REGULATION OF AUTOPHAGY PATHWAY PHOSPHORYLATION AND USES THEREOF
The invention relates to polypeptides and proteins known to function in the autophagy pathway that have novel phosphorylation sites. The invention also relates to antibodies specific to these polypeptides and proteins that are phosphorylated or not phosphorylated at novel phosphorylated sites. The invention also relates to methods of producing these antibodies and use of these antibodies in the treatment of diseases related to autophagocytosis.
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This application is a continuation of U.S. application Ser. No. 12/505,281, now U.S. Pat. No. 8,148,088, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Ser. No. 61/082,174 filed Jul. 18, 2008 and U.S. Provisional Application Ser. No. 61/082,179 filed Jul. 18, 2008, the disclosures of which are herein incorporated by reference in their entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEBThe entire content of the following electronic submission of the sequence listing via the USPTO EFS-WEB server, as authorized and set forth in MPEP §1730 II.B.2(a)(C), is incorporated herein by reference in its entirety for all purposes. The sequence listing is identified on the electronically filed text file as follows:
The claimed compositions, methods, and kits are directed to antibodies to detect autophagy proteins in either the phosphorylated or nonphosphorylated forms.
BACKGROUND ARTAutophagy is a process whereby cells convert proteins and organelles into amino acids as a source of food. Many cells in the human body rely on autophagy to maintain homeostasis, especially when insulin levels are low. Autophagocytosis may play a role in human disease and aging. In eukayrotic cells autophagy occur constitutively at low levels in all cells to perform housekeeping functions such as destruction of dysfunctional organelles. Dramatic upregulation occurs (e.g., cytoplasmic and organelle turnover) in the presence of external stressors (starvation, hormonal imbalance, oxidation, extreme temperature, and infection), and internal needs (generation of source materials for architectural remodeling, removal of protein aggregates). Autophagy is highly regulated through the coordinated action of various kinases, phosphatases, and guanosine triphosphatases (GTPases).
At least three different autophagy mechanisms are known, all of which result in targeting of cytosolic proteins and organelles to the lysosome in order to provide amino acids and energy in the form of catabolites. These types are macroautophagy, microautophagy, and chaperone-mediated autophagy.
Macroautophagy is a major inducible pathway for the general turnover of cytoplasmic constituents in eukaryotic cells and also plays a significant role in the degradation of active cytoplasmic enzymes and organelles during nutrient starvation. Macroautophagy involves the formation of double-membrane bound autophagosomes which enclose the cytoplasmic constituent targeted for degradation in a membrane bound structure, which then fuse with the lysosome (or vacuole) releasing a single-membrane bound autophagic bodies which are then degraded within the lysosome (or vacuole). MAP1A and MAP1B are microtubule-associated proteins which mediate the physical interactions between microtubules and components of the cytoskeleton. These proteins are involved in formation of autophagosomal vacuoles (autophagosomes). MAP1A and MAP1B each consist of a heavy chain subunit and multiple light chain subunits. Apg8a is one of the light chain subunits and can associate with either MAP1A or MAP1B. The precursor molecule is cleaved by APG4B/ATG4B to form the cytosolic form, Apg8a-I. This is activated by APG7L/ATG7, transferred to ATG3 and conjugated to phospholipid to form the membrane-bound form, Apg8a-II.
Microautophagy circumvents the autophagosomic step of macrophagy, and begins with the direct uptake of cytosolic material via invaginations and pinching off of the lysosomal membrane. The internalized cytosolic components are digested by lysosomal enzymes released when the vacuolar membrane disintegrates, as in macroautophagy.
In chaperone-mediated autophagy, specific chaperone proteins bind to target proteins containing a KFERQ (SEQ ID NO: 1) sequence and channel these proteins to the surface of the lysosome. These proteins bind to Lamp2a and are then transported across the lysosomal membrane with the assistance of lysosomal chaperones, after which they are degraded by vacuolar proteases.
Mizushima, et al., describes autophagy as promoting both cell survival and cell death. By maintaining homeostasis during times of cellular stress, autophagy generally promotes survival when it is controlled. See Mizushima, N., et al., “Autophagy fights disease through cellular self-digestion” Nature (2008) 451:1069-1075.
However, dramatic upregulation of autophagy via Beclin 1 overexpression brings about cell death. Mizushima, et al., describe how the autophagy and apoptosis pathways share many common regulatory factors, with the likelihood of significant cross-talk between these pathways in the cell. Since apoptosis is known to be implicated in human disease, autophagy also is likely an important phenomenon to target in order to treat disease.
Proteins that regulate autophagy in cancer cells make attractive therapeutic and diagnostic targets. Cancer cells rely on autophagy in order to evade anti-cancer treatments designed to reduce nutrient supply and enhance the stress on rapidly dividing cells. A compound that downregulates autophagy may be a useful additional drug in cancer treatment. Mizushima, et al., state that since autophagy may help prevent cancer, there is a potential need to target autophagy in a context-specific manner. The targeting of specific autophagy regulatory proteins rather than a targeting of autophagy in general may be critical in developing a treatment of cancer as well as new modes of diagnosing cancer.
Alterations in the autophagy degradation pathway have been described in normal brain aging and in age-related neurodegenerative diseases including Alzheimer's and Parkinson's diseases. See Nixon, R., “Autophagy in neurodegenerative disease: friend, foe, or turncoat?” Trends in Neurosciences (2006) 29(9):528-535. An improper clearance of proteins in these diseases may result either from a compromise in the autophagy degradation pathway or induced alterations in this pathway, and may result in neuron dysfunction and neuron loss. The targeting of specific autophagy regulatory proteins, rather than a targeting of autophagy in general, may be critical in developing a treatment of neurodegenerative diseases as well as new modes of diagnosing neurodegenerative diseases. Therefore, there exists a need to develop an assay to monitor the activity of autophagy proteins that does not rely exclusively on protein localization.
DISCLOSURE OF THE INVENTIONSeveral of the following aspects provide peptides comprising amino acids that are novel phosphorylation sites on various autophagy proteins. Several of the following aspects also provide antibodies that react specifically to the phosphorylated forms of these proteins, and antibodies that react specifically to the non-phosphorylated forms of these proteins.
In one aspect, the present disclosure provides an isolated autophagy peptide that comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 1, wherein the x residue is nonphosphorylated or phosphorylated serine, threonine, or tyrosine, and with the proviso that the peptide is not a full-length autophagy protein comprising an amino acid sequence set forth in Table 2. The peptides of this embodiment are referred to as “autophagy peptides”.
In some embodiments, an isolated peptide of the present disclosure comprises an amino sequence found in Table 3. The peptides of this embodiment are a subset of possible autophagy peptides.
In another embodiment, isolated peptides of the present disclosure comprise an amino acid sequence found in Table 4. In some embodiments, the x residue may be phosphorylated. In some embodiments, the x residue may be nonphosphorylated.
In another aspect, the disclosure provides for an immunogen, which comprises an autophagy peptide and immune response potentiator.
In another aspect, the disclosure provides for a multiple antigenic peptide (MAP), which comprises a branched oligolysine core conjugated with a plurality of isolated autophagy peptides.
In another aspect, the disclosure provides for a method for producing an antibody to an autophagy polypeptide. The method comprises introducing an isolated autophagy peptide comprising a sequence set forth in Table 1 to a mammal in an amount sufficient to produce an antibody to the autophagy peptide; and recovering the antibody from the mammal.
In another aspect, the disclosure provides for a kit for producing an antibody to an autophagy polypeptide. The kit comprises an isolated autophagy peptide comprising a sequence set forth in Table 1, a means for introducing the isolated autophagy peptide to a mammal in an amount sufficient to produce an antibody to the autophagy peptide, and a means for recovering the antibody from the mammal.
In another aspect, the disclosure provides for a method for producing an antibody to an autophagy polypeptide. The method comprises introducing an autophagy protein to a mammal in an amount sufficient to produce an antibody to the autophagy protein, recovering the antibody from the mammal, and affinity purifying an autophagy antibody that specifically binds to an epitope of the sequence in Table 3. This method is referred to below as the “method for producing an affinity-purified autophagy antibody” In one embodiment, the disclosure provides for an antibody to an autophagy polypeptide produced by this method.
In another aspect, the disclosure provides for a kit for producing an antibody to an autophagy polypeptide. The kit comprises an autophagy protein, a means for introducing the autophagy protein to a mammal in an amount sufficient to produce an antibody to the autophagy polypeptide, a means for recovering the antibody from the mammal, and an isolated autophagy peptide comprising a sequence from Table 1.
In another aspect, the disclosure provides for an isolated antibody that specifically binds to an epitope that comprises the amino acid residue x (also referred to herein as the “X residue”) in an amino acid sequence set forth in Table 3, wherein the x residue is either phosphorylated or nonphosphorylated serine, threonine, or tyrosine. In one embodiment, the epitope comprises the amino acid residue x in one amino acid sequence set forth in Table 3 and an amino acid residue of autophagy protein that is outside the same amino acid sequence set forth in Table 3. In another embodiment, the epitope comprises the amino acid residue x in one amino acid sequence set forth in Table 3 and amino acid residues of an autophagy protein that are outside the same amino acid sequence set forth in Table 3.
In another aspect, the disclosure provides for a method for detecting an autophagy protein or fragment comprising an amino acid sequence set forth in Table 4, wherein the x residue is either phosphorylated or nonphosphorylated serine, threonine, or tyrosine, in a sample. The method comprises the following steps. First, a sample containing or suspected of containing an autophagy protein or fragment comprising the amino acid sequence set forth in Table 4, wherein the x residue is either phosphorylated or nonphosphorylated, is contacted with an isolated antibody that specifically binds to an epitope that comprises the amino acid residue x in the amino acid sequence set forth in Table 4, wherein x is either phosphorylated or nonphosphorylated. The next step is assessing a complex formed between the autophagy protein or fragment, if present in the sample, and the antibody, to determine the presence, absence and/or amount of the autophagy protein or fragment in the sample.
In another aspect, the disclosure provides for a kit for detecting an autophagy protein or fragment comprising amino acid sequence set forth in Table 4 wherein x is serine or phosphoserine in a sample, which kit comprises, in a container, an isolated antibody that specifically binds to an epitope that comprises the amino acid residue x in the amino acid sequence set forth in Table 4, wherein x is serine or phosphoserine.
In another aspect, the disclosure provides for a method for treating a disease or disorder associated with abnormal phosphorylation status of an autophagy protein or fragment comprising amino acid sequence set forth in Table 4 wherein the x residue is either phosphorylated or nonphosphorylated, which method comprises administering, to a subject when such a treatment is needed or desired, a sufficient amount of an isolated antibody that specifically binds to an epitope that comprises the x residue in the amino acid sequence set forth in Table 4, wherein x is either phosphorylated or nonphosphorylated.
Another aspect is a method for identifying a kinase that phosphorylates an autophagy protein on the x residue. The method comprises the steps of: (1) providing autophagy polypeptide comprising an amino acid sequence listed in Table 1, wherein x is nonphosphorylated serine, threonine, or tyrosine; (2) contacting the autophagy polypeptide with a test protein and ATP under conditions suitable for the phosphorylation of the x residue of the autophagy polypeptide; and (3) assessing the phosphorylation status of the autophagy polypeptide to determine whether the test protein is a kinase for the autophagy protein on the x residue.
Another aspect is a method for identifying a modulator of a kinase that phosphorylates an autophagy protein on the x residue. The method comprises the steps of: (1) providing an autophagy polypeptide comprising an amino acid sequence selected from a sequence listed in Table 1, wherein the x residue is not phosphorylated; (2) contacting the autophagy polypeptide with a kinase that phosphorylates the protein on the residue indicated by x and ATP under conditions suitable for the phosphorylation of the x residue of the autophagy polypeptide in the presence or absence of a test substance; and assessing and comparing phosphorylation status of the autophagy polypeptide by the kinase to determine whether the test substance modulates the kinase.
Another aspect is a method for identifying a phosphatase that dephosphorylates an autophagy protein on a phosphorylated x residue. The method comprises the steps of: (1) providing an autophagy polypeptide comprising an amino acid sequence selected from the group consisting of a sequence listed in Table 1, wherein x is phosphorylated; (2) contacting the autophagy polypeptide with a test protein and H2O under conditions suitable for the dephosphorylation of the phosphoserine residue of the autophagy polypeptide; and (3) assessing phosphorylation status of the autophagy polypeptide to determine whether the test protein is a phosphatase for the autophagy polypeptide on the x residue.
Another aspect is a method for identifying a modulator of a kinase that phosphorylates an autophagy protein on the x residue. The method comprises the steps of: (1) providing an autophagy polypeptide comprising an amino acid sequence listed in Table 1, wherein x is nonphosphorylated serine, threonine, or tyrosine; (2) contacting the autophagy polypeptide with a kinase that phosphorylates an autophagy polypeptide on the x residue and ATP under conditions suitable for the phosphorylation of the x residue of the autophagy polypeptide in the presence or absence of a test substance; and (3) assessing and comparing the phosphorylation status of the autophagy polypeptide by the kinase to determine whether the test substance modulates the kinase.
Another aspect is an isolated nucleic acid fragment which is comprised of a sequence of nucleotides encoding an autophagy peptide comprising an amino acid sequence selected from the group consisting of sequences set forth in Table 1, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine. The autophagy peptide is not a full-length autophagy protein comprising an amino acid sequence set forth in Table 2. The nucleic acid may be DNA. The nucleic acid may also be RNA.
Other objects, features, and technical advantages of the present invention will become more apparent from a consideration of the detailed description herein and from the accompanying drawings.
All publications, patents, patent applications, GenBank sequences and ATCC deposits, cited herein are hereby expressly incorporated by reference for all purposes.
For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections that follow.
DefinitionsUnless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications (published or unpublished), and other publications referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.
As used herein, “a” or “an” means “at least one” or “one or more.”
As used herein, the term “autophagy protein” refers to one of the following proteins, as indicated by name and an exemplary NCBI accession number: APG-3L (NP—071933), APG4A (NP—443168), APG4B (NP—037457), APG4C(NP—835739), APG4D (NP—116274), APG5L (NP—004840), APG7L (NP—006386), APG8b (NP—073729), APG8c (NP—073729), APG9L1 (NP—076990), APG10L (NP—113670), APG12L (NP—004698), APG16L (NP—110430), and BECN1 (NP—003757).
As used herein, “antibody” is used in the broadest sense. Therefore, an “antibody” can be naturally occurring or man-made such as monoclonal antibodies produced by conventional hybridoma technology and/or a functional fragment thereof. Antibodies of the present invention comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies. Antibodies can be of any isotype including IgM, IgG, IgD, IgA and IgE, and any sub-Isotype, including IgG1, IgG2a, IgG2b, IgG3 and IgG4, IgE1, IgE2, etc. The light chains of the antibodies can either be kappa light chains or lambda light chains.
As used herein, “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts. As used herein, a “monoclonal antibody” further refers to functional fragments of monoclonal antibodies.
As used herein, the phrase “oligoclonal antibodies” refers to a predetermined mixture of distinct monoclonal antibodies. See, e.g., PCT publication WO 95/20401; U.S. Pat. Nos. 5,789,208 and 6,335,163.
As used herein, “peptide” includes all “mimetic” and “peptidomimetic” forms. The terms “mimetic” and “peptidomimetic” refer to a synthetic chemical compound which has substantially the same structural and/or functional characteristics of the polypeptides of the invention. The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity. Routine experimentation will determine whether a mimetic is within the scope of the invention, i.e., that its structure and/or function is not substantially altered.
As used herein, “polypeptide” includes a molecular chain of amino acids linked through peptide bonds. “Polypeptide” does not refer to a specific length of the product. Thus, “peptides,” “oligopeptides,” and “proteins” are included within the definition of polypeptide.
As used herein, an “antigen”, includes any portion of a protein (peptide, protein fragment, full-length protein), wherein the protein is naturally occurring or synthetically derived, a cellular composition (whole cell, cell lysate or disrupted cells), an organism (whole organism, lysate or disrupted cells), a carbohydrate, a lipid, or other molecule, or a portion thereof, wherein the antigen elicits an antigen-specific immune response (humoral and/or cellular immune response).
As used herein, “mammal” refers to any of the mammalian class of species. Frequently, the term “mammal,” as used herein, refers to humans, human subjects or human patients.
As used herein, “treatment” means any manner in which the symptoms of a condition, disorder or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein.
As used herein, “disease or disorder” refers to a pathological condition in an organism resulting from, e.g., infection or genetic defect, and characterized by identifiable symptoms.
As used herein, the term “subject” is not limited to a specific species or sample type. For example, the term “subject” may refer to a patient, and frequently a human patient. However, this term is not limited to humans and thus encompasses a variety of mammalian species.
As used herein, “afflicted” as it relates to a disease or disorder refers to a subject having or directly affected by the designated disease or disorder.
