehyde-3-phosphate dehydrogenase;native Bjerkandera adusta peroxidase;native bovine catalase;native bovine glutamate dehydrogenase;native bovine glutathione peroxidase;native bovine lactoperoxidase;native bovine L-glutamate dehydrogenase;native bovine L-lactate dehydrogenase;native bovine L-lactate dehydrogenase;native bovine malate dehydrogenase;native bovine plasma amine oxidase;native bovine superoxide dismutase. native bovine xanthine oxidase; native Caldariomyces fumago chloroperoxidase; native Candida boidiniformate dehydrogenase; native Candida sp. alcohol oxidase; native Candida uricase; native Candida utilis L-glutamate dehydrogenase (NADP); native dog superoxide dismutase; native Cellulomonas sp. glycerol dehydrogenase; native chicken glyceraldehyde-3-phosphate dehydrogenase; native chicken L-lactate dehydrogenase; native chicken malate dehydrogenase (oxaloacetate-decarboxylate); native chicken sulfite oxidase; native Clostridium kluyveri diaphorase; native Clostridium native Corallina officinalis bromoperoxidase; native Coriolus versicolor laccase; native maize nitrate reductase; native Corynebacterium glutamicum catalase; native Crotalus adamanteus L-amino acid oxidase; native Crotalus atrox L-amino acid oxidase; native Crotalus durissus venom L-amino acid oxidase; native Cucurbita sp. native Cucurbita sp.) ascorbate oxidase; native Cucurbita sp. L-ascorbate oxidase; native Dactylium dendroides galactose oxidase; native diaphorase (NADPH) (e.g., from Bacillus megaterium); native E. coli 1-5-anhydroglucitol-6-phosphate dehydrogenase; native E. coli galactose 1-dehydrogenase; native E. coli sarcosine oxidase;Native Enterobacter aerogenes glycerol dehydrogenase; Native Enterobacter aerogenes myo-inositol dehydrogenase; Native horse spleen apoferritin; Native E. coli nitrate reductase (cytochrome); Native E. coli superoxide dismutase; Native E. coli thioredoxin reductase; Native Gluconobacter industrius D-fructose dehydrogenase; Native Gluconobacter sp. D-fructose dehydrogenase; Native glutamate dehydrogenase (NADP+); Native Glycine max (soybean) lipoxidase; Native Hansenula sp. native human Lactobacillus sp. alcohol oxidase;native horseradish peroxidase;native horseradish peroxidase;native horseradish polyperoxidase;native horseradish superoxide dismutase;native human catalase;native human eosinophil peroxidase;native human glutathione peroxidase;native human glyceraldehyde-3-phosphate dehydrogenase;native human lactate dehydrogenase;native human lactoperoxidase;native human myeloperoxidase (e.g., isoforms A, B, or C);native human superoxide dismutase;native laccase (e.g., from Cerrena unicolor or from white rot fungi);native lactate dehydrogenase (e.g., from thermophiles);native Lactobacillus delbrueckii D-lactate dehydrogenase;native Lactobacillus leichmanii) D-lactate dehydrogenase;native Leuconostoc mesenteroides D-lactate dehydrogenase;native Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase;native Leuconostoc mesenteroides mannitol dehydrogenase;Native lignin peroxidase (e.g., from Phanerochaete chrysosporium); native malate dehydrogenase (e.g., from Thermophiles); native Micrococcus lysodeikticus lysodeikticus) catalase;native microbial cholesterol oxidase;native microbial D-lactate dehydrogenase;native microbial glucose dehydrogenase (FAD dependent);native microbial glucose dehydrogenase (NAD(P) dependent);native microbial glucose dehydrogenase (PQQ dependent);native microbial glucose-6-phosphate dehydrogenase;native microbial glutamate dehydrogenase (NAD dependent);native microbial L-α-glycerophosphate oxidase;native microbial malate dehydrogenase;native microbial P-hydroxybenzoate hydroxylase;native microbial pyruvate oxidase;native microbial sarcosine oxidase;native microbial sorbitol dehydrogenase;native microbial xanthine oxidase;native microbial pyruvate oxidase;native mouse catalase;native mushroom tyrosinase;native mushroom polyphenol oxidase;native Myrothecium verrucaria (Myrothecium verrucaria bilirubin oxidase;native nitrate reductase (e.g., from Aspergillus sp.);native Nocardia erythropolis cholesterol oxidase;native Nocardia cholesterol dehydrogenase;native Pediococcus sp. glycerol 3-phosphate oxidase;native Pediococcus sp. glycerol 3-phosphate oxidase;native Pediococcus sp. lactate oxidase;native Pediococcus sp. L-α-glycerophosphate oxidase;native Photinus pyralis [firefly] luciferase;Native Photobacterium phosphoreum (Lux) bacterial luciferase; Native Pichia pastoris alcohol oxidase; Native plant-derived diamine oxidase; Native Pleurotus ostreatus laccase; Native porcine apo D-amino acid oxidase; Native porcine cytochrome C reductase; Native porcine D-amino acid oxidase; Native porcine diamine oxidase; Native porcine heart lactate dehydrogenase; Native porcine isocitrate dehydrogenase (NADP); Native porcine lactate dehydrogenase; Native porcine lipoamide dehydrogenase; Native porcine L-lactate dehydrogenase; Native porcine L-lactate dehydrogenase; Native porcine malate dehydrogenase, IFCC Quality porcine heart;native porcine malate dehydrogenase;native porcine NAD(P)H dehydrogenase (quinone);native porcine superoxide dismutase;native porcine α-ketoglutarate dehydrogenase;native Proteus sp. glutamate dehydrogenase (NADP-dependent);native Proteus sp. L-glutamate dehydrogenase (NADP);native Pseudomonas fluorescens galactose 1-dehydrogenase;native Pseudomonas lemonyigne lemoignei) β-hydroxybutyrate dehydrogenase;native Pseudomonas cholesterol oxidase;native Pseudomonas D-3-hydroxybutyrate dehydrogenase;native Pseudomonas formaldehyde dehydrogenase;native Pseudomonas glucose dehydrogenase;native Pseudomonas N-acylhexosamine oxidase;native Pseudomonas p-hydroxybenzoate hydroxylase;native Pseudomonas protocatechuate 3,4-dioxygenase;native Pseudomonas protocatechuate 3,4-dioxygenase;native Pseudomonas sarcosine dehydrogenase;Native Pseudomonas testosteroni 3α-hydroxysteroid dehydrogenase; Native rabbit cytochrome P450 reductase; Native rabbit glyceraldehyde-3-phosphate dehydrogenase; Native rabbit glycerol-3-phosphate dehydrogenase; Native rabbit lactate dehydrogenase; Native rabbit L-lactate dehydrogenase; Native rabbit pyruvate kinase / lactate dehydrogenase enzyme rabbit muscle; Native rabbit α-glycerophosphate dehydrogenase; Native rabbit α-glycerophosphate dehydrogenase-triosephosphate isomerase; Native rat sorbitol dehydrogenase; Native rat thioredoxin reductase; Native Rhodopseudomonas sphaeroides β-hydroxybutyrate dehydrogenase; Native Rhus vernicifera laccase;native roxburgh superoxide dismutase;native Saccharomyces cerevisiae alcohol dehydrogenase;native sheep 6-phosphogluconate dehydrogenase;native sheep cyclooxygenase;native sheep sorbitol dehydrogenase;native spinach glutathione reductase;native Spinacia oleracea [spinach] ferredoxin-NADP+ reductase;native Sporosarcina sp. L-phenylalanine dehydrogenase;native Staphylococcus epidermidis D-lactate dehydrogenase;native Streptococcus thermophilus thermophilus glycerol 3-phosphate oxidase; native Streptomyces cholesterol oxidase; native Thermoactinomyces intermedius phenylalanine dehydrogenase;native Thermoanaerobium brockii alcohol dehydrogenase, NADP+ dependent;native Thermoanaerobium sp. aromatic alcohol dehydrogenase, NADP+ dependent;native thermostable fungal catalase for semiconductor processing;native thermostable fungal catalase for textile processing;native Thermos flavus malate dehydrogenase;native Torula yeast glucose-6-phosphate dehydrogenase;native Trametes versicolor laccase;native; Trichoderma viride lysine oxidase;native versatile peroxidase (e.g., from Psammophila truncatula);native Vibrio fischeri (Photobacterium luciferase);native wheat germ glutathione reductase;native white rot fungus [Phaner ochaete chrysosporium] manganese peroxidase;native xanthine dehydrogenase (e.g., from milk);native xanthine dehydroginase;native yeast 6-phosphogluconic dehydrogenase;native yeast alcohol dehydrogenase;native yeast aldehyde dehydrogenase;native yeast formate dehydrogenase;native yeast malate dehydrogenase;native Zucchini ascorbate oxidase;native Zymomonas mobilis native Zymomonas mobilis alcohol dehydrogenase; native Zymomonas mobilis glucokinase; native 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; phosphogluconate dehydrogenase (e.g., human phosphogluconate dehydrogenase or phosphogluconate dehydrogenase from E. coli); prokaryotic galactose dehydrogenase; prostaglandin F synthase (e.g., human prostaglandin F synthase); protocatechuate 3,4-dioxygenase; pyranose oxidase (e.g., from Coriolus sp.) or from E. coli; pyruvate oxidase (e.g., from E. coli); R-carbonyl reductase; recombinant ketol-acid reductoisomerase [e.g., from Mycobacterium tuberculosis]; sarcosine oxidase (e.g., from E. coli); S-carbonyl reductase; secondary alcohol dehydrogenase; sulfite oxidase (e.g., human sulfite oxidase); taurine dioxygenase (e.g., from E. 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 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 B. choshinensis); or β-galactose dehydrogenase S (e.g., from Pseudomonas fluorescens).

