Modified xanthine oxidase-antibody complexes and methods thereof

Abstract

Systems and methods for the use of improved enzyme-antibody complexes for treating cancer or other disease are generally provided. In some embodiments, the application is directed towards complexes comprising an enzyme such as xanthine oxidase or a xanthine oxidase derivative. Some of the enzymes of complexes provided herein have unexpectedly high therapeutic activity, e.g., as demonstrated by the uncharacteristically high portion of the enzyme that remains therapeutically active under physiological conditions.

Description

[0001] MODIFIED XANTHINE OXIDASE- ANTIBODY COMPLEXES AND METHODS THEREOF

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 550,708, filed February 7, 2024, entitled “Modified Xanthine Oxidase-Antibody Complexes and Methods Thereof,” by Fossel, et al., incorporated herein by reference in its entirety.

[0004] TECHNICAL FIELD

[0005] Complexes comprising antibodies and enzymes with high therapeutic potency are generally described.

[0006] BACKGROUND

[0007] Diseases and conditions such as cancer may be multifactorial in their pathogenesis. Treatments of these diseases or conditions are needed.

[0008] SUMMARY

[0009] Systems and methods for the use of improved enzyme-antibody complexes for treating cancer or other disease are generally provided. In some embodiments, the application is directed towards complexes comprising an enzyme such as xanthine oxidase or a xanthine oxidase derivative. Some of the enzymes of complexes provided herein have unexpectedly high therapeutic activity, e.g., as demonstrated by the uncharacteristically high portion of the enzyme that remains therapeutically active under physiological conditions.

[0010] The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0011] In one aspect, a composition is provided. According to some embodiments, the composition comprises: a complex comprising an antibody or antibody fragment, and an enzyme comprising at least one of Seq. ID. Nos. 11-18 and linked to the antibody or antibody fragment.

[0012] In another aspect, a method is provided. According to some embodiments, the method comprises administering, to a subject, a complex comprising: an antibody or antibody fragment, and an enzyme comprising at least one of Seq. ID. Nos. 11-18 and linked to the antibody or antibody fragments.

[0013] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale unless otherwise indicated. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

[0016] The Figure presents an exemplary mechanism for lysosome induced immunogenic cell death induced by reactive oxygen species, according to some embodiments.

[0017] DETAILED DESCRIPTION

[0018] The present disclosure provides improvements in antibody-enzyme (e.g., antibody-oxidase) complexes and associated treatment methods. Some naturally occurring oxidase enzymes (e.g., xanthine oxidase) are known to equilibrate with a therapeutically inactive form (e.g., xanthine hydrogenase). The present disclosure is directed, in some aspects, towards complexes between antibodies and oxidases that unexpectedly shift the active-inactive oxidase equilibrium in favor of active oxidase formation. The complexes provided herein may thus offer improved therapeutic potential, and may thus be used in a number of important disease treatment contexts such as cancer treatment, aging treatment, nonalcoholic steatohepatitis (NASH) treatment, neurodegenerative disease treatment (e.g., Alzheimer’s and Parkinson’s disease treatment), obesity treatment, pulmonary fibrosis treatment, and congestive heart failure treatment, among others.

[0019] As a specific, non-limiting example, discussed here for the sole purpose of providing a specific illustration, in some embodiments, the disclosure is directed towards one of Seq. ID. Nos. 1-5, as provided in Table 1. Each of Seq. ID. Nos. 1-5 is a fusion protein comprising an EGFR-binding antibody suitable for cancer targeting and fused to an oxidase such that the EGFR-binding antibody and the oxidase are fused by a polyamino acid linker having the sequence GGGGSGGGGS (Seq. ID. No. 32) or the sequence GGGSGGGGS (Seq. ID. No. 39). As discussed in greater detail in the description and examples below, each of Seq. ID. Nos. 1-5 preserves or improves upon the oxidase-dehydrogenase equilibrium reaction in native oxidase, with the result that each of Seq. ID. Nos. 1-5 is an anti-cancer therapeutic that provides unexpectedly good oxidase performance, and thus unexpectedly high potency.

[0020] Table 1. Amino acid sequences associated with various compositions described herein.

[0021] Of course, it should be understood that the disclosure is not limited to Seq. ID. Nos. 1-5, as the disclosure is not so limited.

[0022] In one aspect, the disclosure is directed towards a composition comprising a complex that comprises an enzyme. For example, the composition may comprise a complex comprising an enzyme and an antibody. In some embodiments, the enzyme is linked to the antibody. For example, the enzyme may be covalently linked to the antibody. In some cases, the antibody and enzyme may be expressed as a single fusion protein, as discussed in greater detail below.

[0023] The enzyme may be naturally occurring, a synthetically modified enzyme, or a fragment of any of these. In some embodiments, the enzyme is an oxidoreductase. For example, the enzyme may be oxidase in various embodiments. Oxidases, according to some embodiments, are enzymes that catalyze an oxidation-reduction reaction, including reactions involving oxygen (O2) as an electron acceptor. The oxygen may 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 mutants or derivatives thereof. Enzymes described herein may be recombinant enzymes, in some embodiments.

[0024] Without wishing to be bound by any particular theory, it is believed that certain enzymes (e.g., oxidases) create reactive oxygen species such as superoxides, peroxides, hydroxyl radicals, etc., by oxidizing a substrate associated with the enzyme. Accordingly, in some embodiments, a substrate of an enzyme may be optionally added (e.g., may be included with the composition, or may be administered to a subject to be treated with the composition) to facilitate such a reaction. In some embodiments, the reactive oxygen species are endocytosed by a targeted cell. The reactive oxygen species may interact with a membrane of a targeted cell. In some embodiments, oxidation of unsaturated lipids can cause their endocytosis. The Figure presents a cross-sectional schematic diagram of this process, in which reactive oxygen species 101 react with unsaturated lipids 102, creating oxidized unsaturated lipids 104 that are endocytosed into cell 103. Without wishing to be bound by theory, the interaction between the lysosome of the targeted cell and the oxidized unsaturated lipids may induce a lysosomal membrane of the cell to become more permeable. For example, in the Figure, oxidized unsaturated lipids 104 are endocytosed into cell 103, where lipids 104 are taken up by lysosome 105, causing lysosome membrane 107 to become permeable lysosome membrane 109. Thus, an enzyme may be configured to increase lysosomal membrane permeability of a cell, e.g., by creating reactive oxygen species. Changes in lysosomal membrane permeability may be detected one of ordinary skill using a galectin puncta assay.

[0025] Increased lysosomal membrane permeability may cause the cell to leak degradative enzymes. For example, referring again to the Figure, once lysosome membrane 107 becomes permeable lysosome membrane 109, degradative enzymes 111 can leak through permeable lysosome membrane 109. Destabilization of the cell’s lysosomal membrane may disperse lysosomal enzymes throughout the cell. The cell may then create cell-specific neoantigens that can be recognized by the immune system, and / or that may trigger apoptosis and / or cell death. This is presented in the Figure, where the leakage of degradative enzymes 111 creates degradation byproducts 117 and independently causes cell 103 to express neoantigens 113, signaling cell 103 for endocytosis by dendritic cell 115. Thus, reactive oxygen species may be useful in some embodiments to cause lysosome-induced immunogenic cell death (LIICD). Accordingly, it is believed that cells associated with diseases or conditions such as cancer may be targeted (e.g., with an antibody) for the delivery of enzymes that can kill the cells via administration of a composition as described herein.

[0026] According to some embodiments, a complex provided herein comprises an enzyme such as natural xanthine oxidase or a mutant or derivative thereof. In some embodiments, for example, the enzyme comprises at least one of Seq. ID. Nos. 11-18 shown in Table 2, below. For example, in some embodiments, the enzyme consists of at least one of Seq. ID. Nos. 11-18.

[0027] Table 2. Amino acid sequences associated with various enzymes described herein.