As used herein the term “sample” refers to anything which may contain an analyte for which an analyte assay is desired. The sample may be a biological sample, such as a biological fluid or a biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like. Biological tissues are aggregate of cells, usually of a particular kind together with their intercellular substance that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle and nerve tissues. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cell(s).
As used herein, the term “specifically binds” refers to the binding specificity of a specific binding pair. Recognition by an antibody of a particular target in the presence of other potential targets is one characteristic of such binding. “Binding component member” refers to a member of a specific binding pair, i.e., two different molecules wherein one of the molecules specifically binds with the second molecule through chemical or physical means. The two molecules are related in the sense that their binding with each other is such that they are capable of distinguishing their binding partner from other assay constituents having similar characteristics. The members of the binding component pair are referred to as ligand and receptor (antiligand), specific binding pair (sbp) member and sbp partner, and the like. A molecule may also be a sbp member for an aggregation of molecules; for example an antibody raised against an immune complex of a second antibody and its corresponding antigen may be considered to be an sbp member for the immune complex.
As used herein the term “does not specifically bind” refers to the specificity of particular antibodies or antibody fragments. Antibodies or antibody fragments that do not specifically bind a particular moiety generally contain a specificity such that a large percentage of the particular moiety would not be bound by such antibodies or antibody fragments. This percentage generally lies within the acceptable cross reactivity percentage with interfering moieties of assays utilizing antibodies directed to detecting a specific target. Frequently, antibodies or antibody fragments of the present disclosure do not specifically bind greater than about 90% of an interfering moiety, although higher percentages are clearly contemplated and preferred. For example, antibodies or antibody fragments of the present disclosure do not specifically bind about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and about 99% or more of an interfering moiety. Less occasionally, antibodies or antibody fragments of the present disclosure do not specifically bind greater than about 70%, or greater than about 75%, or greater than about 80%, or greater than about 85% of an interfering moiety.
As used herein the term “isolated” refers to material removed from its original environment, and is altered from its natural state. For example, an isolated polypeptide could be coupled to a carrier, and still be “isolated” because that polypeptide is not in its original environment.
As used herein, the term “X residue” refers to residues of autophagy proteins that are phosphorylation sites, as depicted in Tables 4 and 5. Table 4 shows the site of the x residue, with a label “X”. Table 5 shows the x residue in underlined form, with the x residue as threonine, serine, or tyrosine. The phosphorylation state of the x residue is typically described as phosphorylated or nonphosphorylated.
As used herein, the term “autophagy polypeptide” refers to a polypeptide comprising an entire autophagy protein or a fragment of an entire autophagy protein.
As used herein, the term “autophagy peptide” refers to a peptide comprising at least four consecutive amino acid residues of a sequence from Table 4. The “X” residue may be phosphorylated or not phosphorylated.
As used herein, the term “phosphorylated autophagy peptide” refers to an autophagy peptide that is phosphorylated at the x residue.
As used herein, the term “nonphosphorylated” when modifying “autophagy peptide” refer to an autophagy peptide that is not phosphorylated at the x residue.
The titles of each section and the examples of the following specification are not intended to be limiting.
The disclosure is based on the discovery of novel phosphorylation sites in a variety of proteins that play a role in autophagy. Throughout the specification, these proteins are referred to as autophagy proteins, whose names and an exemplary full-length sequence from a species are found in Table 2. The phosphorylation sites are on threonine, serine, and tyrosine residues in the autophagy proteins. Subsequences of these autophagy proteins are listed in Table 5, which further indicates the residue that is phosphorylated as underlined. The phosphorylated residue is represented by the letter x in the peptides shown in Tables 1, 3, and 4.
As a result of the identification of the novel sites of phosphorylation, peptide antigens may now be designed to raise phospho-specific antibodies that bind autophagy proteins when phosphorylated at the residue indicated by the letter x in the peptide (“X residue”). Furthermore, non-phosphospecfic antibodies may be designed that bind autophagy proteins when not phosphorylated at the x residue.
The epitopic site of the antibodies disclosed may be the sequences shown in Tables 1 and 3, with x represented the phosphorylation site as described in these tables. Epitopic sequences may be as short as four bases, with the possible epitopes shown in Table 1. Epitopic sequences may be longer than four bases, with possible epitopes having longer sequence shown in Table 3. Peptides having a sequence that includes the epitopic site are disclosed.
Autophagy Proteins
The following autophagy proteins may play a role in autophagy and are also phosphorylated. Autophagy peptides are comprised of a sequence within the autophagy proteins that encompass the phosphorylation site, which is indicated as an “X”. The autophagy peptide may have sequences that correspond to epitopes having sequence set forth in Table 1, wherein the x residue is either phosphorylated or nonphosphorylated serine, threonine, or tyrosine. The autophagy peptide may be comprised of a sequence having at least five residues that is found in Table 3. For any of the above autophagy peptides, the x residue is either phosphorylated or nonphosphorylated.
The autophagy peptide may be comprised of a sequence found in Table 4, in which the x residue is either phosphorylated or nonphosphorylated serine, threonine, or tyrosine. These sequences, as well as the above shorter sequences of this sequence, may be particularly strong antigens for the production of antibodies.
The autophagy peptide comprising the above sequences may not be the full length autophagy protein in any organism that is comprised of a sequence listed in Table 2.
In another embodiment, an isolated peptide of the present disclosure comprises an amino acid sequence set forth in Table 4. In some embodiments, the x residue of serine, threonine, or tyrosine may be nonphosphorylated. In some embodiments, the x residue of serine, threonine, or tyrosine may be phosphorylated.
Pharmaceutical Compositions
In some embodiments, the disclosure provides for a pharmaceutical composition comprising a pharmaceutically acceptable carrier, an excipient, and an isolated autophagy peptide. The pharmaceutical compositions can be used to promote or otherwise enhance the rate of autophagy in vertebrate animals including mammals. Alternatively, the pharmaceutical compositions can be used to inhibit or reduce the rate of autophagy in vertebrate animals, including mammals. Accordingly, the compositions are considered useful for treating or preventing a variety of conditions including ischemic brain injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion diseases and polyglutamine disorders including Huntington's disease and various spinocerebellar ataxias, transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease, breast cancer, ovarian cancer, brain cancer, pancreatic cancer, esophageal cancer, colorectal cancer, liver cancer, prostate cancer, renal cancer, lung cancer, Myocardial ischemia, cardiac remodeling, cardiomyopathy, hemodynamic stress, myocardial hypertrophy, Neuronal ceroid-lipofuscinosis (adult and juvenile), Multiple Sulfatase Deficiency (MSD) and Mucopolysaccharidosis type IIIA, Batten disease, Niemann-Pick C, Danon disease, Pompe disease, and dysfunction of innate and adaptive immunity against intracellular pathogens.
The peptide compounds may be formulated into the compositions as neutral or salt forms. Pharmaceutically acceptable non-toxic salts include the acid addition salts (formed with the free amino groups) and which are formed by reaction with inorganic acids such as, for example, hydrochloric, sulfuric or phosphoric acids, or organic acids such as, for example, acetic, oxalic, tartaric, mandelic, citric, malic, and the like. Salts formed with the free carboxyl groups may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases such as amines, i.e., isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
An autophagy peptide is suitably administered to a subject, e.g., a human or a non-human mammal, such as a domestic animal. The amount administered may vary depending on various factors including, but not limited to, the agent chosen, the disease, and whether prevention or treatment is to be achieved. The peptides may be administered locally or systemically. Administration of the therapeutic agents may be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the agents of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses.
One or more suitable unit dosage forms comprising an autophagy peptide can be administered by a variety of routes including oral, or parenteral, including by rectal, buccal, vaginal and sublingual, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intrathoracic, intracoronary, intrapulmonary and intranasal routes. The dosage form may optionally be formulated for sustained release. The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to pharmacy. Such methods may include the step of bringing into association the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.
When the autophagy peptide is prepared for oral administration, it is preferably combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. The total active ingredients in such formulations comprise from 0.1 to 99.9% by weight of the formulation. By “pharmaceutically acceptable” it is meant the carrier, diluent, excipient, and/or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof. The active ingredient for oral administration may be present as a powder or as granules; as a solution, a suspension or an emulsion; or in achievable base such as a synthetic resin for ingestion of the active ingredients from a chewing gum. The active ingredient may also be presented as a bolus, electuary or paste. A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991) which is incorporated herein by reference. Supplementary active compounds can also be incorporated into the compositions.
Pharmaceutical formulations containing an autophagy protein can be prepared by procedures known in the art using well known and readily available ingredients. For example, the natriuretic peptide can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, powders, and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include the following fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose, HPMC and other cellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone; moisturizing agents such as glycerol; disintegrating agents such as calcium carbonate and sodium bicarbonate; agents for retarding dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surface active agents such as cetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite; and lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols.
For example, tablets or caplets containing the nucleic acid molecule or peptide of the invention can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate. Caplets and tablets can also include inactive ingredients such as cellulose, pregelatinized starch, silicon dioxide, hydroxy propyl methyl cellulose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, and zinc stearate, and the like. Hard or soft gelatin capsules containing the nucleic acid molecule or peptide of the invention can contain inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, and titanium dioxide, and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil. Moreover, enteric coated caplets or tablets of the nucleic acid molecule or peptide of the invention are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum.
The autophagy peptide may be prepared in an injectable formulation. Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, are formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
Autophagy Polypeptide Immunogens
The autophagy peptide may be used as an immunogen, with an exemplary use being to generate antibodies. The autophagy peptide may be phosphorylated or not at the residue indicated by x in the sequences of Table 5. In some embodiments, the isolated autophagy peptide of the above embodiments may be conjugated to a carrier to enhance the peptide's immunogenicity. Use of carriers while immunizing the animal is preferred when producing antibodies against an autophagy peptide. It is generally appreciated in the art that antigens must be at least 10 kDa in order to elicit a satisfactory immune response. The size of an antigen can be effectively increased by association of the antigen with a carrier.
The carrier may be a carrier protein. Alternatively, the autophagy peptide and the carrier protein may be part of a fusion protein. Exemplary carriers include, but are not limited to, Keyhole limpet cyanin, BSA, cationized BSA, ovalbumin, blue carrier immunogenic protein, avidin, BTG, bovine G globulin, bovine Immunoglobulin G (BlgG), bovine thyroglobulin, conalbumin, colloidal gold, edestin, exoprotein A (recombinant) from P. aeruginosa, hemocyanin from crab P. camtschatica, Helix promatia Hemocyanin (HPH), HSA, KTI (Kunitz trypsin inhibitor from soybeans), LPH (Heamocyanin from Limulus polyphemus), Pam3Cys-Th, polylysine, porcine thyroglobulin (PTG), purified protein derivative (PPD), rabbit serum albumin (RSA), soybean trypsin inhibitor (STI), sunflower globulin (SFG), and Tetanus toxoid. The carrier protein may be coupled to the peptide as described in Lateef, S., et al., J. Biomol. Tech. (2007) 8:173-176.
In another aspect, the immunogen may comprise an autophagy polypeptide and an immune response potentiator. In some embodiments, the immune response potentiator may be Bacille Calmette-Guerin (BCG), Corynebacterium Parvum, Brucella abortus extract, glucan, levamisole, tilorone, an enzyme or a non-virulent virus.
Multiple Antigenic Peptide
In another aspect, a multiple antigenic peptide (MAP) is provided, which MAP comprises a branched oligolysine core conjugated with a plurality of autophagy polypeptides as described herein. On occasion, the branched oligolysine core comprises 3, 7 or 15 lysine residues, also on occasion, the MAP comprises 4, 8 or 16 copies of the autophagy peptide. The plurality of the autophagy peptide comprises the same or different autophagy peptides. In some embodiments, the plurality of the autophagy peptide is conjugated to the branched oligolysine core via a spacer. Frequently, the spacer may be one or more amino acid residues. Multiple antigenic peptides comprise generally known technology. See, e.g., Adermann, K., et al., Innovations and Perspectives in Solid Phase Synthesis 429-432 (R. Epton, ed., Mayflower Worldwide 1994). Furthermore, in some embodiments, the plurality of autophagy peptides is comprised of the same peptide or different peptides.
Method of Producing an Antibody Against an Autophagy Protein
In another aspect is a method for producing an antibody to an autophagy polypeptide. The method comprises introducing an isolated an autophagy peptide into a mammal in an amount sufficient to produce an antibody to the autophagy peptide and recovering the antibody from the mammal. This method and variations thereof in order to enhance antibody specificity can be appreciated by one of skill in the art.
In one embodiment of this aspect, the x residue in the isolated autophagy peptide is not phosphorylated and the method is used to produce an antibody to an autophagy polypeptide that is not phosphorylated at the corresponding serine. In practicing this and subsequent embodiments of this aspect, one of skill in the art may test the specificity of the produced antibody against an autophagy polypeptide phosphorylated or not phosphorylated at the x residue in order to determine specificity.
In another embodiment of this aspect, the x residue in the isolated autophagy peptide is phosphorylated and the method is used to produce an antibody to a phosphorylated autophagy polypeptide. This embodiment may further comprise a step of removing an antibody that binds to an isolated autophagy peptide having a sequence in which the x residue is not phosphorylated.
In some embodiments of the method for producing an autophagy antibody, the isolated autophagy peptide is conjugated to a carrier to enhance the peptide's immunogenicity. Alternatively, in some embodiments, the isolated autophagy peptide is comprised in an immunogen comprised of an isolated autophagy peptide and an immune response potentiator. The carrier, immunogen, and immune response potentiator may be in any of the forms described above.
In another embodiment of the method for producing an autophagy antibody, the isolated autophagy peptide is comprised in a multiple antigenic peptide (MAP), in which the MAP comprises a branched oligolysine core conjugated with a plurality of isolated autophagy peptides. The MAP may be in any of the forms as described above.
Method of Producing an Autophagy Polypeptide Antibody with Subsequent Affinity Purification
Another aspect is a method for producing an antibody to an autophagy polypeptide. The method comprises introducing an autophagy polypeptide to a mammal in an amount sufficient to produce an antibody to the autophagy polypeptide, recovering the antibody from the mammal, and affinity purifying an antibody that specifically binds to an epitope having a sequence listed in Table 1. The autophagy polypeptide may be a full-length sequence listed in Table 4. The autophagy polypeptide may be a truncated form of the full-length sequence listed in Table 4 that at least comprises the sequence of the autophagy peptide used in affinity purification. It will be appreciated by one of skill in the art that affinity purification of the antibody leads to an enrichment of antibodies that specifically bind to the autophagy peptide used in affinity purification, as well as a high possibility of enriching for antibodies that bind to epitopes containing phosphorylated “X residues”.
In some embodiments of this method, the autophagy polypeptide is nonphosphorylated and the method is used to produce an antibody that specifically binds to a nonphosphorylated autophagy polypeptide. As an example, the autophagy polypeptide used to immunize the animal is not phosphorylated at the x residue and affinity purification is performed using a nonphosphorylated autophagy peptide.
In some embodiments of this method, the x residue in the amino acid sequence set forth in Table 4 of is phosphorylated and the method is used to produce an antibody to a phosphorylated autophagy polypeptide. As an example, the autophagy polypeptide used to immunize the animal is phosphorylated at the x residue and affinity purification is performed using the autophagy polypeptide that is phosphorylated at the x residue. Optionally, this method further comprises a step of removing an antibody that binds to an isolated nonphosphorylated autophagy peptide, wherein x is serine, threonine, or tyrosine. As an example, the antibody sample can be passed through a column containing bound nonphosphorylated autophagy peptide such that antibodies specific to nonphosphorylated autophagy peptide are removed by remaining bound to the column.
Kit for Preparing Antibody to an Autophagy polypeptide
A kit may be used to prepare an antibody to an autophagy polypeptide. The kit comprises an autophagy protein and a means for introducing the autophagy protein to a mammal in an amount sufficient to produce an antibody to the autophagy polypeptide. The kit also comprises a means for recovering the antibody from the mammal and an isolated autophagy polypeptide comprised of one of the sequences from Table 1. Another embodiment of the kit comprises an autophagy protein and a means for introducing the autophagy protein to a mammal in an amount sufficient to produce an antibody to an autophagy polypeptide.
One exemplary kit contains a full length autophagy protein and reagents containing appropriate immunogens, adjuvants, and buffers such that the autophagy polypeptide can be prepared for introduction into a mammal. The exemplary kit further contains an autophagy polypeptide as well as affinity purification columns that can be used to enrich for antibodies that specifically bind to the autophagy peptide.
Isolated Antibodies
Another aspect is an isolated antibody that specifically binds to an epitope that comprises the x residue in an amino acid sequence set forth in Table 4, wherein x residue is phosphorylated or nonphosphorylated. Multiple embodiments of this aspect are discussed as follows.
In one further embodiment of this aspect, the antibody is a monoclonal or polyclonal antibody or an antibody fragment.
