Systems and methods for immunogenic cell death applications

Abstract

The present disclosure generally relates to systems and methods for treating and preventing diseases or conditions, such as aging, atherosclerosis, obesity, viral diseases, etc., which may be treated, according to some embodiments, by activation of an immune response in a subject. In some embodiments, for example, aging may be treated or prevented by removal of senescent cells by the immune system. In some embodiments, atherosclerosis may be treated or prevented by removal of plaque by the immune system. In some embodiments, obesity may be treated by removal of fat cells by the immune system. In some embodiments, viral diseases may be treated by removal of viruses by the immune system. Exemplary viral diseases include diseases caused by SARS-CoV-2 and HIV, among others.

Description

[Technical field]

[0001] Vaccines and methods of treatment for various diseases are generally described. The present disclosure generally relates to systems and methods for the treatment and prevention of diseases or conditions, such as aging, atherosclerosis, obesity, and viral diseases, such as those caused by SARS-CoV-2, HIV, influenza, SARS, and others. [Background technology]

[0002] The immune system is designed to recognize and kill substances that are foreign to the body, such as mutant cells, malignant cells, bacteria, and viruses. Both treatment and prevention of diseases or conditions are important. Treatment and prevention of diseases or conditions, such as aging, atherosclerosis, obesity, or viral diseases, can take many forms, and in some cases, vaccines are the main prevention strategy, but treatment is often impractical. Therefore, improvements are still needed. Summary of the Invention [Problem to be solved by the invention]

[0003] Vaccines and treatment methods for various diseases or conditions are generally described. The present disclosure generally relates to systems and methods for the treatment and prevention of diseases or conditions, such as aging, atherosclerosis, obesity, and viral diseases, such as those caused by SARS-CoV-2, HIV, influenza, SARS, and others. For example, treating and / or curing these diseases or conditions is the purpose of certain embodiments of the present disclosure. Some embodiments are directed to vaccinating the body against further recurrence or onset of the disease or condition. As discussed in more detail herein, this can be achieved by a composition comprising a portion recognized by antigen-presenting cells (e.g., dendritic cells) and a portion of the virus that acts as an antigen; a portion recognized by antigen-presenting cells (e.g., dendritic cells) and a portion of plaque proteins and lipids that act as antigens; a portion recognized by antigen-presenting cells (e.g., dendritic cells) and a portion of adipocytes that act as antigens; a portion recognized by antigen-presenting cells (e.g., dendritic cells) and a portion of the virus that acts as an antigen; or other compositions as described herein. In some embodiments, the compositions may be taken up by antigen presenting cells and transported to lymph nodes where the antigens may be processed and presented to T cells.

[0004] Several methods of administering a composition to a subject for the prevention or treatment of a particular condition or disease are disclosed herein. In each such aspect of the disclosure, it should be understood that the disclosure also specifically includes the composition for use in the treatment or prevention of that particular condition or disease, as well as the use of the composition for the manufacture of a medicament for the treatment or prevention of that particular condition or disease. [Means for solving the problem]

[0005] In one aspect, the present disclosure is directed to a composition for treating or preventing a disease or condition. In some embodiments, the composition comprises a fusion protein comprising an eat-me signal and at least a portion of an antibody, wherein at least a portion of the antibody is configured to bind to a cell, virus, or extracellular material associated with the disease or condition.

[0006] In another aspect, the disclosure is directed to a method of treating or preventing a disease or condition. In some embodiments, the method comprises administering to a subject a composition comprising a fusion protein comprising an eat-me signal and at least a portion of an antibody.

[0007] In yet another aspect, the present disclosure is directed to a composition for treating or preventing a disease or condition. In some embodiments, the composition comprises a fusion protein comprising: an eat-me signal and a fragment of a surface antigen of a cell or virus, wherein the composition is configured to induce an immune response against the cell or virus.

[0008] In yet another aspect, the disclosure is directed to a method of treating or preventing a disease or condition. In some embodiments, the method comprises administering to a subject a composition comprising a fusion protein comprising an eat-me signal and a fragment of a cell or viral surface antigen.

[0009] In another aspect, the present disclosure is directed to a composition. In some embodiments, the composition comprises an antibody that recognizes a cell, a virus, or an extracellular substance, and an eat-me signal that is recognized by an antigen-presenting cell, such as a dendritic cell.

[0010] In yet another aspect, the present disclosure is directed to a composition. In some embodiments, the composition comprises an antibody fragment that recognizes a specific protein of a cell, a virus, or an extracellular substance, and an eat-me signal that is recognized by an antigen-presenting cell, such as a dendritic cell.

[0011] In yet another aspect, the disclosure is directed to a method comprising administering to a subject a composition comprising an antibody that recognizes a cell, a virus, or an extracellular substance and an eat-me signal.

[0012] In one aspect, the disclosure is directed to a method comprising administering to a subject a composition comprising an antibody fragment that recognizes a specific protein of a cell or virus and an eat-me signal.

[0013] In another aspect, the disclosure is directed to a composition comprising an eat-me signal and one or more fragments of a cellular or viral surface antigen.

[0014] In yet another aspect, the present disclosure is directed to a composition comprising an eat me signal and one or more DNA fragments of a cell or virus.

[0015] In yet another aspect, the present disclosure is directed to a composition comprising an eat-me signal and one or more lipid fragments of a cell or virus.

[0016] In another embodiment, the present disclosure is directed to a composition comprising a first component that is an eat-me signal, a DNA fragment, a lipid fragment, or a fragment of a cellular or viral surface antigen, and a second component different from the first component that is also a DNA fragment, a lipid fragment, or a fragment of a cellular or viral surface antigen.

[0017] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments thereof, when considered in conjunction with the accompanying drawings. In the event that the present specification and any documents incorporated by reference include conflicting and / or inconsistent disclosure, the present specification will control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present disclosure generally relates to compositions and methods for treating and preventing diseases or conditions, such as aging, atherosclerosis, obesity, viral diseases, etc., which may be treated, according to some embodiments, by activation of an immune response in a subject. In some embodiments, for example, aging may be treated or prevented by removal of senescent cells by the immune system. In some embodiments, atherosclerosis may be treated or prevented by removal of plaque by the immune system. In some embodiments, obesity may be treated by removal of fat cells by the immune system. In some embodiments, viral diseases may be treated by removal of viruses by the immune system. Exemplary viral diseases include diseases caused by SARS-CoV-2 and HIV, among others.

[0019] The compositions and methods of the present disclosure are, in many aspects, directed to the treatment of a disease or condition in a subject (e.g., a human or non-human animal) using the subject's immune system. For example, the subject's immune system may be used in some embodiments to phagocytose a cell, virus, or extracellular substance that causes a disease or condition. In some embodiments, a composition as described herein is configured to target a cell, virus, or extracellular substance for phagocytosis by directly binding to the cell, virus, or extracellular substance. Targeting a cell, virus, or extracellular substance for phagocytosis may treat a disease or condition. Targeting a cell, virus, or extracellular substance for phagocytosis may also train the subject's immune system to recognize and target the cell, virus, or extracellular substance on its own, so that ultimately, in the absence of the composition, the subject's immune system continues to phagocytose the cell, virus, or extracellular substance. In another aspect, a composition is provided for the purpose of training an immune response against a cell, bacteria, or extracellular substance that causes a disease or condition. The composition may include fragments of cells, bacteria, or extracellular substances (e.g., surface antigen fragments) and may be configured to be phagocytosed by immune cells. Such compositions may be used in some embodiments to train an immune response against cells, bacteria, or extracellular substances, thereby preventing or treating a disease or condition caused by the cells, bacteria, or extracellular substances.

[0020] In some circumstances, the composition comprises an "eat me" signal. An eat me signal may refer to a signaling moiety (e.g., a protein or polypeptide) that helps a phagocyte recognize an extracellular cargo (e.g., a cell, a virus, an extracellular material, or a fragment of a cell, a virus, or an extracellular material) for phagocytosis, as known to those skilled in the art. Phagocytosis may refer to the process by which a cell engulfs and breaks down an extracellular material. Without wishing to be bound by theory, phagocytosis may be utilized by the immune system as a mechanism for removing pathogens, dead cells, or other cellular debris. Normally, phagocytosis of a living cell of a subject is relatively unlikely to occur. However, in the context of the present disclosure, it is recognized by the present invention that phagocytosis may be applicable to the treatment or prevention of a disease or condition, including a disease or condition caused by a living cell of a subject.

[0021] The eat me signal may be configured to induce a phagocyte to phagocytose a cell, a virus, or an extracellular material bound to the eat me signal. For example, the eat me signal may be configured to induce an antigen presenting cell to phagocytose a virus, a cell, or an extracellular material.

[0022] Any of a variety of eatme signals may be used. For example, the eatme signal may comprise a biopolymer. In some embodiments, the biopolymer comprises a polypeptide (e.g., a protein). For example, the eatme signal may be a protein (e.g., calreticulin; an annexin such as annexin A1; a pentraxin such as pentraxin-3; or a histone) or a fragment thereof. In some embodiments, the biopolymer is a nucleic acid (e.g., DNA). In some embodiments, the eatme signal is a small molecule (e.g., a phospholipid such as phosphatidylserine; an oxidized phospholipid; or a sugar residue such as galactose). In the context of the present disclosure, it is recognized that the use of a polypeptide eatme signal (e.g., calreticulin) may be advantageous in the treatment or prevention of a disease or condition. In some embodiments, the polypeptide eatme signal forms part of a fusion protein, which may have a number of unexpected advantages, which are described in more detail below.

[0023] It is recognized herein that in certain embodiments, the present invention can target cells, viruses, or extracellular substances that cause any of a variety of diseases or conditions for early phagocytosis by binding an eat-me signal to a cell, virus, or extracellular substance for the purpose of labeling for phagocytosis. In principle, labeling of a cell, virus, or extracellular substance with an eat-me signal can be achieved using any of a variety of known antibodies that recognize cells, viruses, or extracellular substances, as discussed in more detail below.