[0028] According to some embodiments, an oxidase (e.g., natural xanthine oxidase may react (e.g., via a reversable reaction) to produce a therapeutically inactive form of the oxidase. For example, natural xanthine oxidase may react to form natural xanthine dehydrogenase, a protein that does not catalyze formation of reactive oxygen species. Likewise, in some embodiments, a xanthine oxidase mutant or another xanthine oxidase derivative may react (e.g., via a reversable reaction) to form a xanthine dehydrogenase mutant or a xanthine dehydrogenase derivative that does not catalyze formation of reactive oxygen species and is thus therapeutically inactive. Thus, xanthine oxidase (or a derivative thereof) may equilibrate with xanthine dehydrogenase (or a derivative thereof) under at least some solution conditions. Without wishing to be bound by any particular theory, the xanthine oxidase to xanthine dehydrogenase transition is believed to be associated, in at least some cases, with reversable disulfide bond formation as well as reversable conformational changes in the protein associated with the disulfide bond formation. Generally, a complex comprising an enzyme provided herein may equilibrate under a set of physiological conditions to have any of a variety of suitable therapeutically-active:therapeutically-inactive enzyme ratios (e.g., xanthine oxidase: xanthine dehydrogenase ratios). In some embodiments, a complex comprising an enzyme provided herein equilibrates under a set of physiological conditions to have a therapeutically-active:therapeutically-inactive enzyme ratio of greater than or equal to 6:4, greater than or equal to 6.5:3.5, greater than or equal to 7:3, greater than or equal to 7.5:2.5, greater than or equal to 8:2, greater than or equal to 8.5:1.5, greater than or equal to 9:1, greater than or equal to 9.5:0.5, or greater. In some embodiments, a complex comprising an enzyme provided herein equilibrates under a set of physiological conditions to have a therapeutically-active:therapeutically-inactive enzyme ratio of less than or equal to 9.99:0.01, less than or equal to 9.5:5, less than or equal to 9:1, or less. In some embodiments, there may effectively be no therapeutically-active:therapeutically- inactive enzyme ratio, such that 100% of the enzyme is therapeutically active. Combinations of these ranges are possible. For example, in some embodiments, a complex comprising an enzyme provided herein equilibrates under a set of physiological conditions to have a therapeutically-active:therapeutically-inactive enzyme ratio of greater than or equal to 6:4 and less than or equal to 9.99:0.01. Other ranges are also possible. The therapeutically-active:therapeutically-inactive enzyme ratio of a complex comprising xanthine oxidase or a related enzyme may be determined, according to some embodiments, by an assay that determines the production of uric acid (a product of both the therapeutically-active and therapeutically-inactive forms of the xanthine oxidase or related enzyme) in the presence and absence of nicotinamide adenine dinucleotide (NAD+). Relative xanthine oxidase (or related enzyme) activity is uric acid production in the absence of NAD+, in some embodiments. Total relative activity is uric acid production in the presence of NAD+, in some embodiments. The activity of the therapeutically-inactive xanthine dehydrogenase activity is the total relative activity minus the relative xanthine oxidase activity, according to some embodiments. The assay may be performed by incubating the complex for 20 min in a reaction mix containing 50 uM hypoxanthine, 100 uM EDTA, and 50 mM potassium phosphate buffer. To test total relative activity, 500 uM NAD+ may be added to the reaction mix. To test the relative xanthine oxidase (or related enzyme) activity, NAD+ may be omitted from the reaction mix, so that oxygen is the sole electron acceptor available. After incubating equal volumes of the NAD+-included and NAD+-omitted reaction mixes for 20 min, the reaction mixes are added to a commercial uric acid detection kit (Sigma MAK007) and uric acid production is monitored by fluorescence according to the manufacturers instructions, according to some embodiments.

[0029] Although reversable interchange between xanthine dehydrogenase and xanthine oxidase is known, there is no reason to expect this interchange to impact the performance of xanthine oxidase complexes. Protein complexation is highly unpredictable. For example, without wishing to be bound by any particular theory, protein conformations that are commonplace in natural xanthine oxidase may be totally barred in xanthine oxidase complexes, e.g., as a result of steric hinderance arising from a species complexed with the xanthine oxidase. Accordingly, it is surprising that the xanthine oxidase to xanthine dehydrogenase transition has a meaningful impact on the clinical efficacy of xanthine oxidase complexes.

[0030] The present disclosure recognizes, for the first time, that the reaction of complexed xanthine oxidase and xanthine oxidase derivatives to form complexed xanthine dehydrogenase or xanthine dehydrogenase derivatives may reduce the therapeutic potency of at least some xanthine oxidase complexes. Moreover, the present disclosure provides, for the first time, xanthine oxidase complexes (e.g., xanthine oxidase fusion proteins) with lower propensities for interconversion to inactive forms than natural xanthine oxidase has, according to some embodiments.

[0031] Without wishing to be bound by any particular theory, the reaction of xanthine oxidase to form xanthine dehydrogenase may be associated with conformational changes proximate the flavin adenine dinucleotide (“FAD”) site, an active site associated with a structure of the protein called the “FAD cavity”. For example, without wishing to be bound by any particular theory, the formation of xanthine dehydrogenase is associated with insertion of C-terminal residues of xanthine oxidase into the FAD cavity, and / or with formation of a tight amino acid cluster connecting three sequentially separated portions of the protein: an adjacent Arg-Trp residue pair (e.g., Arg334-Trp335); an Arg residue (e.g., Arg426); and a Phe residue (e.g., Phe549). (The precise sequential position of the residues of the complex may vary, depending on the embodiment.)

[0032] In the context of these conformational changes, an unexpected result provided by the present disclosure is the fact that complexes comprising a fusion protein comprising xanthine oxidase or a xanthine oxidase derivative linked at its C-terminus to an antibody may, in some embodiments, exist in an equilibrium that preferentially favors the xanthine oxidase form of the complex over the xanthine dehydrogenase form of the complex. It is known that deletions of C-terminal portions of xanthine oxidase are associated with xanthine oxidase-xanthine dehydrogenase equilibria that favor the xanthine oxidase form of the protein, rather than the xanthine dehydrogenase form of the protein. Without wishing to be bound by any particular theory, this shift is believed to result from the reduced likelihood of the insertion of C-terminal residues of xanthine oxidase into the FAD cavity which, as described above, may favor the xanthine oxidase to xanthine dehydrogenase transition. Thus, it would have been expected that the replacement or addition of C-terminal amino acids would have an unpredictably proxanthine dehydrogenase effect on the xanthine oxidase-xanthine dehydrogenase equilibrium.

[0033] Unexpectedly, however, the present disclosure demonstrates (through experiments provided in the examples below) that, under at least some circumstances, complexes comprising Seq. ID. Nos. 1-2 (each of which is a C-terminal fusion of xanthine oxidase or a xanthine oxidase derivative) can be associated with increased production of reactive oxygen species, relative to natural xanthine oxidase. Without wishing to be bound by any particular theory, the increased production of reactive oxygen species may be associated with increased therapeutic potential of the complexes.

[0034] As mentioned above, in at least some embodiments, the complex comprises an antibody or antibody fragment. The antibody or antibody fragment may be selected to recognize a cell (e.g., a cancer cell, or other cells, such as a senescent cell, a fat cell, or a fibroblast) associated with a disease or condition of a subject. In some embodiments, a complex as described herein may create antigens, e.g., by increasing lysosomal membrane permeability and / or leakage, and / or by creating reactive oxygen species, etc., as discussed herein. Recognition of the cell by the antibody may result in the preferential creation of the antigens at the recognized cell. The creation of antigens may allow the immune system of the subject, e.g., T cells, to recognize the cell (e.g., the cancer cell, the senescent cell, the fat cell, or the fibroblast) associated with the disease or condition of the subject by recognizing the antigens created within the cell. In some embodiments, recognition by T cells may result in lysosome-induced immunogenic cell death of the cell. In some embodiments, the immunogenic cell death of the cell may train an immune response against target cells (e.g., cancer cells, senescent cells, fat cells, or fibroblasts), resulting in long-term treatment of the disease or condition as a result of an ongoing immune response.

[0035] In some embodiments, compositions comprising a complex provided herein may be used to treat the disease of cancer. For example, the enzyme of the composition may be configured to be connected to a cancer cell. Herein, it has been inventively recognized that through the use of compositions comprising enzymes and cancer cell recognizing antibodies, cancer cells may be targeted for immunogenic cell death in some embodiments. The immunogenic cell death of cancer cells may treat the cancer.

[0036] According to certain embodiments, a composition useful for treating cancer comprises at least a portion of an antibody (e.g., an entire antibody or an antibody fragment) that recognizes cancer cells. For example, the antibody may be an antibody for epidermal growth factor receptors (EGFR), which may be overexpressed in cancer cells, or may be an antibody to any of a variety of other antigens or other markers that are uniquely expressed or overexpressed in cancer cells. In some embodiments, a complex comprises an antibody or antibody fragment comprises an amino acid sequence that is at least 70% identical to any one of Seq. ID. Nos. 21-24. For example, a complex comprises an antibody or antibody fragment comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or at least 99.5% identical to any one of Seq. ID. Nos. 21-24. In some embodiments, a complex comprises an antibody or antibody fragment comprises an amino acid sequence that is about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% identical to any one of Seq. ID. Nos. 21-24. In some embodiments, the fusion protein comprises the amino acid sequence of any one of Seq. ID. Nos. 21-24. In some embodiments, the antibody comprises one of Seq. ID. Nos. 21- 24. For example, in some embodiments, the antibody consists of one of Seq. ID. Nos. 21-24.

[0037] Table 3. Amino acid sequences associated with various enzymes described herein.

[0038] An antibody, as described herein, may comprise a biopolymer, such as a polypeptide. For example, the antibody may comprise a protein. In some embodiments, the antibody is a glycoprotein. However, in some embodiments, the antibody comprising a protein is a glycoprotein. The antibody 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 ordinary skill in the art. For example, the antibody may be substantially encoded by immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as myriad immunoglobulin variable region genes and fragments thereof. The antibody may be encoded by a light chain immunoglobulin gene or a fragment thereof. Light chain immunoglobulins may be classified as either kappa or lambda.

[0039] The antibody may be encoded by a heavy chain immunoglobulin gene or a fragment thereof. Heavy chain immunoglobulins may be classified as gamma, mu, alpha, delta, or epsilon(which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively). An immunoglobulin structural unit may comprise a tetramer. Each tetramer may be composed of two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain.