In a further embodiment of this antibody, the epitope of the above antibody comprises both the amino acid residue x in the amino acid sequence set forth in Table 4 and an amino acid residue of the autophagy protein that is outside of the amino acid sequence set forth in Table 4. An exemplary epitope may arise from protein folding in which a residue outside set forth in Table 4 is brought into close proximity with the first epitope. Optionally, a kit may be prepared that is comprised of an antibody of this embodiment, a pharmaceutically acceptable carrier and an excipient.
In a third further embodiment of this antibody, the epitope comprises the amino acid x in the amino acid sequence set forth in Table 4 and amino acid residues of the autophagy protein that are outside of the amino acid sequence set forth in Table 4.
In a fourth further embodiment, the epitope is comprised in the amino acid sequence set forth in Table 4.
In another further embodiment, the epitope is comprised of a sequence set forth in Table 3.
In another further embodiment, the epitope is comprised of a sequence set forth in Table 1.
In another further embodiment, the antibody specifically binds to the epitope that comprises the amino acid residue x in the amino acid sequence set forth in Table 4, wherein the x residue is nonphosphorylated.
In another further embodiment, the antibody specifically binds to the epitope that comprises the amino acid residue x in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated.
In another further embodiment, the antibody specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is nonphosphorylated, but does not specifically bind to an epitope comprised in the amino acid sequence of Table 4, wherein the x residue is phosphorylated.
In another further embodiment, the antibody specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue of serine, threonine, or tyrosine is phosphorylated, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue of serine, threonine, or tyrosine is nonphosphorylated.
In another further embodiment of this antibody, the amino acid sequence set forth in Table 4 is comprised in an autophagy protein or fragment. In one variation of this embodiment, the antibody specifically binds to the full length autophagy protein when the x residue in a sequence set forth in Table 4 is nonphosphorylated, but does not specifically bind to the full length autophagy protein when the x residue is phosphorylated in the amino acid sequence set forth in Table 4. In another variation of this embodiment, the antibody specifically binds to the full length autophagy protein when the x residue is phosphorylated in the amino acid sequence set forth in Table 4, but does not specifically bind to the full length autophagy protein when the x residue is nonphosphorylated in the amino acid sequence set forth in Table 4. In a third variation of this embodiment, the antibody specifically binds to the full length autophagy protein at its natural conformation.
It will be appreciated by one of skill in the art, that there are many approaches to produce antibodies of this aspect. The following are some exemplary techniques of preparing antibodies.
Antibodies may be prepared using recombinant techniques to generate a Fab fragment that binds to an antigen. The Fab fragment may be generated by sequencing the amino acid residues of a desired monoclonal antibody and determining the sequence of the light chain and the variable and constant regions of the heavy chain that are associated with that particular light chain. Then, two peptides may be designed with these sequences. The peptides may be produced recombinantly in any system, preferably E. coli and B. subtilis, by designing a vector that contains sequence encoding these two peptides.
Animals are generally required for at least one stage of antibody production. The following exemplary animals may be used: rabbits, chickens, goats, guinea pigs, hamsters, horses, mice, rats, and sheep. Factors governing which animal to choose include: 1) the amount of antibody needed, 2) phylogenetic relationship between the donor of the antigen and the antibody producing animal and 3) the specific desired characteristics of the antibodies. If large amounts of antibody is the overriding concern, then horses, goats, and sheep may be preferred. If a distant phylogenetic relationship is important, then chickens are preferred. Mice and rats are preferred for monoclonal antibody production since only small amounts of antigen are needed to generate antibody producing cells needed for subsequent cloning. Rabbits are a preferred choice for laboratory scale polyclonal antibody production.
Various embodiments of this and other aspects use the following methods to enhance antibody production in an animal: use of carriers (i.e., keyhole limpet cyanin) and immune response potentiators (i.e., multiple antigen peptide such as branched oligolysine).
The amount of antigen used to immunize the animal may vary. Larger amounts of antigen are correlated with higher titer antibody production. For high titer antibody production, the following exemplary amounts of antigen may be used: 250-1,000 micrograms for a rabbit, 50-200 micrograms for a mouse, 150 to 500 micrograms for a guinea pig, 750 to 5,000 micrograms for a goat. Additionally, antibody production may be enhanced by administering small priming doses to an animal before a larger dose as above. The following exemplary amounts of antigen may be used for priming: 50-200 micrograms for a rabbit, 10-50 micrograms for a mouse, 50-500 micrograms for a guinea pig, 250 to 750 micrograms for a goat. Additional amounts of antigen may be administered in the form of a booster, which may be in an amount roughly equal to the priming doses.
Adjuvants may be combined with the antigen in order to stimulate the immune response. Exemplary adjuvants include Freund's complete, Freund's incomplete, Titermax, and Ribi.
Monoclonal antibodies may be produced using phage display techniques. An autophagy polypeptide may be attached to a support medium, such as a column. Phage that express a plurality of antibodies or fragments of antibodies, such as a phage display library, may be exposed to the autophagy polypeptide-bound support medium. An exemplary phage display library used for this purpose is described in Schofield, D., et al., Genome Biology (2007) 8(11):R254. Phage that bind to the support may then be further analyzed to determine if they express a molecule that may be an antibody to an autophagy polypeptide. Optionally, a phage display library may be used to isolate an antibody fragment that binds to an autophagy polypeptide. See Teunissen, S., et al., RNA (1998) 4:1124-1133, for an example of using phage display to isolate an antibody fragment.
An antibody may also be produced by using a clonal expansion of B cells to isolate an original light chain-heavy chain pairing. An example of this method is described in de Wildt, R., et al., J. Immunol. Methods (1997) 207:61-67.
Variant Antibodies
In some embodiments, the antibody can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgG, IgA or IgD or sub-Isotype including IgG1, IgG2, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain.
In some embodiments, an antibody may have fewer than 4 chains. Such exemplary antibodies include, but are not limited to, single chain antibodies, Camelid antibodies and the like and components of the antibody, including a heavy chain or a light chain. Such exemplary antibodies include small modular immunopharmaceuticals or SMIPs™, Fab and F(ab′)2 fragments, etc. These antibodies may be produced by papain or pepsin digestion of antibodies. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment of an antibody yields an F(ab′)2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
In some embodiments, the antibody may comprise an “Fv” domain. “Fv” usually refers to the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the CDRs confer antigen-binding specificity to the antibody.
In some embodiments, the antibody may have a single variable domain (or half of an Fv comprising three CDRs specific for an antigen), since the single variable domain may have the ability to recognize and bind antigen, although likely at a lower affinity than the entire binding site.
In some embodiments, the antibody comprises a single-chain Fv antibody fragment. “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In certain embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, eds. (Springer-Verlag: New York, 1994), pp. 269-315.
In some embodiments, the antibody may comprise a SMIP. SMIPs are a class of single-chain peptides engineered to include a target binding region and effector domain (CH2 and CH3 domains). See, e.g., U.S. Patent Application Publication No. 20050238646. The target binding region may be derived from the variable region or CDRs of an antibody, e.g., a phosphorylation site-specific antibody of the application. Alternatively, the target binding region is derived from a protein that binds a phosphorylation site.
In some embodiments, a therapeutic agent may be placed on one arm of the antibody. The therapeutic agent can be a drug, toxin, enzyme, DNA, radionuclide, etc.
In some embodiments, the antigen-binding fragment can be a diabody. The term “diabody” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404097; WO 93/11161; and Hollinger, et al., Proc. Natl. Acad. Sci. USA (1993) 90:6444-6448.
In some embodiments, antibodies may comprise camelid antibodies. Camelid antibodies refer to a unique type of antibodies that are devoid of light chain, initially discovered from animals of the camelid family. The heavy chains of camelid antibodies bind their antigen by one single domain, the variable domain of the heavy immunoglobulin chain, referred to as VHH. VHHs show homology with the variable domain of heavy chains of the human VHIII family. The VHHs obtained from an immunized camel, dromedary, or llama have a number of advantages, such as effective production in microorganisms such as Saccharomyces cerevisiae.
In some embodiments, the antibodies can be humanized, chimerized, deimmunized, or fully human. Numerous publications set forth the many types of antibodies and the methods of engineering such antibodies. For example, see U.S. Pat. Nos. 6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539; 6,103,889; and 5,260,203. The chimeric antibody is an antibody having portions derived from different antibodies. For example, a chimeric antibody may have a variable region and a constant region derived from two different antibodies. The donor antibodies may be from different species. In certain embodiments, the variable region of a chimeric antibody is non-human, e.g., murine, and the constant region is human.
The genetically altered antibodies used in the invention include CDR grafted humanized antibodies. In one embodiment, the humanized antibody comprises heavy and/or light chain CDRs of a non-human donor immunoglobulin and heavy chain and light chain frameworks and constant regions of a human acceptor immunoglobulin. The method of making humanized antibody is disclosed in U.S. Pat. Nos. 5,530,101 ; 5,585,089; 5,693,761 ; 5,693,762; and 6,180,370 each of which is incorporated herein by reference in its entirety.
In some embodiments, the antibody may be an “antigen-binding fragment of an antibody”, which is any portion of an antibody that retains specific binding of the intact antibody. An exemplary antigen-binding fragment of an antibody is the heavy chain and/or light chain CDR, or the heavy and/or light chain variable region. Antigen-binding fragments of the antibodies of the invention, which retain the binding specificity of the intact antibody, are also included in the invention. Examples of these antigen-binding fragments include, but are not limited to, partial or full heavy chains or light chains, variable regions, or CDR regions of any phosphorylation site-specific antibodies described herein.
In some embodiments, the antibody may be an immunoglobulin chain comprised in order from 5′ to 3′, of a variable region and a constant region. The variable region may comprise three complementarity determining regions (CDRs), with interspersed framework (FR) regions for a structure FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In these embodiments, the antibody may further comprise heavy or light chain variable regions, framework regions and CDRs.
In some embodiments, the antibody may comprise a heavy chain constant region that comprises some or all of a CH1 region, hinge, CH2 and CH3 region.
In some embodiments, the antibody may have an binding affinity (KD) of 1×107 M or less. In other embodiments, the antibody binds with a KD of 1×108 M, 1×109 M, 1×1010 M, 1×1011 M, 1×1012 M or less. In certain embodiments, the KD is 1 μM to 500 μM, between 500 μM to 1 μM, between 1 μM to 100 nM, or between 100 mM to 10 tiM.
In some embodiments, the antibodies may be genetically-altered, wherein the amino acid sequence of the native antibody has been varied. Because of the relevance of recombinant DNA techniques to this application, one need not be confined to the sequences of amino acids found in natural antibodies; antibodies can be redesigned to obtain desired characteristics. The possible variations are many and range from the changing of just one or a few amino acids to the complete redesign of, for example, the variable or constant region. Changes in the constant region may be made in order to improve or alter characteristics, such as complement fixation, interaction with membranes and other effector functions. Changes in the variable region may be made in order to improve the antigen binding characteristics. Modified antibodies may provide improved stability or/and therapeutic efficacy. Examples of modified antibodies include those with conservative substitutions of amino acid residues, and one or more deletions or additions of amino acids that do not significantly deleteriously alter the antigen binding utility. Substitutions can range from changing or modifying one or more amino acid residues to complete redesign of a region as long as the therapeutic utility is maintained. Antibodies can be modified post-translationally (e.g., acetylation, and/or phosphorylation) or can be modified synthetically (e.g., the attachment of a labeling group).
In preferred embodiments, genetically altered antibodies are functionally equivalent to the above-mentioned natural antibodies.
Oligoclonal antibodies may be used in various embodiments. In some embodiments, oligoclonal antibodies consisting of a predetermined mixture of antibodies against one or more epitopes are generated in a single cell. In other embodiments, oligoclonal antibodies comprise a plurality of heavy chains capable of pairing with a common light chain to generate antibodies with multiple specificities (e.g., PCT publication WO 04/009618). It is appreciated by one of skill in the art that oligoclonal antibodies are particularly useful when it is desired to target multiple epitopes on a single target molecule. In view of the assays and epitopes disclosed herein, those skilled in the art can generate or select antibodies or mixtures of antibodies that are applicable for an intended purpose and desired need.
Recombinant antibodies against the novel phosphorylation sites identified in the disclosure may be used in various embodiments. These recombinant antibodies have the same amino acid sequence as the natural antibodies or have altered amino acid sequences of the natural antibodies in the present application. They can be made in any expression systems including both prokaryotic and eukaryotic expression systems or using phage display methods (see, e.g., Dower, et al., WO91/17271 and McCafferty, et al., WO92/01047; U.S. Pat. No. 5,969,108, which are herein incorporated by reference in their entirety).
In some embodiments, antibodies have variant constant or Fc regions. Such antibodies can be useful in modulating effector functions, i.e., antigen-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Such antibodies may be useful in instances where a parent singling protein is expressed in normal tissue; variant antibodies without effector function in these instances may elicit the desired therapeutic response while not damaging normal tissue.
In some embodiments, the antibody fragments are truncated chains (truncated at the carboxyl end). These truncated chains may possess one or more immunoglobulin activities (e.g., complement fixation activity). Examples of truncated chains include, but are not limited to, Fab fragments (consisting of the VL, VH, CL and CH1 domains); Fd fragments (consisting of the VH and CH1 domains); Fv fragments (consisting of VL and VH domains of a single chain of an antibody); dAb fragments (consisting of a VH domain); isolated CDR regions; (FaW)2 fragments, bivalent fragments (comprising two Fab fragments linked by a disulphide bridge at the hinge region). The truncated chains can be produced by conventional biochemical techniques, such as enzyme cleavage, or recombinant DNA techniques, each of which is known in the art. These polypeptide fragments may be produced by proteolytic cleavage of intact antibodies by methods well known in the art, or by inserting stop codons at the desired locations in the vectors using site-directed mutagenesis, such as after CH1 to produce Fab fragments or after the hinge region to produce (Fab′)2 fragments. Single chain antibodies may be produced by joining VL- and VH-coding regions with a DNA that encodes a peptide linker connecting the VL and VH protein fragments.
In some embodiments, single chain antibodies, and chimeric, humanized or primatized (CDR-grafted) antibodies, as well as chimeric or CDR— grafted single chain antibodies, comprising portions derived from different species, are also encompassed by the present disclosure as antigen-binding fragments of an antibody. The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., U.S. Pat. Nos. 4,816,567 and 6,331,415; U.S. Pat. No. 4,816,397; European Patent No. 0,120,694; WO86/01533; European Patent No. 0,194,276 B1; U.S. Pat. No. 5,225,539; and European Patent No. 0,239,400 B1. See also, Newman, et al., BioTechnology (1992) 10:1455-1460, regarding primatized antibody. See, e.g., Ladner, et al., U.S. Pat. No. 4,946,778; and Bird, et al., Science (1988) 242:423-426), regarding single chain antibodies.
In addition, functional fragments of antibodies, including fragments of chimeric, humanized, primatized or single chain antibodies, can also be produced. Functional fragments of the subject antibodies retain at least one binding function and/or modulation function of the full-length antibody from which they are derived.
Since the immunoglobulin-related genes contain separate functional regions, each having one or more distinct biological activities, the genes of the antibody fragments may be fused to functional regions from other genes (e.g., enzymes, U.S. Pat. No. 5,004,692, which is incorporated by reference in its entirety) to produce fusion proteins or conjugates having novel properties.
In some embodiments, non-Immunoglobulin binding polypeptides are also contemplated. For example, CDRs from an antibody disclosed herein may be inserted into a suitable non-Immunoglobulin scaffold to create a non-immunoglobulin binding polypeptide. Suitable candidate scaffold structures may be derived from, for example, members of fibronectin type III and cadherin superfamilies.
Some embodiments may comprise other equivalent non-antibody molecules, such as protein binding domains or aptamers, which bind, in a phospho-specific manner, to an amino acid sequence comprising a novel phosphorylation site of the invention. See, e.g., Neuberger, et al., Nature (1984) 312:604. Aptamers are oligonucleic acid or peptide molecules that bind a specific target molecule. DNA or RNA aptamers are typically short oligonucleotides, engineered through repeated rounds of selection to bind to a molecular target. Peptide aptamers typically consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint generally increases the binding affinity of the peptide aptamer to levels comparable to an antibody (nanomolar range).
In some embodiments, phosphorylation site-specific antibodies may be conjugated with immunotoxins. Conjugates that are immunotoxins including antibodies have been widely described in the art. The toxins may be coupled to the antibodies by conventional coupling techniques or immunotoxins containing protein toxin portions can be produced as fusion proteins. In certain embodiments, antibody conjugates may comprise stable linkers and may release cytotoxic agents inside cells (see U.S. Pat. Nos. 6,867,007 and 6,884,869). The conjugates of the present application can be used in a corresponding way to obtain such immunotoxins. Illustrative of such immunotoxins are those described by Byers, et al., Seminars Cell Biol (1991) 2:59-70 and by Fanger, et al., Immunol Today (1991) 12:51-54. Exemplary immunotoxins include radiotherapeutic agents, ribosome-Inactivating proteins (RIPs), chemotherapeutic agents, toxic peptides, or toxic proteins.