[0024] In some embodiments, the composition comprising the eat-me signal is suitable for treating a disease. Such a composition can be administered to a subject (e.g., a human) with a disease. In some embodiments, the composition generates an immune response against the cell, virus, or extracellular substance targeted by the antibody. In some embodiments, the immune response treats the disease or condition by attacking the targeted cell, virus, or extracellular substance, limiting the symptoms of the disease or condition. A subject treated with the composition can be at least partially immunized against the disease or condition.

[0025] In a first embodiment, a composition comprising an eat-me signal and an antibody can be used to treat a disease or condition by binding to cells, viruses, or extracellular substances and inducing phagocytosis. Such a composition can be used to treat a disease by inducing an immune response against the cells, viruses, or extracellular substances that cause these diseases. The following paragraphs provide specific examples.

[0026] In some embodiments, the composition (e.g., a composition comprising an eat-me signal) is configured to bind to an extracellular substance that causes atherosclerosis. For example, the eat-me signal of the composition may be configured to bind to plaque. Without wishing to be bound by theory, plaque is generally a multi-component substance that may include cholesterol, plaque cells such as white blood cells, calcium, and other fatty substances. Plaque may form on or around the walls of blood vessels (e.g., arteries), which may cause atherosclerosis.

[0027] It is recognized herein that the use of compositions comprising eat-me-signals can target plaque for phagocytosis. Phagocytosis of plaque can reduce the amount of plaque, thereby treating atherosclerosis. Furthermore, in some embodiments, the immune system can learn to recognize plaque and eventually target plaque for phagocytosis even in the absence of the composition. Thus, in some embodiments, the composition can act as a vaccine against atherosclerosis.

[0028] According to certain embodiments, compositions useful for treating or preventing atherosclerosis include at least a portion of an antibody (e.g., a whole antibody or an antibody fragment) that recognizes plaque. For example, the composition may include at least a portion of the plaque-reactive antibody A12, ASA6, or any of a variety of plaque-recognizing antibodies known to those of skill in the art.

[0029] At least a portion of the antibody (e.g., whole antibody or antibody fragment) and the eatme signal may be linked to each other, as discussed in more detail below. In some embodiments, the eatme signal and the antibody form a fusion protein. The fusion protein may be particularly advantageous for treating or preventing arteriosclerosis. In some embodiments, for example, the fusion protein may bind to plaque with higher affinity than would be expected if the same eatme signal were linked to the same antibody or antibody portion by a linker. In some embodiments, the fusion protein may also have a longer life span in a subject, which may also result in improved performance of the composition.

[0030] The antibody can be selected to bind to any of a variety of suitable extracellular substances within the plaque, for example, the antibody can be selected to bind to cholesterol, plaque cells such as white blood cells, calcium, and / or other fatty substances within the plaque.

[0031] In some embodiments, the composition can be used to treat or prevent aging conditions.For example, the eat-me signal of the composition can be configured to bind to senescent cells.Without wishing to be bound by theory, senescent cells can be incapable of cell division and / or can be associated with aging diseases in certain embodiments.

[0032] It is recognized by the present invention that senescent cells can be targeted for phagocytosis by using compositions that include eat-me signals. Phagocytosis of senescent cells can reduce the amount or percentage of senescent cells in the body. The reduction in the amount or percentage of senescent cells can in turn slow or reverse the aging process. Furthermore, in some embodiments, the immune system can eventually learn to recognize senescent cells and target them for phagocytosis even in the absence of the composition. Thus, in some embodiments, the composition can act as a vaccine against aging and related diseases.

[0033] According to certain embodiments, the composition useful for treating or preventing aging comprises at least a portion of an antibody (e.g., a whole antibody or an antibody fragment) that recognizes senescent cells. For example, the composition may comprise at least a portion of canakinumab, an anti-DPP4 antibody, an anti-CD9 antibody, or another of a variety of suitable antibodies that recognize senescent cells and are known to those skilled in the art.

[0034] At least a portion of the antibody (e.g., a whole antibody or an antibody fragment) and the eat-me signal may be linked to each other, as discussed in more detail below. In some embodiments, the eat-me signal and the antibody form a fusion protein. This fusion protein may be particularly advantageous for treating or preventing aging. In some embodiments, for example, the fusion protein may bind to senescent cells with higher affinity than would be expected from the same eat-me signal linked to the same antibody or antibody portion by a linker. In some embodiments, the fusion protein may also have a longer life span in a subject, which may result in improved performance of the composition.

[0035] In some embodiments, the composition can be used to treat or prevent obesity disorders.For example, the eat-me signal of the composition can be configured to bind to fat cells (also known as adipocytes).Excess of fat cells and / or excess size of fat cells can be associated with obesity and many related disorders, such as insulin resistance / diabetes.

[0036] It is recognized herein that the use of compositions comprising eat-me-signal can target fat cells for phagocytosis. The phagocytosis of fat cells can reduce the number of fat cells in the body, thereby treating obesity. Furthermore, in some embodiments, the immune system can eventually learn to recognize fat cells and target them for phagocytosis even in the absence of the composition. Thus, in some embodiments, the composition can act as a vaccine against obesity and related diseases.

[0037] According to certain embodiments, the composition useful for treating or preventing obesity comprises at least a portion of an antibody (e.g., a whole antibody or an antibody fragment) that recognizes adipocytes. For example, the composition may comprise at least a portion of an adipocyte-specific IgG antibody, an adipocyte-type fatty acid binding protein (A-FABP), an anti-ASC-1 antibody, an anti-PAT2 antibody, an anti-P2RX5 antibody, or another of a variety of suitable antibodies that recognize adipocytes and are known to those skilled in the art.

[0038] At least a portion of the antibody (e.g., a whole antibody or an antibody fragment) and the eat-me signal may be linked to each other, as discussed in more detail below. In some embodiments, the eat-me signal and the antibody form a fusion protein. The fusion protein may be particularly advantageous for treating or preventing obesity. In some embodiments, for example, the fusion protein may bind to adipocytes with higher affinity than would be expected from the same eat-me signal linked to the same antibody or antibody portion by a linker. In some embodiments, the fusion protein may also have a longer life span in a subject, which may also result in improved performance of the composition.

[0039] In some embodiments, the composition may be used to treat or prevent viral diseases. For example, the eat-me signal of the composition may be configured to bind to a virus. Exemplary viruses include coronaviruses (e.g., SARS-CoV-2), rhinoviruses, influenza viruses, HIV, or any of a variety of other virus types. The compositions and methods described herein may have a number of advantages for preventing diseases caused by these viruses, as discussed below.

[0040] It is recognized by the present invention that the composition comprising the eat-me signal can be used to target viruses for phagocytosis. Phagocytosis of viruses can reduce the amount or proportion of viruses in the body. The reduction in the number of viruses can then reduce the intensity of symptoms caused by the virus, thereby treating the virus. Furthermore, in some embodiments, the immune system can eventually learn to recognize and target viruses for phagocytosis even in the absence of the composition. Thus, in some embodiments, the composition can act as a vaccine against viruses and related diseases.

[0041] According to certain embodiments, compositions useful for treating or preventing viral diseases include at least a portion of an antibody (e.g., a whole antibody or an antibody fragment) that recognizes a virus. For example, the composition may include at least a portion of any of the following: an antibody that recognizes SARS CoV-2, such as casirivimab, imdevimab, etesevimab, vanlanivimab, or sotrovimab; an antibody that recognizes HIV, such as ibalizumab, 10-1074, or 3BNC117; or an antibody that recognizes a variety of other viruses known to those of skill in the art.

[0042] At least a portion of the antibody (e.g., a whole antibody or an antibody fragment) and the eat-me signal may be linked to each other, as discussed in more detail below. In some embodiments, the eat-me signal and the antibody form a fusion protein. The fusion protein may be particularly advantageous for treating or preventing viral diseases. In some embodiments, for example, the fusion protein may bind to a virus with higher affinity than would be expected from the same eat-me signal linked to the same antibody or antibody portion by a linker. In some embodiments, the fusion protein may also have a longer life span in a subject, which may result in improved performance of the composition.

[0043] In another aspect, the composition comprises an eat-me signal and a fragment from a disease-causing virus, cell, or extracellular material (e.g., senescent cells, fat cells, plaque, or virus, etc.) that induces phagocytosis of the composition. Any suitable fragment can be used. For example, the fragment can comprise a biopolymer, such as a polypeptide (e.g., a protein or fragment thereof) or a polynucleotide (e.g., DNA, RNA). In some embodiments, the fragment is a small molecule (e.g., a component of plaque). In some embodiments, the fragment is a fragment of a surface antigen of a cell or virus. Such a composition can be used to train the immune system to target a disease-causing virus, cell, or extracellular material by eliciting an immune response against a cell, virus, or extracellular material that comprises a fragment bound to the eat-me signal of the composition. The following paragraphs provide a number of specific examples.

[0044] In some embodiments, the composition (e.g., a composition comprising an eat-me signal) is configured to bind to a fragment of an extracellular substance that causes atherosclerosis. For example, the eat-me signal of the composition may be configured to bind to a fragment of plaque. In some embodiments, the fragment is a fragment of a cellular or viral surface antigen found in plaque.

[0045] It is recognized herein that the use of compositions comprising eat-me-signal and plaque fragments can train phagocytes to target plaque for phagocytosis.Phagocytosis of plaque can reduce the amount of plaque, thereby treating or preventing atherosclerosis.Thus, in some embodiments, the composition can act as a vaccine against atherosclerosis.

[0046] Suitable fragments of plaque that may be constituted by the composition include aldehyde dehydrogenase 4 family member A1 (ALDH4A1) or any of a variety of other plaque fragments known to those of skill in the art.