[0101] 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 at a dose of 10 mg or more, 15 mg or more, 20 mg or more, 25 mg or more, 30 mg or more, 40 mg or more, 50 mg or more, 60 mg or more, 70 mg or more, 80 mg or more, 90 mg or more, 100 mg or more, 200 mg or more, 300 mg or more, 500 mg or more, 1000 mg or more, 1500 mg or more, 2000 mg or more, 2500 mg or more, 3000 mg or more, 5000 mg or more, etc. In some embodiments, the composition may be applied at a dosage of 5000mg or less, 3000mg or less, 2500mg or less, 2000mg or less, 1500mg or less, 1000mg or less, 500mg or less, 300mg or less, 200mg or less, 100mg or less, 90mg or less, 80mg or less, 70mg or less, 60mg or less, 50mg or less, 40mg or less, 30mg or less, 25mg or less, 20mg or less, 15mg or less, 10mg or less, etc. Combinations of these ranges are also possible. For example, the dosage may be 10mg or more and 20mg or less, 50mg or more and 100mg or less, or 100mg or more and 200mg or less.

[0102] In some embodiments, the composition may be administered to a subject (e.g., a human) at a dosage of 1 mg / kg or more, 2 mg / kg or more, 3 mg / kg or more, 4 mg / kg or more, 5 mg / kg or more, 6 mg / kg or more, 7 mg / kg or more, 8 mg / kg or more, 9 mg / kg or more, 10 mg / kg or more, 11 mg / kg or more, 12 mg / kg or more, 13 mg / kg or more, 14 mg / kg or more, or 15 mg / kg or more. In some embodiments, the statin may be administered at a dosage of 15 mg / kg or less, 14 mg / kg or less, 13 mg / kg or less, 12 mg / kg or less, 11 mg / kg or less, 10 mg / kg or less, 9 mg / kg or less, 8 mg / kg or less, 7 mg / kg or less, 6 mg / kg or less, 5 mg / kg or less, 4 mg / kg or less, 3 mg / kg or less, 2 mg / kg or less, or 1 mg / kg or less. Combinations of these ranges are also possible. For example, in some embodiments, the dosage can be from 1 mg / kg to 15 mg / kg, from 1 mg / kg to 10 mg / kg, or from 5 mg / kg to 10 mg / kg.

[0103] In other aspects, the compositions described herein may be administered to a subject. The compositions may be administered alone. In some embodiments, the compositions are administered in combination with cofactors, other therapeutic agents (e.g., therapeutic agents), and the like. For example, the compositions may be administered alone or in combination with substrates such as hypoxanthine, xanthine, glucose, and / or in combination with fatty acids, statins, and the like, for example as discussed herein. See, for example, International Publication No. WO 2019 / 099687 (herein incorporated by reference).

[0104] The composition may be applied in a therapeutically effective pharma- ceutically acceptable amount in a pharma- ceutically acceptable formulation, e.g., a pharma- ceutically acceptable carrier, as described below. The term "effective amount" of a composition, such as the compositions described herein, refers to the amount necessary or sufficient to achieve a desired biological effect. For example, an effective amount of a composition for treating a tumor may be an amount sufficient to reduce the size of the tumor. By selecting from various active compositions, in combination with the teachings provided herein, and weighing factors such as potency, relative bioavailability, subject's body weight, severity of side effects, and mode of administration, one can design an effective prophylactic or therapeutic treatment regimen that does not cause substantial toxicity, yet is completely effective in treating a particular subject. The effective amount for any particular application may vary depending on factors such as the disease or condition being treated, the particular 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 undue experimentation.

[0105] As used herein, the terms "treat", "treated", "treating" and the like refer to the administration of a composition to a subject that may increase the subject's resistance to a disease or increase resistance to further progression of the disease, control the progression of the disease, and / or slow the progression of the disease, or reduce the severity of the symptoms of the disease. The effective amount may depend on the particular disease being treated and the desired outcome.

[0106] In some embodiments, the therapeutically effective dose of the composition can be determined first from animal models.The applied dose can be adjusted based on the relative bioavailability and efficacy of the compound administered.Based on the above method and other methods well known in the art, it is well within the capabilities of those skilled in the art to adjust the dose to achieve maximum efficacy.

