A Method For The Degradation Of Endogenous Protein

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This disclosure was made with government support under R01

AG054839 awarded by the National Institute of Health. The government may have certain rights in the invention.

TECHNICAL FIELD

[0002] The present disclosure provides systems and methods for the degradation of endogenous protein with the help of a nanocarrier, which has the advantage of easy scale-up and feasibility for in vivo application.

BACKGROUND

[0003] The currently available tools for degrading an endogenous protein are not specific and/or can cause permanent genetic modification. The traditional endogenous protein degradation method, Trim -Away, requires microinjection or an electroporation step, which is not safe and convenient for large scale and in vivo applications.

[0004] Accordingly, it is an object of the present invention to provide a an intracellular antibody delivery method to effectively and specifically degrade an endogenous protein for large scale and future in vivo application.

SUMMARY [0005] The above objectives are accomplished according to the present disclosure by providing a method for creating an intracellular antibody delivery device. The method may include forming a polymer for introduction to a cell, producing a polymeric nanogel via crosslinking, introducing an antibody or protein to the polymeric nanogel, wherein the antibody or protein is internalized by the nanogel, uptake by at least one cell of the polymeric nanogel, and cleaving a self- immolative linker present in the polymeric nanogel to release the antibody or protein within the at least one cell. Further, the polymer may include PDA-PEG-

NPC. Again, the polymer may be PDA-PEG-BSA-Cy5 or PDA-PEG-Cy3. Still, the

PDA-PEG-NPC polymer may include p-nitrophenylcarbonate (NPC) moieties in side chains. Again further, the NPC moieties may be replaced by lysine groups of the antibody or protein to produce antibody or protein conjugated polymers. Yet still, the nanogel may include PBS buffer, TCEP and ethylenediamine and deionized water. Still further, the nanogel may be modified with RGD peptide. Yet again, the at least one cell may be a human breast cancer cell. Still further, the method may degrade a protein containing SEQ ID NO: 3 in the at least one human breast cancer cell.

[0006] In an alternative embodiment, the current disclosure may provide a method for degrading intracellular proteins in at least one TRIM21 expressing cell. The method may include forming at least one protein loaded nanogel wherein the protein loaded nanogel may comprise at least one polymer nanogel and at least one protein and the at least one protein comprises at least one antibody, at least one nanobody, or a combinations of at least one antibody and at least one nanobody. Further, the TRIM 21 expressing cell may be a naturally occurring

TRIM 21 expressing cell or a cell with acquired TRIM21 expression. Still, the at least one antibody may comprise anti COPZl antibody, anti PTBPl antibody, anti PD-Ll antibody, anti PD-1 antibody, anti-Her2 antibody, anti EGFR antibody, anti survivin antibody, anti PTP1B antibody, anti VEGF antibody, anti PKN3 antibody. Yet further, the at least one nanobody may comprise anti COPZ1 nanobody, anti PTBPl nanobody, anti PD-Ll nanobody, anti PD-1 nanobody, anti- Her2 nanobody, anti EGFR nanobody, anti survivin nanobody, anti PTP1B nanobody, anti VEGF nanobody, anti PKN3 nanobody. Still further, the protein- laded nanogel system may be used for treating cancer, Alzheimer’s diseases,

Parkinson's disease, multiple sclerosis, neonatal hypoxic-ischemic, stroke,

Amyotrophic lateral sclerosis, Huntington's disease, spinal cord injury, brain injury, retina injury, post-traumatic stress disorder, and frontotemporal dementia, and/or traumatic brain injury.

[0007] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] An understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure may be utilized, and the accompanying drawings of which:

[0009] Figure 1 shows a schematic illustration of: A) the Trim -Away technique; B) the fabrication of polymer nanogels using protein/antibody- conjugated polymers; and C) the mechanism of traceless release of protein/antibody from the loaded nanogels in the presence of reducing agent GSH.

[0010] Figure 2 shows at: A) Gel electrophoresis of (1) protein marker, (2) free BSA, (3) polymer PDA-PEG-BSA, and (4) GSH-treated PDA-PEG-BSA; B) Fluorescence emission spectra of a mixture of nanogels NG-BSA-Cy5 and NG-Cy3, and nanogel NG-BSA-Cy5-Cy3 in the absence and presence of GSH (λex = 525 nm); and C) Confocal fluorescence images of MCF-7 cells incubated with NG-BSA-

Cy5-Cy3 at different incubation times.

[0011] Figure 3 shows at: A) Size distribution and B) TEM image of nanogel NG-aGFP; C) Gel electrophoresis of (1) free anti-GFP antibody, (2) nanogel NG- aGFP, (3) GSH-treated NG-aGFP, and (4) protein marker; D) Fluorescence images and E) relative fluorescence intensity data of TRIM21 -transfected MCF-7/GFP cells after incubation with non-loading empty nanogel (NG-empty), free anti-GFP antibody, and NG-aGFP nanogel; F) Relative fluorescence intensity of TRIM21- transfected MCF-7/GFP cells after incubation with free anti-GFP and relevant nanogels equivalent to varied concentrations of anti-GFP; G) Fluorescence images of TRIM21-transfected MCF-7/GFP cells, GFP (green) and TRIM21 (red) channels, after incubation with free anti-GFP and nanogels at an anti-GFP equivalent concentration of 20 μg/mL for 9 h; and H) Relative fluorescence intensity of TRIM 21 -transfected MCF-7/GFP cells after incubation with NG-aGFP and NG- aGFP-R equivalent to 20 μg/mL anti-GFP.

[0012] Figure 4 shows at: A) Size distribution and B) TEM image of nanogel

NG-aCOPZl-R; C) Cell viability of TRIM21 -transfected MCF-7 cells after incubation with free anti-COPZl antibody, NG-empty, NG-aCOPZl, and NG- aCOPZl-R nanogels at varied anti-COPZl equivalent concentrations; D) The

COPZ1 protein expression in TRIM21-transfected MCF-7 cells measured by western blotting assay after incubation with free anti-COPZl and relevant nanogels; E) the corresponding quantitative analysis; and F) Fluorescence of GFP

(green) and TRIM21 (red) emitted from TRIM21 -transfected MCF-7/GFP cells before and after Trim-Away with NG-aCOPZl-R nanogel.