[0047] As discussed elsewhere herein, in some embodiments, the composition may comprise fusion protein. Fusion protein may be particularly advantageous for preventing arteriosclerosis. In some embodiments, fusion protein may also have a longer life span in the subject, which may result in improved performance of the composition.

[0048] In some embodiments, the composition (e.g., a composition comprising an eat-me signal) is configured to bind to a fragment of a senescent cell. For example, the fragment can be a fragment of a surface antigen of a senescent cell. It is recognized herein that the use of a composition comprising an eat-me signal and a fragment of a senescent cell can train phagocytes to target senescent cells for phagocytosis. Phagocytosis of senescent cells can reduce the amount of senescent cells, thereby treating or preventing aging. Thus, in some embodiments, the composition can act as a vaccine against aging.

[0049] Suitable senescent cell fragments that may be comprised by the composition include the CD9 receptor, DPP4, or any of a variety of other senescent cell fragments known to those of skill in the art.

[0050] As discussed elsewhere herein, in some embodiments, the composition may include a fusion protein. Fusion proteins may be particularly advantageous for preventing aging. In some embodiments, fusion proteins may also have a longer life span in a subject, which may result in improved performance of the composition.

[0051] In some embodiments, the composition (e.g., a composition comprising an eat-me signal) is configured to bind to a fragment of an adipocyte. For example, the fragment may be a fragment of a surface antigen of an adipocyte. It is recognized herein that the use of a composition comprising an eat-me signal and a fragment of an adipocyte can train phagocytes to target adipocytes for phagocytosis. Phagocytosis of adipocytes can reduce the amount of adipocytes (and / or the amount of excess-sized adipocytes), thereby treating or preventing obesity. Thus, the composition, in some embodiments, can act as a vaccine against obesity and / or associated diseases such as diabetes.

[0052] Suitable fragments of adipocytes that may be comprised by the composition include adipocyte-type fatty acids, ASC-1, PAT2, P2RX5, or any of a variety of other fragments of adipocytes known to those of skill in the art.

[0053] As discussed elsewhere herein, in some embodiments, the composition may include a fusion protein. Fusion proteins may be particularly advantageous for preventing obesity. In some embodiments, fusion proteins may also have a longer life span in a subject, which may result in improved performance of the composition.

[0054] In some embodiments, the composition (e.g., the composition comprising the eat-me signal) is configured to bind to a fragment of a virus. For example, the fragment can be a fragment of a surface antigen of the virus. Here, it is recognized by the present invention that the use of the composition comprising the eat-me signal and a fragment of the virus can train phagocytes to target the virus for phagocytosis. Phagocytosis of the virus can reduce the amount of the virus, thereby treating or preventing viral disease. Thus, in some embodiments, the composition can act as a vaccine against viral disease.

[0055] Suitable viral fragments that may be comprised by the composition include the SARS-CoV-2 spike protein, the CD4 receptor of the HIV virus, or any of a variety of other viral fragments known to those of skill in the art.

[0056] As discussed elsewhere herein, in some embodiments, the composition may include a fusion protein. Fusion proteins may be particularly advantageous for preventing viral diseases. In some embodiments, fusion proteins may also have a longer life span in a subject, which may result in improved performance of the composition.

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

[0058] A "fusion protein" generally refers to a hybrid polypeptide that contains protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein, or at the carboxy-terminal (C-terminal) portion of the protein, thereby forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein", respectively. A fusion protein may contain different domains, such as an eat-me signal and an antibody; an eat-me signal and an antibody fragment; a disease-causing cell fragment; a viral fragment; or an extracellular protein fragment. In some embodiments, the eat-me signal is fused at the N-terminus of an antibody, an antibody fragment, a disease-causing cell fragment, a viral fragment, or an extracellular protein fragment. In some embodiments, the eat-me signal is fused at the C-terminus of an antibody, an antibody fragment, a disease-causing cell fragment, a viral fragment, or an extracellular protein fragment.

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

[0060] Further provided herein are eat me signal mutants and fusion proteins comprising such eat me signal mutants.In some embodiments, the fusion proteins described herein comprise modifications.When fusion proteins are referred to herein, they encompass all of their mutants and derivatives.Polypeptides that comprise modifications have additional characteristics other than amino acid content. As used herein, a "modification" or "derivative" of a protein or polypeptide (e.g., a fusion protein as described herein) produces a modified or derivatized polypeptide, which is a form of a given peptide that is chemically modified relative to a reference peptide, including, but not limited to, oligomerization or polymerization, modification of amino acid residues or the peptide backbone, crosslinking, cyclization, conjugation, PEGylation, glycosylation, acetylation, phosphorylation, acylation, carboxylation, lipidation, thioglycolamidation, alkylation, methylation, polyglycylation, glycosylation, polysialylation, adenylation, PEGylation, fusion to additional heterologous amino acid sequences, or other modifications that substantially alter the stability, solubility or other properties of the peptide while substantially retaining the activity of the polypeptide as described herein. It should be understood that the fusion protein containing such modifications is crosslinked, cyclized, conjugated, acylated, carboxylated, lipidated, acetylated, thioglycolamidated, alkylated, methylated, polyglycylated, glycosylated, polysialylated, phosphorylated, adenylated, PEGylated, or a combination thereof. In some embodiments, the modified fusion protein of the present disclosure may contain non-amino acid elements such as polyethylene glycol, lipids, polysaccharides or monosaccharides, and phosphates. The fusion protein of the present disclosure may contain the modifications disclosed herein at the C-terminus (e.g., C-terminal amidation), N-terminus (e.g., N-terminal acetylation). Terminal modifications are useful and well known to reduce susceptibility to proteinase digestion, and thus serve to extend the half-life of the polypeptide in solution, especially in biological fluids where proteases may be present.In some embodiments, the fusion proteins described herein are further modified in sequence by terminal NH3 acylation, e.g., acetylation, or thioglycolic acid amidation, terminal carboxylamidation, e.g., modification with ammonia, methylamine, and similar terminal modifications.

[0061] Terminal modifications are useful for reducing susceptibility to proteinase digestion and therefore may play a role in extending the half-life of a polypeptide in solution, particularly in biological fluids where proteases may be present. Amino-terminal modifications include methylation (e.g., --NHCH3 or --N(CH3)2), acetylation (e.g., with acetic acid or its halogenated derivatives, such as a-chloroacetic acid, a-bromoacetic acid, or a-iodoacetic acid), addition of a benzyloxycarbonyl (Cbz) group, or blocking of the amino terminus with any blocking group, including a carboxylate functional group defined by RCOO-- or a sulfonyl functional group defined by R--SO2--, where R is selected from the group consisting of alkyl, aryl, heteroaryl, alkylaryl, etc. Also, des amino acids can be incorporated at the N-terminus (so that the N-terminal amino group is absent) to reduce susceptibility to proteases or restrict the conformation of the polypeptide. In certain embodiments, the N-terminus is acetylated with acetic acid or acetic anhydride.

[0062] Modifications of the carboxy terminus include the introduction of structural constraints by replacing free acids with carboxamide groups or forming cyclic lactams at the carboxy terminus. The peptides described herein can also be cyclized or incorporate des amino or des carboxy residues at the terminus of the peptide to reduce susceptibility to proteases or restrict peptide conformation, eliminating the presence of amino or carboxyl groups at the terminus. Methods for cyclic peptide synthesis are known in the art. C-terminal functional groups of the peptides described herein include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and lower ester derivatives thereof, as well as pharmaceutically acceptable salts thereof.

[0063] In some embodiments, the fusion proteins described herein are phosphorylated. Peptides can also be easily modified by phosphorylation and other methods. In some embodiments, the naturally occurring side chains of the genetically encoded amino acids (or stereoisomeric D amino acids) can also be replaced with other side chains, such as alkyl, lower (C 1-6 ) alkyl, cyclic 4-, 5-, 6- to 7-membered alkyl, amido, amido lower alkyl amido di(lower alkyl), lower alkoxy, hydroxy, carboxy and their lower ester derivatives, and 4-, 5-, 6- to 7-membered heterocycles. For example, proline analogs in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members can be employed.

[0064] The cyclic group may be saturated or unsaturated, and if unsaturated, may be aromatic or non-aromatic. Heterocyclic groups preferably contain one or more nitrogen, oxygen, and / or sulfur heteroatoms. Examples of such groups include furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g., morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl groups. These heterocyclic groups may be substituted or unsubstituted. When a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.

[0065] In some embodiments, the fusion proteins described herein may be linked to one or more polymer moieties. In some embodiments, these polymers are covalently attached to the fusion proteins of the present disclosure. In some embodiments, the polymer is pharma- ceutically acceptable for therapeutic use of the final product preparation. Those skilled in the art will be able to select the desired polymer based on such considerations as whether the polymer-peptide conjugate will be used therapeutically, if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations.

[0066] All combinations of the different modifications and derivatizations are envisioned for the fusion proteins described herein.

[0067] Another aspect of the present disclosure provides a method for producing a fusion protein. The fusion protein can be produced and isolated by expression form recombinant nucleic acid in a suitable cell (e.g., bacterial cell or eukaryotic cell). To produce the fusion protein, the nucleic acid encoding the fusion protein can be introduced into a cell (e.g., bacterial cell or eukaryotic cell such as yeast cell or insect cell). The cell can be cultured under conditions that allow the fusion protein to be expressed from the nucleic acid encoding the fusion protein. The fusion protein including the signal peptide can be secreted, for example, into the culture medium and then recovered. The fusion protein can be isolated using any method of purifying proteins known in the art.