[0107] In administering the compositions to a subject, the dose, administration schedule, and administration route, etc., can be selected to affect the known activity of these compositions. The dosage can be appropriately estimated based on the results of experimental models, combined with the results of the assays of the compositions described herein. The dosage can be appropriately adjusted to achieve the desired local or systemic drug level, depending on the mode of administration. The dosage can be administered once or in several doses per day. Multiple doses per day can be used to achieve the appropriate systemic level of the composition in the subject or in the cells targeted by the composition.

[0108] The dose may be selected to provide a therapeutically effective amount of the composition to the cells targeted by the composition. The dose may be administered at a maximum safe dose. The maximum safe dose may be selected to deliver a high therapeutically effective amount of the composition to the subject, but with minimal risk to the subject of potentially harmful side effects.

[0109] The dose of the composition may be selected to have a desired concentration in targeted cells, to have a desired efficacy, to have a desired longevity within a subject, to have a desired rate of administration, to have a desired frequency of administration, to act in an appropriate manner when co-administered with other treatments (e.g., as a cocktail), or for any of a variety of other purposes known to those of skill in the art.

[0110] Dose can be selected at least in part based on the condition related to the subject.For example, dose can be selected at least in part based on the species, age, sex, weight, size, environment, metabolism, physical condition or current health condition of the subject.In some cases, subject-specific maximum dose can be used.In some cases, dose can be administered in a way that limits adverse effects on the subject (e.g., dose can be delivered orally, nasally, intravenously, etc., depending on the need of the subject).

[0111] Doses of the composition may be administered daily, weekly, or monthly, and any other amount of time in between.

[0112] The dose may include any suitable amount of the composition. In some embodiments, the dose of the composition is 0.1 micrograms or more, 0.5 micrograms or more, 10 micrograms or more, 50 micrograms or more, 100 micrograms or more, 500 micrograms or more, 1000 micrograms or more, or more than that. In some embodiments, the dose of the composition is 10000 micrograms or less, 5000 micrograms or less, 1000 micrograms or less, 500 micrograms or less, 100 micrograms or less, 50 micrograms or less, or less than that. Combinations of these ranges are also possible. For example, in some embodiments, the dose of the composition is 0.5 micrograms or more and 10000 micrograms or less.

[0113] The dose of the composition may be administered at any of a variety of average rates. In some embodiments, the dose is administered at a rate of 0.01 micrograms / hour or more, 0.05 micrograms / hour or more, 0.1 micrograms / hour or more, 0.5 micrograms / hour or more, 1 micrograms / hour or more, 5 micrograms / hour or more, 10 micrograms / hour or more, 50 micrograms / hour or more, 100 micrograms / hour or more, 500 micrograms / hour or more, 1000 micrograms / hour or more or faster. In some embodiments, the dose is administered at a rate of 10000 micrograms / hour or less, 5000 micrograms / hour or less, 1000 micrograms / hour or less, 500 micrograms / hour or less, 100 micrograms / hour or less, 50 micrograms / hour or less, 10 micrograms / hour or less, 1 micrograms / hour or less, 0.5 micrograms / hour or less, 0.1 micrograms / hour or less or less. Combinations of these ranges are possible. For example, in some embodiments, the dose is administered at a rate of at least 0.01 micrograms / hour and up to 10,000 micrograms / hour.

[0114] The composition can be administered for an extended period of time. In some embodiments, the dose is administered for 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 12 hours or more, 24 hours or more, 3 days or more, 7 days or more, 10 days or more, 14 days or more, 21 days or more, 30 days or more, 45 days or more, 60 days or more, 90 days or more, or longer. In some embodiments, the dose is administered for 365 days or less, 200 days or less, 90 days or less, 60 days or less, 45 days or less, 30 days or less, 21 days or less, 14 days or less, 10 days or less, 7 days or less, 3 days or less, 24 hours or less, 12 hours or less, or less. Combinations of these ranges are possible. For example, in some embodiments, the dose is administered for 1 hour or more and 365 days or less.

[0115] 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, cat, horse, rabbit, cow, pig, sheep, goat, rat (e.g., Rattus Norvegicus), mouse (e.g., Mus musculus), guinea pig, non-human primate (e.g., monkey, chimpanzee, baboon, ape, gorilla, etc.), etc.

[0116] Administration of the compositions of the present disclosure can be accomplished by any of a variety of medically acceptable methods that allow the composition to reach its target cells. The particular mode selected can of course depend on factors such as those described above, such as the particular composition, the severity of the condition of the subject being treated, the dosage required for therapeutic effect, etc. As used herein, a "medically acceptable" therapeutic mode is one that can produce an effective level 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, the medically acceptable mode may be administration via cerebrospinal fluid, i.e., intrathecal administration.

[0117] The composition may be administered to a subject by any of a variety of medically acceptable methods. Administration may be localized (e.g., to a specific area, physiological system, tissue, organ, or cell type) or systemic, depending on the condition to be treated. For example, the composition may be administered orally, vaginally, rectally, buccal, pulmonary, topically, nasally, transdermally, by parenteral injection or implantation, via surgical administration, or by any other administration method that achieves access to the tumor. In some cases, for example, when more than one composition is administered, more than one administration method may be used.

[0118] 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 can be advantageous due to convenience for the subject as well as administration schedule. Compositions suitable for oral administration can be administered using separate units such as hard or soft capsules, pills, cachets, tablets, troches, dissolving films, or lozenges. Other oral compositions suitable for use include liquids or suspensions such as syrups, elixirs, or emulsions. Liquids or suspensions can include aqueous or non-aqueous liquids. In some embodiments, the composition can be used to fortify food or beverages.

[0119] When it is desired to deliver the composition systemically, the composition can be formulated for parenteral administration by injection, for example, bolus injection or continuous infusion.The preparation for injection can be provided in a unit dosage form, for example, in ampoules or multi-dose containers, with the addition of preservatives.The composition can take the form of a suspension, solution, or emulsion in an oily or aqueous medium, and can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents.

[0120] Pharmaceutical preparations for parenteral administration may include aqueous solutions of the active compounds in water-soluble form. Suspensions of the active compounds may in some cases be prepared as appropriate oily injection suspensions. 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 include substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. The suspension may include suitable stabilizers. In some embodiments, the suspension includes agents that increase the solubility of the compound. Stabilizers or solubility enhancers may allow the preparation of highly concentrated solutions.

[0121] The composition may in some embodiments be formulated as a depot preparation. Such long-acting preparations may in some cases be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as poorly soluble derivatives, e.g., as poorly soluble salts.

[0122] The composition may be prepared in the form of a liquid pharmaceutical preparation. In some embodiments, the liquid pharmaceutical preparation comprises a solution, a suspension, or an emulsion. The composition may be included in the liquid pharmaceutical preparation in any of a variety of forms. For example, the composition may be included in an aqueous solution or saline solution (e.g., suitable for inhalation), may be microencapsulated, may be encochleated, may be coated on fine gold particles, may be contained in liposomes, may be nebulized, may be aerosolized, or may be dried on a sharp object and scraped into the skin.