[0040] In some embodiments, the light chain has a molecular weight of greater than or equal to 15 kDa, greater than or equal to 20 kDa, greater than or equal to 25 kDa, or greater. In some embodiments, the light chain has a molecular weight of less than or equal to 35 kDa, less than or equal to 30 kDa, less than or equal to 25 kDa, or less. Combinations of these ranges are possible. For example, in some embodiments, the light chain has a molecular weight of greater than or equal to 15 kDa and less than or equal to 35 kDa.

[0041] In some embodiments, the heavy chain has a molecular weight of greater than or equal to 50 kDa, greater than or equal to 55 kDa, greater than or equal to 60 kDa, or greater. In some embodiments, the heavy chain has a molecular weight of less than or equal to 70 kDa, less than or equal to 65 kDa, less than or equal to 60 kDa, or less. Combinations of these ranges are possible. For example, in some embodiments, the heavy chain has a molecular weight of greater than or equal to 50 kDa and less than or equal to 70 kDa.

[0042] The N-terminus of each polypeptide chain of an antibody or antibody fragment may define a variable region of the immunoglobulin structural unit. The variable region may be primarily responsible for antigen recognition. In some embodiments, the variable region comprises greater than or equal to 95, greater than or equal to 98, greater than or equal to 100, greater than or equal to 103, greater than or equal to 105, or more amino acids. In some embodiments, the variable region comprises less than or equal to 115, less than or equal to 113, less than or equal to 110, less than or equal to 108, less than or equal to 105, or fewer amino acids. Combinations of these ranges are possible. For example, in some embodiments, the variable region comprises of greater than or equal to 95 and less than or equal to 115 amino acids.

[0043] Antibodies may exist as intact immunoglobulins. However, in some embodiments, antibodies exist as any of a number of immunoglobulin fragments.

[0044] The immunoglobulin fragment 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 using pepsin. In some, exemplary embodiments, pepsin is used to digest the Fc domain of an antibody, e.g., by degrading disulfide linkages in a hinge region of the Fc domain to produce F(ab)’2. The F(ab)’2 is, according to certain embodiments, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)’2 may be reduced to break the disulfide linkage in the hinge region, thereby converting the (Fab’)2 dimer into a Fab’ monomer. The Fab’ monomer, according to some embodiments, comprises Fab and a part of the hinge region of the Fc domain.

[0045] In some embodiments, the immunoglobulin fragment is synthesized de novo. The immunoglobulin fragment may be produced by any of a variety of methods known to those of ordinary skill in the art, such as by chemical synthesis, by utilizing recombinant DNA methodology, or by “phage display” methods.

[0046] Examples of antibodies include single chain antibodies, e.g., single chain Fv (scFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide. In one embodiment, the antibody is a monoclonal antibody.

[0047] In some embodiments, the composition comprising the antibody or antibody fragment is configured such that the antibody or antibody fragment retains a relatively high affinity for a target molecule. Without wishing to be bound by theory, the affinity of the antibody or antibody fragment for the target may be inversely related to the dissociation constant (KD) between the antibody or antibody fragment and the target, so that a lower KD value corresponds to a higher affinity. In some embodiments, the KD value of the antibody or antibody fragment is less than or equal to 10'6M, less than or equal to 10'7M, less than or equal to 10'8M, less than or equal to 10'9M, less than or equal to IO40M, or less under physiological conditions. In some embodiments, the KD value of the antibody or antibody fragment is greater than or equal to 1043M, greater than or equal to IO2M, greater than or equal to IO1M, or more. Combinations of these ranges are also possible. For example, in some embodiments, the KD value of the antibody or the antibody fragment is greater than or equal to 1042M and less than or equal to 10'6M. The KD value of the composition may be determined by a test that would be well-known to one of ordinary skill in the art. In some embodiments, the KD of the composition may be relatively close to the KD of the pure antibody or antibody fragment analogous to the antibody or antibody fragment of the composition. This relatively high affinity of the composition for its target may be associated with the fact that the composition is a fusion protein.

[0048] In this context, the term “antibody fragment” can refer to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing / destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, any antibody fragments described elsewhere herein and including Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VE or VH), camelid VHH domains, multi- specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v- NAR and bis-scFv.

[0049] In some embodiments, the antibody may be a minibody or a nanobody. In the context of the present disclosure, it has been inventively recognized that use of a minibody or a nanobody may advantageously increase the binding affinity of the antibody, relative to a full-sized antibody.

[0050] The enzyme may be connected to another portion of the composition (e.g., an antibody or an antibody portion, etc.) covalently to form a complex. The enzyme and the antibody may be connected at any of a variety of suitable positions. For example, the antibody or antibody fragment could be linked to the N-terminal end of the enzyme, the C-terminal end of the enzyme, or to a side-chain of an amino acid residue of the enzyme. Unexpectedly, certain C-terminal fusion proteins of xanthine oxidase have been shown to be particularly well-suited for use in therapeutics, despite the fact that, as discussed above, the impact of C-terminal modifications of xanthine oxidase on xanthine oxidase potency would have been highly unpredictable. In some embodiments, an enzyme and an antibody are directly connected (e.g., directly covalently linked). However, the enzyme and the antibody may be covalently linked via a linker (e.g., another amino acid chain or another compound covalently bound to both the enzyme and the antibody or antibody fragment). For example, the enzyme and the antibody or antibody fragment may be covalently linked such that the enzyme and the other portion are part of a single polypeptide chain. For example, the composition may be expressed as a single fusion protein containing both an enzyme (e.g., an oxidase, such as xanthine oxidase or a xanthine oxidase derivative) and another portion of the composition (e.g., an antibody or an antibody portion). According to some embodiments, other methods may be used to connect the enzyme to the composition, for example, directly bound to each other, or bound via one or more linking agents. Non-limiting examples include glutaraldehyde, NHS-esters (A-hydroxysuccinimide) (e.g., dithiobis(succinimidylpropionate), dithiobis(sulfosuccinimidylpropionate), etc.), PEG groups, imidoesters (e.g., dimethyl adipimidate, dimethyl suberimidate, dimethyl pimelimidate, etc.), maleimides, pyridyls, carbodiimide, isocyanate, or the like. The antibody and the enzyme may be coupled through any suitable system, e.g., amine-to-amine, sulfhydryl-to-sulfhydryl, amine-to- sulfhydryl, carboxyl-to-amine, sulfhydryl-to-carbohydrate, hydroxyl-to- sulfhydryl, or the like. Those of ordinary skill in the art will be familiar with methods of cross -linking or conjugating proteins to each other.

[0051] In some embodiments, the complex between the enzyme and the antibody is a fusion protein comprising the enzyme and the antibody. A “fusion protein” generally refers to a hybrid polypeptide which comprises 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 thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. A fusion protein may comprise different domains, for example, an enzyme and an antibody; or an enzyme and an antibody fragment. In some embodiments, the enzyme is fused at the N-terminus of the antibody or antibody fragment. In some embodiments, the enzyme is fused at the C-terminus of the antibody or antibody fragment.

[0052] The use of a fusion protein comprising an enzyme and an antibody or antibody fragment may be particularly advantageous for treating cancer. In some embodiments, for example, the fusion protein may bind to cancer cells with a higher affinity than would be expected from an identical enzyme connected to an identical antibody or antibody portion by a linker. In some embodiments, the fusion protein also has a longer lifetime in the subject, which may also result in an improved performance of the composition.

[0053] In some embodiments, fusion proteins may advantageously resist degradation, relative to other complexes of antibodies and enzymes. Fusion proteins may also result in high performance of the complex. For example. For example, the enzyme, the antibody, or the antibody fragment may have an unexpectedly high activity when comprised by a fusion protein of a composition, relative to the activity of compositions where the enzyme is cross-linked with the antibody or the antibody fragment. This may advantageously improve the potency of compositions comprising fusion proteins.

[0054] In some embodiments, the enzyme is separated from the antibody or antibody fragment by a linker region. The linker region may be configured to separate the antibody from the enzyme spatially. In some embodiments, the linker region may have an amino acid sequence that comprises repeated subsequences. For example, in some embodiments the linker region has an amino acid sequence that comprises (GGGGS)n, where is an integer greater than or equal to 1 (e.g., 1, 2, 3, 4, 5, 6, 8, 10, or more, e.g., as reported in Table 4). For example, the linker may be GGGGSGGGGS (Seq. ID. No. 32, also expressible as (GGGGS ). In other embodiments, the composition does not comprise a linker region. Of course, other linkers (e.g., Seq. ID. No. 39) not according to this motif are also possible

[0055] In some embodiments, the fusion protein comprises a linker that includes the amino acid sequence of any one of Seq. ID. Nos. 31-39. In some embodiments, the fusion protein comprises a linker that consists of the amino acid sequence of any one of Seq. ID. Nos. 31-39.

[0056] Table 4. Amino acid sequences associated with various linkers described herein.