[0068] The nucleic acid encoding the fusion protein described herein can be obtained by any method known in the art, and the nucleotide sequence of the nucleic acid can be determined. Those skilled in the art can identify the nucleotide sequence encoding the fusion protein from the amino acid sequence of the fusion protein. The nucleic acid encoding the fusion protein of the present disclosure can be DNA or RNA, double-stranded or single-stranded. In some embodiments, the nucleotide sequence encoding the fusion protein can be codon-optimized to accommodate different expression systems (e.g., for mammalian expression).

[0069] In some embodiments, the nucleic acid is contained within a vector, such as an expression vector. In some embodiments, the vector comprises a promoter operably linked to the nucleic acid.

[0070] A variety of promoters can be used for expression of the fusion proteins described herein, including, but not limited to, the cytomegalovirus (CMV) intermediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Simian Virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and herpes simplex tk virus promoter.

[0071] Controllable promoters can also be used, including those that use the lac repressor from E. coli as a transcriptional modulator to control transcription from mammalian cell promoters with the lac operator, and those that use the tetracycline repressor (tetR). Other systems include FK506 dimers, VP16 or p65 with astroradiol, RU486, diphenol murislerone, or rapamycin.

[0072] A controllable promoter containing a repressor may be used in conjunction with an operon. In one embodiment, the lac repressor from E. coli may function as a transcriptional modulator to control transcription from a mammalian cell promoter with a lac operator. In one embodiment, a tetracycline-inducible switch is used.

[0073] In addition, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene, for the selection of stable or transient transfectants in mammalian cells; an enhancer / promoter sequence from the immediate early gene of human CMV for high-level transcription; a transcription termination signal and an RNA processing signal from SV40 for mRNA stability; an SV40 polyoma origin of replication and ColE1 for proper episomal replication; an internal ribosome binding site (IRES), a versatile multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Methods for making suitable vectors and vectors containing transgenes are well known and available in the art.

[0074] An expression vector containing the nucleic acid can be transfected into a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation), and the transfected cells can then be cultured by conventional techniques to produce the fusion proteins described herein. In some embodiments, expression of the fusion proteins described herein is controlled by a constitutive, inducible, or tissue-specific promoter.

[0075] Host cells used to express the fusion proteins described herein may include bacterial cells such as E. coli, eukaryotic cells, etc. In one embodiment, mammalian cells such as Chinese hamster ovary cells (CHO) may be used.

[0076] A variety of host-expression vector systems may be utilized to express the fusion proteins described herein. Such host-expression systems represent vehicles in which the coding sequences of the isolated fusion proteins described herein may be produced and subsequently purified, but also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequences, may express the fusion proteins described herein in situ. These include microorganisms such as bacteria [e.g., Escherichia coli and B. subtilis] transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the coding sequences of the fusion proteins described herein; yeast [e.g., Saccharomyces pichia] transformed with recombinant yeast expression vectors containing the coding sequences of the fusion proteins described herein; pichia)]; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing sequences encoding the fusion proteins described herein; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)] or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing sequences encoding the fusion proteins described herein; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphocytic cells, Per C.6 cells) harboring recombinant expression constructs containing promoters derived from the genome of a mammalian cell (e.g., metallothionein promoter) or from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0077] In bacterial systems, a number of expression vectors may be conveniently selected depending on the intended use of the expressed fusion protein. For example, when producing large quantities of such proteins, a vector that induces expression of a high level of a fusion protein product that can be easily purified may be desirable for the production of pharmaceutical compositions of the fusion proteins described herein. Such vectors include, but are not limited to, the E. coli expression vector pUR278; pIN vectors, in which coding sequences can be individually ligated into the vector in frame with the lac Z coding region to produce a fusion protein. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0078] In mammalian host cells, a number of viral-based expression systems are available. When adenovirus is used as an expression vector, the coding sequence of interest may be ligated to the adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., regions E1 or E3) results in recombinant viruses that are viable and capable of expressing immunoglobulin molecules in infected hosts. Specific initiation signals may also be required for efficient translation of the inserted antibody coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, to ensure translation of the entire insert, the initiation codon must be in phase with the reading frame of the desired coding sequence. These exogenous translational control signals and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be improved by the inclusion of appropriate transcription enhancer elements, transcription terminators, and the like.

[0079] In addition, a host cell line can be selected that modulates the expression of the inserted sequence or modifies and processes the gene product in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Purification and modification of recombinant proteins are well known in the art, so that the design of polyprotein precursors can include a number of embodiments that are readily understood by those skilled in the art. Any known protease or peptidase known in the art can be used for the described modifications of precursor molecules, such as thrombin or factor Xa, enterokinase, furin, and AcTEV, and foot and mouth disease virus protease C3.

[0080] Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. To ensure the correct modification and processing of expressed foreign proteins, appropriate cell lines or host systems can be selected. For this purpose, eukaryotic host cells that have the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, HeLa, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.

[0081] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines can be engineered that stably express the fusion proteins described herein. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. After introduction of the foreign DNA, engineered cells can be grown in enriched medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express the fusion proteins described herein. Such engineered cell lines can be particularly useful for screening and evaluating fusion proteins that interact directly or indirectly with the fusion proteins described herein.

[0082] The expression level of the fusion described herein can be increased by vector amplification.If the marker in the vector system expressing the fusion protein described herein is amplifiable, the increase in the level of inhibitor present in the culture of the host cell increases the copy number of the marker gene.Since the amplified region is associated with the fusion protein described herein or the nucleotide sequence of the fusion protein described herein, the production of the fusion protein also increases.

[0083] Once a fusion described herein is recombinantly expressed, it may be purified by any method known in the art for the purification of polypeptides, polyproteins or antibodies (e.g., similar to antibody purification schemes based on antigen selectivity), such as, for example, chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen (optionally after Protein A selection if the polypeptide contains an Fc domain (or portion thereof)), and sizing column chromatography), centrifugation, differential lysis, or any other standard technique for the purification of polypeptides or antibodies.

[0084] In some embodiments, fusion proteins can be advantageously resistant to degradation compared to other compositions that contain bound antibodies and eat-me signals. Fusion proteins can also provide unexpectedly high molecular performance. For example, an eat-me signal, an antibody, an antibody fragment, a disease-causing cell fragment, a virus fragment, or an extracellular protein fragment can have unexpectedly high activity when included in a fusion protein of a composition compared to the activity of a composition in which an eat-me signal is crosslinked with an antibody, an antibody fragment, a disease-causing cell fragment, a virus fragment, or an extracellular protein fragment. This can advantageously improve the efficacy of a composition that contains a fusion protein.

[0085] The treatments disclosed herein may be administered to any subject, e.g., a human, or a non-human animal, such as a dog, cat, horse, rabbit, cow, pig, sheep, goat, rat (e.g., Rattus Norvegicus), mouse (e.g., Mus musculus), guinea pig, or non-human primate (e.g., monkey, chimpanzee, baboon, ape, gorilla, etc.).

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

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

[0088] The compositions described herein can be administered to a subject (e.g., a human) using any of a variety of suitable techniques, as discussed in more detail below. The compositions can be administered to a subject at any suitable dose. For example, the compositions can be administered at a dose of 10 mg or more, 15 mg or more, 20 mg or more, 25 mg or more, 30 mg or more, 40 mg or more, 50 mg or more, 60 mg or more, 70 mg or more, 80 mg or more, 90 mg or more, 100 mg or more, 200 mg or more, 300 mg or more, 500 mg or more, 1000 mg or more, 1500 mg or more, 2000 mg or more, 2500 mg or more, 3000 mg or more, 5000 mg or more, etc. In some embodiments, the composition may be applied at a dosage of 5000mg or less, 3000mg or less, 2500mg or less, 2000mg or less, 1500mg or less, 1000mg or less, 500mg or less, 300mg or less, 200mg or less, 100mg or less, 90mg or less, 80mg or less, 70mg or less, 60mg or less, 50mg or less, 40mg or less, 30mg or less, 25mg or less, 20mg or less, 15mg or less, 10mg or less, etc. Combinations of these ranges are also possible. For example, the dosage may be 10mg or more and 20mg or less, 50mg or more and 100mg or less, or 100mg or more and 200mg or less.

[0089] In some embodiments, the composition may be administered to a subject (e.g., a human) at a dosage of 1 mg / kg or more, 2 mg / kg or more, 3 mg / kg or more, 4 mg / kg or more, 5 mg / kg or more, 6 mg / kg or more, 7 mg / kg or more, 8 mg / kg or more, 9 mg / kg or more, 10 mg / kg or more, 11 mg / kg or more, 12 mg / kg or more, 13 mg / kg or more, 14 mg / kg or more, or 15 mg / kg or more. In some embodiments, the statin may be administered at a dosage of 15 mg / kg or less, 14 mg / kg or less, 13 mg / kg or less, 12 mg / kg or less, 11 mg / kg or less, 10 mg / kg or less, 9 mg / kg or less, 8 mg / kg or less, 7 mg / kg or less, 6 mg / kg or less, 5 mg / kg or less, 4 mg / kg or less, 3 mg / kg or less, 2 mg / kg or less, or 1 mg / kg or less. Combinations of these ranges are also possible. For example, in some embodiments, the dosage can be from 1 mg / kg to 15 mg / kg, from 1 mg / kg to 10 mg / kg, or from 5 mg / kg to 10 mg / kg.

[0090] In other aspects, the compositions described herein may be administered to a subject. The compositions may be administered alone. In some embodiments, the compositions are administered together with cofactors, other therapeutic agents, etc.

[0091] The composition may be applied in a therapeutically effective pharma- ceutically acceptable amount in a pharma- ceutically acceptable formulation, e.g., a pharma- ceutically acceptable carrier, as described below. The term "effective amount" of a composition, such as the compositions described herein, refers to the amount necessary or sufficient to achieve a desired biological effect. For example, an effective amount of a composition for treating a tumor may be an amount sufficient to reduce the size of the tumor. By selecting from various active compositions, in combination with the teachings provided herein, and weighing factors such as potency, relative bioavailability, patient weight, severity of side effects, and mode of administration, one can design an effective prophylactic or therapeutic treatment regimen that does not cause substantial toxicity, yet is completely effective in treating a particular subject. The effective amount for any particular application may vary depending on factors such as the disease or condition being treated, the particular composition being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of the composition without undue experimentation.