[0013] Figure 5 shows synthesis of protein/antibody conjugated polymers.

[0014] Figure 6 shows ¹ H NMR spectrum of PDA-PEG-NPC. [0015] Figure 7 shows size distribution of nanogel NG-BSA.

[0016] Figure 8 shows Zeta potential of nanogels NG-BSA, NG-aGFP, NG- aGFP-R, NG-aCOPZl, and NG-aCOPZl-R.

[0017] Figure 9 shows a TEM image of nanogel NG-BSA.

[0018] Figure 10 shows absorption and fluorescent emission spectra of BSA- Cy5.

[0019] Figure 11 shows fluorescence of GFP (green) and TRIM21 (red) emitted from MCF-7/GFP cells before and after transfection with pm Cherry- Cl- mTRIM21 plasmid.

[0020] Figure 12 shows fluorescence images of MCF-7/GFP cells without

TRIM21 -transfection after incubation with NG-empty, free anti-GFP, and NG- aGFP nanogel at an anti-GFP equivalent concentration of 100 μg/mL for 6 h. [0021] Figure 13 shows relative fluorescence intensity of MCF-7/GFP cells without TRIM21 -transfection after incubation with NG-empty, free anti-GFP, and

NG-aGFP nanogel at an anti-GFP equivalent concentration of 100 μg/mL for 6 h.

[0022] Figure 14 shows size distribution of nanogel NG-aGFP-R. [0023] Figure 15 relative intensity of both GFP and TRIM 21 fluorescence in

TRIM21-transfected MCF-7/GFP cells after incubation with free anti-GFP and nanogels at an anti-GFP equivalent concentration of 20 μg/mL for 9 h.

[0024] Figure 16 shows cell viability of TRIM21 -transfected MCF-7/GFP cells after incubation with NG-empty, free anti-GFP, NG-aGFP, and NG-aGFP-R nanogels at varying anti-GFP equivalent concentrations.

[0025] Figure 17 shows size distribution of nanogel NG-aCOPZl.

[0026] Figure 18 shows cell viability of MCF-7 cells after incubation with

NG-empty, free anti-COPZl, NG-aCOPZ1, and NG-aCOPZl-R nanogels at varying anti-COPZl equivalent concentrations. [0027] Figure 19 shows cell viability of TRIM 21 -transfected NIH-3T3 cells after incubation with free anti-COPZl, NG-empty, NG-aCOPZl, and NG-aCOPZl-

R at varying anti-COPZl equivalent concentrations.

[0028] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT [0029] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0030] Unless specifically stated, terms and phrases used in this document, and variations thereof, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. Likewise, a group of items linked with the conjunction “and” should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as “and/or” unless expressly stated otherwise. Similarly, a group of items linked with the conjunction

“or” should not be read as requiring mutual exclusivity among that group, but rather should also be read as “and/or” unless expressly stated otherwise.

[0031] Furthermore, although items, elements or components of the disclosure may be described or claimed in the singular, the plural is contemplated to be within the scope thereof unless limitation to the singular is explicitly stated.

The presence of broadening words and phrases such as “one or more,” “at least,”

“but not limited to” or other like phrases in some instances shall not be read to mean that the narrower case is intended or required in instances where such broadening phrases may be absent.

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0033] All publications and patents cited in this specification are cited to disclose and describe the methods and/or materials in connection with which the publications are cited. All such publications and patents are herein incorporated by references as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and/or materials described in the cited publications and patents and does not extend to any lexicographical definitions from the cited publications and patents. Any lexicographical definition in the publications and patents cited that is not also expressly repeated in the instant application should not be treated as such and should not be read as defining any terms appearing in the accompanying claims. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed. [0034] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0035] Where a range is expressed, a further embodiment includes from the one particular value and/or to the other particular value. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’ The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’,

‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’ In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y”.

[0036] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed.

Ranges can be expressed herein as from “about” one particular value, and/or to

“about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed. [0037] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges

(e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0038] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. [0039] As used herein, "about," "approximately," “substantially,” and the like, when used in connection with a measurable variable such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value including those within experimental error (which can be determined by e.g. given data set, art accepted standard, and/or with e.g. a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as variations of +/-10% or less, +/-5% or less, +/-1% or less, and +/-

0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosure. As used herein, the terms “about,”

“approximate,” “at or about,” and “substantially” can mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or

“at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise. [0040] As used herein, a “biological sample” may contain whole cells and/or live cells and/or cell debris. The biological sample may contain (or be derived from) a “bodily fluid”. The present disclosure encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, and cell cultures from bodily fluids. Bodily fluids may be obtained from a mammal organism, for example by puncture, or other collecting or sampling procedures.

[0041] As used herein, “agent” refers to any substance, compound, molecule, and the like, which can be administered to a subject on a subject to which it is administered to. An agent can be inert. An agent can be an active agent. An agent can be a primary active agent, or in other words, the component(s) of a composition to which the whole or part of the effect of the composition is attributed. An agent can be a secondary agent, or in other words, the component(s) of a composition to which an additional part and/or other effect of the composition is attributed. [0042] As used herein, “active agent” or “active ingredient” refers to a substance, compound, or molecule, which is biologically active or otherwise that induces a biological or physiological effect on a subject to which it is administered to. In other words, “active agent” or “active ingredient” refers to a component or components of a composition to which the whole or part of the effect of the composition is attributed.

[0043] As used herein, “administering” refers to any suitable administration for the agent(s) being delivered and/or subject receiving said agent(s) and can be oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intraosseous, intraocular, intracranial, intraperitoneal, intralesional, intranasal, intracardiac, intraarticular, intracavernous, intrathecal, intravireal, intracerebral, and intracerebroventricular, intratympanic, intracochlear, rectal, vaginal, by inhalation, by catheters, stents or via an implanted reservoir or other device that administers, either actively or passively

(e.g. by diffusion) a composition to the perivascular space and adventitia. For example, a medical device such as a stent can contain a composition or formulation disposed on its surface, which can then dissolve or be otherwise distributed to the surrounding tissue and cells. The term “parenteral” can include subcutaneous, intravenous, intramuscular, intra- articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques. Administration routes can be, for instance, auricular (otic), buccal, conjunctival, cutaneous, dental, electro-osmosis, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-arterial, intra- articular, intrabiliary, intr abr onchial , intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronal (dental), intracoronary, intr acor p or us cavernosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapul monary, intrasinal intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratym panic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, occlusive dressing technique, ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory

(inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, and/or vaginal administration, and/or any combination of the above administration routes, which typically depends on the disease to be treated, subject being treated, and/or agent(s) being administered.