[0123] The composition may be prepared in the form of a solid pharmaceutical preparation. For example, the composition may be included in granules; powders; tablets; coated tablets; (micro)capsules; suppositories; syrups; emulsions; suspensions; creams; or drops or preparations with extended release of the active compound. In some embodiments, the composition is prepared in the form of a solid pharmaceutical preparation that is configured to be dissolved or otherwise converted into a pharmaceutical liquid preparation. For example, the composition may be prepared in the form of a dry powder that 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 additives include disintegrants, binders, coating agents, swelling agents, lubricants, flavors, sweeteners, or solubilizers, although any of a variety of additives may be used, and the disclosure is not so limited.

[0124] In some embodiments, the composition can be administered to provide continuous exposure to the composition over a period of time, for example, hours, days, weeks, months, or years.

[0125] For example, the composition can be administered by repeated administration or controlled release.For example, the composition can be controlled release over a period of time so as to maintain the dosage of the composition during that period.Controlled release can be achieved, for example, by oral dosage form, bolus injection, transdermal patch, subcutaneous implant, or other methods as described herein.In some cases, it may be desirable to maintain a substantially constant concentration of the composition.

[0126] Other delivery systems suitable for use in certain embodiments may include time-release, delayed release, sustained release, or controlled release delivery systems. Such systems can advantageously avoid the need for repeated administration and increase the convenience of the subject. Many types of delivery systems are available and known to those skilled in the art. The composition can be administered using delivery systems such as, for example, polymer-based systems such as polylactic acid and / or polyglycolic acid, polyanhydrides, polycaprolactones, and / or combinations thereof; lipid-based non-polymeric systems including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di-, and triglycerides; hydrogel release systems; liposome-based systems; phospholipid-based systems; silastic systems; peptide-based systems; wax coatings; compressed tablets with conventional binders and excipients; or partially fused implants. Specific examples include, but are not limited to, erosion systems in which the composition is contained in a form within a matrix, or diffusion systems in which the active ingredient controls the release rate. The formulation may be present, for example, as microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, or polymeric systems. In some embodiments, the system may allow for sustained or controlled release of the composition, for example, through control of the diffusion or erosion / degradation rate of the formulation. Additionally, in some embodiments, a pump-based hardware delivery system may be used.

[0127] In some embodiments, the composition is delivered using an implant. The implant can be configured for short-term or long-term release of the composition. In some embodiments, the delivery system is a long-term release implant. The use of a long-term release implant can be particularly suitable in some embodiments. As used herein, "long-term release" means that the implant containing the composition described herein is constructed and arranged to deliver therapeutically effective levels. In some embodiments, the composition is delivered at therapeutically effective levels for 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 12 hours or more, 24 hours or more, 3 days or more, 7 days or more, 10 days or more, 14 days or more, 21 days or more, 30 days or more, 45 days or more, 60 days or more, 90 days or more, or longer. In some embodiments, the composition is delivered at therapeutically effective levels for 365 days or less, 200 days or less, 90 days or less, 60 days or less, 45 days or less, 30 days or less, 21 days or less, 14 days or less, 10 days or less, 7 days or less, 3 days or less, 24 hours or less, 12 hours or less, or less. Combinations of these ranges are possible. For example, in some embodiments, the composition is delivered at therapeutically effective levels for a period of at least one hour and up to 365 days. Long-term release implants are well known to those skilled in the art and include some of the release systems described herein.

[0128] In certain embodiments, the composition can be combined with a suitable pharma- ceutically acceptable carrier. In general, pharma- ceutically acceptable carriers suitable for use are well known to those skilled in the art. As used herein, "pharma- ceutically acceptable carrier" refers to a non-toxic substance that does not significantly inhibit the effectiveness of the biological activity of the active compound(s) administered, but is used as a formulation component, for example, to stabilize or protect the active compound(s) in the composition prior to use. For example, the composition can be incorporated into liposomes, polymeric release systems, suspended in liquid (e.g., in dissolved or colloidal form), or used in other ways as described herein. The carrier can include one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other vertebrates. The term "carrier" refers to an organic or inorganic component, which may be natural or synthetic, with which one or more active compounds of the present disclosure are combined to facilitate application. The carrier can be mixed or otherwise mixed with one or more compositions described herein, and / or with each other, in a manner such that there is no interaction that would substantially impair the desired medicinal effect. Carrier can be either soluble or insoluble depending on the application.Well-known examples of carrier include, but are not limited to, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite.Carrier can be either soluble or insoluble in nature.

[0129] In some embodiments, the formulations described herein can be administered in pharmaceutically acceptable solution.Pharmaceutically acceptable solution can routinely contain pharmaceutically acceptable concentrations of salt, buffer, preservative, compatible carrier, adjuvant, emulsifier, diluent, excipient, chelating agent, filler, desiccant, antioxidant, antibacterial agent, preservative, binder, bulking agent, silica, solubilizer, stabilizer, and other therapeutic ingredients that can be used with the composition optionally.For example, when the formulation is liquid, carrier can be solvent, partial solvent, or non-solvent, and can be aqueous 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 algenic acid; binding agents such as starch, gelatin, or acacia; lubricating agents such as magnesium stearate, stearic acid, or talc; time delay materials such as glycerol 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 phosphatides; thickening agents such as cetyl alcohol or beeswax; buffering agents 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 may be formulated into solid, semi-solid, liquid or gaseous form preparations such as tablets, capsules, elixirs, powders, granules, ointments, solutions, deposits, inhalants or injectables.

[0130] Suitable buffering agents include, but are not limited to: acetic acid and salts (1-2% w / v), citric acid and salts (1-3% w / v), boric acid and salts (0.5-2.5% w / v), and phosphoric acid and salts (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).

[0131] The preparation may include a solution, suspension, or emulsion as described above. The solution, suspension, or emulsion may be sterile. In some embodiments, the solution, suspension, or emulsion is aqueous. In some embodiments, the solution, suspension, or emulsion is non-aqueous. The sterile aqueous or non-aqueous solution, suspension, or emulsion may be isotonic with the blood of the subject in certain embodiments. Non-limiting examples of non-aqueous solvents are polypropylene glycol, polyethylene glycol, vegetable oils such as olive oil, sesame oil, coconut oil, arachis oil, peanut oil, mineral oil, injectable organic esters such as ethyl oleate, or fixed oils including synthetic mono- or diglycerides. The solution, suspension, or emulsion may include an aqueous carrier. Exemplary aqueous carriers that may be used include, but are not limited to, water, alcoholic / aqueous solutions, emulsions or suspensions including saline and buffered media. The preparation may further comprise a parenteral vehicle configured to improve the suitability of the solution, suspension, or emulsion 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 replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. In some embodiments, preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0132] In some embodiments, the compositions described herein can be associated or contacted with a suitable carrier. A suitable carrier can include one or more accessory ingredients. The preparation can be prepared by any of a variety of suitable techniques. For example, the preparation can be prepared by uniformly and intimately associating the composition with a liquid carrier, a finely divided solid carrier, or both. In some embodiments, the resulting preparation can be shaped (e.g., into a tablet, pellet, etc.).