[0092] As used herein, the terms "treat", "treated", "treating" and the like refer to the administration of a composition to a subject that may increase the subject's resistance to a disease or increase resistance to further progression of the disease, control the progression of the disease, and / or slow the progression of the disease, or reduce the severity of the symptoms of the disease. The effective amount may depend on the particular disease being treated and the desired outcome.

[0093] In some embodiments, the therapeutically effective dose of the composition can be determined first from animal models.The applied dose can be adjusted based on the relative bioavailability and efficacy of the compound administered.Based on the above method and other methods well known in the art, it is well within the capabilities of those skilled in the art to adjust the dose to achieve maximum efficacy.

[0094] In administering the compositions to a subject, the dose, administration schedule, and administration route, etc., can be selected to affect the known activity of these compositions. The dosage can be appropriately estimated based on the results of experimental models in combination with the results of the assays of the compositions described herein. The dosage can be appropriately adjusted to achieve the desired local or systemic drug level depending on the mode of administration. The dosage can be administered once or in several doses per day. Multiple doses per day can be used to achieve the appropriate systemic level of the composition in the subject or in the cells or viruses targeted by the composition.

[0095] The dose may be selected to provide a therapeutically effective amount of the composition to the cell or virus targeted by the composition. The dose may be administered at a maximum safe dose. The maximum safe dose may be selected to deliver a high therapeutically effective amount of the composition to the subject, but with minimal risk to the subject of potentially harmful side effects.

[0096] The dose of the composition may be selected to have a desired concentration in the targeted virus or 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 manner when co-administered with other treatments (e.g., as a cocktail), or for any of a variety of other purposes known to those of skill in the art.

[0097] Dose can be selected at least in part based on the condition related to the subject.For example, dose can be selected at least in part based on the species, age, sex, weight, size, environment, metabolism, physical condition or current health condition of the subject.In some cases, subject-specific maximum dose can be used.In some cases, dose can be administered in a way that limits adverse effects on the subject (e.g., dose can be delivered orally, nasally, intravenously, etc., depending on the need of the subject).

[0098] Doses of the composition may be administered daily, weekly, or monthly, and any other amount of time in between.

[0099] The dose may include any suitable amount of the composition. In some embodiments, the dose of the composition is 0.1 micrograms or more, 0.5 micrograms or more, 10 micrograms or more, 50 micrograms or more, 100 micrograms or more, 500 micrograms or more, 1000 micrograms or more, or more than that. In some embodiments, the dose of the composition is 10000 micrograms or less, 5000 micrograms or less, 1000 micrograms or less, 500 micrograms or less, 100 micrograms or less, 50 micrograms or less, or less than that. Combinations of these ranges are also possible. For example, in some embodiments, the dose of the composition is 0.5 micrograms or more and 10000 micrograms or less.

[0100] The dose of the composition may be administered at any of a variety of average rates. In some embodiments, the dose is administered at a rate of 0.01 micrograms / hour or more, 0.05 micrograms / hour or more, 0.1 micrograms / hour or more, 0.5 micrograms / hour or more, 1 micrograms / hour or more, 5 micrograms / hour or more, 10 micrograms / hour or more, 50 micrograms / hour or more, 100 micrograms / hour or more, 500 micrograms / hour or more, 1000 micrograms / hour or more or faster. In some embodiments, the dose is administered at a rate of 10000 micrograms / hour or less, 5000 micrograms / hour or less, 1000 micrograms / hour or less, 500 micrograms / hour or less, 100 micrograms / hour or less, 50 micrograms / hour or less, 10 micrograms / hour or less, 1 micrograms / hour or less, 0.5 micrograms / hour or less, 0.1 micrograms / hour or less or less. Combinations of these ranges are possible. For example, in some embodiments, the dose is administered at a rate of at least 0.01 micrograms / hour and up to 10,000 micrograms / hour.

[0101] The composition can be administered for an extended period of time. In some embodiments, the dose is administered for 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 12 hours or more, 24 hours or more, 3 days or more, 7 days or more, 10 days or more, 14 days or more, 21 days or more, 30 days or more, 45 days or more, 60 days or more, 90 days or more, or longer. In some embodiments, the dose is administered for 365 days or less, 200 days or less, 90 days or less, 60 days or less, 45 days or less, 30 days or less, 21 days or less, 14 days or less, 10 days or less, 7 days or less, 3 days or less, 24 hours or less, 12 hours or less, or less. Combinations of these ranges are possible. For example, in some embodiments, the dose is administered for 1 hour or more and 365 days or less.

[0102] The subject can be any suitable subject. For example, the subject can be a mammal, such as a human, or a non-human animal, such as a dog, cat, horse, rabbit, cow, pig, sheep, goat, rat (e.g., Rattus Norvegicus), mouse (e.g., Mus musculus), guinea pig, non-human primate (e.g., monkey, chimpanzee, baboon, ape, gorilla, etc.), etc.

[0103] Administration of the compositions of the present disclosure can be accomplished by any of a variety of medically acceptable methods that allow the composition to reach its target cells or viruses. The particular mode selected can of course depend on factors such as those described above, such as the particular composition, the severity of the condition of the subject being treated, the dosage required for therapeutic effect, etc. As used herein, a "medically acceptable" therapeutic mode is one that can produce an effective level of the composition in the subject without causing clinically unacceptable side effects. In the case of neurodegenerative diseases, the blood-brain barrier may prevent the drug from reaching senescent cells. In this case, the medically acceptable mode can be administration via cerebrospinal fluid, i.e., intrathecal administration.

[0104] The composition may be administered to a subject by any of a variety of medically acceptable methods. Administration may be localized (e.g., to a specific area, physiological system, tissue, organ, or cell type) or systemic, depending on the condition to be treated. For example, the composition may be administered orally, vaginally, rectally, buccal, pulmonary, topically, nasally, transdermally, by parenteral injection or implantation, via surgical administration, or by any other administration method that achieves access to the tumor. In some cases, for example, when more than one composition is administered, more than one administration method may be used.

[0105] Examples of parenteral administration methods that can be used include intravenous, intradermal, subcutaneous, intracavitary, intramuscular, intraperitoneal, epidural, or intrathecal administration. Examples of implantation administration methods include implantation or injection of any implantable or injectable drug delivery system. Oral administration can be used in some embodiments. Oral administration can be advantageous due to convenience for the subject as well as administration schedule. Compositions suitable for oral administration can be administered using separate units such as hard or soft capsules, pills, cachets, tablets, troches, dissolving films, or lozenges. Other oral compositions suitable for use include liquids or suspensions such as syrups, elixirs, or emulsions. Liquids or suspensions can include aqueous or non-aqueous liquids. In some embodiments, the composition can be used to fortify food or beverages.

[0106] When it is desired to deliver the composition systemically, the composition can be formulated for parenteral administration by injection, for example, bolus injection or continuous infusion.The preparation for injection can be provided in a unit dosage form, for example, in ampoules or multi-dose containers, with the addition of preservatives.The composition can take the form of a suspension, solution, or emulsion in an oily or aqueous medium, and can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents.

[0107] Pharmaceutical preparations for parenteral administration may include aqueous solutions of the active compounds in water-soluble form. Suspensions of the active compounds may in some cases be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles may include, but are not limited to, fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may include substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. The suspension may include suitable stabilizers. In some embodiments, the suspension includes agents that increase the solubility of the compound. Stabilizers or solubility enhancers may allow the preparation of highly concentrated solutions.

[0108] The composition may in some embodiments be formulated as a depot preparation. Such long-acting preparations may in some cases be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as poorly soluble derivatives, e.g., as poorly soluble salts.

[0109] The composition may be prepared in the form of a liquid pharmaceutical preparation. In some embodiments, the liquid pharmaceutical preparation comprises a solution, a suspension, or an emulsion. The composition may be included in the liquid pharmaceutical preparation in any of a variety of forms. For example, the composition may be included in an aqueous solution or saline solution (e.g., suitable for inhalation), may be microencapsulated, may be encochleated, may be coated on fine gold particles, may be contained in liposomes, may be nebulized, may be aerosolized, or may be dried on a sharp object and scraped into the skin.

[0110] The composition may be prepared in the form of a solid pharmaceutical preparation. For example, the composition may be included in granules; powders; tablets; coated tablets; (micro)capsules; suppositories; syrups; emulsions; suspensions; creams; or drops or preparations with extended release of the active compound. In some embodiments, the composition is prepared in the form of a solid pharmaceutical preparation that is configured to be dissolved or otherwise converted into a pharmaceutical liquid preparation. For example, the composition may be prepared in the form of a dry powder that can be dissolved in water to form an aqueous solution. The liquid or solid composition may include additives such as excipients. In some embodiments, the additives include disintegrants, binders, coating agents, swelling agents, lubricants, flavors, sweeteners, or solubilizers, although any of a variety of additives may be used, and the disclosure is not so limited.

[0111] In some embodiments, the composition can be administered to provide continuous exposure to the composition over a period of time, for example, hours, days, weeks, months, or years.

[0112] For example, the composition can be administered by repeated administration or controlled release.For example, the composition can be controlled release over a period of time so as to maintain the dosage of the composition during that period.Controlled release can be achieved, for example, by oral dosage form, bolus injection, transdermal patch, subcutaneous implant, or other methods as described herein.In some cases, it may be desirable to maintain a substantially constant concentration of the composition.