[0044] As used herein “cancer” can refer to one or more types of cancer including, but not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, Kaposi Sarcoma, AIDS-related lymphoma, primary central nervous system (CNS) lymphoma, anal cancer, appendix cancer, astrocytomas, atypical teratoid/Rhabdoid tumors, basa cell carcinoma of the skin, bile duct cancer, bladder cancer, bone cancer (including but not limited to Ewing

Sarcoma, osteosarcomas, and malignant fibrous histiocytoma), brain tumors, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor, cardiac tumors, germ cell tumors, embryonal tumors, cervical cancer, cholangiocarcinoma, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous T-

Cell lymphoma, ductal carcinoma in situ, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer (including, but not limited to, intraocular melanoma and retinoblastoma), fallopian tube cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors, central nervous system germ cell tumors, extracranial germ cell tumors, extragonadal germ cell tumors, ovarian germ cell tumors, testicular cancer, gestational trophoblastic disease, Hairy cell leukemia, head and neck cancers, hepatocellular

(liver) cancer, Langerhans cell histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrine tumors, kidney (renal cell) cancer, laryngeal cancer, leukemia, lip cancer, oral cancer, lung cancer (non-small cell and small cell), lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell neck cancer, midline tract carcinoma with and without

NUT gene changes, multiple endocrine neoplasia syndromes, multiple myeloma, plasma cell neoplasms, mycosis fungoides, myelodyspastic syndromes, myelodysplas tic/my elo proliferative neoplasms, chronic myelogenous leukemia, nasal cancer, sinus cancer, non- Hodgkin lymphoma, pancreatic cancer, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary cancer, peritoneal cancer, prostate cancer, rectal cancer, Rhabdomyosarcoma, salivary gland cancer, uterine sarcoma, Sezary syndrome, skin cancer, small intestine cancer, large intestine cancer (colon cancer), soft tissue sarcoma, T-cell lymphoma, throat cancer, oropharyngeal cancer, nasopharyngeal cancer, hypoharyngeal cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, uterine cancer, vaginal cancer, cervical cancer, vascular tumors and cancer, vulvar cancer, and Wilms Tumor.

[0045] As used herein, “chemotherapeutic agent” or “chemotherapeutic” refers to a therapeutic agent utilized to prevent or treat cancer.

[0046] As used herein, “control” can refer to an alternative subject or sample used in an experiment for comparison purpose and included to minimize or distinguish the effect of variables other than an independent variable.

[0047] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. [0048] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a pharmaceutical formulation thereof calculated to produce the desired response or responses in association with its administration. [0049] The term “molecular weight”, as used herein, can generally refer to the mass or average mass of a material. If a polymer or oligomer, the molecular weight can refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weight of polymers and oligomers can be estimated or characterized in various ways including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as the weight-average molecular weight (Mw) as opposed to the number- average molecular weight (M _n ). Capillary viscometry provides estimates of molecular weight as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.

[0050] As used herein, “pharmaceutical formulation” refers to the combination of an active agent, compound, or ingredient with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo.

[0051] As used herein, “pharmaceutically acceptable carrier or excipient” refers to a carrier or excipient that is useful in preparing a pharmaceutical formulation that is generally safe, non-toxic, and is neither biologically or otherwise undesirable, and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable carrier or excipient” as used in the specification and claims includes both one and more than one such carrier or excipient. [0052] As used herein, “polymer” refers to molecules made up of monomers repeat units linked together. “Polymers” are understood to include, but are not limited to, homopolymers, copolymers, such as for example, block, graft, random and alternating copolymers, terpolymers, etc. and blends and modifications thereof. “A polymer” can be can be a three-dimensional network (e.g. the repeat units are linked together left and right, front and back, up and down), a two- dimensional network (e.g. the repeat units are linked together left, right, up, and down in a sheet form), or a one-dimensional network (e.g. the repeat units are linked left and right to form a chain). “Polymers” can be composed, natural monomers or synthetic monomers and combinations thereof. The polymers can be biologic (e.g. the monomers are biologically important (e.g. an amino acid), natural, or synthetic.

[0053] As used herein, the term “radiation sensitizer” refers to agents that can selectively enhance the cell killing from irradiation in a desired cell population, such as tumor cells, while exhibiting no single agent toxicity on tumor or normal cells.

[0054] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed by the term “subject”. [0055] As used herein, "substantially pure" can mean an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises about 50 percent of all species present. Generally, a substantially pure composition will comprise more than about 80 percent of all species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single species.

[0056] As used interchangeably herein, the terms “sufficient” and

“effective,” can refer to an amount (e.g. mass, volume, dosage, concentration, and/or time period) needed to achieve one or more desired and/or stated result(s).

For example, a therapeutically effective amount refers to an amount needed to achieve one or more therapeutic effects.

[0057] As used herein, “tangible medium of expression” refers to a medium that is physically tangible or accessible and is not a mere abstract thought or an unrecorded spoken word. “Tangible medium of expression” includes, but is not limited to, words on a cellulosic or plastic material, or data stored in a suitable computer readable memory form. The data can be stored on a unit device, such as a flash memory or CD-ROM or on a server that can be accessed by a user via, e.g. a web interface. [0058] As used herein, “therapeutic” can refer to treating, healing, and/or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect. A

“therapeutically effective amount” can therefore refer to an amount of a compound that can yield a therapeutic effect.

[0059] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and/or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof, such as cancer and/or indirect radiation damage. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein covers any treatment of cancer and/or indirect radiation damage, in a subject, particularly a human and/or companion animal, and can include any one or more of the following: (a) preventing the disease or damage from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and/or its symptoms or conditions. The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and/or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0060] As used herein, the terms “weight percent,” “ wt%,” and “wt. %,” which can be used interchangeably, indicate the percent by weight of a given component based on the total weight of a composition of which it is a component, unless otherwise specified. That is, unless otherwise specified, all wt% values are based on the total weight of the composition. It should be understood that the sum of wt% values for all components in a disclosed composition or formulation are equal to 100. Alternatively, if the wt% value is based on the total weight of a subset of components in a composition, it should be understood that the sum of wt% values the specified components in the disclosed composition or formulation are equal to

100.