Antibody Pharmaceutical Compositions
Another aspect is a pharmaceutical composition that comprises an antibody of any other aspect disclosed. The pharmaceutical composition may further comprise a carrier. Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences, 16th Edition, A. Osal., Ed., 1980). PEG may be used as a pharmaceutical carrier. PEG may be covalently attached to the antibody in the pharmaceutical composition.
Method for Detecting an Autophagy Protein
Another aspect is a method for detecting an autophagy protein or fragment comprising an amino acid sequence set forth in Table 4, wherein the x residue is either phosphorylated or nonphosphorylated, in a sample. The method comprises contacting a sample containing or suspected of containing an autophagy protein or fragment comprising the amino acid sequence set forth in Table 4, wherein the x residue is either phosphorylated or nonphosphorylated serine, threonine, or tyrosine, with an isolated antibody that specifically binds to an epitope that comprises the x residue of the sequence set forth in Table 4, wherein x is either phosphorylated or nonphosphorylated; and assessing a complex formed between the autophagy protein or fragment, if present in the sample, and the antibody, to determine the presence, absence and/or amount of the autophagy protein or fragment in the sample.
In an embodiment of this method, the epitope comprises the amino acid residue x in the sequence set forth in Table 4 and an amino acid residue of the autophagy protein that is outside the amino acid sequence set forth in Table 4.
In another embodiment of this method, the epitope comprises the amino acid residue x in the amino acid sequence set forth in Table 4 and amino acid residues of the autophagy protein that are outside the amino acid sequence set forth in Table 4.
In some embodiments, the epitope is comprised in the amino acid sequence set forth in Table 4.
In some embodiments, the epitope is comprised in an amino acid sequence set forth in Table 3.
In some embodiments, the epitope is comprised in an amino acid sequence selected from the group consisting of a sequence set forth in Table 1.
In another embodiment, the isolated antibody specifically binds to the epitope that comprises the x residue in the amino acid sequence set forth in Table 4, wherein x is not phosphorylated.
In another embodiment, the isolated antibody specifically binds to the epitope that comprises the amino acid residue x in the amino acid sequence set forth in Table 4, wherein x is phosphorylated.
In another embodiment, the isolated antibody specifically binds to an epitope comprised in an amino acid sequence set forth in Table 4, wherein the x residue is not phosphorylated. The antibody does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated.
In another embodiment, the isolated antibody specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated. The antibody does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is not phosphorylated.
In another embodiment, the isolated antibody specifically binds to the full length autophagy protein at its natural conformation.
In another embodiment, the isolated antibody is a monoclonal or polyclonal antibody or antibody fragment.
In another embodiment, the complex is assessed by a format selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immunofluorescent assay (IFA), nephelometry, flow cytometry assay, plasmon resonance assay, chemiluminescence assay, lateral flow immunoassay, u-capture assay, inhibition assay and avidity assay.
In another embodiment, the complex is assessed in a homogeneous or a heterogeneous assay format.
Immunoassay
Although a variety of assay types are contemplated, the present methods frequently assess the complex formed between a full-length autophagy protein and the antibody via a sandwich or competitive assay format. On occasion, the complex is assessed in a homogeneous or a heterogeneous assay format. Also frequently, the complex is assessed by a format selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immunofluorescent assay (IFA), nephelometry, flow cytometry assay, plasmon resonance assay, chemiluminescence assay, lateral flow immunoassay, u-capture assay, inhibition assay and avidity assay.
In a sandwich assay format, the antibody that that specifically binds to an epitope that comprises the amino acid residue x in the amino acid sequence set forth in Table 4 is used as a first antibody. The antibody that is capable of binding to a portion of full-length autophagy protein other than a sequence set forth in Table 4, which binds to the first antibody, is used as a second antibody. Alternatively, in a sandwich assay, the antibody that that specifically binds to an epitope that comprises the amino acid residue x in an amino acid sequence set forth in Table 4 is used as a first antibody. The antibody that is capable of binding to a portion of whole autophagy polypeptide other than the sequence set forth in Table 4, which binds to the first antibody, is used as a second antibody.
Either the first antibody or the second antibody is frequently attached to a surface and functions as a capture antibody. The attachment can be direct or indirect. In a preferred embodiment, the attachment is provided via a biotin-avidin (or streptavidin) linking pair.
Determination of Phosphorylation Status
Phosphorylation site-specific binding molecules are disclosed that specifically bind at novel phosphorylation sites in various autophagy protein molecules set forth in Table 2. These molecules distinguish between the phosphorylated and unphosphorylated forms. In one embodiment, the binding molecule is an antibody or an antigen-binding fragment thereof. The antibody may specifically bind to an amino acid sequence comprising a phosphorylation site identified by “X” in each of the sequences set forth in Table 3.
In this aspect is a method to determine the phosphorylation status of an autophagy protein or fragment by detecting a polypeptide comprising the amino acid sequence set forth in Table 4. The method comprises contacting a sample containing or suspected of containing this protein with an isolated antibody that specifically binds to an epitope that comprises the x residue in the sequence set forth in Table 4. The antibody may recognize either the phosphorylated or nonphosphorylated form of this epitope, i.e., serine, threonine, tyrosine or phosphoserine, phosphoserine, or phosphotyrosine. The method further comprises a step of assessing a complex formed between the autophagy protein or fragment, if present in the sample, to determine the phosphorylation status of the autophagy protein.
An example of this embodiment involves using two antibodies, one that specifically recognizes serine and one that specifically recognizes phosphoserine. It can be appreciated that one of skill in the art can measure the phosphorylation status by determining the relative degree of staining of the two antibodies in various standards containing known amounts of phosphorylated and nonphosphorylated autophagy peptides to determine the phosphorylation status of an autophagy protein.
A further embodiment of this aspect is to determine the phosphorylation status of a full length autophagy protein. Optionally, this embodiment is used to determine the phosphorylation status of a full length autophagy protein in a biological sample. The biological sample may be a clinical sample. The determination of the phosphorylation status of an autophagy protein, or optionally a full length autophagy protein, in a biological sample may be conducted for the prognosis, diagnosis and/or treatment monitoring of a disease or disorder associated with abnormal phosphorylation status of a autophagy protein or fragment comprising an amino acid sequence set forth in Table 4.
Exemplary diseases associated with abnormal phosphorylation level of an autophagy protein or fragment may include ischemic brain injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion diseases and polyglutamine disorders including Huntington's disease and various spinocerebellar ataxias, transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease, breast cancer, ovarian cancer, brain cancer, pancreatic cancer, esophageal cancer, colorectal cancer, liver cancer, prostate cancer, renal cancer, lung cancer, Myocardial ischemia, cardiac remodeling, cardiomyopathy, hemodynamic stress, myocardial hypertrophy, Neuronal ceroid-lipofuscinosis (adult and juvenile), Multiple Sulfatase Deficiency (MSD) and Mucopolysaccharidosis type IIIA, Batten disease, Niemann-Pick C, Danon disease, Pompe disease, and dysfunction of innate and adaptive immunity against intracellular pathogens.
Method for Identifying a Phosphatase
Another aspect is a method for identifying a phosphatase that dephosphorylates an autophagy polypeptide on the x residue. The method comprises the following steps:
(1) providing an autophagy polypeptide comprising an amino acid sequence set forth in Table 1, wherein the x residue is phosphorylated, (2) contacting the autophagy polypeptide with a test protein and H2O under conditions suitable for the dephosphorylation of the phosphoserine residue of the autophagy polypeptide; and (3) assessing the phosphorylation status of the autophagy polypeptide to determine whether the test protein is a phosphatase for the autophagy protein at the x residue.
In a further embodiment, the autophagy protein or fragment comprises an amino acid sequence set forth in Table 4 wherein the x residue is phosphorylated. Optionally, the phosphorylation status of the autophagy polypeptide is assessed by an isolated antibody that specifically binds to an epitope comprised in an amino acid sequence set forth in Table 4 wherein the x residue is not phosphorylated, but does not specifically bind to an epitope comprised of the same amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated. An example of this embodiment is the detection of a nonphosphorylated autophagy protein by detecting binding of an antibody that specifically binds to the nonphosphorylated epitope and does not bind to the phosphorylated epitope.
Method for Identifying a Phosphatase Modulator
Another aspect is a method for identifying a modulator of a phosphatase that dephosphorylates an autophagy protein on the x residue. The method comprises the following steps: (1) providing an autophagy polypeptide comprising an amino acid sequence selected from Table 1, wherein the x residue is phosphorylated; (2) contacting the autophagy polypeptide with a phosphatase that dephosphorylates the autophagy polypeptide on the x residue and H2O under conditions suitable for the dephosphorylation of the phosphorylated x residue of the autophagy polypeptide in the presence or absence of a test substance; and (3) assessing and comparing dephosphorylation status of the autophagy polypeptide by the phosphatase to determine whether the test substance modulates the phosphatase.
In a further embodiment of this aspect, the autophagy protein or fragment comprises an amino acid sequence from Table 4 wherein the x residue is phosphorylated. Optionally, the phosphorylation status of the autophagy polypeptide is assessed by an isolated antibody that specifically binds to an epitope comprised in the amino acid sequence from Table 4, wherein the x residue is not phosphorylated, but does not specifically bind to an epitope comprised in the amino acid sequence from Table 4, wherein the x residue is phosphorylated.
Kit for Protein Detection
In another aspect is a kit for detecting an autophagy protein or fragment comprising an amino acid sequence from Table 4, wherein the x residue is either phosphorylated or nonphosphorylated, in a sample. The kit comprises, in a container, an isolated antibody that specifically binds to an epitope that comprises the amino acid residue x in the amino acid sequence from Table 4, wherein x is serine or phosphoserine.
An exemplary use of the kit is to specifically detect a native autophagy protein that is phosphorylated at the x residue. Another exemplary use of the kit is to specifically detect native autophagy protein that is not phosphorylated at the x residue.
Method of Treating DiseaseThe following aspect relates to a method of treating a disease or disorder associated with abnormal phosphorylation status of an autophagy protein or fragment comprising amino acid sequence from Table 4, wherein the x residue of serine, threonine, or tyrosine is phosphorylated or nonphosphorylated. The method comprises administering, to a subject when such a treatment is needed or desired, a sufficient amount of an isolated antibody that specifically binds to an epitope that comprises the amino acid residue x in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated or nonphosphorylated. The subject may be a mammal. The subject may be a human.
In some embodiments, the isolated antibody specifically binds to the full length autophagy protein at its natural conformation.
In some embodiments, the isolated antibody is a monoclonal or polyclonal antibody or antibody fragment.
Some embodiments further comprise the step of administering a pharmaceutically acceptable carrier and excipient.
In one embodiment of the above aspect the isolated antibody specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein x is not phosphorylated, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4, wherein x is phosphorylated. Optionally, the disease or disorder is associated with abnormal phosphorylation level of an autophagy protein or fragment, and the binding of the antibody to the autophagy protein or fragment prevents or reduces the phosphorylation level of the autophagy protein or fragment. As a further option, the diseases or disorders associated with abnormal phosphorylation level of an autophagy protein or fragment are ischemic brain injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion diseases and polyglutamine disorders including Huntington's disease and various spinocerebellar ataxias, transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease, breast cancer, ovarian cancer, brain cancer, pancreatic cancer, esophageal cancer, colorectal cancer, liver cancer, prostate cancer, renal cancer, lung cancer, Myocardial ischemia, cardiac remodeling, cardiomyopathy, hemodynamic stress, myocardial hypertrophy, Neuronal ceroid-lipofuscinosis (adult and juvenile), Multiple Sulfatase Deficiency (MSD) and Mucopolysaccharidosis type IIIA, Batten disease, Niemann-Pick C, Danon disease, Pompe disease, and dysfunction of innate and adaptive immunity against intracellular pathogens.
In another embodiment of the above aspect, the isolated antibody specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is not phosphorylated. Optionally, the disease or disorder is associated with abnormally low phosphorylation level of an autophagy protein or fragment, and the binding of the antibody to the autophagy protein or fragment prevents or reduces dephosphorylation of the autophagy protein or fragment. As a further option, the disease or disorder associated with abnormally low phosphorylation level of an autophagy protein or fragment are ischemic brain injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion diseases and polyglutamine disorders including Huntington's disease and various spinocerebellar ataxias, transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease, breast cancer, ovarian cancer, brain cancer, pancreatic cancer, esophageal cancer, colorectal cancer, liver cancer, prostate cancer, renal cancer, lung cancer, Myocardial ischemia, cardiac remodeling, cardiomyopathy, hemodynamic stress, myocardial hypertrophy, Neuronal ceroid-lipofuscinosis (adult and juvenile), Multiple Sulfatase Deficiency (MSD) and Mucopolysaccharidosis type IIIA, Batten disease, Niemann-Pick C, Danon disease, Pompe disease, and dysfunction of innate and adaptive immunity against intracellular pathogens.
In another embodiment of the above aspect, the amino acid sequence of a sequence from Table 4 is comprised in a full length autophagy protein and the isolated antibody specifically binds to the full length autophagy protein. As a first option, the isolated antibody specifically binds to the full length autophagy protein when the x residue is not phosphorylated in the amino acid sequence set forth in Table 4, but does not specifically bind to the full length autophagy protein when the x residue is phosphorylated in the amino acid sequence set forth in Table 4. As a second option, the isolated antibody specifically binds to the full length autophagy protein when the x residue is phosphorylated in the amino acid sequence set forth in Table 4, but does not specifically bind to the full length autophagy protein when the x residue is not phosphorylated in the amino acid sequence set forth in Table 4.
Identification of a Kinase
Another aspect is a method for identifying a kinase that phosphorylates an autophagy protein on the phosphorylation site indicated by the x residue. The method comprises the steps of (1) providing an autophagy polypeptide comprising an amino acid sequence selected from Table 1, wherein the x residue is not phosophorylated; (2) contacting the autophagy polypeptide with a test protein and ATP under conditions suitable for the phosphorylation of the serine residue of the autophagy polypeptide; and (3) assessing the phosphorylation status of the autophagy polypeptide to determine whether the test protein is a kinase for the autophagy protein on the x residue.
In another embodiment of this aspect, the autophagy protein or fragment comprises an amino acid sequence set forth in Table 4 wherein the x residue is not phosphorylated.
In another embodiment, the phosphorylation status of the autophagy polypeptide is assessed by an isolated antibody that specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is not phosphorylated.
Identification of a Kinase Modulator
Another aspect is a method for identifying a modulator of a kinase that phosphorylates an autophagy protein on the x residue. The method comprises the steps of: (1) providing an autophagy polypeptide comprising an amino acid sequence selected from Table 1, wherein x is not phosphorylated; (2) contacting the autophagy polypeptide with a kinase that phosphorylates the autophagy polypeptide on the x residue and ATP under conditions suitable for the phosphorylation of the x residue of the autophagy polypeptide in the presence or absence of a test substance; and (3) assessing and comparing phosphorylation status of the autophagy polypeptide by the kinase to determine whether the test substance modulates the kinase.
In one embodiment of this aspect, the autophagy protein or fragment comprises an amino acid sequence set forth in Table 4, wherein x is serine.
In another embodiment of this aspect, the phosphorylation status of the autophagy polypeptide is assessed by an isolated antibody that specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4, wherein x is not phosphorylated.
Isolated Nucleic Acid Fragment
Another aspect is an isolated nucleic acid fragment which is comprised of a sequence of nucleotides encoding an autophagy peptide comprising an amino acid sequence selected from Table 1, wherein x is not phosphorylated. The autophagy peptide is not a full-length autophagy protein comprising an amino acid sequence set forth in Table 2. The nucleic acid may be DNA. The nucleic acid may also be RNA.
In another embodiment, the autophagy peptide comprises an amino acid sequence set forth in Table 4.
In another embodiment there is a plasmid which comprises the nucleic acid fragments of this aspect.
In another embodiment there is a vector which comprises the nucleic acid fragments of this aspect.
In another embodiment, there may be a cell which comprises the plasmids or vectors of this aspect. The cell may be a bacterial cell, a yeast cell, a fungal cell, a plant cell, an insect cell, an animal cell or a human cell. The cell may be used in a method for producing an autophagy peptide, comprising growing the cell of claim 112 under conditions whereby the autophagy peptide is expressed by the cell, and recovering the expressed autophagy peptide. Optionally, the method further comprises a step of phosphorylating the autophagy peptide at the x residue (as indicated in Tables 4 and 5).