[0057] Further provided herein are enzyme variants and fusion proteins comprising such enzyme variants. In some embodiments, the fusion protein described herein comprises a modification. When the fusion protein is referred to herein, it encompasses all its variants and derivatives. Polypeptides comprising modifications have additional features other than amino acid contents. As used herein, a “modification” or “derivative” of a protein or polypeptide (e.g., the fusion protein described herein) produces a modified or derivatized polypeptide, which is a form of a given peptide that is chemically modified relative to the reference peptide, the modification including, but not limited to, oligomerization or polymerization, modifications of amino acid residues or peptide backbone, cross-linking, cyclization, conjugation, PEGylation, glycosylation, acetylation, phosphorylation, acylation, carboxylation, lipidation, thioglycolic acid amidation, alkylation, methylation, polyglycylation, glycosylation, polysialylation, adenylylation, 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 polypeptides described herein. It is to be understood that the fusion protein comprising such modifications, are crosslinked, cyclized, conjugated, acylated, carboxylated, lipidated, acetylated, thioglycolic acid amidated, alkylated, methylated, polyglycylated, glycosylated, polysialylated, phosphorylated, adenylylated, PEGylated, or combination thereof. In some embodiments, the modified fusion protein of the present disclosure may contain nonamino acid elements, such as polyethylene glycols, lipids, poly- or mono-saccharide, and phosphates. The fusion protein of the present disclosure, may comprise 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 are well known, to reduce susceptibility to proteinase digestion, and therefore serve to prolong half-life of the polypeptides in solutions, particularly biological fluids where proteases may be present. In some embodiments, the fusion proteins described herein are further modified within the sequence, such as, modification by tcrminal-NH acylation, e.g., acetylation, or thioglycolic acid amidation, by terminal-carboxylamidation, e.g., with ammonia, methylamine, and the like terminal modifications.

[0058] Terminal modifications are useful, to reduce susceptibility by proteinase digestion, and therefore can serve to prolong half-life of the polypeptides in solution, particularly in biological fluids where proteases may be present. Amino terminus modifications include methylation (e.g., — NHCH3 or — N(CH3)2), acetylation (e.g., with acetic acid or a halogenated derivative thereof such as a-chloroacetic acid, a-bromoacetic acid, or a-iodoacetic acid), adding a benzyloxycarbonyl (Cbz) group, or blocking the amino terminus with any blocking group containing a carboxylate functionality defined by RCOO— or sulfonyl functionality defined by R— SO2— , where R is selected from the group consisting of alkyl, aryl, heteroaryl, alkyl aryl, and the like, and similar groups. One can also incorporate a desamino acid at the N-terminus (so that there is no N- terminal amino group) to decrease susceptibility to proteases or to restrict the conformation of the polypeptide. In certain embodiments, the N-terminus is acetylated with acetic acid or acetic anhydride.

[0059] Carboxy terminus modifications include replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxy terminus to introduce structural constraints. One can also cyclize the peptides described herein, or incorporate a desamino or descarboxy residue at the termini of the peptide, so that there is no terminal amino or carboxyl group, to decrease susceptibility to proteases or to restrict the conformation of the peptide. Methods of circular 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 the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof.

[0060] In some embodiments, the fusion proteins described herein are phosphorylated. One can also readily modify peptides by phosphorylation, and other methods. In some embodiments, one can also replace the naturally occurring side chains of the genetically encoded amino acids (or the stereoisomeric D amino acids) with other side chains, for instance with groups such as alkyl, lower (Ci-e) alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocycles. For example, proline analogues in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members can be employed.

[0061] Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic. Heterocyclic groups preferably contain one or more nitrogen, oxygen, and / or sulfur heteroatoms. Examples of such groups include the 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 can be substituted or unsubstituted. Where a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.

[0062] In some embodiments, the fusion proteins described herein may be attached to one or more polymer moieties. In some embodiments, these polymers are covalently attached to the fusion proteins of the disclosure. In some embodiments, for therapeutic use of the end product preparation, the polymer is pharmaceutically acceptable. One 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, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations.

[0063] All combinations of the different modifications and derivatizations are envisioned for the fusion protein described herein.

[0064] Other aspects of the present disclosure provide methods of producing the fusion protein. The fusion protein may be produced by expression form recombinant nucleic acids in appropriate cells (e.g., bacterial cell or eukaryotic cells) and isolated. To produce the fusion protein, nucleic acids encoding the fusion protein may be introduced to a cell (e.g., a bacterial cell or a eukaryotic cell such as a yeast cell or an insect cell. The cells may be cultured under conditions that allow the fusion protein to express from the nucleic acids encoding the fusion protein. Fusion proteins comprising a signal peptide can be secreted, e.g., into the culturing media and can subsequently be recovered. The fusion protein may be isolated using any methods of purifying a protein known in the art.

[0065] The nucleic acids encoding the fusion protein described herein may be obtained, and the nucleotide sequence of the nucleic acids determined, by any method known in the art. One skilled in the art is able to identify the nucleotide sequence encoding the fusion protein from the amino acid sequence of the fusion protein. The nucleic acids encoding the fusion protein of the present disclosure, may be DNA or RNA, doublestranded or single stranded. In some embodiments, the nucleotide sequence encoding the fusion protein may be codon optimized to adapt to different expression systems e.g., for mammalian expression).

[0066] In some embodiments, the nucleic acid is comprised within a vector, such as an expression vector. In some embodiments, the vector comprises a promoter operably linked to the nucleic acid.

[0067] A variety of promoters can be used for expression of the fusion proteins described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and the herpes simplex tk virus promoter.

[0068] Regulatable promoters can also be used. Such regulatable promoters include those using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters, those using the tetracycline repressor (tetR), etc. Other systems include FK506 dimer, VP16 or p65 using astradiol, RU486, diphenol murislerone, or rapamycin.

[0069] Regulatable promoters that include a repressor with the operon can be used. In one embodiment, the lac repressor from Escherichia coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters. In one embodiment, a tetracycline inducible switch is used.

[0070] Additionally, the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer / promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

[0071] An expression vector comprising the nucleic acid can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the fusion proteins described herein. In some embodiments, the expression of the fusion proteins described herein is regulated by a constitutive, an inducible or a tissue-specific promoter.

[0072] The host cells used to express the fusion proteins described herein may include bacterial cells such as Escherichia coli, eukaryotic cells, or he like. In one embodiment, mammalian cells, such as Chinese hamster ovary cells (CHO) can be used.

[0073] A variety of host-expression vector systems may be utilized to express the fusion proteins described herein. Such host-expression systems represent vehicles by which the coding sequences of the isolated fusion proteins described herein may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the fusion proteins described herein in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences for the fusion proteins described herein; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing sequences encoding the fusion proteins described herein; insect cell systems infected with recombinant virus expression vectors (e.g., baclovirus) containing the sequences encoding the fusion proteins described herein; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing sequences encoding the fusion proteins described herein; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphotic cells, Per C.6 cells harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

[0074] In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the fusion proteins being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of fusion proteins described herein, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278, in which the coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors, and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0075] In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the coding sequence of interest may be ligated to an adenovirus transcription / translation control composition, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts. Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc.

[0076] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Purification and modification of recombinant proteins is well known in the art such that the design of the polyprotein precursor could include a number of embodiments readily appreciated by a skilled worker. Any known proteases or peptidases known in the art can be used for the described modification of the precursor molecule, e.g., thrombin or factor Xa, enterokinase, furin, and AcTEV, and the Foot and Mouth Disease Virus Protease C3, etc.

[0077] Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may 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.

[0078] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express fusion proteins described herein may be engineered. Rather than using expression vectors which contain 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. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the fusion proteins described herein. Such engineered cell lines may be particularly useful in screening and evaluation of fusion proteins that interact directly or indirectly with the fusion proteins described herein.

[0079] The expression levels of the fusion described herein can be increased by vector amplification. When a marker in the vector system expressing a fusion protein described herein is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of a fusion protein described herein or a fusion protein described herein, production of the fusion protein will also increase.

[0080] Once a fusion described herein has been recombinantly expressed, it may be purified by any method known in the art for purification of polypeptides, polyproteins or antibodies (e.g., analogous to antibody purification schemes based on antigen selectivity) for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen (optionally after Protein A selection where the polypeptide comprises an Fc domain (or portion thereof)), and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of polypeptides or antibodies.

[0081] The composition may include, or may be administered along with, a treatment agent in certain embodiments. The treatment agent may be chosen to increase the susceptibility of cells to the composition. In some embodiments, the subject is pretreated with the treatment agent. In some embodiments, the subject is administered the treatment agent as a part of, or concurrently with the administration of, a composition as described herein. The subject may also receive the treatment agent after administration of the composition and the disclosure is not so limited. Treatment of the subject with the treatment agent may prepare the subject for successful attack on the lysosome membrane, which may be useful for treating the disease. Treatments using a treatment agent may provide for more efficient attack on the lysosome membrane. However, it should also be understood that use of a treatment agent is not required, and in some cases, a composition may be administered without use of a treatment agent.

[0082] In some embodiments, the treatment agent is configured to increase a concentration of reactive oxygen species near a cell targeted by the composition. Without wishing to be bound by theory, it is believed that, as discussed above, oxidization processes (e.g., those caused by the action of oxidases or peroxidases) may be useful for treating diseases or conditions. Thus, the treatment agent may increase a rate of oxidation, or may increase a total amount of oxidation, by increasing the concentration of reactive oxygen species near the cell.