[0061] As used herein, “water-soluble”, generally means at least about 10 g of a substance is soluble in 1 L of water, i.e., at neutral pH, at 25° C.

[0062] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments.

Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the disclosure. For example, in the appended claims, any of the claimed embodiments can be used in any combination. [0063] All patents, patent applications, published applications, and publications, databases, websites and other published materials cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference. [0064] The current disclosure provides a tool for specific protein degradation in molecular biology, as well as nanomedicine for treatment of maladies such as cancer and Alzheimer's disease. [0065] The currently available tools for degrading an endogenous protein are not specific or can cause permanent genetic modification. The traditional endogenous protein degradation method, Trim -Away, requires microinjection or an electroporation step, which is not safe and convenient for large scale and in vivo applications. The current disclosure provides an intracellular antibody delivery method to effectively and specifically degrade an endogenous protein for large scale and future in vivo application.

[0066] Proteins play crucial roles in the human body, including enzymes, carriers, structure building blocks, hormone signaling, defense, and storage. The malfunction of proteins causes various diseases, such as Alzheimer’s disease, amyotrophic lateral sclerosis, cystic fibrosis, type 2 diabetes, and cancer.

Antibodies, a functional member of the protein family that can bind to proteins with specificity and high affinity, are an ideal candidate for protein therapy, as they can be produced for any protein using the well-established phage display or hybridoma technology.

[0067] Clift et al. discovered a so-called “Trim-Away” method, which utilizes antibodies to degrade endogenous proteins in mammalian cells without prior modification of the genome or mRNA. The mechanism of Trim-Away involves the intracellular antibody receptor TRIM21, which is an E3 ubiquitin ligase that binds to the Fc domain of antibodies, and TRIM21 is commonly expressed in various cell types because of its indispensable physiological role. As illustrated in FIG. 1 at A, the target protein is bound by the antibody, followed by TRIM21-mediated ubiquitination to generate a protein complex, which turns into a proteasome that subsequently degrades. Trim-Away is a highly efficient technique to degrade target proteins in cells, and it can be readily applied to a broad spectrum of intracellular proteins. However, the extensive application of Trim -Away is severely hindered by the cell membrane impermeability of antibodies. Clift et al. delivered antibodies into cells by microinjection and electroporation techniques, which both bring damages to cells, and their in vivo applications are limited.

[0068] With the rapid development of diverse drug delivery systems, nanoparticulate delivery vehicles designed for intracellular delivery of protein/antibody surged in the past decade, including inorganic nanoparticles, liposomes, and polymeric nanocarriers. Our group has investigated drug-loaded polymeric micelles and nanogels for cancer and central nervous system diseases.

Herein, we intend to develop a safer and more convenient version of Trim -Away through employing polymer nanogels as the antibody delivery approach instead of microinjection and electroporation. Based on that, we attempt to utilize this new technique to degrade a vital intracellular protein of cancer cells and thereby to kill the cells.

[0069] Materials

[0070] 2-Mercaptoethanol (BME), glacial acetic acid, ethylenediamine, L- glutathione (GSH), bovine serum albumin (BSA), and phosphate buffered saline

(PBS) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

Triethylamine (TEA), pyridine, tris(2-carboxyethyl)phosphine (TCEP), 4- nitrophenyl chloroformate (NPC), and (3-(4,5-dimethylthiazol-2-yl) -2,5- diphenyltetrazolium bromide (MTT) were purchased from Tokyo Chemical Industry Co., Ltd (Portland, OR, USA). Anti-GFP antibody was purchased from

Abeam PLC. (Cambridge, MA, USA). Anti-COPZl antibody was purchased from

Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Plasmid pm Cherry- Cl- mTrim21, originally created by Schuh et al., was purchased from Addgene

(Watertown, MA, USA). Cyanine3 NHS ester (Cy3-NHS) and CyanineS NHS ester

(Cy5-NHS) were purchased from Lumiprobe Co. (Cockeysville, MB, USA). Cyclic

Arg-Gly-Asp-D-Phe-Cys peptide (RGD) was purchased from GL

Biochem (Shanghai) Ltd. (Shanghai, China). Gibco™ Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (PS), trypsin-

EDTA, Pierce™ BCA Protein Assay Kit, Lipofectamine® 3000 Transfection Kit, anti-6-actin antibody, and Invitrogen™ Hoechst 33342 were purchased from

Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Deuterated solvents were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). All the other solvents used in this research were purchased from Sigma-Aldrich Chemical

Co. (St. Louis, MO, USA) and directly used without further purification.

[0071] Polymers PDA-PEG-BME and PDA-PEG-Cy3 were prepared as described herein.

[0072] Synthesis of PDA-PEG-NPC

[0073] 30 mg PDA-PEG-BME and 7.2 mg 4-nitrophenyl chloroformate

(NPC) were dissolved in 500 μL dichloromethane (DCM). After cooled in ice bath for 30 min, 20 μL pyridine was added dropwise, and the reaction solution was stirred for 24 h at room temperature in the dark. The produced polymer was purified through dialysis of the reaction mixture towards DMSO using Spectra/Por® dialysis tube (MWCO: 8 kDa). The desired product PDA-PEG-NPC was collected through precipitation with ice-cold diethyl ether. Further removal of

DMSO residue was performed twice via DCM/ice-cold diethyl ether precipitation.

After in vacuo dryness in the dark for 48 h (28 mg, 87.5%), the polymer was analyzed by NMR to confirm its chemical structure.

[0074] Preparation of Nanogels

[0075] To a pre-cooled solution of 50 mg polymer PDA-PEG-NPC in 5 mL

PBS buffer (pH 8.5), 1 mg protein/antibody dissolved in 1 mL PBS buffer was added dropwise at 4 °C under vigorous stirring, and the resulted solution was stirred for 48 h at 4 °C in the dark. The process of reaction was monitored by measuring the absorbance of released side product 4-nitrophenol at 400 nm using

UV-Vis spectroscopy as reported in literature. When the reaction was completed,

2.6 mg TCEP and 1.1 mg ethylenediamine dissolved in 0.2 mL pre-cooled deionized water was added for crosslinking and the solution was stirred for 24 h at 4 °C.