Screening for Compounds
In another aspect is a method for identifying a compound that binds to specifically to an autophagy polypeptide phosphorylated at the x residue. The method comprises the following steps: 1) providing a phosphorylated autophagy polypeptide comprising an amino acid sequence selected from Table 1, wherein the x residue is phosphorylated; 2) contacting the phosphorylated autophagy polypeptide with a test compound and H2O under conditions suitable for binding of the test protein with the autophagy polypeptide; 3) assessing binding of the phosphorylated autophagy polypeptide with the test compound; 4) providing a nonphosphorylated autophagy polypeptide comprising an amino acid sequence selected from Table 1, wherein the x residue is phosphorylated; 5) contacting the nonphosphorylated autophagy polypeptide with a test compound and H2O under conditions suitable for binding of the test protein with the autophagy polypeptide; and 6) assessing binding of the nonphosphorylated autophagy polypeptide with the test compound. In the method, the compound that binds specifically to a phosphorylated autophagy polypeptide is a test compound which binds to phosphorylated autophagy polypeptide according to step 3 but does not bind to nonphosphorylated autophagy polypeptide according to step 5. In some embodiments, the test compound may be a polypeptide. In some embodiments, the test compound may be a peptide. In some embodiments the test compound may be a nucleic acid. In some embodiments, the compound may be a small molecule.
In another aspect is a method for identifying a compound that binds to specifically to an autophagy polypeptide that is not phosphorylated at the x residue. The method comprises the following steps: 1) providing a nonphosphorylated autophagy polypeptide comprising an amino acid sequence selected from Table 1, wherein x is serine; 2) contacting the nonphosphorylated autophagy polypeptide with a test compound and H2O under conditions suitable for binding of the test protein with the autophagy polypeptide; 3) assessing binding of the nonphosphorylated autophagy polypeptide with the test compound; 4) providing a phosphorylated autophagy polypeptide comprising an amino acid sequence selected from Table 1, wherein the x residue is phosphorylated; 5) contacting the phosphorylated autophagy polypeptide with a test compound and H2O under conditions suitable for binding of the test protein with the autophagy polypeptide; and 6) assessing binding of the phosphorylated autophagy polypeptide with the test compound. In the method, the compound that binds specifically to a nonphosphorylated autophagy polypeptide is a test compound which binds to nonphosphorylated autophagy polypeptide according to step 3, but does not bind to the phosphorylated autophagy polypeptide according to step 5. In some embodiments, the test compound may be a polypeptide. In some embodiments, the test compound may be a peptide. In some embodiments the test compound may be a nucleic acid. In some embodiments, the compound may be a small molecule.
Method of Treating Disease by Modulating Phosphorylation of an Autophagy Polypeptide
Diseases may be treated by modulating the phosphorylation of an autophagy polypeptide. These diseases may arise from abnormal phosphorylation of the autophagy polypeptide. Alternatively, the phosphorylation status of an autophagy polypeptide may be normal, but the disease may be treated by modulating the phosphorylation of an autophagy polypeptide to enhance or reduce autophagy. Such exemplary diseases include ischemic brain injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion diseases and polyglutamine disorders including Huntington's disease and various spinocerebellar ataxias, transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease, breast cancer, ovarian cancer, brain cancer, pancreatic cancer, esophageal cancer, colorectal cancer, liver cancer, prostate cancer, renal cancer, lung cancer, Myocardial ischemia, cardiac remodeling, cardiomyopathy, hemodynamic stress, myocardial hypertrophy, Neuronal ceroid-lipofuscinosis (adult and juvenile), Multiple Sulfatase Deficiency (MSD) and Mucopolysaccharidosis type IIIA, Batten disease, Niemann-Pick C, Danon disease, Pompe disease, and dysfunction of innate and adaptive immunity against intracellular pathogens.
By increasing the rate of autophagy, there can be enhanced cleaning and turnover of damaged organelles, protein aggregates, and other unwanted cellular features that can be removed by autophagy. By decreasing the rate of autophagy, a diseased cell, such as a cancer cell, may be more susceptible to treatment by chemotherapeutic and other agents.
Autophagy polypeptide phosphorylation may be increased in order to increase autophagy.
Alternatively, autophagy polypeptide phosphorylation may be decreased in order to decrease autophagy.
Autophagy polypeptide phosphorylation may be decreased in order to increase autophagy.
Alternatively, autophagy polypeptide phosphorylation may be increased in order to decrease autophagy.
Antibody Microarrays
In another aspect is a microarray comprising an antibody disclosed above. Microarrays are useful for screening potential binding partners for binding to proteins on an array, in a high-throughput manner. Microarrays are typically comprised of a large number of a library of target or capture reagents robotically arrayed or spotted in high density onto a solid support. Potential binding partners for screening are labeled, usually with fluorescence, and contacted with to a target or capture reagent immobilized on the array under conditions to allow for binding. Following a wash step, binding to the individual targets may be measured and quantified. Each collection of binding partners may be tested individually, with results from individual arrays compared.
An antibody microarray using antibodies of the present invention has substantial utility. The microarray may be used as a diagnostic tool to monitor the phosphorylation of autophagy proteins in a variety of tissue samples from a patient in order to determine whether the patient has a disease that is correlated with an abnormal degree of autophagy. Additionally, the microarray may be used to screen for drugs and other compounds that affect the phosphorylation status of autophagy proteins.
Example 1 Production of an APG3L Tyrosine-18 Phosphospecific Polyclonal AntibodyA 12 amino acid phospho-peptide antigen, LEVAEY*LTPVLC (where Y*=phosphotyrosine) (SEQ ID NO: 2), corresponding to residues 13-23 of human APG3L plus cysteine on the C-terminal for coupling, was constructed according to standard synthesis techniques using a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See, e.g., Antibodies: A Laboratory Manual, Chapter 5, pages 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology (1991) 201:264-283; Merrifield, J. Am. Chem. Soc. (1962) 85:21-49.
This peptide was coupled to KLH, and rabbits were injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (200 μg antigen per rabbit). The rabbits were boosted with same antigen in incomplete Freund adjuvant (100 μg antigen per rabbit) weekly for eight. Coincident with the dates of the third through eighth boosts, 15-20 mL bleed per rabbit was collected. One week after the eight boost, a terminating bleed of 40-60 mL per rabbit was collected. The sera were purified by Protein A-affinity chromatography as previously described (see Antibodies: A Laboratory Manual, Cold Spring Harbor, supra.). The eluted immunoglobulins were further loaded onto LEVAEYLTPVLC (SEQ ID NO: 3)-resin Knotes column. The flow through fraction was collected and applied onto LEVAEY*LTPVLC (SEQ ID NO: 2)-resin column. After washing the column extensively, the phospho-APG3L-Y18 antibodies were eluted and kept in antibody storage buffer.
The antibody was confirmed for phosphospecificity against the peptide antigen using Dot blot assay. 50 ng of phosphopeptide and 50 ng of the nonphosphorylated version of the peptide were spotted separately onto a nitrocellulose membrane. The membrane was allowed to dry. Non-specific sites were blocked by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). A 10 cm petri dish was used for the reaction chamber. The membrane was incubated with the purified APG3L-Y18 phosphospecific polyclonal antibody at a concentration of 0.5 μg/mL dissolved in BSA/TBS-T for 30 min at room temperature. The membrane was washed three times with TBS-T (3×5 min). The membrane was incubated with secondary antibody conjugated to HRP using manufacturer's recommended dilution for 30 min at room temperature. The membrane was then washed three times with TBS-T (15 min×1, 5 min×2), then once with TBS (5 min). The membrane was then incubated with ECL reagent for 1 min, covered with Saran-wrap after removing excess solution from the surface, and then exposed to X-ray film in a dark room at several different lengths of exposure. The results of the dot blot is shown in
A 12 amino acid phospho-peptide antigen, LGRDWS*WEKQKC (SEQ ID NO: 4) (where S*=phosphoserine), corresponding to residues 95-105 of human APG4A plus cysteine on the C-terminal for coupling, was constructed according to standard synthesis techniques using a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See, e.g., Antibodies: A Laboratory Manual, Chapter 5, pages 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology (1991) 201:264-283; Merrifield, J. Am. Chem. Soc. (1962) 85:21-49.
This peptide was coupled to KLH, and rabbits were injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (200 μg antigen per rabbit). The rabbits were boosted with same antigen in incomplete Freund adjuvant (100 μg antigen per rabbit) weekly for eight. Coincident with the dates of the third through eighth boosts, 15-20 mL bleed per rabbit was collected. One week after the eight boost, a terminating bleed of 40-60 mL per rabbit was collected. The sera were purified by Protein A-affinity chromatography as previously described (see Antibodies: A Laboratory Manual, Cold Spring Harbor, supra.). The eluted immunoglobulins were further loaded onto LGRDWSWEKQKC (SEQ ID NO: 5)-resin Knotes column. The flow through fraction was collected and applied onto LGRDWS*WEKQKC (SEQ ID NO: 4)-resin column. After washing the column extensively, the phospho-APG4A-S100 antibodies were eluted and kept in antibody storage buffer.
The antibody was confirmed for phosphospecificity against the peptide antigen using Dot blot assay. 50 ng of phosphopeptide and 50 ng of the nonphosphorylated version of the peptide were spotted separately onto a nitrocellulose membrane. The membrane was allowed to dry. Non-specific sites were blocked by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). A 10 cm petri dish was used for the reaction chamber. The membrane was incubated with the purified APG4A-S100 phosphospecific polyclonal antibody at a concentration of 0.5 μg/mL dissolved in BSA/TBS-T for 30 min at room temperature. The membrane was washed three times with TBS-T (3×5 min). The membrane was incubated with secondary antibody conjugated to HRP using manufacturer's recommended dilution for 30 min at room temperature. The membrane was then washed three times with TBS-T (15 min×1, 5 min×2), then once with TBS (5 min). The membrane was then incubated with ECL reagent for 1 min, covered with Saran-wrap after removing excess solution from the surface, and then exposed to X-ray film in a dark room at several different lengths of exposure. The results of the dot blot is shown in
A 12 amino acid phospho-peptide antigen, FEASLS*GEREFC (SEQ ID NO: 6) (where S*=phosphoserine), corresponding to residues 161-171 of human APG4C plus cysteine on the C-terminal for coupling, was constructed according to standard synthesis techniques using a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See, e.g., Antibodies: A Laboratory Manual, Chapter 5, pages 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology (1991) 201:264-283; Merrifield, J. Am. Chem. Soc. (1962) 85:21-49.
This peptide was coupled to KLH, and rabbits were injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (200 μg antigen per rabbit). The rabbits were boosted with same antigen in incomplete Freund adjuvant (100 μg antigen per rabbit) weekly for eight. Coincident with the dates of the third through eighth boosts, 15-20 mL bleed per rabbit was collected. One week after the eight boost, a terminating bleed of 40-60 mL per rabbit was collected. The sera were purified by Protein A-affinity chromatography as previously described (see Antibodies: A Laboratory Manual, Cold Spring Harbor, supra.). The eluted immunoglobulins were further loaded onto FEASLSGEREFC (SEQ ID NO: 7)-resin Knotes column. The flow through fraction was collected and applied onto FEASLS*GEREFC (SEQ ID NO: 6)-resin column. After washing the column extensively, the phospho-APG4C-S166 antibodies were eluted and kept in antibody storage buffer.
The antibody was confirmed for phosphospecificity against the peptide antigen using Dot blot assay. 50 ng of phosphopeptide and 50 ng of the nonphosphorylated version of the peptide were spotted separately onto a nitrocellulose membrane. The membrane was allowed to dry. Non-specific sites were blocked by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). A 10 cm petri dish was used for the reaction chamber. The membrane was incubated with the purified APG4C-S166 phosphospecific polyclonal antibody at a concentration of 0.5 μg/mL dissolved in BSA/TBS-T for 30 min at room temperature. The membrane was washed three times with TBS-T (3×5 min). The membrane was incubated with secondary antibody conjugated to HRP using manufacturer's recommended dilution for 30 min at room temperature. The membrane was then washed three times with TBS-T (15 min×1, 5 min×2), then once with TBS (5 min). The membrane was then incubated with ECL reagent for 1 min, covered with Saran-wrap after removing excess solution from the surface, and then exposed to X-ray film in a dark room at several different lengths of exposure. The results of the dot blot is shown in
A 12 amino acid phospho-peptide antigen, KTPTIS*LKETIC (SEQ ID NO: 8) (where S*=phosphoserine), corresponding to residues 172-182 of human APG4C plus cysteine on the C-terminal for coupling, was constructed according to standard synthesis techniques using a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See, e.g., Antibodies: A Laboratory Manual, Chapter 5, pages 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology (1991) 201:264-283; Merrifield, J. Am. Chem. Soc. (1962) 85:21-49.
This peptide was coupled to KLH, and rabbits were injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (200 μg antigen per rabbit). The rabbits were boosted with same antigen in incomplete Freund adjuvant (100 μg antigen per rabbit) weekly for eight. Coincident with the dates of the third through eighth boosts, 15-20 mL bleed per rabbit was collected. One week after the eight boost, a terminating bleed of 40-60 mL per rabbit was collected. The sera were purified by Protein A-affinity chromatography as previously described (see Antibodies: A Laboratory Manual, Cold Spring Harbor, supra.). The eluted immunoglobulins were further loaded onto KTPTISLKETIC (SEQ ID NO: 9)-resin Knotes column. The flow through fraction was collected and applied onto KTPTIS*LKETIC (SEQ ID NO: 8)-resin column. After washing the column extensively, the phospho-APG4C-S177 antibodies were eluted and kept in antibody storage buffer.
The antibody was confirmed for phosphospecificity against the peptide antigen using Dot blot assay. 50 ng of phosphopeptide and 50 ng of the nonphosphorylated version of the peptide were spotted separately onto a nitrocellulose membrane. The membrane was allowed to dry. Non-specific sites were blocked by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). A 10 cm petri dish was used for the reaction chamber. The membrane was incubated with the purified APG4C-S177 phosphospecific polyclonal antibody at a concentration of 0.5 μg/mL dissolved in BSA/TBS-T for 30 min at room temperature. The membrane was washed three times with TBS-T (3×5 min). The membrane was incubated with secondary antibody conjugated to HRP using manufacturer's recommended dilution for 30 min at room temperature. The membrane was then washed three times with TBS-T (15 min×1, 5 min×2), then once with TBS (5 min). The membrane was then incubated with ECL reagent for 1 min, covered with Saran-wrap after removing excess solution from the surface, and then exposed to X-ray film in a dark room at several different lengths of exposure. The results of the dot blot is shown in
A 12 amino acid phospho-peptide antigen, CDFKRAS*EEITK (SEQ ID NO: 10) (where S*=phosphoserine), corresponding to residues 393-403 of human APG4C plus cysteine on the N-terminal for coupling, was constructed according to standard synthesis techniques using a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See, e.g., Antibodies: A Laboratory Manual, Chapter 5, pages 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology (1991) 201:264-283; Merrifield, J. Am. Chem. Soc. (1962) 85:21-49.
This peptide was coupled to KLH, and rabbits were injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (200 μg antigen per rabbit). The rabbits were boosted with same antigen in incomplete Freund adjuvant (100 μg antigen per rabbit) weekly for eight. Coincident with the dates of the third through eighth boosts, 15-20 mL bleed per rabbit was collected. One week after the eight boost, a terminating bleed of 40-60 mL per rabbit was collected. The sera were purified by Protein A-affinity chromatography as previously described (see Antibodies: A Laboratory Manual, Cold Spring Harbor, supra.). The eluted immunoglobulins were further loaded onto CDFKRASEEITK (SEQ ID NO: 11)-resin Knotes column. The flow through fraction was collected and applied onto CDFKRAS*EEITK (SEQ ID NO: 10)-resin column. After washing the column extensively, the phospho-APG4C-5398 antibodies were eluted and kept in antibody storage buffer.
The antibody was confirmed for phosphospecificity against the peptide antigen using Dot blot assay. 50 ng of phosphopeptide and 50 ng of the nonphosphorylated version of the peptide were spotted separately onto a nitrocellulose membrane. The membrane was allowed to dry. Non-specific sites were blocked by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). A 10 cm petri dish was used for the reaction chamber. The membrane was incubated with the purified APG4C-5398 phosphospecific polyclonal antibody at a concentration of 0.5 μg/mL dissolved in BSA/TBS-T for 30 min at room temperature. The membrane was washed three times with TBS-T (3×5 min). The membrane was incubated with secondary antibody conjugated to HRP using manufacturer's recommended dilution for 30 min at room temperature. The membrane was then washed three times with TBS-T (15 min×1, 5 min×2), then once with TBS (5 min). The membrane was then incubated with ECL reagent for 1 min, covered with Saran-wrap after removing excess solution from the surface, and then exposed to X-ray film in a dark room at several different lengths of exposure. The results of the dot blot is shown in
A 12 amino acid phospho-peptide antigen, CQLKRFS*TEEFV (SEQ ID NO: 12) (where S*=phosphoserine), corresponding to residues 446-456 of human APG4C plus cysteine on the N-terminal for coupling, was constructed according to standard synthesis techniques using a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See, e.g., Antibodies: A Laboratory Manual, Chapter 5, pages 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology (1991) 201:264-283; Merrifield, J. Am. Chem. Soc. (1962) 85:21-49.