[0133] The compositions discussed herein may be administered neat or in the form of a pharma- ceutically acceptable salt. Although non-pharma-ceutically acceptable salts may be used to prepare the compositions, when used in medicine, the salts should be pharma-ceutically acceptable. The term "pharma-ceutically acceptable salts" includes salts of the compositions described herein, which are prepared, for example, in combination with an acid or base. Pharmaceutically acceptable salts can be prepared as alkali metal salts, such as lithium, sodium, or potassium salts; or alkaline earth metal salts, such as magnesium or calcium salts. Examples of suitable bases that can be used to form salts include ammonium or suitable bases, such as sodium hydroxide, lithium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, etc. 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, phosphoric acid, etc. 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-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, glucuronic acid, galacturonic acid, salicylic acid, formic acid, naphthalene-2-sulfonic acid, and the like. Still other suitable acids include amino acids such as alginic acid, aspartic acid, glutamic acid, and the like. Such salts can also be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium, or calcium salts of the carboxylic acid group.

[0134] In another aspect, the present disclosure also provides any of the above-mentioned compositions in a kit, which optionally includes instructions for using the composition for treating a disease or condition (e.g., by targeting senescent cells, adipocytes, cancer cells, or fibroblasts). In some cases, the kit may include instructions for using the composition as discussed herein. The kit may also include instructions for using a combination of two or more compositions. Instructions for use may also be provided for administering the composition by any suitable technique as described above, for example, forally, intravenously, via a pump or implantable delivery device, or via another known route of drug delivery.

[0135] The kits described herein can also include one or more containers. The containers can contain compositions and other components as described above. The kits can also, in some cases, include instructions for mixing, diluting, and / or administering the compositions of the present disclosure. The kits can also include other containers that contain one or more solvents, surfactants, preservatives, and / or diluents (e.g., normal saline (0.9% NaCl), or 5% dextrose), as well as containers for mixing, diluting, or administering the components in the sample to a subject in need of such treatment.

[0136] The composition of the kit may be provided in any suitable form, for example, as a liquid or solid pharmaceutical preparation. If the composition provided is a dry powder, the composition may be reconstituted by adding a suitable solvent, which may also be provided in some cases. In embodiments in which a liquid form of the composition is used, the liquid form may be concentrated or ready to use. The solvent depends on the composition and the mode of use or administration. Solvents suitable for pharmaceutical compositions are well known and available in the literature, for example as described above. The solvent depends on the composition and the mode of use or administration.

[0137] In yet other aspects, the disclosure includes promoting one or more of the above-mentioned embodiments, e.g., promoting the treatment or prevention of a tumor in vitro or in vivo, e.g., by administering a composition as described herein to a subject.

[0138] The treatments disclosed herein may be administered to any subject, e.g., a human, or a non-human animal, such as a dog, cat, horse, rabbit, cow, pig, sheep, goat, rat (e.g., Rattus Norvegicus), mouse (e.g., Mus musculus), guinea pig, or non-human primate (e.g., monkey, chimpanzee, baboon, ape, gorilla, etc.).

[0139] The antibodies described herein may include biopolymers such as polypeptides. For example, the antibodies may include proteins. In some embodiments, the antibodies are glycoproteins. However, in some embodiments, the antibodies that include proteins are glycoproteins. The antibodies may be substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Any of a variety of immunoglobulin genes or fragments thereof are known to those of skill in the art. For example, the antibodies may be substantially encoded by immunoglobulin genes, including kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes and fragments thereof. The antibodies may be encoded by light chain immunoglobulin genes or fragments thereof. Light chain immunoglobulins may be classified as either kappa or lambda.

[0140] Antibodies may be encoded by heavy chain immunoglobulin genes or fragments thereof. Heavy chain immunoglobulins can be classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.

[0141] An immunoglobulin structural unit may comprise a tetramer. Each tetramer may be composed of two pairs of identical polypeptide chains, each pair having one light and one heavy chain.

[0142] In some embodiments, the light chain has a molecular weight of 15 kDa or more, 20 kDa or more, 25 kDa or more, or more. In some embodiments, the light chain has a molecular weight of 35 kDa or less, 30 kDa or less, 25 kDa or less, or less. Combinations of these ranges are possible. For example, in some embodiments, the light chain has a molecular weight of 15 kDa or more and 35 kDa or less.

[0143] In some embodiments, the heavy chain has a molecular weight of 50 kDa or more, 55 kDa or more, 60 kDa or more, or more. In some embodiments, the heavy chain has a molecular weight of 70 kDa or less, 65 kDa or less, 60 kDa or less, or less. Combinations of these ranges are possible. For example, in some embodiments, the heavy chain has a molecular weight of 50 kDa or more and 70 kDa or less.

[0144] The N-terminus of each polypeptide chain may 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 95 or more, 98 or more, 100 or more, 103 or more, 105 or more, or more amino acids. In some embodiments, the variable region comprises 115 or less, 113 or less, 110 or less, 108 or less, 105 or less, or fewer amino acids. Combinations of these ranges are also possible. For example, in some embodiments, the variable region comprises 95 or more and 115 or less amino acids.

[0145] Antibodies may exist as intact immunoglobulins, however, in some embodiments, antibodies exist as any of a number of immunoglobulin fragments.

[0146] Immunoglobulin fragments may be produced by digestion with any of a variety of peptidases (e.g., pepsin). For example, in some embodiments, immunoglobulin fragments may be formed by digesting an antibody with pepsin. In some exemplary embodiments, pepsin is used to digest the Fc domain of an antibody, for example by breaking down disulfide bonds in the hinge region of the Fc domain, to generate F(ab)'2. F(ab)'2 is, according to certain embodiments, a dimer of Fab, which is itself a light chain linked to VH-CH1 by a disulfide bond. F(ab)'2 may be reduced to break the disulfide bond in the hinge region, thereby converting the (Fab') dimer into a Fab' monomer. According to some embodiments, the Fab' monomer comprises a Fab and a portion of the hinge region of the Fc domain.

[0147] In some embodiments, immunoglobulin fragments are synthesized de novo. Immunoglobulin fragments can be produced by any of a variety of methods known to those of skill in the art, such as chemical synthesis, using recombinant DNA methodology, or "phage display" methods.

[0148] Exemplary antibodies include single chain antibodies, such as single chain Fv (scFv) antibodies in which a variable heavy chain and a variable light chain are linked (directly or via a peptide linker) to form a contiguous polypeptide, hi one embodiment, the antibody is a monoclonal antibody.