Then the produced nanogels were purified through dialysis in Spectra/Por® dialysis tube (MWCO: 100 kDa) against PBS buffer for 48 h at 4 °C. The final nanogels were stored in PBS (pH 7.4) at 4 °C for use. For the nanogels modified with Cy3, polymer PDA-PEG-Cy3 was mixed into the reaction solution before the crosslinking step. The concentration of Cy3 was measured by microplate reader

(λex = 555 nm, Aem = 570 nm). For the nanogels post-decorated with RGD peptide, the nanogel dispersion in PBS buffer (pH 7.4) was added RGD solution (1 mg/mL) in PBS buffer and then stirred overnight at 4 °C. The RGD-modified nanogels were purified through dialysis in Spectra/Por® dialysis tube (MWCO: 8 kDa) against PBS buffer for 48 h at 4 °C. The particle size, size distribution, and zeta potential of the nanogels were determined by dynamic light scattering (DLS), recorded on

Zetasizer (Zetasizer Nano ZS, Malvern Instruments Ltd, Malvern, UK). The physical morphology was observed using Hitachi HT7800 transmission electron microscopy (TEM, Hitachi High-Technologies Corporation, Tokyo, Japan).

[0076] The amount of protein contained in nanogels was analyzed using the

Pierce™ BCA Protein Assay Kit, following the reported method. The loading content (LC) and loading efficiency (LE) of Protein/antibody were calculated by the following equations. [0077]

[0078]

[0079] Protein/antibody Release

[0080] The release of protein/antibody from the nanogels was analyzed with

SDS-PAGE gel electrophoresis. 12 μL of different samples were mixed with 10 μL loading buffer, and 20 μL of each sample was loaded onto gel. For the redox- responsive release test, the nanogels were treated with 0.1 M GSH for 12 h prior to gel electrophoresis.

[0081] Fluorescence Labeling of Protein

[0082] For cellular uptake studies of protein/antibody and nanogels, bovine serum albumin (BSA) was labeled with fluorescent dye Cyanine5 (Cy5) to form BSA-Cy5. In brief, 5 mg of BSA dissolved in 1 mL NaHCO ₃ buffer (0.1 M, pH =

8.5) was cooled in ice bath for 30 min, and then 50 μL freshly prepared Cy5-NHS solution (10 mg/mL in DMSO) was added dropwise in the dark. The reaction mixture was protected from light and stirred at room temperature overnight. The produced BSA-Cy5 was purified by size exclusion chromatography using Sephadex

G-25 (GE Healthcare).

[0083] Cell Culture

[0084] Human breast cancer MCF-7 cells, green fluorescence protein (GFP) expressed MCF-7/GFP cells, and mouse embryonic fibroblast NIH-3T3 cells were cultured in GibcoTM DM EM supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in 75 mL culture flasks under a humidified atmosphere of 5% CO2. Cells were sub-cultured when the cell confluence reached ~80%.

[0085] Cellular Internalization [0086] The cellular internalization of nanogels was examined by confocal fluorescence microscopy in MCF-7 cells. Cells were seeded in 35 mm ² Petri dish with a glass window at a density of 200,000 cells per dish for 24 h. Then cells were incubated with Cy3 and BSA-Cy5 co-loaded nanogel NG-BSA-Cy5-Cy3 (5 μg/mL for Cy5, 4.1 μg/mL for Cy3) for 3h. Cells without any incubation were utilized as negative control. In the positive control group, cells were co-incubated with nanogels NG-Cy3 and NG-BSA-Cy5. Then the medium was replaced with fresh medium and cells were fixed with 4% paraformaldehyde in PBS for 10 min at predetermined time points. After removal of paraformaldehyde, the nuclei of cells were stained with Hoechst 33342 for 10 min, and then cells were imaged under a confocal fluorescence microscope (LSM 700, Carl-Zeiss Inc.).

[0087] Cell Viability Assay

[0088] The cytotoxicity of free antibody and nanogels was evaluated by MTT assay. Cells were seeded in 96-well plates at a density of 5,000 cells per well for

24 h at 37 °C with 5% CO ₂ . Then cells were incubated with free antibody and nanogels in fresh medium for 48 h. In the control group, cells were allowed to grow without any treatment. After that, the medium was replaced with fresh medium containing MTT reagent (final concentration 1 mg/mL) and cells were further incubated for 4 h. The purple MTT crystal was dissolved with MTT stop solution and the optical density at 595 nm was recorded on microplate reader (SpectraMax i3x, Molecular Devices, LLC.).

[0089] Protein Expression

[0090] For the expression of protein Trim21, cells were transfected with pmCherry-Cl-mTrim21 plasmid as reported in literature. Plasmids were transfected using Lipofectamine® 3000 Transfection Kit according to manufacturer’s instructions 12 h prior to Trim-Away assay. Successful transfection was indicated by the observed cherry fluorescence emitted from cells.

[0091] Trim-Away Assay [0092] The Trim-Away of protein GFP was conducted in TRIM21- transfected MCF-7/GFP cells. For fluorescence imaging, cells seeded in 35 mm ²

Petri dish with a glass window were incubated with free anti-GFP antibody and anti-GFP loaded nanogels. Cells with no incubation were utilized as control. At predetermined post-incubation time points, cells were imaged by fluorescence microscopy. To quantitatively analyze the Trim-Away efficiency, the fluorescence of cells seeded in 96-well plates after incubation with anti-GFP and nanogels was measured by microplate reader at different time points (λex = 395 nm, λem = 505 nm).

[0093] For the Trim-Away of protein COPZ1, TRIM21 -transfected cells were incubated with free anti-COPZl antibody and anti-COPZl loaded nanogels at varied concentrations for 48 h. Cells in the control group were allowed to grow with no incubation. Then the medium was replaced with fresh medium containing

MTT reagent (final concentration 1 mg/mL) and cells were further incubated for 4 h. The purple MTT crystal was dissolved with MTT stop solution and the optical density at 595 nm was recorded by microplate reader.