This peptide was coupled to KLH, and rabbits were injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (200 μg antigen per rabbit). The rabbits were boosted with same antigen in incomplete Freund adjuvant (100 μg antigen per rabbit) weekly for eight. Coincident with the dates of the third through eighth boosts, 15-20 mL bleed per rabbit was collected. One week after the eight boost, a terminating bleed of 40-60 mL per rabbit was collected. The sera were purified by Protein A-affinity chromatography as previously described (see Antibodies: A Laboratory Manual, Cold Spring Harbor, supra.). The eluted immunoglobulins were further loaded onto CQLKRFSTEEFV (SEQ ID NO: 13)-resin Knotes column. The flow through fraction was collected and applied onto CQLKRFS*TEEFV (SEQ ID NO: 12)-resin column. After washing the column extensively, the phospho-APG4C-5451 antibodies were eluted and kept in antibody storage buffer.
The antibody was confirmed for phosphospecificity against the peptide antigen using Dot blot assay. 50 ng of phosphopeptide and 50 ng of the nonphosphorylated version of the peptide were spotted separately onto a nitrocellulose membrane. The membrane was allowed to dry. Non-specific sites were blocked by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). A 10 cm petri dish was used for the reaction chamber. The membrane was incubated with the purified APG4C-S451 phosphospecific polyclonal antibody at a concentration of 0.5 μg/mL dissolved in BSA/TBS-T for 30 min at room temperature. The membrane was washed three times with TBS-T (3×5 min). The membrane was incubated with secondary antibody conjugated to HRP using manufacturer's recommended dilution for 30 min at room temperature. The membrane was then washed three times with TBS-T (15 min×1, 5 min×2), then once with TBS (5 min). The membrane was then incubated with ECL reagent for 1 min, covered with Saran-wrap after removing excess solution from the surface, and then exposed to X-ray film in a dark room at several different lengths of exposure. The results of the dot blot is shown in
A 12 amino acid phospho-peptide antigen, QYRSSS*PEDARC (SEQ ID NO: 14) (where S*=phosphoserine), corresponding to residues 10-20 of human APG4D (SEQ ID NO:196) plus cysteine on the C-terminal for coupling, was constructed according to standard synthesis techniques using a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See, e.g., Antibodies: A Laboratory Manual, Chapter 5, pages 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology (1991) 201:264-283; Merrifield, J. Am. Chem. Soc. (1962) 85:21-49.
This peptide was coupled to KLH, and rabbits were injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (200 μg antigen per rabbit). The rabbits were boosted with same antigen in incomplete Freund adjuvant (100 μg antigen per rabbit) weekly for eight. Coincident with the dates of the third through eighth boosts, 15-20 mL bleed per rabbit was collected. One week after the eight boost, a terminating bleed of 40-60 mL per rabbit was collected. The sera were purified by Protein A-affinity chromatography as previously described (see Antibodies: A Laboratory Manual, Cold Spring Harbor, supra.). The eluted immunoglobulins were further loaded onto QYRSSSPEDARC (SEQ ID NO: 15)-resin Knotes column. The flow through fraction was collected and applied onto QYRSSS*PEDARC (SEQ ID NO: 14)-resin column. After washing the column extensively, the phospho-APG4D-S15 antibodies were eluted and kept in antibody storage buffer.
The antibody was confirmed for phosphospecificity against the peptide antigen using Dot blot assay. 50 ng of phosphopeptide and 50 ng of the nonphosphorylated version of the peptide were spotted separately onto a nitrocellulose membrane. The membrane was allowed to dry. Non-specific sites were blocked by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). A 10 cm petri dish was used for the reaction chamber. The membrane was incubated with the purified APG4D-S15 phosphospecific polyclonal antibody at a concentration of 0.5 μg/mL dissolved in BSA/TBS-T for 30 min at room temperature. The membrane was washed three times with TBS-T (3×5 min). The membrane was incubated with secondary antibody conjugated to HRP using manufacturer's recommended dilution for 30 min at room temperature. The membrane was then washed three times with TBS-T (15 min×1, 5 min×2), then once with TBS (5 min). The membrane was then incubated with ECL reagent for 1 min, covered with Saran-wrap after removing excess solution from the surface, and then exposed to X-ray film in a dark room at several different lengths of exposure. The results of the dot blot is shown in
A 12 amino acid phospho-peptide antigen, CGKPRHS*LYFIG (SEQ ID NO: 16) (where S*=phosphoserine), corresponding to residues 336-346 of human APG4D plus cysteine on the N-terminal for coupling, was constructed according to standard synthesis techniques using a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See, e.g., Antibodies: A Laboratory Manual, Chapter 5, pages 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology (1991) 201:264-283; Merrifield, J. Am. Chem. Soc. (1962) 85:21-49.
This peptide was coupled to KLH, and rabbits were injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (200 μg antigen per rabbit). The rabbits were boosted with same antigen in incomplete Freund adjuvant (100 μg antigen per rabbit) weekly for eight. Coincident with the dates of the third through eighth boosts, 15-20 mL bleed per rabbit was collected. One week after the eight boost, a terminating bleed of 40-60 mL per rabbit was collected. The sera were purified by Protein A-affinity chromatography as previously described (see Antibodies: A Laboratory Manual, Cold Spring Harbor, supra.). The eluted immunoglobulins were further loaded onto CGKPRHSLYFIG (SEQ ID NO: 17)-resin Knotes column. The flow through fraction was collected and applied onto CGKPRHS*LYFIG (SEQ ID NO: 16)-resin column. After washing the column extensively, the phospho-APG4D-5341 antibodies were eluted and kept in antibody storage buffer.
The antibody was confirmed for phosphospecificity against the peptide antigen using Dot blot assay. 50 ng of phosphopeptide and 50 ng of the nonphosphorylated version of the peptide were spotted separately onto a nitrocellulose membrane. The membrane was allowed to dry. Non-specific sites were blocked by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). A 10 cm petri dish was used for the reaction chamber. The membrane was incubated with the purified APG4D-5341 phosphospecific polyclonal antibody at a concentration of 0.5 μg/mL dissolved in BSA/TBS-T for 30 min at room temperature. The membrane was washed three times with TBS-T (3×5 min). The membrane was incubated with secondary antibody conjugated to HRP using manufacturer's recommended dilution for 30 min at room temperature. The membrane was then washed three times with TBS-T (15 min×1, 5 min×2), then once with TBS (5 min). The membrane was then incubated with ECL reagent for 1 min, covered with Saran-wrap after removing excess solution from the surface, and then exposed to X-ray film in a dark room at several different lengths of exposure. The results of the dot blot is shown in
A 12 amino acid phospho-peptide antigen, CRAKRPS*SEDFV (SEQ ID NO: 18) (where S*=phosphoserine), corresponding to residues 462-472 of human APG4D plus cysteine on the N-terminal for coupling, was constructed according to standard synthesis techniques using a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See, e.g., Antibodies: A Laboratory Manual, Chapter 5, pages 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology (1991) 201:264-283; Merrifield, J. Am. Chem. Soc. (1962) 85:21-49.
This peptide was coupled to KLH, and rabbits were injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (200 μg antigen per rabbit). The rabbits were boosted with same antigen in incomplete Freund adjuvant (100 μg antigen per rabbit) weekly for eight. Coincident with the dates of the third through eighth boosts, 15-20 mL bleed per rabbit was collected. One week after the eight boost, a terminating bleed of 40-60 mL per rabbit was collected. The sera were purified by Protein A-affinity chromatography as previously described (see Antibodies: A Laboratory Manual, Cold Spring Harbor, supra.). The eluted immunoglobulins were further loaded onto CRAKRPSSEDFV (SEQ ID NO: 19)-resin Knotes column. The flow through fraction was collected and applied onto CRAKRPS*SEDFV (SEQ ID NO: 18)-resin column. After washing the column extensively, the phospho-APG4D-5467 antibodies were eluted and kept in antibody storage buffer.
The antibody was confirmed for phosphospecificity against the peptide antigen using Dot blot assay. 50 ng of phosphopeptide and 50 ng of the nonphosphorylated version of the peptide were spotted separately onto a nitrocellulose membrane. The membrane was allowed to dry. Non-specific sites were blocked by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). A 10 cm petri dish was used for the reaction chamber. The membrane was incubated with the purified APG4D-5467 phosphospecific polyclonal antibody at a concentration of 0.5 μg/mL dissolved in BSA/TBS-T for 30 min at room temperature. The membrane was washed three times with TBS-T (3×5 min). The membrane was incubated with secondary antibody conjugated to HRP using manufacturer's recommended dilution for 30 min at room temperature. The membrane was then washed three times with TBS-T (15 min×1, 5 min×2), then once with TBS (5 min). The membrane was then incubated with ECL reagent for 1 min, covered with Saran-wrap after removing excess solution from the surface, and then exposed to X-ray film in a dark room at several different lengths of exposure. The results of the dot blot is shown in
A 12 amino acid phospho-peptide antigen, TNTLES*FKTADC (SEQ ID NO: 20) (where S*=phosphoserine), corresponding to residues 90-100 of human APG7L plus cysteine on the C-terminal for coupling, was constructed according to standard synthesis techniques using a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See, e.g., Antibodies: A Laboratory Manual, Chapter 5, pages 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology (1991) 201:264-283; Merrifield, J. Am. Chem. Soc. (1962) 85:21-49.
This peptide was coupled to KLH, and rabbits were injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (200 μg antigen per rabbit). The rabbits were boosted with same antigen in incomplete Freund adjuvant (100 μg antigen per rabbit) weekly for eight. Coincident with the dates of the third through eighth boosts, 15-20 mL bleed per rabbit was collected. One week after the eight boost, a terminating bleed of 40-60 mL per rabbit was collected. The sera were purified by Protein A-affinity chromatography as previously described (see Antibodies: A Laboratory Manual, Cold Spring Harbor, supra.). The eluted immunoglobulins were further loaded onto TNTLESFKTADC (SEQ ID NO: 21)-resin Knotes column. The flow through fraction was collected and applied onto TNTLES*FKTADC (SEQ ID NO: 20)-resin column. After washing the column extensively, the phospho-APG7-S95 antibodies were eluted and kept in antibody storage buffer.
The antibody was confirmed for phosphospecificity against the peptide antigen using Dot blot assay. 50 ng of phosphopeptide and 50 ng of the nonphosphorylated version of the peptide were spotted separately onto a nitrocellulose membrane. The membrane was allowed to dry. Non-specific sites were blocked by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). A 10 cm petri dish was used for the reaction chamber. The membrane was incubated with the purified APG7-S95 phosphospecific polyclonal antibody at a concentration of 0.5 μg/mL dissolved in BSA/TBS-T for 30 min at room temperature. The membrane was washed three times with TBS-T (3×5 min). The membrane was incubated with secondary antibody conjugated to HRP using manufacturer's recommended dilution for 30 min at room temperature. The membrane was then washed three times with TBS-T (15 min×1, 5 min×2), then once with TBS (5 min). The membrane was then incubated with ECL reagent for 1 min, covered with Saran-wrap after removing excess solution from the surface, and then exposed to X-ray film in a dark room at several different lengths of exposure. The results of the dot blot is shown in
A 12 amino acid phospho-peptide antigen, WHRRES*DESGEC (SEQ ID NO: 22) (where S*=phosphoserine), corresponding to residues 730-740 of human APG9L1 plus cysteine on the C-terminal for coupling, was constructed according to standard synthesis techniques using a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See, e.g., Antibodies: A Laboratory Manual, Chapter 5, pages 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology (1991) 201:264-283; Merrifield, J. Am. Chem. Soc. (1962) 85:21-49.
This peptide was coupled to KLH, and rabbits were injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (200 μg antigen per rabbit). The rabbits were boosted with same antigen in incomplete Freund adjuvant (100 μg antigen per rabbit) weekly for eight. Coincident with the dates of the third through eighth boosts, 15-20 mL bleed per rabbit was collected. One week after the eight boost, a terminating bleed of 40-60 mL per rabbit was collected. The sera were purified by Protein A-affinity chromatography as previously described (see Antibodies: A Laboratory Manual, Cold Spring Harbor, supra.). The eluted immunoglobulins were further loaded onto WHRRESDESGEC (SEQ ID NO: 23)-resin Knotes column. The flow through fraction was collected and applied onto WHRRES*DESGEC (SEQ ID NO: 22)-resin column. After washing the column extensively, the phospho-APG9L1-5735 antibodies were eluted and kept in antibody storage buffer.
The antibody was confirmed for phosphospecificity against the peptide antigen using Dot blot assay. 50 ng of phosphopeptide and 50 ng of the nonphosphorylated version of the peptide were spotted separately onto a nitrocellulose membrane. The membrane was allowed to dry. Non-specific sites were blocked by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). A 10 cm petri dish was used for the reaction chamber. The membrane was incubated with the purified APG9L1-5735 phosphospecific polyclonal antibody at a concentration of 0.5 μg/mL dissolved in BSA/TBS-T for 30 min at room temperature. The membrane was washed three times with TBS-T (3×5 min). The membrane was incubated with secondary antibody conjugated to HRP using manufacturer's recommended dilution for 30 min at room temperature. The membrane was then washed three times with TBS-T (15 min×1, 5 min×2), then once with TBS (5 min). The membrane was then incubated with ECL reagent for 1 min, covered with Saran-wrap after removing excess solution from the surface, and then exposed to X-ray film in a dark room at several different lengths of exposure. The results of the dot blot is shown in
A 12 amino acid phospho-peptide antigen, CENEKDS*RRRQA (SEQ ID NO: 24) (where S*=phosphoserine), corresponding to residues 208-218 of human APG16L plus cysteine on the N-terminal for coupling, was constructed according to standard synthesis techniques using a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See, e.g., Antibodies: A Laboratory Manual, Chapter 5, pages 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology (1991) 201:264-283; Merrifield, J. Am. Chem. Soc. (1962) 85:21-49.
This peptide was coupled to KLH, and rabbits were injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (200 μg antigen per rabbit). The rabbits were boosted with same antigen in incomplete Freund adjuvant (100 μg antigen per rabbit) weekly for eight. Coincident with the dates of the third through eighth boosts, 15-20 mL bleed per rabbit was collected. One week after the eight boost, a terminating bleed of 40-60 mL per rabbit was collected. The sera were purified by Protein A-affinity chromatography as previously described (see Antibodies: A Laboratory Manual, Cold Spring Harbor, supra.). The eluted immunoglobulins were further loaded onto CENEKDSRRRQA (SEQ ID NO: 25)-resin Knotes column. The flow through fraction was collected and applied onto CENEKDS*RRRQA (SEQ ID NO: 24)-resin column. After washing the column extensively, the phospho-APG16L-S213 antibodies were eluted and kept in antibody storage buffer.
The antibody was confirmed for phosphospecificity against the peptide antigen using Dot blot assay. 50 ng of phosphopeptide and 50 ng of the nonphosphorylated version of the peptide were spotted separately onto a nitrocellulose membrane. The membrane was allowed to dry. Non-specific sites were blocked by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). A 10 cm petri dish was used for the reaction chamber. The membrane was incubated with the purified APG16L-S213 phosphospecific polyclonal antibody at a concentration of 0.5 μg/mL dissolved in BSA/TBS-T for 30 min at room temperature. The membrane was washed three times with TBS-T (3×5 min). The membrane was incubated with secondary antibody conjugated to HRP using manufacturer's recommended dilution for 30 min at room temperature. The membrane was then washed three times with TBS-T (15 min×1, 5 min×2), then once with TBS (5 min). The membrane was then incubated with ECL reagent for 1 min, covered with Saran-wrap after removing excess solution from the surface, and then exposed to X-ray film in a dark room at several different lengths of exposure. The results of the dot blot is shown in
A 12 amino acid phospho-peptide antigen, IFGRRS*VSSFPC (SEQ ID NO: 26) (where S*=phosphoserine), corresponding to residues 282-292 of human APG16L plus cysteine on the C-terminal for coupling, was constructed according to standard synthesis techniques using a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See, e.g., Antibodies: A Laboratory Manual, Chapter 5, pages 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology (1991) 201:264-283; Merrifield, J. Am. Chem. Soc. (1962) 85:21-49.
This peptide was coupled to KLH, and rabbits were injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (200 μg antigen per rabbit). The rabbits were boosted with same antigen in incomplete Freund adjuvant (100 μg antigen per rabbit) weekly for eight. Coincident with the dates of the third through eighth boosts, 15-20 mL bleed per rabbit was collected. One week after the eight boost, a terminating bleed of 40-60 mL per rabbit was collected. The sera were purified by Protein A-affinity chromatography as previously described (see Antibodies: A Laboratory Manual, Cold Spring Harbor, supra.). The eluted immunoglobulins were further loaded onto IFGRRSVSSFPC (SEQ ID NO: 27)-resin Knotes column. The flow through fraction was collected and applied onto IFGRRS*VSSFPC (SEQ ID NO: 26)-resin column. After washing the column extensively, the phospho-APG16L-5287 antibodies were eluted and kept in antibody storage buffer.