[0149] In some embodiments, compositions comprising an antibody or antibody fragment are configured such that the antibody or antibody fragment retains a relatively high affinity for a target molecule. Without wishing to be bound by theory, the affinity of an antibody or antibody fragment for a target is determined by the dissociation constant (K D ) may be inversely proportional to K D The lower the value, the higher the affinity. In some embodiments, the K D Values ​​are 10 under physiological conditions. -6 M or less, 10 -7M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 In some embodiments, the K of the antibody or antibody fragment is equal to or less than M. D The value is 10 -13 M or above, 10 -12 M or above, 10 -11 M or higher. Combinations of these ranges are possible. For example, in some embodiments, the K D The value is 10 -12 M or more 10 -6 M or less. D The value can be determined by tests that would be well known to one skilled in the art.

[0150] In some embodiments, the K of the composition D is the K of a pure antibody or antibody fragment similar to the antibody or antibody fragment of the composition. D This relatively high affinity of the composition for its target may be related to the fact that the composition is a fusion protein.

[0151] In this context, the term "antibody fragment" can refer to at least a portion of an antibody that retains the ability to specifically interact (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution) with an epitope of an antigen. Examples of antibody fragments are described elsewhere herein and include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFvs), Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies (either VL or VH) such as sdAbs, camelid VHH domains, multispecific antibodies formed from antibody fragments such as bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, and any antibody fragment comprising an isolated CDR or other epitope-binding fragment of an antibody. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs.

[0152] In some embodiments, the antibody may be a minibody or nanobody. In the context of the present disclosure, it is recognized by the present invention that the use of a minibody or nanobody may advantageously increase the binding affinity of the antibody compared to a full-sized antibody.

[0153] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure. EXAMPLES

[0154] [Example 1] In this 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 this example, the antibody is a commercially available antibody, e.g., a senescent cell-recognizing antibody or a monoclonal antibody.

[0155] The enzyme causes lysosomal leakage, which leads to the production of antigens. In this example, the enzyme is an oxidase that produces reactive oxygen species, which damages or destroys lysosomes, causing their leakage. The oxidase produces superoxide, an exemplary reactive oxygen species. In this example, the oxidase is xanthine oxidase.

[0156] Xanthine oxidase is covalently linked to the antibody and administered intravenously or intramuscularly to the subject. Upon reaching the composition's target cells, the enzyme reacts with lysosomes, causing damage or destruction, allowing the production of antigens, which boosts the subject's immune response. In this way, the subject's immune system can more effectively target the subject's senescent cells. [Example 2]

[0157] In this prophetic example, a composition is provided to a human subject with NASH, comprising an antibody that recognizes senescent cells and an enzyme. In some cases, the antibody is commercially available, e.g., a senescent cell-recognizing antibody or a monoclonal antibody.

[0158] The enzyme causes lysosomal leakage, which leads to the production of antigens. In this example, the enzyme is an oxidase that produces reactive oxygen species, which damages or destroys lysosomes, causing their leakage. The oxidase produces superoxide, an exemplary reactive oxygen species. In this example, the oxidase is xanthine oxidase.

[0159] Xanthine oxidase is covalently linked to the antibody and administered intravenously or intramuscularly to the subject. Upon reaching the composition's target cells, the enzyme reacts with lysosomes, causing damage or destruction, allowing the production of antigens, which boosts the subject's immune response. In this way, the subject's immune system can more effectively target the subject's senescent cells. [Example 3]

[0160] In this prophetic example, a human subject with Alzheimer's disease is provided with a composition comprising an antibody that recognizes senescent cells and an enzyme, in some cases, the antibody is commercially available, e.g., a senescent cell-recognizing antibody or a monoclonal antibody.

[0161] The enzyme is one that causes lysosomal leakage, which leads to the production of antigens. In this example, the enzyme is an oxidase that produces reactive oxygen species, which damages or destroys lysosomes, causing them to leak. The oxidase can generate reactive oxygen species, such as superoxide. In this example, the oxidase is xanthine oxidase.

[0162] Xanthine oxidase is covalently linked to the antibody and administered intravenously or intramuscularly to the subject. Upon reaching the composition's target cells, the enzyme reacts with lysosomes, causing damage or destruction, allowing the production of antigens, which boosts the subject's immune response. In this way, the subject's immune system can more effectively target the subject's senescent cells. [Example 4]

[0163] In this prophetic example, a human subject is treated as in Example 1, but in addition, the subject is provided with a therapeutic agent that removes and / or inhibits antioxidants that affect the action of the composition. In one example, the subject is provided with an unsaturated fatty acid, such as CH2=CHCOOH. The fatty acid acts to inhibit the action of the antioxidants, resulting in a greater response to the composition. [Example 5]

[0164] In this prophetic example, human subjects are treated similarly to Example 1, but in addition, the subjects are exposed to a statin, such as atorvastatin, which acts to inhibit the action of the antioxidants and may result in a greater response to the composition. [Example 6]

[0165] In this prophetic example, a composition comprising a senescent cell-recognizing antibody and a xanthine oxidase enzyme is prepared using an amino-amino crosslinker. Senescent cells recognized by the antibody are cultured in 96-well plates and treated with the conjugate at an appropriate concentration between 10 micrograms / ml and 100 micrograms / mL. Hypoxanthine, an exemplary enzyme substrate for xanthine oxidase, was provided to the cell culture medium at a concentration of 1 mM. Markers of immunogenic cell death (ICD), including calreticulin exposure, extracellular ATP formation, and HMGB1 formation, were assayed 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, and 24 hours after hypoxanthine provision. A graph of the results is shown in Figure 3. Calreticulin is appropriately formed early, followed by ATP formation, followed by HMGB1 formation. This establishes that cells produce markers of ICD and are expected to form neoantigens that activate T cells and induce senescent cells in the process of dying. [Example 7]

[0166] Once senescent cells are killed in the process of inducing ICD, the subject bearing those cells becomes "immunized" against such cells, and ICD therefore protects the subject from senescent cells in the future.

[0167] To demonstrate this, in this prophetic example, senescent cell fragments killed in Example 2 are harvested and injected into the left flank of BALB / c mice. Eight days after injection, live senescent cells are injected into the right flank of each mouse. Mice are monitored for senescent cells in the right flank for 120 days. Mice treated with ICD dead cells are compared to mice injected with saline. At day 120, 92% of mice injected with ICD dead cells were senescent cell-free, whereas only 7% of mice injected with saline were senescent cell-free. [Example 8]

[0168] In this prophetic example, an 80 year old human subject with a BMI of 55 is provided with a composition comprising an antibody that recognizes oversized adipocytes and an enzyme. In some cases, the antibody is commercially available, e.g., an adipocyte-recognizing antibody or a monoclonal antibody.

[0169] The enzyme causes lysosomal leakage, which leads to the production of antigens. In this example, the enzyme is an oxidase that generates reactive oxygen species, which damages or destroys lysosomes, causing their leakage. The oxidase generates superoxide, an exemplary reactive oxygen species. In this example, the oxidase is xanthine oxidase.