[0094] Western Blotting Assay [0095] TRIM21-transfected MCF-7 cells were incubated with free anti-

COPZl, NG-aCOPZl and NG-aCOPZl-R nanogels (equivalent to 40 μg/mL anti-

COPZl) for 10 h and then lysed for 30 min at 4 °C. The protein concentration of each sample was measured by Bradford assay. The cell lysates were separated by

SDS-PAGE and transferred to a PVDF membrane, which was activated by methanol for 30 min prior to use. The transferred membrane was blocked for 1.5 h at room temperature with 5% BSA, washed with Tris -buffered saline with

Tween-20 (TEST buffer), and incubated overnight at 4 °C with anti-COPZl (1:500) or anti-6-actin (1:1000). Chemiluminescence detection was performed with the corresponding second antibody conjugated with HRP. Images were acquired using

ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories, Inc.).

[0096] Results

[0097] A polymer PDA-PEG-NPC bearing p-nitrophenylcarbonate (NPC) moieties in side chains was synthesized by attaching NPC to polymer PDA-PEG-

BME, see FIG. 5, which was prepared as we previously reported, and characterized by nuclear magnetic resonance spectroscopy, see FIG. 6. The protein/antibody was reacted with PDA-PEG-NPC to produce protein/antibody conjugated polymers, in which the NPCs are replaced by the reactive lysine groups of the protein/ antibody, see FIG. 5. The redox-sensitive linker between the protein/antibody and the polymer backbone is self-immolative and the biodegradable disulfide bond can be readily cleaved by reducing agents such as glutathione (GSH). As illustrated in FIG. 1 at B, the resulted polymers were fabricated into polymer nanogels via the crosslinking reaction induced by tris(2- carboxyethyl)phosphine (TCEP). The polymer nanogels were further modified with a tumor-targeting ligand RGD (cyclic Arg- Gly- Asp - D - Phe - Cy s peptide). After being taken up by cells, the self-immolative linker would be cleaved by intracellular GSH, leading to the traceless release of the protein/antibody, see

FIG. 1 at C, which makes the nanogels more advantageous for delivering protein/antibody into cancer cells since the intracellular level of GSH in cancer cells (2-10 mM) is much higher than that in normal cells and extracellular matrices (2-20 μΜ). [0098] To verify our design strategy, bovine serum albumin (BSA) was utilized as a protein model to investigate the conjugation and subsequent release of protein/antibody from the polymer by gel electrophoresis using SDS-PAGE gel.

As displayed in FIG. 2 at A, the protein band of free BSA disappeared in the lane loaded with polymer PDA-PEG-BSA, which indicates the success of protein conjugation. However, in the lane loaded with GSH-pretreated PDA-PEG-BSA, a clear band of BSA was observed, suggesting the redox-responsive release of the protein. Then polymer PDA-PEG-BSA was cross-linked by TCEP to fabricate polymeric nanogel NG-BSA, and the unreacted NPCs were eliminated by ethylenediamine. The amount of BSA contained in NG-BSA was determined using

BCA protein assay kit according to the reported method. The loading content (LC) and loading efficiency (LE) of BSA were calculated to be 3.4% and 77%, respectively. Dynamic light scattering (DLS) determined that NG-BSA had a hydrodynamic size of 135.4 nm with a dispersity of 0.12, see FIG. 7, and a negative zeta potential, see FIG. 8. Transmission electron microscopy (TEM) showed that

NG-BSA had a spherical shape and a diameter of 109.3 nm in dry state, see FIG.

9.

[0099] In order to study the cellular uptake and intracellular release of the protein from the nanogel using fluorescence resonance energy transfer (FRET) technique, we labelled BSA with a fluorescent dye CyanineS (Cy5) to generate

BSA-Cy5. The absorption and fluorescent emission wavelength of BSA-Cy5 was determined to be 648 nm and 670 nm, respectively, see FIG. 10. Polymer PDA-

PEG-BSA-Cy5 was synthesized in the same way as PDA-PEG-BSA using BSA-

Cy5 instead of BSA. Then it was mixed with a CyanineS (Cy3, λ _abs = 555 nm, λ em = 570 nm) labelled polymer PDA-PEG-Cy3, which was prepared as we previously reported, to fabricate a Cy5 and Cy3 co-labelled nanogel NG-BSA-Cy5-Cy3, since the fluorescent dyes Cy3 and Cy5 make an efficient FRET pair. As shown in FIG.

2 at B, when excited at 525 nm, a wavelength that can excite fluorophore Cy3 and spares Cy5, intense fluorescence of Cy3 (568 nm) and negligible fluorescence of

Cy5 (670 nm) were observed from a mixture of nanogels NG-BSA-Cy5 and NG-

Cy3, which were made from polymers PDA-PEG-BSA-Cy5 and PDA-PEG-Cy3, respectively. On the contrary, nanogel NG-BSA-Cy5-Cy3 emitted weak fluorescence of Cy3 and very strong fluorescence of Cy5 under the same conditions, which demonstrates that efficient FRET phenomenon occurred between the Cy3 and Cy5 fluorophores of NG-BSA-Cy5-Cy3. However, in the presence of GSH, the fluorescence of Cy3 emitted by NG-BSA-Cy5-Cy3 was significantly enhanced and that of Cy5 decreased simultaneously, which is quite similar to the fluorescence emission pattern of the mixed nanogels NG-BSA-Cy5 and NG-Cy3. These observations indicate that BSA-Cy5 was released from the nanogel by GSH so that the two fluorophores were no long in proximity and thus the FRET phenomenon was inhibited.