[0113] Other delivery systems suitable for use in certain embodiments may include time-release, delayed release, sustained release, or controlled release delivery systems. Such systems can advantageously avoid the need for repeated administration and increase the convenience of the subject. Many types of delivery systems are available and known to those skilled in the art. The composition can be administered using delivery systems such as, for example, polymer-based systems such as polylactic acid and / or polyglycolic acid, polyanhydrides, polycaprolactones, and / or combinations thereof; lipid-based non-polymeric systems including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di-, and triglycerides; hydrogel release systems; liposome-based systems; phospholipid-based systems; silastic systems; peptide-based systems; wax coatings; compressed tablets with conventional binders and excipients; or partially fused implants. Specific examples include, but are not limited to, erosion systems in which the composition is contained in a form within a matrix, or diffusion systems in which the active ingredient controls the release rate. The formulation may be present, for example, as microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, or polymeric systems. In some embodiments, the system may allow for sustained or controlled release of the composition, for example, through control of the diffusion or erosion / degradation rate of the formulation. Additionally, in some embodiments, a pump-based hardware delivery system may be used.

[0114] In some embodiments, the composition is delivered using an implant. The implant can be configured for short-term or long-term release of the composition. In some embodiments, the delivery system is a long-term release implant. The use of a long-term release implant can be particularly suitable in some embodiments. As used herein, "long-term release" means that the implant containing the composition described herein is constructed and arranged to deliver therapeutically effective levels. In some embodiments, the composition is delivered at therapeutically effective levels for 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 12 hours or more, 24 hours or more, 3 days or more, 7 days or more, 10 days or more, 14 days or more, 21 days or more, 30 days or more, 45 days or more, 60 days or more, 90 days or more, or longer. In some embodiments, the composition is delivered at therapeutically effective levels for 365 days or less, 200 days or less, 90 days or less, 60 days or less, 45 days or less, 30 days or less, 21 days or less, 14 days or less, 10 days or less, 7 days or less, 3 days or less, 24 hours or less, 12 hours or less, or less. Combinations of these ranges are possible. For example, in some embodiments, the composition is delivered at therapeutically effective levels for a period of at least one hour and up to 365 days. Long-term release implants are well known to those skilled in the art and include some of the release systems described herein.

[0115] In certain embodiments, the composition can be combined with a suitable pharma- ceutically acceptable carrier. In general, pharma- ceutically acceptable carriers suitable for use are well known to those skilled in the art. As used herein, "pharma- ceutically acceptable carrier" refers to a non-toxic substance that does not significantly inhibit the effectiveness of the biological activity of the active compound(s) administered, but is used as a formulation component, for example, to stabilize or protect the active compound(s) in the composition prior to use. For example, the composition can be incorporated into liposomes, polymeric release systems, suspended in liquid (e.g., in dissolved or colloidal form), or used in other ways as described herein. The carrier can include one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other vertebrates. The term "carrier" refers to an organic or inorganic component, which may be natural or synthetic, with which one or more active compounds of the present disclosure are combined to facilitate application. The carrier can be mixed or otherwise mixed with one or more compositions described herein, and / or with each other, in a manner such that there is no interaction that would substantially impair the desired medicinal effect. Carrier can be either soluble or insoluble depending on the application.Well-known examples of carrier include, but are not limited to, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite.Carrier can be either soluble or insoluble in nature.

[0116] In some embodiments, the formulations described herein can be administered in pharmaceutically acceptable solution.Pharmaceutically acceptable solution can routinely contain pharmaceutically acceptable concentrations of salt, buffer, preservative, compatible carrier, adjuvant, emulsifier, diluent, excipient, chelating agent, filler, desiccant, antioxidant, antibacterial agent, preservative, binder, bulking agent, silica, solubilizer, stabilizer, and other therapeutic ingredients that can be used with the composition optionally.For example, when the formulation is liquid, carrier can be solvent, partial solvent, or non-solvent, and can be aqueous or organic based. Non-limiting examples of suitable formulation ingredients include diluents such as calcium carbonate, sodium carbonate, lactose, kaolin, calcium phosphate, or sodium phosphate; granulating and disintegrating agents such as corn starch or algenic acid; binding agents such as starch, gelatin, or acacia; lubricating agents such as magnesium stearate, stearic acid, or talc; time delay materials such as glycerol monostearate or glyceryl distearate; suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone; dispersing or wetting agents such as lecithin or other naturally occurring phosphatides; thickening agents such as cetyl alcohol or beeswax; buffering agents such as acetic acid and its salts, citric acid and its salts, boric acid and its salts, or phosphoric acid and its salts; or preservatives such as benzalkonium chloride, chlorobutanol, parabens, or thimerosal. The compositions of the present disclosure may be formulated into solid, semi-solid, liquid or gaseous form preparations such as tablets, capsules, elixirs, powders, granules, ointments, solutions, deposits, inhalants or injectables.

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

[0118] The preparation may include a solution, suspension, or emulsion as described above. The solution, suspension, or emulsion may be sterile. In some embodiments, the solution, suspension, or emulsion is aqueous. In some embodiments, the solution, suspension, or emulsion is non-aqueous. The sterile aqueous or non-aqueous solution, suspension, or emulsion may be isotonic with the blood of the subject in certain embodiments. Non-limiting examples of non-aqueous solvents are polypropylene glycol, polyethylene glycol, vegetable oils such as olive oil, sesame oil, coconut oil, arachis oil, peanut oil, mineral oil, injectable organic esters such as ethyl oleate, or fixed oils including synthetic mono- or diglycerides. The solution, suspension, or emulsion may include an aqueous carrier. Exemplary aqueous carriers that may be used include, but are not limited to, water, alcoholic / aqueous solutions, emulsions or suspensions including saline and buffered media. The preparation may further comprise a parenteral vehicle configured to improve the suitability of the solution, suspension, or emulsion for parenteral delivery. Parenteral vehicles include sodium chloride solution, 1,3-butanediol, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. In some embodiments, preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases and the like.

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

[0120] The compositions discussed herein may be administered neat or in the form of a pharma- ceutically acceptable salt. Although non-pharma-ceutically acceptable salts may be used to prepare the compositions, when used in medicine, the salts should be pharma-ceutically acceptable. The term "pharma-ceutically acceptable salts" includes salts of the compositions described herein, which are prepared, for example, in combination with an acid or base. Pharmaceutically acceptable salts can be prepared as alkali metal salts, such as lithium, sodium, or potassium salts; or alkaline earth metal salts, such as magnesium or calcium salts. Examples of suitable bases that can be used to form salts include ammonium or suitable bases, such as sodium hydroxide, lithium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, etc. Examples of suitable acids that can be used to form salts include inorganic or mineral acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, phosphoric acid, etc. Other suitable acids include organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, glucuronic acid, galacturonic acid, salicylic acid, formic acid, naphthalene-2-sulfonic acid, and the like. Still other suitable acids include amino acids such as alginic acid, aspartic acid, glutamic acid, and the like. Such salts can also be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium, or calcium salts of the carboxylic acid group.

[0121] In another embodiment, the present disclosure also provides any of the above-mentioned compositions in a kit, which optionally includes instructions for use of the composition for treatment of a disease or condition (e.g., by targeting senescent cells, fat cells, plaques, or viruses such as HIV or SARS-CoV-2). In some cases, the kit may include instructions for use of the compositions discussed herein. The kit may also include instructions for use of a combination of two or more compositions. Instructions for use may also be provided for administering the composition by any suitable technique, such as those described above, for example, orally, intravenously, via a pump or implantable delivery device, or another known route of drug delivery.

[0122] The kits described herein can also include one or more containers. The containers can contain compositions and other components as described above. The kits can also, in some cases, include instructions for mixing, diluting, and / or administering the compositions of the present disclosure. The kits can also include other containers that contain one or more solvents, surfactants, preservatives, and / or diluents (e.g., normal saline (0.9% NaCl), or 5% dextrose), as well as containers for mixing, diluting, or administering the components in the sample to a subject in need of such treatment.

[0123] The composition of the kit may be provided in any suitable form, for example, as a liquid or solid pharmaceutical preparation. If the composition provided is a dry powder, the composition may be reconstituted by adding a suitable solvent, which may also be provided in some cases. In embodiments in which a liquid form of the composition is used, the liquid form may be concentrated or ready to use. The solvent depends on the composition and the mode of use or administration. Solvents suitable for pharmaceutical compositions are well known and available in the literature, for example as described above. The solvent depends on the composition and the mode of use or administration.

[0124] In yet other aspects, the disclosure includes promoting one or more of the above-mentioned embodiments, e.g., promoting the treatment or prevention of a tumor in vitro or in vivo, e.g., by administering a composition as described herein to a subject.

[0125] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

[0126] The antibodies described herein may include biopolymers such as polypeptides. For example, the antibodies may include proteins. In some embodiments, the antibodies are glycoproteins. However, in some embodiments, the antibodies that include proteins are glycoproteins. The antibodies may be substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Any of a variety of immunoglobulin genes or fragments thereof are known to those of skill in the art. For example, the antibodies may be substantially encoded by immunoglobulin genes, including kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes and fragments thereof. The antibodies may be encoded by light chain immunoglobulin genes or fragments thereof. Light chain immunoglobulins may be classified as either kappa or lambda.

[0127] Antibodies may be encoded by heavy chain immunoglobulin genes or fragments thereof. Heavy chain immunoglobulins can be classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.

[0128] An immunoglobulin structural unit may comprise a tetramer. Each tetramer may be composed of two pairs of identical polypeptide chains, each pair having one light and one heavy chain.

[0129] In some embodiments, the light chain has a molecular weight of 15 kDa or more, 20 kDa or more, 25 kDa or more, or more. In some embodiments, the light chain has a molecular weight of 35 kDa or less, 30 kDa or less, 25 kDa or less, or less. Combinations of these ranges are possible. For example, in some embodiments, the light chain has a molecular weight of 15 kDa or more and 35 kDa or less.