[0170] Xanthine oxidase is covalently attached to the antibody and administered intravenously or intramuscularly to the subject. Upon reaching the composition's target cells, the enzyme reacts with lysosomes, causing damage or destruction, allowing for the production of antigens, which boost the subject's immune response. In this way, the subject's immune system can more effectively target the subject's oversized fat cells. After three months of treatment, the subject's BMI was reduced to 37. [Example 9]

[0171] In this prophetic example, a conjugate between an oversized adipocyte-recognizing antibody and the enzyme xanthine oxidase is prepared using an amino-amino crosslinker. Oversized adipocytes recognized by the antibody are cultured in 96-well plates and treated with the conjugate at an appropriate concentration between 10 micrograms / ml and 100 micrograms / mL. Hypoxanthine is provided in the cell culture medium at a concentration of 1 mM. Markers of immunogenic cell death (ICD), including calreticulin exposure, extracellular ATP formation, and HMGB1 formation, are assayed at 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, and 24 hours. A graph of the results is shown in Figure 3. Appropriately, calreticulin is formed early, followed by ATP formation, and then HMGB1 formation. This establishes that cells produce markers of ICD and, in the process of dying, are expected to form neoantigens to activate T cells and direct them to oversized adipocytes. [Example 10]

[0172] In this prophetic example, an 80 year old human subject with respiratory distress is provided with a composition comprising an antibody that recognizes pulmonary fibroblasts and an enzyme. In some cases, the antibody is commercially available, e.g., a fibroblast-recognizing antibody or a monoclonal antibody.

[0173] The enzyme causes lysosomal leakage, which leads to the production of antigens. In this example, the enzyme is an oxidase that produces reactive oxygen species, which damage or destroy the lysosomes, causing them to leak. The oxidase produces superoxide, an exemplary reactive oxygen species. In this example, the oxidase is xanthine oxidase.

[0174] Xanthine oxidase is covalently linked to the antibody and administered intravenously or intramuscularly to the subject. Upon reaching the composition's target cells, the enzyme reacts with lysosomes, causing damage or destruction, allowing the production of antigens, which boosts the subject's immune response. In this way, the subject's immune system can more effectively target the subject's fibroblasts. After treatment, the subject is more able to breathe. [Example 11]

[0175] Once fibroblasts are killed in the process of inducing ICD, animals with fibrotic lungs become "immunized" against such cells, and ICD thus protects the animals from future pulmonary fibrosis.

[0176] To demonstrate this, in this prophetic example, fibroblast fragments killed in Example 2 are harvested and injected into the left flank of BALB / c mice. Eight days after injection, live fibroblasts are injected into the right flank of each mouse. The mice are monitored for pulmonary fibrosis for 120 days. Mice treated with ICD dead cells are compared to mice injected with saline. After 120 days, 92% of the mice injected with ICD dead cells are free of pulmonary fibroblasts, compared to only 7% of the mice injected with saline. [Example 12]

[0177] In this prophetic example, an 80 year old human subject with congestive heart failure is provided with a composition comprising an antibody that recognizes cardiac fibroblasts and an enzyme. In some cases, the antibody is commercially available, e.g., an antibody that recognizes fibroblasts or a monoclonal antibody.

[0178] The enzyme is what causes lysosomal leakage, resulting in the production of antigens. In this example, the enzyme is an oxidase that produces reactive oxygen species, which damage or destroy lysosomes, causing their leakage. The oxidase produces superoxide, an exemplary reactive oxygen species. In this example, the oxidase is xanthine oxidase.

[0179] Xanthine oxidase is covalently bound to the antibody and administered intravenously or intramuscularly to the subject. Upon reaching the composition's target cells, the enzyme reacts with lysosomes, causing damage or destruction, allowing the production of antigens, which boosts the subject's immune response. In this way, the subject's immune system can more effectively target the subject's fibroblasts. After treatment, the subject's discharge volume increases significantly. [Example 13]

[0180] In this prophetic example, a conjugate between a fibroblast-recognizing antibody and the enzyme xanthine oxidase is prepared using an amino-amino crosslinker. Fibroblasts recognized by the antibody are cultured in 96-well plates and treated with the conjugate at an appropriate concentration between 10 micrograms / ml and 100 micrograms / mL. Hypoxanthine is provided in the cell culture medium at a concentration of 1 mM. Markers of immunogenic cell death (ICD), including calreticulin exposure, extracellular ATP formation, and HMGB1 formation, are assayed at 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, and 24 hours. Calreticulin is formed appropriately early, followed by ATP formation, followed by HMGB1 formation. This establishes that cells produce markers of ICD and, in the process of dying, are expected to form neoantigens to activate and direct T cells to fibroblasts. [Example 14]

[0181] This example describes the generation and analysis of two exemplary compositions comprising a xanthine oxidase enzyme fragment with a C-terminal truncation ("XO"; SEQ ID NO:2) linked to an epidermal growth factor receptor nanobody ("VHH 9G8"; SEQ ID NO:1) suitable for targeting cancer cells. The compositions were linked in the form of a fusion protein. The fusion protein also includes a linker region between XO and VHH 9G8, the linker region having the amino acid sequence GGGGSGGGGS (SEQ ID NO:3). In one of the exemplary compositions, the xanthine oxidase was located at the N-terminus of the nanobody ("XO-VHH 9G8"; SEQ ID NO:5). In the other exemplary composition, the xanthine oxidase was 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 experiments. Table 1 shows the amino acid sequences associated with each of these species.

[0182] [Table 1] TIFF2025509986000003.tif75153

[0183] Each fusion protein was prepared by cloning into the pCZN4b expression vector and amplifying in TOP10 E. coli. HEK293 cells were cultured and harvested, and the supernatants were collected - each fusion protein was purified from the supernatant by a process of column purification (Ni-NTA column) with a wash buffer of 20 mM imidazole, 50 mM Tris, 300 mM NaCl, pH 8 (1 mL / min). The fusion protein was then eluted from the column with an elution buffer of 200 mM imidazole, 50 mM Tris, 300 mM NaCl, pH 8 at 1 mL / min. The fusion protein was dialyzed overnight against PBS and the protein concentration was determined using a BCA assay. Each fusion protein and control was His-tagged.

[0184] Figure 2 shows the binding of fusion protein 202 to epidermal growth factor receptor 205 ("EGFR"). In Figure 2, fusion protein 202 is VHH 9G8-XO, which comprises nanobody 203 (VHH 9G8) and enzyme fragment 201 (XO) linked by linker 207 (GGGGSGGGGS).

[0185] Table 1 shows the dissociation constants (K ) associated with the binding of each fusion protein and the VHH9G8 control to EGFR, as determined by surface plasmon resonance (SPR). D ), and the association rate constant (k a ) and the dissociation rate constant (k d ) reported. 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 / min. The results showed that the exemplary fusion proteins (XO-VHH 9G8 and VHH 9G8-XO) bound nearly as well as the nanobody control (VHH 9G8). These results demonstrate the utility of the compositions described herein and demonstrate the surprising result that compositions including fusion proteins containing nanobodies such as XO-VHH 9G8 and VHH 9G8-XO can have unexpectedly high affinities, comparable to those of pure antibodies.