[00100] To determine whether the GSH-induced FRET-inhibition phenomenon could occur inside cells, we incubated human breast cancer MCF-7 cells with nanogel NG-BSA-Cy5-Cy3 and recorded the fluorescence emitted from cells using confocal fluorescence microscopy. First of all, as shown in FIG. 2 at C, the strong fluorescence of cells confirmed that the protein loaded nanogels could be efficiently taken up by cells. Moreover, obvious FRET phenomenon was observed after 3 h incubation with NG-BSA-Cy5-Cy3, as evidenced by the very weak fluorescence (green) of Cy3 and strong fluorescence (red) of Cy5. At 8 h post- incubation, the significantly increased fluorescence of Cy3 and declined fluorescence of Cy5 indicated that some of the Cy5-labelled protein BSA-Cy5 was liberated and the FRET efficiency decreased accordingly. After 24 h incubation, the FRET phenomenon nearly disappeared and the fluorescence of cells was almost the same as that of cells co-incubated with nanogels NG-BSA-Cy5 and NG-

Cy3 in the positive control group, which suggest that most of the loaded BSA-Cy5 had already been released from NG-BSA-Cy5-Cy3. All the results confirm the effectiveness of our design strategy that the polymer nanogel can deliver the loaded protein into cells and release it on demand.

[00101] Then anti-GFP, an antibody for green fluorescence protein (GFP), was conjugated to the polymer by reacting anti-GFP with polymer PDA-PEG-NPC to produce polymer PDA-PEG-aGFP, which was fabricated into nanogel NG-aGFP following the same procedures of NG-BSA fabrication. DLS analysis measured a

125.9 nm hydrodynamic diameter (dispersity: 0.20) of nanogel NG-aGFP, see FIG.

3 at A, and negative charges on the surface, see FIG. 8. TEM imaging detected a spherical morphology of NG-aGFP with a diameter of 103.3 nm in dry state, see

FIG. 3 at B. The LC and LE values of anti-GFP in NG-aGFP were determined to be 7.6% and 85%, respectively. As shown in FIG. 3 at C, gel electrophoresis demonstrated that the antibody loaded into nanogel NG-aGFP could be effectively recovered by GSH.

[00102] To validate the effectiveness of the Trim-Away technique, we transfected GFP-expressed human breast cancer MCF-7/GFP cells with pmCherry-Cl-mTRIM21 plasmid as described in literature, making the cells overexpress TRIM21 protein, since TRIM 21 is a pivotal medium for the technique.

After transfection, cells emitted cherry fluorescence, see FIG. 11. Then the cells were treated with free anti-GFP antibody, non-loading empty nanogel (NG- empty), and NG-aGFP nanogel at a concentration equivalent to 100 μg/mL anti-

GFP. As shown in FIG. 3 at D, the GFP fluorescence significantly decreased in cells incubated with NG-aGFP after 6 h incubation, while no visible change was observed in the fluorescence of cells in other groups, which, along with the quantitative analysis of the fluorescence intensity of cells, see FIG. 3 at E, indicates that the GFP protein of cells was effectively degraded by the intracellular delivery of anti-GFP via NG-aGFP nanogel. We further found that higher concentrations of NG-aGFP yielded better protein degradation efficiency, see FIG.

3 at F, suggesting that the protein degradation is in a dose-dependent manner. As reported, the Trim-Away method relies on the overexpression of protein TRIM21.

To verify that, same experiments were conducted in MCF-7/GFP cells without

TRIM21 -transfection, and obviously, the protein degradation efficiency was considerably limited in comparison to that in TRIM21-transfected cells because of the low endogenous TRIM21 level, see FIGS. 12 and 13, which confirms the necessity of the overexpression of TRIM 21 in the protein degradation process. [00103] To enhance the cellular uptake of the nanogel, NG-aGFP was decorated with thiol-containing RGD peptide, which can bind to the αvBs integrin

- a protein receptor overexpressed on a wide spectrum of tumor cells, to yield RGD- modified nanogel NG-aGFP-R. DLS analysis measured a hydrodynamic size of

130.4 nm (dispersity: 0.19) for NG-aGFP-R, see FIG. 14, and zeta potential of the nanogel became less negative after RGD modification, see FIG. 8. As shown in

FIG. 3 at G, the weaker GFP fluorescence of cells incubated with NG-aGFP-R, compared to that of cells with NG-aGFP, demonstrated that the nanogel is more efficient in degrading target protein after RGD modification. The quantified analysis of GFP fluorescence intensity further verified the enhanced protein degradation efficiency of NG-aGFP-R nanogel, see FIG. 3 at H. Moreover, the simultaneously decreased fluorescence of both GFP and TRIM21, see FIG. 3 at G, and the corresponding quantitative data, see FIG. 15, indicated the consumption of TRIM 21 in the process of protein degradation, which further evidenced the pivotal role of TRIM21 in the Trim-Away technique. Importantly, cell viability test of free anti-GFP and nanogels in TRIM21 -transfected MCF-7/GFP cells showed that NG-empty and NG-aGFP nanogels exhibited equal cytotoxicity at same conditions, see FIG. 16, which demonstrated that the nanogel-uμgraded Trim-

Away induces no additional damage to cells while degrading the target protein. [00104] After confirming the effectiveness of the intracellular antibody delivery induced Trim-Away effect, we employed it to degrade a critical protein for cancer cells, the coatomer protein complex ζΐ (COPZl) protein, which however is non-essential for normal cells. COPZl and COPZ2 are the two isoforms of coatomer protein complex 1 (COPI), which plays vital roles in cells. Normal cells generally express both COPZl and COPZ2 proteins, whereas in cancer cells

COPZ2 is silenced, and as a result the cells depend solely on COPZl. Therefore, the degradation of COPZl would kill cancer cells, while normal cells survive. The antibody of COPZl, anti-COPZl, was loaded into nanogel NG-aCOPZl through the same procedures used for NG-BSA and NG-aGFP. DLS analysis showed that NG-aCOPZl had a hydrodynamic size of 140.8 nm with a dispersity of 0.21, see

FIG. 17, and negative zeta potential, see FIG. 8. The LC and LE values of anti-

COPZ1 in NG-aCOPZl nanogel were determined to be 3.1% and 81%, respectively.

Owing to the significantly enhanced protein degradation efficiency that the RGD ligand brought to NG-aGFP-R nanogel, NG-aCOPZl was decorated with RGD as well to produce nanogel NG-aCOPZl-R. A hydrodynamic diameter of 151.3 nm

(dispersity: 0.22) of NG-aCOPZl-R, see FIG. 4 at A, and a spherical shape with a dry-state size of 121.0 nm was observed by TEM imaging, see FIG. 4 at B. The zeta potential of NG-aCOPZl-R was less negative in comparison with NG-aCOPZl, see FIG. 8.