The antibody was confirmed for phosphospecificity against the peptide antigen using Dot blot assay. 50 ng of phosphopeptide and 50 ng of the nonphosphorylated version of the peptide were spotted separately onto a nitrocellulose membrane. The membrane was allowed to dry. Non-specific sites were blocked by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). A 10 cm petri dish was used for the reaction chamber. The membrane was incubated with the purified APG16L-5287 phosphospecific polyclonal antibody at a concentration of 0.5 μg/mL dissolved in BSA/TBS-T for 30 min at room temperature. The membrane was washed three times with TBS-T (3×5 min). The membrane was incubated with secondary antibody conjugated to HRP using manufacturer's recommended dilution for 30 min at room temperature. The membrane was then washed three times with TBS-T (15 min×1, 5 min×2), then once with TBS (5 min). The membrane was then incubated with ECL reagent for 1 min, covered with Saran-wrap after removing excess solution from the surface, and then exposed to X-ray film in a dark room at several different lengths of exposure. The results of the dot blot is shown in
The following are further exemplary embodiments:
1. An isolated autophagy peptide comprising an amino acid sequence selected from the group consisting of sequences set forth in Table 1, wherein the x residue is nonphosphorylated or phosphorylated serine, threonine, or tyrosine, and with the proviso that the peptide is not a full-length autophagy protein.
2. An isolated autophagy peptide of embodiment 1 wherein the peptide does not comprise a sequence set forth in Table 2.
3. The isolated autophagy peptide of embodiment 1, which comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 3.
4. The isolated autophagy peptide of embodiment 1, which comprises an amino acid sequence selected from the group consisting of sequences set forth in Table 1.
5. The isolated autophagy peptide of embodiment 4, wherein the x residue is nonphosphorylated or phosphorylated serine, threonine, or tyrosine.
6. The isolated autophagy peptide of embodiment 1, wherein the x residue is phosphorylated serine, threonine, or tyrosine.
7. A pharmaceutical composition, which comprises an isolated autophagy peptide of embodiment 1 and a pharmaceutically acceptable carrier and excipient.
8. The isolated autophagy peptide of embodiment 1, which is conjugated to a carrier to enhance the peptide's immunogenicity.
9. The isolated autophagy peptide of embodiment 8, wherein the carrier is a carrier protein.
10. The isolated autophagy peptide of embodiment 9, wherein the autophagy peptide and the carrier protein are parts of a fusion protein.
11. An immunogen, which immunogen comprises:
(a) an isolated autophagy peptide of embodiment 1; and
(b) an immune response potentiator.
12. The immunogen of embodiment 11, wherein the immune response potentiator is selected from the group consisting of Bacille Calmette-Guerin (BCG), Corynebacterium Parvum, Brucella abortus extract, glucan, levamisole, tilorone, an enzyme and a non-virulent virus.
13. A multiple antigenic peptide (MAP), which MAP comprises a branched oligolysine core conjugated with a plurality of an isolated autophagy peptide of embodiment 1.
14. The MAP of embodiment 13, wherein the branched oligolysine core comprises 3, 7 or 15 lysine residues.
15. The MAP of embodiment 13, wherein the plurality of the autophagy peptide is conjugated to the branched oligolysine core via a spacer.
16. The MAP of embodiment 13, wherein the spacer is one or more amino acid residues.
17. The MAP of embodiment 13, which comprises 4, 8 or 16 copies of the autophagy peptide.
18. The MAP of embodiment 13, wherein the plurality of the autophagy peptide comprises same or different peptides.
19. A method for producing an antibody to an autophagy polypeptide, which method comprises:
(a) introducing an isolated autophagy peptide of embodiment 1 to a mammal in an amount sufficient to produce an antibody to said autophagy peptide; and
(b) recovering said antibody from said mammal.
20. The method of embodiment 19, wherein the x residue in the isolated autophagy peptide is nonphosphorylated serine, threonine, or tyrosine and the method is used to produce an antibody to a nonphosphorylated autophagy polypeptide.
21. The method of embodiment 19, wherein the x residue in the isolated autophagy peptide is phosphorylated serine, threonine, or tyrosine and the method is used to produce an antibody to a phosphorylated autophagy polypeptide.
22. The method of embodiment 21, which further comprises a step of removing an antibody that binds to an isolated autophagy peptide of embodiment 1 wherein x is nonphosphorylated serine, threonine, or tyrosine.
23. The method of embodiment 19, wherein the isolated autophagy peptide is conjugated to a carrier to enhance the peptide's immunogenicity.
24. The method of embodiment 19, wherein the isolated autophagy peptide is comprised in an immunogen of embodiment 10 or a MAP of embodiment 12.
25. An antibody to an autophagy polypeptide produced by the method of embodiment 19.
26. A kit for producing an antibody to an autophagy polypeptide, which kit comprises:
(a) an isolated autophagy peptide of embodiment 1;
(b) means for introducing said isolated autophagy peptide to a mammal in an amount sufficient to produce an antibody to said autophagy peptide; and
(c) means for recovering said antibody from said mammal.
27. A method for producing an antibody to an autophagy polypeptide, which method comprises:
(a) introducing an autophagy polypeptide to a mammal in an amount sufficient to produce an antibody to said autophagy polypeptide;
(b) recovering said antibody from said mammal; and
(c) affinity purifying an autophagy antibody that specifically binds to an epitope comprised in an autophagy peptide of embodiment 1 using said autophagy peptide.
28. The method of embodiment 27, wherein the autophagy polypeptide comprises a full-length autophagy protein.
29. The method of embodiments 27 and 28, wherein the x residue of the autophagy polypeptide is nonphosphorylated serine, threonine, or tyrosine and the method is used to produce an antibody to a nonphosphorylated autophagy polypeptide.
30. The method of embodiments 27 and 28, wherein the autophagy polypeptide is an autophagy polypeptide comprising a sequence set forth in Table 4, the x residue in any sequence set forth in Table 4 is phosphorylated serine, threonine, or tyrosine, and the method is used to produce an antibody to a phosphorylated autophagy polypeptide.
31. The method of embodiment 30, which further comprises a step of removing an antibody that binds to an isolated autophagy peptide of embodiment 1 wherein the x residue is nonphosphorylated serine, threonine, or tyrosine.
32. An antibody to an autophagy polypeptide produced by the method of embodiment 26.
33. A kit for producing an antibody to an autophagy polypeptide, which kit comprises:
(a) an autophagy protein;
(b) means for introducing said an autophagy protein to a mammal in an amount sufficient to produce an antibody to said autophagy polypeptide;
(c) means for recovering said antibody from said mammal; and
(d) an isolated autophagy peptide of embodiment 1.
34. An isolated antibody that specifically binds to an epitope that comprises the amino acid residue x in an amino acid sequence set forth in Table 4, wherein x is serine, threonine, tyrosine, phosphoserine, phosphothreonine or phosphotyrosine.
35. The isolated antibody of embodiment 34, wherein the epitope comprises the amino acid residue x in the amino acid sequence set forth in Table 4 and an amino acid residue of an autophagy protein that is outside the amino acid sequence set forth in Table 4.
36. The isolated antibody of embodiment 34, wherein the epitope comprises the amino acid residue x in an amino acid sequence set forth in Table 4 and amino acid residues of an autophagy protein that are outside the amino acid sequence set forth in Table 4.
37. The isolated antibody of embodiment 34, wherein the epitope is comprised in an amino acid sequence set forth in Table 4.
38. The isolated antibody of embodiment 34, wherein the epitope is comprised of an amino acid sequence set forth in Table 3.
39. The isolated antibody of embodiment 34, wherein the epitope is comprised of an amino acid sequence set forth in Table 1.
40. The isolated antibody of embodiment 34, which specifically binds to the epitope that comprises the x residue in the amino acid sequence set forth in Table 4, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine.
41. The isolated antibody of embodiment 34, which specifically binds to the epitope that comprises the x residue in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated serine, threonine, or tyrosine.
42. The isolated antibody of embodiment 34, which specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated serine, threonine, or tyrosine.
43. The isolated antibody of embodiment 34, which specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated serine, threonine, or tyrosine, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine.
44. The isolated antibody of embodiment 43, wherein the amino acid sequence set forth in Table 4 is comprised in an autophagy protein or fragment.
45. The isolated antibody of embodiment 44, wherein the amino acid sequence set forth in Table 4 is comprised in a full length autophagy protein and the isolated antibody specifically binds to the full length autophagy protein.
46. The isolated antibody of embodiment 45, which specifically binds to the full length autophagy protein when the x residue is nonphosphorylated serine, threonine, or tyrosine in the amino acid sequence set forth in Table 4 but does not specifically bind to the full length autophagy protein when the x residue is phosphorylated serine, threonine, or tyrosine in the amino acid sequence set forth in Table 4.
47. The isolated antibody of embodiment 45, which specifically binds to the full length autophagy protein when the x residue is phosphorylated serine, threonine, or tyrosine in the amino acid sequence set forth in Table 4 but does not specifically bind to the full length autophagy protein when the x residue is nonphosphorylated serine, threonine, or tyrosine in the amino acid sequence set forth in Table 4.
48. The isolated antibody of embodiment 45, which specifically binds to the full length autophagy protein at its natural conformation.
49. The isolated antibody of embodiment 34, which is a monoclonal or polyclonal antibody or an antibody fragment.
50. A method for detecting an autophagy protein or fragment comprising an amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated or nonphosphorylated serine, threonine, or tyrosine in a sample, which method comprises:
(a) contacting a sample containing or suspected of containing an autophagy protein or fragment comprising the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated or nonphosphorylated serine, threonine, or tyrosine, with an isolated antibody that specifically binds to an epitope that comprises the amino acid residue x in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated or nonphosphorylated serine, threonine, or tyrosine; and
(b) assessing a complex formed between said autophagy protein or fragment, if present in said sample, and said antibody, to determine the presence, absence and/or amount of said autophagy protein or fragment in said sample.
51. The method of embodiment 50, wherein the epitope comprises the amino acid residue x in the amino acid sequence set forth in Table 4 and an amino acid residue of the autophagy protein that is outside the amino acid sequence set forth in Table 4.
52. The method of embodiment 50, wherein the epitope comprises the amino acid residue x in the amino acid sequence set forth in Table 4 and amino acid residues of the autophagy protein that are outside the amino acid sequence set forth in Table 4.
53. The method of embodiment 50, wherein the epitope is comprised in the amino acid sequence set forth in Table 4.
54. The method of embodiment 50, wherein the epitope is comprised in an amino acid sequence set forth in Table 3.
55. The method of embodiment 50, wherein the epitope is comprised in an amino acid sequence selected from the sequence set forth in Table 1.
56. The method of embodiment 50, wherein the isolated antibody specifically binds to the epitope that comprises the x residue in the amino acid sequence set forth in Table 4, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine.
57. The method of embodiment 50, wherein the isolated antibody specifically binds to the epitope that comprises the x residue in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated serine, threonine, or tyrosine.
58. The method of embodiment 50, wherein the isolated antibody specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated serine, threonine, or tyrosine.
59. The method of embodiment 50, wherein the isolated antibody specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated serine, threonine, or tyrosine, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine.
60. The method of embodiment 50, wherein the amino acid sequence set forth in Table 4 is comprised in a full length autophagy protein and the isolated antibody specifically binds to the full length autophagy protein.
61. The method of embodiment 60, wherein the isolated antibody specifically binds to the full length autophagy protein when the x residue is nonphosphorylated serine, threonine, or tyrosine in the amino acid sequence set forth in Table 4 but does not specifically bind to the full length autophagy protein when the x residue is phosphorylated serine, threonine, or tyrosine in the amino acid sequence set forth in Table 4 to determine the presence, absence and/or amount of nonphosphorylated full length autophagy protein in the sample.
62. The method of embodiment 60, wherein the isolated antibody specifically binds to the full length autophagy protein when the x residue is phosphorylated serine, threonine, or tyrosine in the amino acid sequence set forth in Table 4 but does not specifically bind to the full length autophagy protein when the x residue is nonphosphorylated serine, threonine, or tyrosine in the amino acid sequence set forth in Table 4 to determine the presence, absence and/or amount of phosphorylated full length autophagy protein in the sample.
63. The method of embodiment 50, wherein the isolated antibody specifically binds to the full length autophagy protein at its natural conformation.
64. The method of embodiment 50, wherein the isolated antibody is a monoclonal or polyclonal antibody or antibody fragment.
65. The method of embodiment 50, wherein the complex is assessed by a sandwich or competitive assay format.
66. The method of embodiment 65, wherein the antibody that that specifically binds to an epitope that comprises the amino acid residue x in the amino acid sequence set forth in Table 4 is used as a first antibody and an antibody that is capable of binding to a portion of an autophagy protein or fragment other than the portion that binds to the first antibody is used as a second antibody in a sandwich assay format.
67. The method of embodiment 66, wherein either the first antibody or the second antibody is attached to a surface and functions as a capture antibody.
68. The method of embodiment 67, wherein the capture antibody is attached to the surface directly or indirectly.
69. The method of embodiment 67, wherein the capture antibody is attached to the surface via a biotin-avidin (or streptavidin) linking pair.
70. The method of embodiment 50, wherein the complex is assessed by a format selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immunofluorescent assay (IFA), nephelometry, flow cytometry assay, plasmon resonance assay, chemiluminescence assay, lateral flow immunoassay, u-capture assay, inhibition assay and avidity assay.
71. The method of embodiment 50, wherein the complex is assessed in a homogeneous or a heterogeneous assay format.
72. The method of embodiment 50, which is used to determine the phosphorylation status of an autophagy protein or fragment comprising the amino acid sequence set forth in Table 4.
73. The method of embodiment 72, which is used to determine the phosphorylation status of a full length autophagy protein.
74. The method of embodiment 73, which is used to determine the phosphorylation status of a full length autophagy protein in a biological sample.
75. The method of embodiment 74, wherein the biological sample is a clinical sample.
76. The method of embodiment 74, which is used for the prognosis, diagnosis and/or treatment monitoring of a disease or disorder associated with abnormal phosphorylation status of an autophagy polypeptide comprising the amino acid sequence set forth in Table 4.
77. The method of embodiment 76, wherein the autophagy protein is a full length autophagy protein.
78. The method of embodiment 76, wherein the disease or disorder associated with abnormal phosphorylation status of an autophagy protein or fragment is selected from the group consisting of ischemic brain injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion diseases and polyglutamine disorders including Huntington's disease and various spinocerebellar ataxias, transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease, breast cancer, ovarian cancer, brain cancer, pancreatic cancer, esophageal cancer, colorectal cancer, liver cancer, prostate cancer, renal cancer, lung cancer, Myocardial ischemia, cardiac remodeling, cardiomyopathy, hemodynamic stress, myocardial hypertrophy, Neuronal ceroid-lipofuscinosis (adult and juvenile), Multiple Sulfatase Deficiency (MSD) and Mucopolysaccharidosis type IIIA, Batten disease, Niemann-Pick C, Danon disease, Pompe disease, and dysfunction of innate and adaptive immunity against intracellular pathogens.
79. A kit for detecting an autophagy protein or fragment comprising amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated or nonphosphorylated serine, threonine, or tyrosine, in a sample, which kit comprises, in a container, an isolated antibody that specifically binds to an epitope that comprises the x residue in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated or nonphosphorylated serine, threonine, or tyrosine.
80. A method for treating a disease or disorder associated with abnormal phosphorylation status of an autophagy protein or fragment comprising amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated or nonphosphorylated serine, threonine, or tyrosine, which method comprises administering, to a subject when such a treatment is needed or desired, a sufficient amount of an isolated antibody that specifically binds to an epitope that comprises the amino acid residue x in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated or nonphosphorylated serine, threonine, or tyrosine.
81. The method of embodiment 80, wherein the isolated antibody specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein x residue is nonphosphorylated serine, threonine, or tyrosine, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated serine, threonine, or tyrosine.
82. The method of embodiment 81, wherein the disease or disorder is associated with abnormally high phosphorylation level of an autophagy protein or fragment, and the binding of the antibody to the autophagy protein or fragment prevents or reduces phosphorylation level of the autophagy protein or fragment.
84. The method of embodiment 80, wherein the isolated antibody specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated serine, threonine, or tyrosine, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine.
85. The method of embodiment 84, wherein the disease or disorder is associated with abnormally low phosphorylation level of an autophagy protein or fragment, and the binding of the antibody to the autophagy protein or fragment prevents or reduces dephosphorylation of the autophagy protein or fragment.