[0083] The increase in the concentration of reactive oxygen species is, in some embodiments, related to a level of antioxidants in the subject, because antioxidants may reduce or inhibit such tumor treatments. In some embodiments, the treatment agent lowers a level of antioxidants near the targeted cell. For example, the treatment agent may remove or suppress antioxidants of the subject, as discussed in greater detail below.

[0084] It should be understood that one or more treatment agents may be used, separately or together, in accordance with some embodiments. For example, to a subject may be administered statins, fatty acids, or both, as discussed in greater detail below. Treatment agents may be administered separately and / or together. If administered separately, treatment agents may be administered simultaneously and / or sequentially, in any suitable order. Any suitable method of administration and dosing schedule may be used, including those discussed herein. Additionally, one or more treatment agents may be present in a suitable, pharmaceutically acceptable carrier. Antioxidants may be removed or suppressed from a subject, with or without the use of a treatment agent.

[0085] In some embodiments, the treatment agent comprises a fatty acid. The fatty acid may comprise an unsaturated fatty acid. For example, the treatment agent may comprise an n-3 through n-6 fatty acid. In some embodiments, the treatment agent is the fatty acid. In some embodiments, administration of the fatty acid causes an antioxidant status of the subject to be lowered. Unsaturated fatty acids, including n-3 through n-6 fatty acids, may be fed to the subject, or may be administered by any of a variety of other appropriate methods. In the context of the present disclosure, it has been inventively recognized that treating a subject with unsaturated fatty acids may increase a local concentration of unsaturated fatty acids that reactive oxygen species can oxidize. This may accelerate lysosome-induced cell-death, by accelerating degradation of lysosomal membranes.

[0086] One or more fatty acids may be administered to a subject. The one or more fatty acids may be administered simultaneously and / or sequentially. The unsaturated fatty acid may include one or more double bonds and / or triple bonds within the fatty acid chain. The fatty acid may be a relatively small-chain fatty acids. Non-limiting examples of such unsaturated fatty acids include n-3 through n-8 (e.g., n-3, n-4, n-5, n-6, n-7, or n- 8) fatty acids. Unsaturated fatty acids may be monounsaturated and / or polyunsaturated fatty acids. Non-limiting examples of unsaturated fatty acids include, but are not limited to, CH2=CHCOOH, CH3CH=CHCOOH, CH3CH2CH=CHCOOH, CH3CH2CH2CH=CHCOOH, CH2=CHCH2COOH, CH2=CH CH2CH2COOH, CH2=CHCH2CH2CH2COOH, CH2=CHCH2CH=CHCOOH, CH2=CHCH=CHCOOH, etc. In addition, in some cases, the fatty acids may come from naturally occurring sources, such as krill oil, fish oil, safflower oil, soybean oil, flaxseed oil, canola oil, algal oil, etc.

[0087] A wide range of dosing of fatty acids may be used. In some embodiments, fatty acids may be given to a subject, such as a human, at a dosage of at least 0.1 g, at least 0.2 g, at least 0.3 g, at least 0.4 g, at least 0.5 g, at least 0.6 g, at least 0.7 g, at least 0.8 g, at least 0.9 g, at least 1 g, at least 2 g, at least 3 g, at least 4 g, at least 5 g, at least 6 g, at least 7 g, at least 8 g, at least 9 g, at least 10 g, at least 11 g, at least 12 g, at least 13 g, at least 14 g, or at least 15 g. In some embodiments, the fatty acids may be given at a dosage of no more than 15 g, no more than 14 g, no more than 13 g, no more than 12 g, no more than 11 g, no more than 10 g, no more than 9 g, no more than 8 g, no more than 7 g, no more than 6 g, no more than 5 g, no more than 4 g, no more than 3 g, no more than 2 g, no more than 1 g, no more than 0.9 g, no more than 0.8 g, no more than 0.7 g, no more than 0.6 g, no more than 0.5 g, no more than 0.4 g, no more than 0.3 g, no more than 0.2 g, or no more than 0.1 g. Combinations of any of these are also possible in some embodiments, e.g., the dosage may be between 1 and 15 g, between 1 and 10 g, between 5 and 10 g, between 0.5 and 1 g, etc.

[0088] In some embodiments, the fatty acids may be given 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 acids may be given at a dosage of no more than 2 g / kg, no more than 1 g / kg, no more than 500 mg / kg, no more than 300 mg / kg, no more than 200 mg / kg, no more than 100 mg / kg, no more than 50 mg / kg, no more than 30 mg / kg, no more than 20 mg / kg, no more than 10 mg / kg, no more than 5 mg / kg, no more than 3 mg / kg, no more than 2 mg / kg, or no more than 1 mg / kg. Combinations of any of these are also possible in some embodiments, e.g., the dosage may be 1 to 2 g / kg, 500 mg / kg to 1 g / kg, 400 to 800 mg / kg, etc. Appropriate doses, dose rates, and treatments using the fatty acids may be determined using the methods described herein.

[0089] In some embodiments, the treatment agent comprises a statin. The statin may be co-administered with fatty acids, or may be administered without administration of fatty acids. Statins, also known as HMG-CoA reductase inhibitors, may 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, e.g., simultaneously and / or sequentially.

[0090] A wide range of dosing of statins may be used. For example, the statins may be given 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, the statins may be given at a dosage of no more than 100 mg, no more than 90 mg, no more than 80 mg, no more than 70 mg, no more than 60 mg, no more than 50 mg, no more than 40 mg, no more than 30 mg, no more than 20 mg, no more than 10 mg, no more than 5 mg, no more than 3 mg, no more than 2 mg, or no more than 1 mg. Combinations of any of these are also possible in some embodiments, e.g., the dosage may be 10 to 20 mg, 20 to 40 mg, 40 to 80 mg, 5 to 10 mg, etc.

[0091] In addition, in some embodiments, statins may be given 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 statins may be given at a dosage of no more than 15 mg / kg, no more than 14 mg / kg, no more than 13 mg / kg, no more than 12 mg / kg, no more than 11 mg / kg, no more than 10 mg / kg, no more than 9 mg / kg, no more than 8 mg / kg, no more than 7 mg / kg, no more than 6 mg / kg, no more than 5 mg / kg, no more than 4 mg / kg, no more than 3 mg / kg, no more than 2 mg / kg, or no more than 1 mg / kg. Combinations of any of these are also possible in some embodiments, e.g., the dosage may be between 1 and 15 mg / kg, between 1 and 10 mg / kg, between 5 and 10 mg / kg, etc. Appropriate doses, dose rates, and treatments using the fatty acids may be determined using the methods described herein.

[0092] The compositions described herein may be administered to the subject (e.g., a human) using any of a variety of suitable techniques, as discussed in greater detail below. The composition may be administered to the subject in any suitable dose. For example, the composition may be administered in a dosage of greater than or equal to 10 mg, greater than or equal to 15 mg, greater than or equal to 20 mg, greater than or equal to 25 mg, greater than or equal to 30 mg, greater than or equal to 40 mg, greater than or equal to 50 mg, greater than or equal to 60 mg, greater than or equal to 70 mg, greater than or equal to 80 mg, greater than or equal to 90 mg, greater than or equal to 100 mg, greater than or equal to 200 mg, greater than or equal to 300 mg, greater than or equal to 500 mg, greater than or equal to 1000 mg, greater than or equal to 1500 mg, greater than or equal to 2000 mg, greater than or equal to 2500 mg, greater than or equal to 3000 mg, greater than or equal to 5000 mg, etc. In some embodiments, the composition may be applied at a dosage of less than or equal to 5000 mg, less than or equal to 3000 mg, less than or equal to 2500 mg, less than or equal to 2000 mg, less than or equal to 1500 mg, less than or equal to 1000 mg, less than or equal to 500 mg, less than or equal to 300 mg, less than or equal to 200 mg, less than or equal to 100 mg, less than or equal to 90 mg, less than or equal to 80 mg, less than or equal to 70 mg, less than or equal to 60 mg, less than or equal to 50 mg, less than or equal to 40 mg, less than or equal to 30 mg, less than or equal to 25 mg, less than or equal to 20 mg, less than or equal to 15 mg, less than or equal to 10 mg, etc. Combinations of these ranges are also possible. For example, the dosage may be greater than or equal to 10 mg and less than or equal to 20 mg, greater than or equal to 50 mg and less than or equal to 100 mg, or greater than or equal to 100 mg and less than or equal to 200 mg.