[0130] In some embodiments, the heavy chain has a molecular weight of 50 kDa or more, 55 kDa or more, 60 kDa or more, or more. In some embodiments, the heavy chain has a molecular weight of 70 kDa or less, 65 kDa or less, 60 kDa or less, or less. Combinations of these ranges are possible. For example, in some embodiments, the heavy chain has a molecular weight of 50 kDa or more and 70 kDa or less.

[0131] The N-terminus of each polypeptide chain may define the variable region of the immunoglobulin structural unit. The variable region may be primarily responsible for antigen recognition. In some embodiments, the variable region comprises 95 or more, 98 or more, 100 or more, 103 or more, 105 or more, or more amino acids. In some embodiments, the variable region comprises 115 or less, 113 or less, 110 or less, 108 or less, 105 or less, or fewer amino acids. Combinations of these ranges are also possible. For example, in some embodiments, the variable region comprises 95 or more and 115 or less amino acids.

[0132] Antibodies may exist as intact immunoglobulins, however, in some embodiments, antibodies exist as any of a number of immunoglobulin fragments.

[0133] Immunoglobulin fragments may be produced by digestion with any of a variety of peptidases (e.g., pepsin). For example, in some embodiments, immunoglobulin fragments may be formed by digesting an antibody with pepsin. In some exemplary embodiments, pepsin is used to digest the Fc domain of an antibody, for example by breaking down disulfide bonds in the hinge region of the Fc domain, to generate F(ab)'2. F(ab)'2 is, according to certain embodiments, a dimer of Fab, which is itself a light chain linked to VH-CH1 by a disulfide bond. F(ab)'2 may be reduced to break the disulfide bond in the hinge region, thereby converting the (Fab') dimer into a Fab' monomer. According to some embodiments, the Fab' monomer comprises a Fab and a portion of the hinge region of the Fc domain.

[0134] In some embodiments, immunoglobulin fragments are synthesized de novo. Immunoglobulin fragments can be produced by any of a variety of methods known to those of skill in the art, such as chemical synthesis, using recombinant DNA methodology, or "phage display" methods.

[0135] Exemplary antibodies include single chain antibodies, such as single chain Fv (scFv) antibodies in which a variable heavy chain and a variable light chain are linked (directly or via a peptide linker) to form a contiguous polypeptide, hi one embodiment, the antibody is a monoclonal antibody.

[0136] In some embodiments, compositions comprising an antibody or antibody fragment are configured such that the antibody or antibody fragment retains a relatively high affinity for a target molecule. Without wishing to be bound by theory, the affinity of an antibody or antibody fragment for a target is determined by the dissociation constant (K D ) may be inversely proportional to K D The lower the value, the higher the affinity. In some embodiments, the K D Values ​​are 10 under physiological conditions. -6 M or less, 10 -7M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 In some embodiments, the K of the antibody or antibody fragment is equal to or less than M. D The value is 10 -13 M or above, 10 -12 M or above, 10 -11 M or higher. Combinations of these ranges are possible. For example, in some embodiments, the K D The value is 10 -12 M or more 10 -6 M or less. D The value can be determined by tests that would be well known to one skilled in the art.

[0137] In some embodiments, the K of the composition D is the K of a pure antibody or antibody fragment similar to the antibody or antibody fragment of the composition. D This relatively high affinity of the composition for its target may be related to the fact that the composition is a fusion protein.

[0138] In this context, the term "antibody fragment" can refer to at least a portion of an antibody that retains the ability to specifically interact (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution) with an epitope of an antigen. Examples of antibody fragments are described elsewhere herein and include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFvs), Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies (either VL or VH) such as sdAbs, camelid VHH domains, multispecific antibodies formed from antibody fragments such as bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, and any antibody fragment comprising an isolated CDR or other epitope-binding fragment of an antibody. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs.

[0139] In some embodiments, the antibody may be a minibody or nanobody. In the context of the present disclosure, it is recognized by the present invention that the use of a minibody or nanobody may advantageously increase the binding affinity of the antibody compared to a full-sized antibody.

[0140] The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention. EXAMPLES

[0141] [Example 1] In one prophetic example, a 60-year-old man with reduced stamina (e.g., as a symptom of aging) is injected with a composition comprising a fusion protein that includes calreticulin and an antibody against senescent muscle cells. The fusion protein binds to the senescent cells, and the senescent cell / fusion protein composition is recognized by antigen-presenting cells, such as dendritic cells, and is taken up by such cells and transported to lymph nodes, where the virus educates T cells to recognize it. Such T cells are then effective in seeking out and killing other senescent cells. Subjects thus treated regain the ability to walk longer distances. [Example 2]

[0142] In one prophetic example, a 60-year-old male with a 90% stenosis of the left anterior descending artery (LAD) is injected with a composition comprising a fusion protein comprising calreticulin and an antibody against plaque. The fusion protein binds to the plaque, and the plaque / fusion protein composition is recognized by antigen-presenting cells such as dendritic cells, taken up by such cells, and transported to lymph nodes, where the virus educates T cells to recognize it. Such T cells are then effective in seeking out and eliminating the plaque. Subjects thus treated were able to reduce LAD stenosis from 90% to 45% in 60 days. [Example 3]

[0143] In one prophetic example, a 60-year-old man with an obesity index of 38 is injected with a composition comprising a fusion protein that includes calreticulin and an antibody against adipocytes. The fusion protein binds to adipocytes, and the adipocyte / fusion protein composition is recognized by antigen-presenting cells, such as dendritic cells, taken up by such cells, and transported to lymph nodes, where the virus educates T cells for recognition. Such T cells are then effective in seeking out and killing other adipocytes. A subject thus treated was able to reduce his obesity index to 34 in two months. [Example 4]

[0144] In one prophetic example, a human infected with SARS-CoV-2 is injected with a composition comprising a fusion protein that includes calreticulin and an antibody against SARS-CoV-2. The fusion protein binds to the virus, and the virus / fusion protein composition is recognized by antigen-presenting cells, such as dendritic cells, and transported to lymph nodes, where the virus educates and recognizes T cells. Such T cells are then effective in seeking out and killing the virus. Subjects so treated are cured of the disease and immunized against future exposure to the same virus. [Example 5]

[0145] In this prophetic example, a subject is injected with a composition comprising a fusion protein comprising an eat-me signal, such as calreticulin, and a surface antigen of a senescent cell. Antigen-presenting cells, such as dendritic cells, recognize the composition, and the composition is taken up by the dendritic cells. The dendritic cells transport the composition to lymph nodes, where they educate T cells to recognize senescent cells. The educated T cells then kill the senescent cells whenever they encounter them. Such a subject is immunized against aging. [Example 6]

[0146] In this prophetic example, a subject is injected with a composition comprising a fusion protein that includes an eat-me signal, such as calreticulin, and a surface antigen of a plaque. Antigen-presenting cells, such as dendritic cells, recognize the composition, and the composition is taken up by the dendritic cells. The dendritic cells transport the composition to lymph nodes, where they educate T cells to recognize plaque. The educated T cells then remove plaque whenever they encounter it. Such a subject is immunized against atherosclerosis. [Example 7]

[0147] In this prophetic example, a subject is injected with a composition comprising a fusion protein comprising an eat-me signal, such as calreticulin, and a surface antigen on an adipocyte. Antigen presenting cells, such as dendritic cells, recognize the composition, and the composition is taken up by the dendritic cells. The dendritic cells transport the composition to lymph nodes, where they educate T cells to recognize adipocytes. The educated T cells then kill the adipocytes whenever they encounter them. Such a subject is immunized against obesity. [Example 8]

[0148] In this prophetic example, a subject is injected with a composition that includes a fusion protein that includes an eat-me signal, such as calreticulin, and a fragment of a virus, such as SARS-CoV-2. Antigen-presenting cells, such as dendritic cells, recognize the composition, and the composition is taken up by the dendritic cells. The dendritic cells transport the composition to lymph nodes, where they educate T cells to recognize the virus. The educated T cells then kill the virus whenever they encounter it. Such a subject is immunized against a particular virus, such as SARS-CoV-2. [Example 9]

[0149] In this prophetic example, a composition includes a fusion protein that includes an eat-me signal, such as calreticulin, and an mRNA that expresses a surface antigen of a senescent cell, and the fusion protein is taken up by a dendritic cell via the dendritic cell receptor LPR. [Example 10]

[0150] In this prophetic example, a composition is provided that includes a fusion protein comprising an eat-me signal, such as calreticulin, and an mRNA that expresses a surface antigen of a plaque, and the fusion protein is taken up by dendritic cells via the dendritic cell receptor LPR. [Example 11]

[0151] In this prophetic example, a composition includes a fusion protein that includes an eat-me signal, such as calreticulin, and an mRNA that expresses an adipocyte surface antigen, and the fusion protein is taken up by dendritic cells via the dendritic cell receptor LPR. [Example 12]

[0152] In this prophetic example, a composition comprising a fusion protein between an eat-me signal, such as calreticulin, and an mRNA, where the mRNA expresses the spike protein of SARS-CoV-2, is taken up by dendritic cells via the dendritic cell receptor LPR. [Example 13]

[0153] In this prophetic example, a composition comprising a fusion protein comprising one or more fragments of calreticulin and a surface antigen of a senescent cell is dissolved in a deep eutectic solvent, such as a 1:2 composition of choline chloride and propylene glycol. The composition comprising the deep eutectic solvent and the fusion protein is incorporated into a thin molten film and applied to the inside of the subject's cheek. The fusion protein vaccine is transferred transbuccally into the subject's blood. [Example 14]

[0154] In this prophetic example, a composition comprising a fusion protein comprising one or more fragments of calreticulin and a plaque surface antigen is dissolved in a deep eutectic solvent, such as a 1:2 composition of choline chloride and propylene glycol. The composition comprising the deep eutectic solvent and the fusion protein is incorporated into a thin melt film and applied to the inside of the subject's cheek. The fusion protein vaccine is transferred transbuccally into the subject's bloodstream. [Example 15]

[0155] In this prophetic example, a composition comprising a fusion protein comprising one or more fragments of calreticulin and an adipocyte surface antigen is dissolved in a deep eutectic solvent, such as a 1:2 composition of choline chloride and propylene glycol. The composition comprising the deep eutectic solvent and the fusion protein is incorporated into a thin melt film and applied to the inside of the subject's cheek. The fusion protein vaccine is transferred transbuccally into the subject's bloodstream. [Example 16]

[0156] In this prophetic example, a composition comprising a fusion protein comprising calreticulin and one or more fragments of the spike protein of SARS-CoV-2 is dissolved in a deep eutectic solvent, such as a 1:2 composition of choline chloride and propylene glycol. The composition comprising the deep eutectic solvent and the fusion protein is incorporated into a thin melt film and applied to the inside of the subject's cheek. The fusion protein vaccine is transferred transbuccally into the subject's bloodstream.