86. The method of embodiments 82 and 85, wherein the disease or disorder associated with abnormally low phosphorylation level of an autophagy protein is selected from the group consisting of ischemic brain injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion diseases and polyglutamine disorders including Huntington's disease and various spinocerebellar ataxias, transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease, breast cancer, ovarian cancer, brain cancer, pancreatic cancer, esophageal cancer, colorectal cancer, liver cancer, prostate cancer, renal cancer, lung cancer, Myocardial ischemia, cardiac remodeling, cardiomyopathy, hemodynamic stress, myocardial hypertrophy, Neuronal ceroid-lipofuscinosis (adult and juvenile), Multiple Sulfatase Deficiency (MSD) and Mucopolysaccharidosis type IIIA, Batten disease, Niemann-Pick C, Danon disease, Pompe disease, and dysfunction of innate and adaptive immunity against intracellular pathogens
87. The method of embodiment 80, wherein the amino acid sequence set forth in Table 4 is comprised in a full length autophagy protein and the isolated antibody specifically binds to the full length autophagy protein.
88. The method of embodiment 87, wherein the isolated antibody specifically binds to the full length autophagy protein when the x residue is nonphosphorylated serine, threonine, or tyrosine in the amino acid sequence set forth in Table 4 but does not specifically bind to the full length autophagy protein when the x residue is phosphorylated serine, threonine, or tyrosine in the amino acid sequence set forth in Table 4.
89. The method of embodiment 87, wherein the isolated antibody specifically binds to the full length autophagy protein when the x is phosphorylated serine, threonine, or tyrosine in the amino acid sequence set forth in Table 4 but does not specifically bind to the full length autophagy protein when the x residue is nonphosphorylated serine, threonine, or tyrosine in the amino acid sequence set forth in Table 4.
90. The method of embodiment 80, wherein the isolated antibody specifically binds to the full length autophagy protein at its natural conformation.
91. The method of embodiment 80, wherein the isolated antibody is a monoclonal or polyclonal antibody or antibody fragment.
92. The method of embodiment 80, which further comprises administering a pharmaceutically acceptable carrier and excipient.
93. The method of embodiment 80, wherein the subject is a mammal.
94. The method of embodiment 93, wherein the mammal is a human.
95. A pharmaceutical composition, which comprises an isolated antibody of embodiment 80 and a pharmaceutically acceptable carrier and excipient.
96. A method for identifying a kinase that phosphorylates an autophagy protein on the nonphosphorylated serine, threonine, or tyrosine at the x residue , which method comprises:
(a) providing an autophagy polypeptide comprising an amino acid sequence selected from a sequence set forth in Table 1, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine;
(b) contacting said autophagy polypeptide with a test protein and ATP under conditions suitable for the phosphorylation of said x residue of said autophagy polypeptide; and
(c) assessing phosphorylation status of said autophagy polypeptide to determine whether said test protein is a kinase for said autophagy protein on the nonphosphorylated serine, threonine, or tyrosine at the x residue.
97. The method of embodiment 96, wherein the autophagy protein or fragment comprises an amino acid sequence set forth in Table 4 wherein the x residue is nonphosphorylated serine, threonine, or tyrosine.
98. The method of embodiment 96, wherein the phosphorylation status of the autophagy polypeptide is assessed by an isolated antibody that specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated serine, threonine, or tyrosine, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine.
99. A method for identifying a modulator of a kinase that phosphorylates an autophagy protein on the x residue, which method comprises:
(a) providing an autophagy polypeptide comprising an amino acid sequence in set forth in Table 1, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine;
(b) contacting said autophagy polypeptide with a kinase that phosphorylates an autophagy protein on the nonphosphorylated x residue of serine, threonine, or tyrosine and ATP under conditions suitable for the phosphorylation of said x residue of said autophagy polypeptide in the presence or absence of a test substance; and
(c) assessing and comparing phosphorylation status of said autophagy polypeptide by said kinase to determine whether said test substance modulates said kinase.
100. The method of embodiment 99, wherein the autophagy protein or fragment comprises an amino acid sequence set forth in Table 4 wherein the x residue is nonphosphorylated serine, threonine, or tyrosine.
101. The method of embodiment 99, wherein the phosphorylation status of the autophagy polypeptide is assessed by an isolated antibody that specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein x is phosphoserine, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine.
102. A method for identifying a phosphatase that dephosphorylates an autophagy protein on the phosphorylated x residue of serine, threonine, or tyrosine, which method comprises:
(a) providing an autophagy polypeptide comprising an amino acid sequence set forth in Table 1, wherein the x residue is phosphorylated serine, threonine, or tyrosine;
(b) contacting said autophagy polypeptide with a test protein and H2O under conditions suitable for the dephosphorylation of said x residue of said autophagy polypeptide; and
(c) assessing phosphorylation status of said autophagy polypeptide to determine whether said test protein is a phosphatase for said autophagy protein on the phosphorylated x residue of serine, threonine, or tyrosine.
103. The method of embodiment 102, wherein the autophagy protein or fragment comprises an amino acid sequence set forth in Table 4 wherein the x residue is phosphorylated serine, threonine, or tyrosine.
104. The method of embodiment 103, wherein the phosphorylation status of the autophagy polypeptide is assessed by an isolated antibody that specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated serine, threonine, or tyrosine.
105. A method for identifying a modulator of a phosphatase that dephosphorylates an autophagy protein on the phosphorylated x residue of serine, threonine, or tyrosine, which method comprises:
(a) providing said autophagy polypeptide comprising an amino acid sequence in set forth in Table 1, wherein the x residue is phosphorylated serine, threonine, or tyrosine;
(b) contacting said autophagy polypeptide with a phosphatase that dephosphorylates an autophagy protein on the phosphorylated x residue of serine, threonine, or tyrosine and H2O under conditions suitable for the dephosphorylation of said phosphoserine residue of said autophagy polypeptide in the presence or absence of a test substance; and
(c) assessing and comparing dephosphorylation status of said autophagy polypeptide by said phosphatase to determine whether said test substance modulates said phosphatase.
106. The method of embodiment 105, wherein the autophagy protein or fragment comprises an amino acid sequence set forth in Table 4 wherein the x residue is phosphorylated serine, threonine, or tyrosine.
107. The method of embodiment 106, wherein the phosphorylation status of the autophagy polypeptide is assessed by an isolated antibody that specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein x is nonphosphorylated serine, threonine, or tyrosine, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated serine, threonine, or tyrosine.
108. An isolated nucleic acid fragment, which isolated nucleic acid fragment comprises a sequence of nucleotides encoding an autophagy peptide comprising an amino acid sequence set forth in Table 1, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine, and with the proviso that the peptide is not a full-length autophagy protein comprising an amino acid sequence set forth in Table 2.
109. The isolated nucleic acid fragment of embodiment 108, wherein the autophagy peptide comprises an amino acid sequence set forth in Table 4.
110. The isolated nucleic acid fragment of embodiment 108, wherein the nucleic acid is DNA.
111. The isolated nucleic acid fragment of embodiment 108, wherein the nucleic acid is RNA.
112. A plasmid, which plasmid comprises the nucleic acid fragment of embodiment 108.
113. A vector, which vector comprises the nucleic acid fragment of embodiment 108.
114. A cell, which cell comprises the plasmid of embodiment 112 or the vector of embodiment 113.
115. The cell of embodiment 114, which is selected from the group consisting of a bacterial cell, a yeast cell, a fungal cell, a plant cell, an insect cell, an animal cell and a human cell.
116. A method for producing an autophagy peptide, which method comprises growing the cell of embodiment 114 under conditions whereby said autophagy peptide is expressed by the cell, and recovering said expressed autophagy peptide.
117. The method of embodiment 116, further comprises a step of phosphorylating the autophagy peptide at the nonphosphorylated x residue of serine, threonine, or tyrosine.
118. A method for identifying a compound that binds specifically to a phosphorylated autophagy polypeptide, which method comprises:
a) providing a phosphorylated autophagy polypeptide comprising an amino acid sequence set forth in Table 1, wherein the x residue is phosphorylated serine, threonine, or tyrosine;
b) contacting said phosphorylated autophagy polypeptide with a test compound under conditions suitable for binding of the test protein with the autophagy polypeptide;
c) assessing binding of the phosphorylated autophagy polypeptide with the test compound;
d) providing a nonphosphorylated autophagy polypeptide comprising an amino acid sequence selected set forth in Table 1, wherein the x residue is phosphorylated serine, threonine, or tyrosine;
e) contacting said nonphosphorylated autophagy polypeptide with the test compound under conditions suitable for binding of the test protein with the autophagy polypeptide;
f) assessing binding of the nonphosphorylated autophagy polypeptide with the test compound
wherein the compound that binds specifically to a phosphorylated autophagy polypeptide is a test compound which binds to phosphorylated autophagy polypeptide according to step c) but does not bind to nonphosphorylated autophagy polypeptide according to step e).
119. A method for identifying a compound that binds to specifically to a nonphosphorylated autophagy polypeptide, which method comprises:
a) providing a nonphosphorylated autophagy polypeptide comprising an amino acid sequence set forth in Table 1, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine;
b) contacting said nonphosphorylated autophagy polypeptide with a test compound under conditions suitable for binding of the test protein with autophagy polypeptide;
c) assessing binding of the nonphosphorylated autophagy polypeptide with the test compound;
d) providing a phosphorylated autophagy polypeptide comprising an amino acid sequence set forth in Table 1, wherein the x residue is phosphorylated serine, threonine, or tyrosine;
e) contacting said phosphorylated autophagy polypeptide with the test compound and under conditions suitable for binding of the test protein with autophagy polypeptide;
f) assessing binding of the phosphorylated autophagy polypeptide with the test compound
wherein the compound that binds specifically to a nonphosphorylated autophagy polypeptide is a test compound which binds to nonphosphorylated autophagy polypeptide according to step c) but does not bind to phosphorylated autophagy polypeptide according to step e).
120. The methods of embodiments 118 and 119, wherein the test compound is a protein.
121. The methods of embodiments 118 and 119, wherein the test compound is a peptide.
122. The methods of embodiments 118 and 119, wherein the test compound is a nucleic acid.
123. The methods of embodiments 118 and 119, wherein the test compound is a small molecule.
124. A method of treating a disease comprising modulating phosphorylation status of an autophagy polypeptide by administering an autophagy antibody, an autophagy peptide, or compounds from the screening process of embodiments 107 and 118-123, wherein the disease is selected from the group consisting of ischemic brain injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion diseases and polyglutamine disorders including Huntington's disease and various spinocerebellar ataxias, transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease, breast cancer, ovarian cancer, brain cancer, pancreatic cancer, esophageal cancer, colorectal cancer, liver cancer, prostate cancer, renal cancer, lung cancer, Myocardial ischemia, cardiac remodeling, cardiomyopathy, hemodynamic stress, myocardial hypertrophy, Neuronal ceroid-lipofuscinosis (adult and juvenile), Multiple Sulfatase Deficiency (MSD) and Mucopolysaccharidosis type IIIA, Batten disease, Niemann-Pick C, Danon disease, Pompe disease, and dysfunction of innate and adaptive immunity against intracellular pathogens.
125. A array comprising an antibody of embodiments 25, 32, and 34-49.
126. A method of array detection comprising use of an antibody of embodiments 25, 32, and 34-39.
127. A microarray comprising a solid support and a plurality of antibodies of embodiments 25, 32, and 34-49 immobilized on the solid support at a known predetermined position.
128. A microarray comprising a solid support and a plurality of polypeptides of embodiments 1-10.
129. The use of a microarray of embodiments 127-128 for assessing the phosphorylation status of a plurality of autophagy polypeptides in the autophagy pathway.
130. A method of monitoring the phosphorylation of an autophagy polypeptide, wherein the monitoring uses a microarray of embodiments 127-128.
131. A method of treating a disease comprising modulating the phosphorylation of an autophagy polypeptide at the x residue set forth in Table 4.
132. The method of embodiment 131, wherein the disease is selected from the group consisting of ischemic brain injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion diseases and polyglutamine disorders including Huntington's disease and various spinocerebellar ataxias, transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease, breast cancer, ovarian cancer, brain cancer, pancreatic cancer, esophageal cancer, colorectal cancer, liver cancer, prostate cancer, renal cancer, lung cancer, Myocardial ischemia, cardiac remodeling, cardiomyopathy, hemodynamic stress, myocardial hypertrophy, Neuronal ceroid-lipofuscinosis (adult and juvenile), Multiple Sulfatase Deficiency (MSD) and Mucopolysaccharidosis type IIIA, Batten disease, Niemann-Pick C, Danon disease, Pompe disease, and dysfunction of innate and adaptive immunity against intracellular pathogens.
133. The method of embodiments 131 and 132, wherein phosphorylation of the autophagy polypeptide is increased.
134. The method of embodiments 131 and 132, wherein phosphorylation of the autophagy polypeptide is decreased.
135. The method of embodiments 131, 133, and 134, wherein the phosphorylation status of the autophagy polypeptide is abnormal.
136. The method of embodiments 131, 133, and 134, wherein the phosphorylation status of the autophagy polypeptide is normal and wherein the disease is treatable by modulating the phosphorylation status of the autophagy polypeptide.
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Claims
1. An isolated autophagy peptide comprising an amino acid sequence selected from the group consisting of sequences set forth in Table 1, wherein the x residue is nonphosphorylated or phosphorylated serine, threonine, or tyrosine, and with the proviso that the peptide is not a full-length autophagy protein.
2. An isolated autophagy peptide of claim 1 wherein the peptide does not comprise a sequence set forth in Table 2.
3. The isolated autophagy peptide of claim 1, which comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 3.
4. The isolated autophagy peptide of claim 1, which comprises an amino acid sequence selected from the group consisting of sequences set forth in Table 1.
5. The isolated autophagy peptide of claim 4, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine.
6. The isolated autophagy peptide of claim 1, wherein the x residue is phosphorylated serine, threonine, or tyrosine.
7. A method for producing an antibody to an autophagy polypeptide, which method comprises:
- (a) introducing an isolated autophagy peptide of claim 1 to a mammal in an amount sufficient to produce an antibody to said autophagy peptide; and
- (b) recovering said antibody from said mammal.
8. The method of claim 7, wherein the x residue in the isolated autophagy peptide is nonphosphorylated serine, threonine, or tyrosine and the method is used to produce an antibody to a nonphosphorylated autophagy polypeptide.
9. The method of claim 7, wherein the x residue in the isolated autophagy peptide is phosphorylated serine, threonine, or tyrosine and the method is used to produce an antibody to a phosphorylated autophagy polypeptide.
10. A method for detecting an autophagy protein or fragment comprising an amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated or nonphosphorylated serine, threonine, or tyrosine in a sample, which method comprises:
- (a) contacting a sample containing or suspected of containing an autophagy protein or fragment comprising the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated or nonphosphorylated serine, threonine, or tyrosine, with an isolated antibody that specifically binds to an epitope that comprises the amino acid residue x in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated or nonphosphorylated serine, threonine, or tyrosine; and
- (b) assessing a complex formed between said autophagy protein or fragment, if present in said sample, and said antibody, to determine the presence, absence and/or amount of said autophagy protein or fragment in said sample.
11. The method of claim 10, wherein the epitope comprises the amino acid residue x in the amino acid sequence set forth in Table 4 and an amino acid residue of the autophagy protein that is outside the amino acid sequence set forth in Table 4.
12. The method of claim 10, wherein the epitope comprises the amino acid residue x in the amino acid sequence set forth in Table 4 and amino acid residues of the autophagy protein that are outside the amino acid sequence set forth in Table 4.
13. The method of claim 10, wherein the epitope is comprised in the amino acid sequence set forth in Table 4.
14. The method of claim 10, wherein the epitope is comprised in an amino acid sequence set forth in Table 3.
15. The method of claim 10, wherein the epitope is comprised in an amino acid sequence selected from the sequence set forth in Table 1.
16. The method of claim 10, wherein the isolated antibody specifically binds to the epitope that comprises the x residue in the amino acid sequence set forth in Table 4, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine.
17. The method of claim 10, wherein the isolated antibody specifically binds to the epitope that comprises the x residue in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated serine, threonine, or tyrosine.
18. The method of claim 10, wherein the isolated antibody specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4 wherein the x residue is phosphorylated serine, threonine, or tyrosine.
19. The method of claim 10, wherein the isolated antibody specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated serine, threonine, or tyrosine, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4 wherein the x residue is nonphosphorylated serine, threonine, or tyrosine.
Type: Application
Filed: Apr 3, 2012
Publication Date: Oct 11, 2012
Applicant: ABGENT (San Diego, CA)
Inventors: Chun Wu (San Diego, CA), John A. Mountzouris (San Diego, CA), Bingren Hu (Baltimore, MD), Chunli Liu (Palmetto Bay, FL)
Application Number: 13/438,770
International Classification: G01N 33/566 (20060101); C07K 16/08 (20060101); C07K 5/117 (20060101); C07K 5/107 (20060101); C07K 5/103 (20060101); C07K 5/113 (20060101); C07K 7/06 (20060101); C07K 5/11 (20060101);