[0093] In some embodiments, the compositions may be given to a subject (e.g., a human) at a dosage of greater than or equal to 1 mg / kg, greater than or equal to 2 mg / kg, greater than or equal to 3 mg / kg, greater than or equal to 4 mg / kg, greater than or equal to 5 mg / kg, greater than or equal to 6 mg / kg, greater than or equal to 7 mg / kg, greater than or equal to 8 mg / kg, greater than or equal to 9 mg / kg, greater than or equal to 10 mg / kg, greater than or equal to 11 mg / kg, greater than or equal to 12 mg / kg, greater than or equal to 13 mg / kg, greater than or equal to 14 mg / kg, or greater than or equal to 15 mg / kg. In some embodiments, the statins maybe given at a dosage of less than or equal to 15 mg / kg, less than or equal to 14 mg / kg, less than or equal to 13 mg / kg, less than or equal to 12 mg / kg, less than or equal to 11 mg / kg, less than or equal to 10 mg / kg, less than or equal to 9 mg / kg, less than or equal to 8 mg / kg, less than or equal to 7 mg / kg, less than or equal to 6 mg / kg, less than or equal to 5 mg / kg, less than or equal to 4 mg / kg, less than or equal to 3 mg / kg, less than or equal to 2 mg / kg, or less than or equal to 1 mg / kg. Combinations of these ranges are also possible. For example, in some embodiments, the dosage is greater than or equal to 1 mg / kg and less than or equal to 15 mg / kg, greater than or equal to 1 mg / kg and less than or equal to 10 mg / kg, greater than or equal to 5 mg / kg or less than or equal to 10 mg / kg.

[0094] In another aspect, a composition as described herein may be administered to a subject. The composition may be administered by itself. In some embodiments, the composition is administered in conjunction with co-factors, other therapeutics (e.g., treatment agents), or the like. For example, a composition may be administered alone, or in conjunction with substrates such as hypoxanthine, xanthine, glucose, or the like, e.g., as discussed herein, and / or in conjunction with fatty acids, statins, etc. See, for example, Int. Pat. Apl. Pub. No. WO 2019 / 099687, incorporated herein by reference.

[0095] The compositions may be applied in a therapeutically effective, pharmaceutically acceptable amount as a pharmaceutically acceptable formulation, for example, a pharmaceutically acceptable carrier such as those described below. The term “effective amount” of a composition, such as the compositions described herein, refers to the amount necessary or sufficient to realize a desired biologic effect. For example, an effective amount of a composition to treat a tumor may be an amount sufficient to reduce the tumor’s size. Combined with the teachings provided herein, by choosing among the various active compositions and weighing factors such as potency, relative bioavailability, subject body weight, severity of adverse side effects and mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular compositions being administered the size of the subject, or the severity of the diseaseor condition. One of ordinary skill in the art can empirically determine the effective amount of the compositions without necessitating undue experimentation.

[0096] The terms “treat,” “treated,” “treating,” and the like, when used herein, refer to administration of the compositions to a subject which may increase the resistance of the subject to a disease, or to further progression of the disease, to control progression of the disease, and / or slow the progression of or to reduce the severity of symptoms of the disease. The effective amount may depend on the particular disease being treated and the desired outcome.

[0097] In some embodiments, a therapeutically effective dose of a composition can be initially determined from an animal model. The applied dose may be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.

[0098] In administering the composition to a subject, dosing amounts, dosing schedules, routes of administration, and the like may be selected so as to affect known activities of these compositions. Dosages may be estimated based on the results of experimental models, optionally in combination with the results of assays of compositions described herein. Dosage may be adjusted appropriately to achieve desired drug levels, local or systemic, depending upon the mode of administration. The doses may be given in one or several administrations per day. Multiple doses per day may be used to achieve appropriate systemic levels of the compositions within the subject or within the cell targeted by the composition.

[0099] The dose may be chosen to provide a therapeutically effective amount of the composition to the cell targeted by the composition. The dosage may be given in a maximum safe dose. The maximum safe dose may be chosen such that a high therapeutically effective amount of the composition is delivered to the subject but such that a risk to the subject of potentially detrimental side effects is minimal. The dose of the composition may be chosen to have a desired concentration at the targeted cell, to have a desired efficacy, to have a desired longevity within the subject, to have a desired rate of administration, to have a desired frequency of administration, to act in an appropriate fashion when administered concurrently with other treatments (e.g., as in a cocktail), or for any of a variety of other purposes known to those of ordinary skill in the art.

[0100] The dose may be chosen based, at least in part, on conditions associated with the subject. For example, the dose may be chosen based at least in part on the species, age, sex, weight, size, environment, metabolism, physical condition, or current state of health of the subject. In some cases, a subject- specific maximum dose may be used. In some cases, the dose may be administered in a way that limits deleterious effects to the subject (e.g., the dose may be delivered orally, nasally, intravenously, etc., depending on the needs of the subject).

[0101] Doses of the composition may be administered daily, weekly, or monthly and any other amount of time therebetween.

[0102] A dose may include the composition in any appropriate amount. In some embodiments, the dose of the composition is greater than or equal to 0.1 micrograms, greater than or equal to 0.5 micrograms, greater than or equal to 10 micrograms, greater than or equal to 50 micrograms, greater than or equal to 100 micrograms, greater than or equal to 500 micrograms, greater than or equal to 1000 micrograms, or more. In some embodiments, the dose of the composition is less than or equal to 10000 micrograms, less than or equal to 5000 micrograms, less than or equal to 1000 micrograms, less than or equal to 500 micrograms, less than or equal to 100 micrograms, less than or equal to 50 micrograms, or less. Combinations of these ranges are also possible. For example, in some embodiments the dose of the composition is greater than or equal to 0.5 micrograms and less than or equal to 10000 micrograms.

[0103] Doses of the composition may be administered with any of a variety of average rates. In some embodiments, the doses are administered at a rate of greater than or equal to 0.01 micrograms / hour, greater than or equal to 0.05 micrograms / hour, greater than or equal to 0.1 micrograms / hour, greater than or equal to 0.5 micrograms / hour, greater than or equal to 1 micrograms / hour, greater than or equal to 5 micrograms / hour, greater than or equal to 10 micrograms / hour, greater than or equal to 50 micrograms / hour, greater than or equal to 100 micrograms / hour, greater than or equal to 500 micrograms / hour, greater than or equal to 1000 micrograms / hour, or greater. In some embodiments, the doses are administered at a rate of less than or equal to 10000 micrograms / hour, less than or equal to 5000 micrograms / hour, less than or equal to 1000 micrograms / hour, less than or equal to 500 micrograms / hour, less than or equal to 100 micrograms / hour, less than or equal to 50 micrograms / hour, less than or equal to 10 micrograms / hour, less than or equal to 1 micrograms / hour, less than or equal to 0.5 micrograms / hour, less than or equal to 0.1 micrograms / hour, or less. Combinations of these ranges are possible. For example, in some embodiments, the doses are administered at a rate of greater than or equal to 0.01 micrograms / hour, and less than or equal to 10000 micrograms / hour.

[0104] The composition may be administered over extended period of time. In some embodiments, the dose is administered over a period of greater than or equal to Ih, greater than or equal to 2h, greater than or equal to 4h, greater than or equal to 6h, greater than or equal to 12h, greater than or equal to 24h, greater than or equal to 3 days, greater than or equal to 7 days, greater than or equal to 10 days, greater than or equal to 14 days, greater than or equal to 21 days, greater than or equal to 30 days, greater than or equal to 45 days, greater than or equal to 60 days, greater than or equal to 90 days, or greater. In some embodiments, the dose is administered over a period of less than or equal to 365 days, less than or equal to 200 days, less than or equal to 90 days, less than or equal to 60 days, less than or equal to 45 days, less than or equal to 30 days, less than or equal to 21 days, less than or equal to 14 days, less than or equal to 10 days, less than or equal to 7 days, less than or equal to 3 days, less than or equal to 24h, less than or equal to 12h, or less. Combinations of these ranges are possible. For example, in some embodiments, the dose is administered over a period of greater than or equal to Ih and less than or equal to 365 days.

[0105] The subject may be any appropriate subject. For example the subject may be a mammal, such as a human, or a non-human animal, such as a dog, a cat, a horse, a rabbit, a cow, a pig, a sheep, a goat, a rat (e.g., Rattus Norvegicus), a mouse (e.g., Mus musculus), a guineapig, a non-human primate (e.g., a monkey, a chimpanzee, a baboon, an ape, a gorilla, etc.), or the like. Administration of a composition of the disclosure may be accomplished by any of a variety of medically acceptable methods that allows the composition to reach its target cell. The particular mode selected may depend of course, upon factors such as those previously described, for example, the particular composition, the severity of thestate of the subject being treated, the dosage required for therapeutic efficacy, etc. As used herein, a “medically acceptable” mode of treatment is a mode able to produce effective levels of the compositions within the subject without causing clinically unacceptable adverse effects. In the case of neurodegenerative diseases the blood brain barrier may prevent the agent from reaching the senescent cells. In this case themedically acceptable mode may be administration through the cerebral spinal fluid, i.e., intrathecally.

[0106] Any medically of a variety of acceptable methods may be used to administer the compositions to the subject. The administration may be localized (e.g., to a particular region, physiological system, tissue, organ, or cell type) or systemic, depending on the condition to be treated. For example, the compositions may be administered orally, vaginally, rectally, buccally, pulmonary, topically, nasally, transdermally, through parenteral injection or implantation, via surgical administration, or any other method of administration where access to the tumor is achieved. In some cases, more than one method of administration may be used, e.g., if two or more compositions are to be administered.