[0157] Although several embodiments of the present disclosure have been described and illustrated herein, those skilled in the art can readily envision various other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and each such variation and / or modification is considered to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular use or application in which the teachings of the present disclosure are used. Those skilled in the art will recognize, or be able to ascertain with no more than routine experimentation, many equivalents to the specific embodiments of the present disclosure described herein. Thus, the foregoing embodiments are presented by way of example only, and it will be understood that within the scope of the appended claims and their equivalents, the present disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods is included within the scope of the present disclosure, if such features, systems, articles, materials, and / or methods are not mutually inconsistent.

[0158] The indefinite articles "a" and "an," as used herein in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0159] The term "and / or" as used herein and in the claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctive in some cases and disjunctive in other cases. Other elements, whether related or unrelated to those elements specifically identified, may be present as appropriate other than the elements specifically identified by the "and / or" clause, unless expressly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and / or B," when used in combination with open-ended language such as "comprising," may refer in one embodiment to A without B (which may include elements other than B); in another embodiment to B without A (which may include elements other than A); in yet another embodiment to both A and B (which may include other elements); and so forth.

[0160] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive, i.e., including at least one of the elements of the number or list, but also including more than one, and may further include items not in the list. Only terms clearly indicating the contrary, such as "only one of," or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of the number or list. In general, the term "or" as used herein shall be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") only when preceded by an exclusive term, such as "either," "one of," "only one of," or "exactly one of." As used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0161] As used herein and in the claims, the phrase "at least one" referring to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that elements other than those specifically identified in the list of elements to which the phrase "at least one" refers may be present as appropriate, whether or not related to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") may refer in one embodiment to A, which may include at least one, and may include more than one, with B absent (and which may include elements other than B); in another embodiment to B, which may include at least one, and may include more than one, with A absent (and which may include elements other than A); in yet another embodiment to A, which may include at least one, and may include more than one, and B, which may include at least one, and may include more than one (and may include other elements); etc.

[0162] As used herein, "wt%" is an abbreviation for weight percentage. As used herein, "at%" is an abbreviation for atomic percentage.

[0163] Some embodiments may be embodied as methods, various examples of which are described. The acts performed as part of the methods may be ordered in any suitable manner. Thus, embodiments may be configured in such a way that acts are performed in an order different from that shown, may include different (e.g., more or less) acts than those described, and / or may include performing some acts simultaneously, even though the acts have been shown as being performed sequentially in the specifically described embodiments above.

[0164] In the claims, the use of ordinal terms such as "first," "second," "third," etc. to modify claim elements does not, in itself, indicate any priority, precedence, or order of one claim element relative to another claim element, or the temporal order in which acts of a method are performed, but is merely used as a label to distinguish one claim element having a particular name from another element having the same name (but which uses ordinal terms).

[0165] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like, are understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as provided in the United States Patent Office Manual of Patent Examining Procedure, Section 211.03.

Claims

1. 1. A composition for treating or preventing a disease or condition comprising: a fusion protein comprising an eat me signal and at least a portion of an antibody, A composition, wherein at least a portion of the antibody is configured to bind to a cell, virus, or extracellular material associated with a disease or condition.

2. The composition of claim 1 , wherein the eat me signal induces an immune response against a cell, a virus, or an extracellular substance.

3. 1. A method of treating or preventing a disease or condition comprising: A method comprising administering to a subject a composition comprising a fusion protein comprising an eat me signal and at least a portion of an antibody.

4. The method of claim 3, wherein at least a portion of the antibodies recognize a cell, virus, or extracellular substance associated with a disease or condition of the subject and induce an immune response against the cell, virus, or extracellular substance.

5. 1. A composition for treating or preventing a disease or condition comprising: a fusion protein comprising an eat-me signal and a fragment of a cell or viral surface antigen, A composition configured to induce an immune response against a cell or a virus.

6. 1. A method of treating or preventing a disease or condition comprising: A method comprising administering to a subject a composition comprising a fusion protein comprising an eat-me signal and a fragment of a cell or viral surface antigen.

7. The method of claim 6 , wherein the composition induces an immune response against a cell or a virus.

8. The composition or method of any one of claims 1 to 4, wherein at least a portion of the antibody is a complete antibody.

9. The composition or method of any one of claims 1 to 4, wherein at least a portion of the antibody is an antibody fragment.

10. The composition or method of any one of claims 1 to 4 and 8 to 9, wherein at least a portion of the antibody recognizes a cell.

11. The composition or method of any one of claims 1 to 4 and 8 to 9, wherein at least a portion of the antibodies recognize a virus.

12. The composition or method according to any one of claims 1 to 4 and 8 to 9, wherein at least a portion of the antibody recognizes an extracellular substance.

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

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

15. The composition or method of any one of claims 1 to 4 and 8 to 14, wherein at least a portion of the antibody recognizes a cell, and the cell is a senescent cell or an adipocyte.

16. The composition or method of any one of claims 1 to 4 and 8 to 12, wherein at least a portion of the antibody recognizes an extracellular substance, and the extracellular substance is plaque.

17. The composition or method of any one of claims 1 to 4 and 8 to 11, wherein at least a portion of the antibodies recognize a virus, the virus being a coronavirus or HIV.

18. The composition or method of any one of claims 1-4 and 8-17, wherein at least a portion of the antibody is a minibody or nanobody.

19. At least a portion of the antibodies of the composition are －６ K less than M Ｄ The composition or method according to any one of claims 1 to 4 and 8 to 18, which recognizes a cell, a virus or an extracellular substance at a specific value.

20. At least a portion of the antibodies of the composition are －７ K less than M Ｄ The composition or method according to any one of claims 1 to 4 and 8 to 19, which recognizes a cell, a virus, or an extracellular substance at a specific value.

21. At least some of the antibodies of the composition have a K Ｄ Value smaller than K Ｄ The composition or method according to any one of claims 1 to 4 and 8 to 20, which recognizes a cell, a virus, or an extracellular substance at a specific value.

22. Eat-me signals are recognized by antibodies that recognize cells, viruses, or extracellular substances, and by antigen-presenting cells such as dendritic cells A composition comprising:

23. Antibody fragments that recognize specific proteins on cells, viruses, or extracellular substances, and eat-me signals that are recognized by antigen-presenting cells such as dendritic cells A composition comprising:

24. The composition according to any one of claims 22 to 23, wherein the antibody or antibody fragment recognizes a cell, and the cell is a senescent cell or an adipocyte, or a plaque cell.

25. The composition of any one of claims 22 to 24, wherein the eat me signal is calreticulin.

26. The composition of any one of claims 1 to 25, wherein the antibody or antibody fragment is directly covalently linked to the eat-me signal.

27. The composition of any one of claims 1 to 26, wherein the fusion protein comprises both an antibody or antibody fragment and an eat-me signal.

28. The composition according to any one of claims 1 to 25, wherein the antibody and the eat-me signal are linked via a linking agent.

29. administering to a subject a composition comprising an antibody that recognizes a cell, virus, or extracellular substance and an eat-me signal; A method comprising:

30. Administering to a subject a composition comprising an antibody fragment that recognizes a specific protein of a cell or virus and an eat-me signal. A method comprising:

31. The method of any one of claims 29 to 30, wherein the antibody or antibody fragment recognizes a cell, and the cell is a senescent cell, an adipocyte, or a plaque.

32. Eat me signal and one or more fragments of a cell or viral surface antigen A composition comprising:

33. Eat me signal and one or more DNA fragments of a cell or virus A composition comprising:

34. Eat me signal and one or more lipid fragments of a cell or virus A composition comprising:

35. a first component which is an eat-me-signal, a DNA fragment, a lipid fragment, or a fragment of a cell or viral surface antigen, and a second component different from the first component which is also a DNA fragment, a lipid fragment, or a fragment of a cell or viral surface antigen. A composition comprising:

36. The composition according to any one of claims 32 to 35, wherein the cell is a senescent cell, an adipocyte, or a plaque.

37. The composition of any one of claims 32 to 36, wherein the eat me signal is calreticulin.

38. The composition of any one of claims 32 to 37, wherein the eat-me signal is a moiety recognized by an antigen-presenting cell.

39. Administering a composition according to any one of claims 32 to 38 to a subject. A method comprising:

40. Dissolving the composition of any one of claims 22 to 38 in a deep eutectic solvent such as choline chloride / propylene glycol to produce a liquid pharmaceutical preparation, and administering the liquid pharmaceutical preparation transbuccally or transdermally to the cheek of a subject. A method comprising:

41. 41. The method of claim 40, wherein the liquid pharmaceutical preparation is administered using a dissolvable thin film comprising the liquid pharmaceutical preparation.

42. 41. The method of claim 40, wherein the liquid pharmaceutical preparation is part of a transdermal patch configured to be applied to the skin of a subject.

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