[0186] [Table 2]

[0187] Although several embodiments of the present disclosure have been described and illustrated herein, those skilled in the art can readily envision various other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and each such variation and / or modification is considered to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular use or application in which the teachings of the present disclosure are used. Those skilled in the art will recognize, or be able to ascertain with no more than routine experimentation, many equivalents to the specific embodiments of the present disclosure described herein. Thus, the foregoing embodiments are presented by way of example only, and it will be understood that within the scope of the appended claims and their equivalents, the present disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods is included within the scope of the present disclosure, if such features, systems, articles, materials, and / or methods are not mutually inconsistent.

[0188] The indefinite articles "a" and "an," as used herein in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0189] The term "and / or" as used herein and in the claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctive in some cases and disjunctive in other cases. Other elements, whether related or unrelated to those elements specifically identified, may be present as appropriate other than the elements specifically identified by the "and / or" clause, unless expressly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and / or B," when used in combination with open-ended language such as "comprising," may refer in one embodiment to A without B (which may include elements other than B); in another embodiment to B without A (which may include elements other than A); in yet another embodiment to both A and B (which may include other elements); and so forth.

[0190] As used herein and in the claims, "or" should 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 interpreted as inclusive, i.e., including at least one of the elements of the number or list, but also including more than one, and may further include items not in the list. Only terms clearly indicating the contrary, such as "only one of," or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of the number or list. In general, the term "or" as used herein shall be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") only when preceded by an exclusive term, such as "either," "one of," "only one of," or "exactly one of." As used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0191] As used herein and in the claims, the phrase "at least one" referring to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that elements other than those specifically identified in the list of elements to which the phrase "at least one" refers may be present as appropriate, whether or not related to those elements specifically identified. 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 A, which may include at least one, and may include more than one, with B absent (and which may include elements other than B); in another embodiment to B, which may include at least one, and may include more than one, with A absent (and which may include elements other than A); in yet another embodiment to A, which may include at least one, and may include more than one, and B, which may include at least one, and may include more than one (and may include other elements); etc.

[0192] As used herein, "wt%" is an abbreviation for weight percentage. As used herein, "at%" is an abbreviation for atomic percentage.

[0193] Some embodiments may be embodied as methods, various examples of which are described. The acts performed as part of the methods may be ordered in any suitable manner. Thus, embodiments may be configured in such a way that acts are performed in an order different from that shown, may include different (e.g., more or less) acts than those described, and / or may include performing some acts simultaneously, even though the acts have been shown as being performed sequentially in the specifically described embodiments above.

[0194] In the claims, the use of ordinal terms such as "first," "second," "third," etc. to modify claim elements does not, in itself, indicate any priority, precedence, or order of one claim element relative to another claim element, or the temporal order in which acts of a method are performed, but is merely used as a label to distinguish one claim element having a particular name from another element having the same name (but which uses ordinal terms).

[0195] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like, are understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as provided in the United States Patent Office Manual of Patent Examining Procedure, Section 211.03.

Claims

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

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

3. 20. A composition or method according to any preceding claim, wherein the enzyme is configured to generate reactive oxygen species.

4. 20. A composition or method according to any one of the preceding claims, wherein the enzyme is an oxidase or a peroxidase.

5. 20. A composition or method according to any one of the preceding claims, wherein the enzyme is an oxidase.

6. 2. A composition or method according to any one of the preceding claims, wherein the oxidase is glucose oxidase.

7. 2. A composition or method according to any one of the preceding claims, wherein the oxidase is xanthine oxidase.

8. 20. A composition or method according to any one of the preceding claims, wherein the enzyme is a peroxidase.

9. 20. The composition or method of any one of the preceding claims, wherein at least a portion of the antibody is a complete antibody.

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

11. 2. A composition or method according to any preceding claim, wherein the antibody recognises a cell.

12. 2. A composition or method according to any preceding claim, wherein the antibody recognises a virus.

13. A composition or method according to any one of the preceding claims, wherein the antibody recognises an extracellular substance.

14. 20. A composition or method according to any one of the preceding claims, wherein the disease or condition is a disease, and the disease is atherosclerosis or obesity.

15. 20. A composition or method according to any one of the preceding claims, wherein the disease or condition is a condition of aging.

16. 2. A composition or method according to any one of the preceding claims, wherein the antibody recognises a cell, and the cell is a senescent cell or an adipocyte.

17. 2. A composition or method according to any one of the preceding claims, wherein the antibody recognises extracellular material, and the extracellular material is plaque.

18. 2. A composition or method according to any one of the preceding claims, wherein the antibody recognises a virus, the virus being a coronavirus or HIV.

19. 2. A composition or method according to any one of the preceding claims, wherein the antibody is a nanobody.

20. The antibody of the composition is －６ K less than M Ｄ 4. A composition or method according to any preceding claim, which recognises cells, viruses or extracellular substances at a specific value.

21. The antibody of the composition is －７ K less than M Ｄ 4. A composition or method according to any preceding claim, which recognises cells, viruses or extracellular substances at a specific value.

22. The antibody of the composition has a K Ｄ K smaller than the value Ｄ 4. A composition or method according to any preceding claim, which recognises cells, viruses or extracellular substances at a specific value.

23. Antibodies that recognize senescent cells, adipocytes, cardiac fibroblasts, or pulmonary fibroblasts, and Enzymes configured to increase lysosomal membrane permeability in recognized senescent cells, adipocytes, cardiac fibroblasts, or pulmonary fibroblasts A composition comprising:

24. Antibodies that recognize senescent cells, adipocytes, cardiac fibroblasts, or pulmonary fibroblasts, and enzymes capable of generating reactive oxygen species A composition comprising:

25. 25. The composition of claim 23 or 24, wherein the enzyme is an oxidase.

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

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

28. 25. The composition of claim 23 or 24, wherein the enzyme is a peroxidase.

29. The composition according to any one of claims 23 to 28, wherein the antibody and the enzyme are directly covalently bound.

30. The composition according to any one of claims 23 to 29, wherein the antibody and the enzyme are linked via a linking agent.

31. administering to a subject a composition comprising an antibody that recognizes senescent cells, adipocytes, cardiac fibroblasts, or pulmonary fibroblasts, and an enzyme that can increase the lysosomal membrane permeability of the recognized senescent cells, adipocytes, cardiac fibroblasts, or pulmonary fibroblasts; A method comprising:

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

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

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

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

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

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

38. The method according to any one of claims 31 to 37, wherein the antibody and the enzyme are linked via a linking agent.

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

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

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

42. 42. The method according to any one of claims 39 to 41, wherein the administration of the composition and the administration of the fatty acid are carried out simultaneously.

43. 43. The method according to any one of claims 39 to 42, wherein the administration of the composition and the administration of the fatty acid are carried out sequentially.

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

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

46. 46. ​​The method of any one of claims 44 or 45, wherein administration of the composition and administration of the statin are performed simultaneously.

47. The method of any one of claims 45 to 46, wherein administration of the composition and administration of the statin are performed sequentially.

48. The method of any one of claims 39 to 47, wherein after administration of the composition, a substrate for the enzyme is administered.

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