[0107] Examples of parenteral methods of administration that can be used include intravenous, intradermal, subcutaneous, intracavity, intramuscular, intraperitoneal, epidural, or intrathecal administration. Examples of implantation methods of administration include implantation or injection of any implantable or injectable drug delivery system. Oral administration may be used in some embodiments. Oral administration may be advantageous because of the convenience to the subject as well as the dosing schedule. A composition suitable for oral administration may be administered using discrete units, such as hard or soft capsules, pills, cachettes, tablets, troches, dissolving films or lozenges. Other oral compositions suitable for use include solutions or suspensions such as a syrup, an elixir, or an emulsion. The solutions or suspensions may comprise aqueous or non-aqueous liquids. In some embodiments, a composition may be used to fortify a food or a beverage. The compositions, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0108] Pharmaceutical formulations for parenteral administration may include aqueous solutions of the active compounds in water soluble form. Suspensions of the active compounds may be prepared, in some cases, 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 contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension may contain suitable stabilizers. In some embodiments, the suspension comprises agents that increase the solubility of the compounds. The stabilizers or solubility increasers may allow for the preparation of highly concentrated solutions.

[0109] The composition may be formulated as a depot preparation in some embodiments. Such long-acting formulations may be formulated, in some cases, with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0110] 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 comprised by an aqueous or saline solution (e.g., suitable for inhalation), may be microencapsulated, may be encochleated, may be coated onto microscopic gold particles, may be contained in liposomes, may be nebulized, may be aerosolized, or may be dried onto a sharp object to be scratched into the skin. The composition may be prepared in the form of a solid pharmaceutical preparation. For example, the composition may be included within granules; powders; tablets; coated tablets; (micro)capsules; suppositories; syrups; emulsions; suspensions; creams; or dropsor preparations with protracted release of active compounds. In some embodiments, the composition is prepared in the form of a solid pharmaceutical preparation configured to be dissolved or otherwise converted to a pharmaceutical liquid preparation. For example, the composition may be prepared in the form of a dry powder that may be dissolved in water to form an aqueous solution. Liquid or solid compositions may include an additive such as an excipient. In some embodiments, the additive comprises disintegrants, binders, coatingagents, swelling agents, lubricants, flavorings, sweeteners or solubilizers, although any of a variety of additives may be used and the disclosure is not so limited.

[0111] In some embodiments, a composition may be administered to provide sequential exposures to a composition over a certain time period, for example, hours, days, weeks, months or years.

[0112] For example, the composition may be administered by repeated administrations or by controlled release. For example, the composition may be control-released over the time-period such that it sustains a dosage of the composition during that time-period. Control release may be achieved, for example, by oral dosage forms, bolus injections, transdermal patches, subcutaneous implants, or other methods such as those described herein. Maintaining a substantially constant concentration of a composition may be desired insome cases.

[0113] Other delivery systems suitable for use in certain embodiments may include timerelease, delayed release, sustained release, or controlled release delivery systems. Such systems may advantageously avoid the need for repeated administrations, increasing convenience to the subject. Many types of delivery systems are available and known to those of ordinary skill in the art. The composition may be administered, for example, using a delivery system such as a polymer-based systems such as polylactic and / or polyglycolic acids, polyanhydrides, polycaprolactonesand / or combinations of these; nonpolymer systems that are lipid-based 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 using conventional binders and excipients; or partially fused implants. Specific examples include, but are not limited to, erosional systems in which the composition is contained in a form within a matrix, or diffusional systems in which an active componentcontrols the release rate. The formulation may be present as, for example, microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, or polymeric systems, etc. In some embodiments, the system may allow sustained or controlled release of a composition to occur, for example, through control of the diffusion or erosion / degradation rate of the formulation. In addition, a pump-based hardware delivery system may be used in some embodiments.

[0114] In some embodiments, the composition is delivered using an implant. The implant may be configured for short-term or long-term release of the composition. In some embodiments, the delivery system is a long-term release implant. Use of a longterm release implant may be particularly suitable in some embodiments. “Long-term release,” as used herein, means that an implant containing a composition as described herein is constructed and arranged to deliver therapeutically effective levels for In some embodiments, the composition is delivered at therapeutically effective levels over a period of greater than or equal to Ih, greater than or equal to 2h, greater than or equal to 4h, greater than or equal to 6h, greater than or equal to 12h, greater than or equal to 24h, greater than or equal to 3 days, greater than or equal to 7 days, greater than or equal to 10 days, greater than or equal to 14 days, greater than or equal to 21 days, greater than or equal to 30 days, greater than or equal to 45 days, greater than or equal to 60 days, greater than or equal to 90 days, or greater. In some embodiments, the composition is delivered at therapeutically effective levels over a period of less than or equal to 365 days, less than or equal to 200 days, less than or equal to 90 days, less than or equal to 60 days, less than or equal to 45 days, less than or equal to 30 days, less than or equal to 21 days, less than or equal to 14 days, less than or equal to 10 days, less than or equal to 7 days, less than or equal to 3 days, less than or equal to 24h, less than or equal to 12h, or less. Combinations of these ranges are possible. For example, in some embodiments, In some embodiments, the composition is delivered at therapeutically effective levels over a period of greater than or equal to Ih and less than or equal to 365 days. Long-term release implants are well known to those of ordinary skill in the art, and include some of the release systems described herein.

[0115] In certain embodiments a composition can be combined with a suitable pharmaceutically acceptable carrier. In general, pharmaceutically acceptable carriers suitable for use are well-known to those of ordinary skill in the art. As used herein, a “pharmaceutically acceptable carrier” refers to a non-toxic material that does not significantly interfere with the effectiveness of the biological activity of the active compound(s) to be administered, but is used as a formulation ingredient, for example, to stabilize or protect the active compound(s) within a composition before use. For example the composition may be incorporated into a liposome, incorporated into a polymer release system, suspended in a liquid (e.g., in a dissolved form or a colloidal form), or other methods such as those described herein. The carrier may include one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administrationto a human or other vertebrate animal. The term “carrier” denotes an organic or inorganic ingredient, which may be natural or synthetic, with which one or more active compounds of the disclosure are combined to facilitate application. The carrier may be co-mingled or otherwise mixed with one or more compositions as described herein, and / or with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy. The carrier may be either soluble or insoluble, depending on the application. Examples of well-known carriers include, but are not limited to, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite. The nature of the carrier may be either soluble or insoluble.

[0116] The formulations described herein may be administered in pharmaceutically acceptable solutions, in some embodiments. Pharmaceutically acceptable solutions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, emulsifiers, diluents, excipients, chelating agents, fillers, drying agents, antioxidants, antimicrobials, preservatives, binding agents, bulking agents, silicas, solubilizers, stabilizers and optionally other therapeutic ingredients, that may be used with the composition. For example, if the formulation is a liquid, the carrier may be a solvent, partial solvent, or non- solvent, and may be aqueous or organically 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 cornstarch 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 asglycerol monostearate or glycerol distearate; suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, 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 salts thereof, citric acid and salts thereof, boric acid and salts thereof, or phosphoric acid and salts thereof; or preservatives such as benzalkonium chloride, chlorobutanol, parabens, or thimerosal. The compositions of the disclosure may be formulated into preparations in solid, semi-solid, liquid or gaseous forms such as tablets, capsules, elixirs, powders, granules, ointments, solutions, depositories, inhalants or injectables, etc.

[0117] Suitable buffering agents include, but are not limited to: acetic acid and a salt (1- 2% w / v); citric acid and a salt (1-3% w / v); boric acid and a salt (0.5-2.5% w / v); and phosphoric acid and a salt (0.8-2% w / v). Suitable preservatives include, but are not limited to, benzalkonium chloride (0.003-0.03% w / v); chlorobutanol (0.3-0.9% w / v); parabens (0.01-0.25% w / v) and thimerosal (0.004-0.02% w / v).

[0118] Preparations may include solutions, suspensions, or emulsions as described above. The solutions, suspensions, or emulsions may be sterile. In some embodiments, the solutions, suspensions, or emulsions are aqueous. In some embodiments the solutions, suspensions or emulsions are non-aqueous. The sterile aqueous or nonaqueous solutions, suspensions, or emulsions may be isotonic with the blood of the subject in certain embodiments. Non-limiting examples of nonaqueous solvents are polypropylene glycol, polyethylene glycol, vegetable oil 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 di-glycerides. 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 suitability of a solution, suspension, or emulsion for parenteral delivery. Parenteral vehicles include sodium chloride solution, 1,3-butandiol, 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. Preservatives and other additives may also be present in some embodiments, such as, for example, antimicrobials, antioxidants, chelating agents and inert gases and the like.

[0119] In some embodiments, a composition as described herein may be brought into association or contact with a suitable carrier. The suitable carrier may comprise one or more accessory ingredients. The preparation may be prepared by any of a variety of suitable techniques. For example, the preparation may be prepared by uniformly and intimately associating a composition with a liquid carrier, a finely divided solid carrier or both. In some embodiments, the resulting preparation may be shaped (e.g., into a tablet, a pellet, etc.).

[0120] The compositions as discussed herein may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. Although non-pharmaceutically acceptable salts may be used for preparing compositions, when used in medicine salts should be pharmaceutically acceptable. The term “pharmaceutically acceptable salts” includes salts of compositions described herein, prepared in combination with, for example, acids or bases. Pharmaceutically acceptable salts can be prepared as alkaline metal salts, such as lithium, sodium, or potassium salts; or as alkaline earth salts, such as magnesium or calcium salts. Examples of suitable bases that may be used to form salts include ammonium, or mineral bases such as sodium hydroxide, lithium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and the like. Examples of suitable acids that may be used to form saltsinclude inorganic or mineral acids such as hydrochloric, hydrobromic, hydroiodic, hydrofluoric, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, phosphorous acids and the like. Other suitable acids include organic acids, for example, acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, glucuronic, galacturonic, salicylic, formic, naphthalene- 2-sulfonic, and the like. Still other suitable acids include amino acids such as arginate, aspartate, glutamate, and the like. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

[0121] In another aspect, the present disclosure also provides any of the above- mentioned compositions in kits, optionally including instructions for use of the composition for the treatment of a disease or condition (e.g., by targeting a senescent cell, a fat cell, a cancer cell, or a fibroblast). In some cases, the kit can include a description of use of the compositions as discussed herein. The kit also can include instructions for use of a combination of two or more compositions. Instructions also may be provided for administering the compositions by any suitable technique as previously described, for example, forally, intravenously, pump or implantable delivery device, or via another known route of drug delivery.

[0122] The kits described herein may also contain one or more containers. The container may contain compositions and other ingredients as previously described. The kits also may contain instructions for mixing, diluting, and / or administrating the compositions of the disclosure in some cases. The kits also can include other containers with one or more solvents, surfactants, preservative 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 a sample or to a subject in need of such treatment.

[0123] The compositions of the kit may be provided as any suitable form, for example, as liquid pharmaceutical preparations or as solid pharmaceutical preparations. When the composition provided is a dry powder, the composition may be reconstituted by the addition of a suitable solvent, which may also be provided in some cases. In embodiments where liquid forms of the composition are used, the liquid form may be concentrated or ready to use. The solvent will depend on the composition and the mode of use or administration. Suitable solvents for drug compositions are well known, for example as previously described, and are available in the literature. The solvent will depend on the composition and themode of use or administration. In still another aspect, the disclosure includes the promotion of one or more of the above-described embodiments, e.g., in vitro or in vivo, promotion of treatment or prevention of a tumor, e.g., by administering, to a subject, compositions such as those described herein.

[0124] The treatments disclosed herein may be given to any subject, for example, a human, or a non-human animal, such as a dog, a cat, a horse, a rabbit, a cow, a pig, a sheep, a goat, a rat (e.g., Rattus Norvegicus), a mouse (e.g., Mus musculus), a guinea pig, a non-human primate (e.g., a monkey, a chimpanzee, a baboon, an ape, a gorilla, etc.), or the like.

[0125] U.S. Pat. Apl. Ser No. 63 / 550,708, filed February 7, 2024, entitled “Modified Xanthine Oxidase- Antibody Complexes and Methods Thereof,” by Fossel, et al., is incorporated herein by reference in its entirety.

[0126] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

[0127] EXAMPLE 1

[0128] This example compares the therapeutically-active:therapeutically inactive enzyme ratios of non-limiting complexes in the form of fusion proteins having Seq. ID. Nos. 1-2 with the therapeutically-active:therapeutically inactive ratio of native xanthine oxidase consisting of Seq. ID. No. 12. Native xanthine oxidase and the fusion proteins were each incubated for 20 min in a reaction mix containing 50 uM hypoxanthine, 100 uM EDTA, and 50 mM potassium phosphate buffer. To test total activity, 500 uM NAD+ was added to the reaction mix. To test xanthine oxidase activity, NAD+ was omitted from the reaction mix, so that oxygen was the sole electron acceptor available. After incubating for 20 min, the reaction mix was added to a commercial uric acid detection kit (Sigma MAK007) and uric acid production is monitored by fluorescence according to the manufacturers instructions. The difference between total relative activity and xanthine oxidase activity was attributed to xanthine dehydrogenase activity. Comparison of xanthine oxidase activity to xanthine dehydrogenase activity was used to compute the relative amounts of xanthine oxidase and xanthine dehydrogenase to determine the therapeutically-active:therapeutically inactive xanthine oxidase ratios. Table 5 reports the results for each protein (described both by Seq. ID. No. and in terms of the fused proteins that comprise it, listed in the “Protein” column). The linker used in each fusion protein was Seq. ID. No. 32 and the antibody was a VHH9G8 antibody with Seq. ID. No. 21.

[0129] As demonstrated in Table 5, the fusion proteins produced comparable or improved XO:XDH ratios relative to native xanthine oxidase, indicating unexpectedly improved therapeutic potential despite the C-terminal fusion of the linker and antibody to the xanthine oxidase enzyme.

[0130] Table 5. Uric acid production with and without NAD+ and computed xanthine oxidase: xanthine dehydrogenase (XO:XDH) ratios of various sequences.

[0131] While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed 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 upon the specific application or applications for which the teachings of the present disclosure is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the 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, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present disclosure.

[0132] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0133] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0134] As used herein in the specification 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 being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0135] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated 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”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0136] As used herein, “wt%” is an abbreviation of weight percentage. As used herein, “at%” is an abbreviation of atomic percentage.

[0137] Some embodiments may be embodied as a method, of which various examples have been described. The acts performed as part of the methods may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include different (e.g., more or less) acts than those that are described, and / or that may involve performing some acts simultaneously, even though the acts are shown as being performed sequentially in the embodiments specifically described above.

[0138] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0139] 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 to be 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 set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

CLAIMSWhat is claimed is:

1. A composition, comprising: a complex comprising an antibody or antibody fragment, and an enzyme comprising at least one of Seq. ID. Nos. 11-18 and linked to the antibody or antibody fragment.

2. The composition of claim 1, wherein the antibody or antibody fragment and the enzyme are directly covalently linked at the C-terminus of the enzyme.

3. The composition of claim 1, wherein the antibody or antibody fragment and the enzyme are directly covalently linked in a fusion protein.

4. The composition of any one of claims 1-2, wherein the antibody or antibody fragment and the enzyme are linked via a linking agent.

5. The composition of any one of claims 1-3, further comprising a fatty acid.

6. The composition of claim 4, wherein the fatty acid comprises an unsaturated n-3 through n-6 fatty acid.

7. The composition of any one of the preceding clams, further comprising a statin.

8. The composition of any one of claims 1-5, wherein the complex equilibrates under physiological conditions to have a therapeutically-active:therapeutically-inactive enzyme ratio of greater than or equal to 6:4.

9. The composition of any one of the preceding claims, wherein the antibody or antibody fragment is an antibody or antibody fragment that recognizes EGFR.

10. The composition of any one of the preceding claims, wherein the antibody or antibody fragment comprises Seq. ID. No. 21.

11. The composition of any one of the preceding claims, wherein the complex comprises one of Seq. ID. Nos. 1-5.

12. The composition of any one of the preceding claims, wherein the complex consists of one of Seq. ID. Nos. 1-5.

13. A method, comprising: administering, to a subject, a complex comprising: an antibody or antibody fragment, and an enzyme comprising at least one of Seq. ID. Nos. 11-18 and linked to the antibody or antibody fragment.

14. The method of claim 13, wherein the antibody or antibody fragment and the enzyme are directly covalently linked in a fusion protein.

15. The method of any one of claims 13-14, wherein the antibody or antibody fragment and the enzyme are linked via a linking agent.

16. The method of any one of claims 13-14, further comprising administering a fatty acid to the subject.

17. The method of claim 16, wherein the fatty acid comprises an unsaturated n-3 through n-6 fatty acid.

18. The method of any one of claims 16 or 17, wherein the composition comprises the fatty acid.

19. The method of any one of claims 16-18, wherein administering the composition and administering the fatty acid occur simultaneously.

20. The method of any one of claims 16-19, wherein administering the composition and administering the fatty acid occur sequentially.

21. The method of any one of claims 13-20, further comprising administering a statin to the subject.

22. The method of claim 21, wherein the composition comprises the statin.

23. The method of any one of claims 21 or 22, wherein administering the composition and administering the statin occur simultaneously.

24. The method of any one of claims 21-23, wherein administering the composition and administering the statin occur sequentially.

25. The method of any one of claims 13-24 where following administration of the composition, a substrate of the enzyme is administered.

26. The method of any one of claims 13-25, wherein the complex equilibrates under physiological conditions to have a therapeutically-active:therapeutically-inactive enzyme ratio of greater than or equal to 6:4.

27. The method of any one of the preceding claims, wherein the antibody or antibody fragment is an antibody or antibody fragment that recognizes EGFR.

28. The method of any one of the preceding claims, wherein the antibody or antibody fragment comprises Seq. ID. No. 21.

29. The method of any one of the preceding claims, wherein the complex comprises one of Seq. ID. Nos. 1-5.

30. The method of any one of the preceding claims, wherein the complex consists of one of Seq. ID. Nos. 1-5.

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