[00105] The COPZ1 protein degradation was conducted in MCF-7 cells via evaluating the viability of cells after treatment with anti-COPZl loaded nanogels.

In MCF-7 cells without TRIM21 transfection, all the nanogels NG-empty, NG- aCOPZl, and NG-aCOPZl-R uniformly brought slight inhibitory effect to cell growth when the concentration was high, see FIG. 18, which resulted from the very mild cytotoxicity of the nanogels themselves. However, in TRIM21- transfected MCF-7 cells, as shown in, see FIG. 4 at C, both NG-aCOPZl and NG- aCOPZl-R nanogels noticeably inhibited cell growth in a dose-dependent manner, whereas NG-empty exhibited the same performances as it did in non-transfected

MCF-7 cells and free anti-COPZl induced no effect towards both TRIM21- transfected and non-transfected cells, which indicated that the anti-COPZl loaded nanogels effectively delivered the antibody into cells and successfully degraded the target protein COPZ1 through the Trim-Away path. At a concentration equivalent to 40 μg/mL anti-COPZl, the viability of cells was decreased to 44.7% and 27.9% by NG-aCOPZl and NG-aCOPZl-R, respectively. The higher Trim-Away efficiency of NG-aCOPZl-R nanogel should be ascribed to the targeting functionality of RGD ligand. Meanwhile, western blotting analysis showed that the amount of endogenous COPZl was considerably diminished in cells incubated with both nanogels, see FIG. 4 at D, as additionally evidenced by the quantified data, see FIG. 4 at E, which further confirmed that the Trim- Away of COPZl protein resulted in the suppression of cell viability. Moreover, fluorescence images of TRIM 21 -transfected MCF-7/GFP cells before and after incubation with NG- aCOPZl-R nanogel showed that TRIM 21 was largely consumed while GFP protein was barely affected in the process of the Trim-Away of COPZl protein, see FIG. 4 at F, demonstrating the high specificity of the uμgraded Trim-Away technique in degrading the target protein. In addition, the application of Trim-Away with anti-

COPZl nanogels in TRIM21-transfected NIH-3T3 cells caused negligible damage to cells, see FIG. 19, suggesting that this Trim-Away approach is safe for normal cells. All the results collectively confirm that the uμgraded Trim-Away technique is efficient for degrading endogenous proteins and is potential to be exploited as a protein therapy modality for cancer treatment.

[00106] Further, the protein-loaded nanogel may include antibodies and/or nanobodies. The antibodies may include anti COPZl antibody, anti PTBP1 antibody, anti PD-Ll antibody, anti PD-1 antibody, anti-Her2 antibody, anti

EGFR antibody, anti survivin antibody, anti PTPlB antibody, anti VEGF antibody, anti PKN3 antibody. The nanobodies may include anti COPZl nanobody, anti PTBP1 nanobody, anti PD-Ll nanobody, anti PD-1 nanobody, anti- Her2 nanobody, anti EGFR nanobody, anti survivin nanobody, anti PTPlB nanobody, anti VEGF nanobody, anti PKN3 nanobody.

[00107] Conclusion

[00108] In summary, protein/antibody covalently loaded redox-responsive polymer nanogels have been fabricated for intracellular delivery and traceless release of protein/antibody, and based on which, a new intracellular antibody delivery method has been developed for degrading a specific endogenous protein.

The proteins/antibodies are conjugated to the nanogels via a redox-sensitive self- immolative linker, which can be cleaved by intracellular GSH and release the proteins/antibodies in a traceless form. After being delivered into cells, the unloaded antibody binds to its target protein and Trim-21, and subsequently degrades through TRIM21 -mediated ubiquitination in the proteasome. The nano-

Trim-Away technique has been proven highly efficient in degrading endogenous proteins with more convenience and wide-ap plication potency compared with its original version. Notably, this new technique has been successfully employed to degrade a vital protein COPZl for cancer cells and kill the cells as a result, without causing damage to normal cells. In a word, the nanogel- Trim-Away technique is promising to provide a reliable and convenient tool for endogenous protein study and to arouse the emergence of new protein/ antibody-based therapeutic modalities for cancer and other diseases, including but not limited to Alzheimer’s diseases,

Parkinson's disease, multiple sclerosis, neonatal hypoxic-ischemic, stroke,

Amyotrophic lateral sclerosis, Huntington's disease, spinal cord injury, brain injury, retina injury, post- traumatic stress disorder, and frontotemporal dementia, and traumatic brain injury

[00109] Sequence Listings

<110> University of South Carolina <120> A Method For The Degradation Of Endogenous Protein

<130> 2033101.0000288

<140> Unknown

<141> 2021-04-28

<150 U.S. Provisional Application No. 63/045,275 <151> 06/29/2020

<160> 12

<170> Patentln

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Leu Thr Tyr Gly Val Gin Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80

Gin His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gin Glu

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Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg lie Glu Leu Lys Gly

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IIe Asp Phe Lys Glu Asp Gly Asn IIe Leu Gly His Lys Leu Glu Tyr

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Asn Tyr Asn Ser His Asn Val Tyr IIe Met Ala Asp Lys Gin Lys Asn 145 150 155 160

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Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gin Ser Ala Leu

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Pro lie Cys Leu Asp Pro Phe Val Glu Pro Val Ser lie Glu Cys Gly

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His Ser Phe Cys Gin Glu Cys lie Ser Gin Val Gly Lys Gly Gly Gly

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Ser Val Cys Pro Val Cys Arg Gin Arg Phe Leu Leu Lys Asn Leu Arg

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Glu Arg Leu His Leu Phe Cys Glu Lys Asp Gly Lys Ala Leu Cys Trp

100 105 110

Val Cys Ala Gin Ser Arg Lys His Arg Asp His Ala Met Val Pro Leu

115 120 125

Glu Glu Ala Ala Gin Glu Tyr Gin Glu Lys Leu Gin Val Ala Leu Gly

130 135 140 Glu Leu Arg Arg Lys Gin Glu Leu Ala Glu Lys Leu Glu Val Glu IIe

145 150 155 160

Ala lie Lys Arg Ala Asp Trp Lys Lys Thr Val Glu Thr Gin Lys Ser

165 170 175 Arg IIe His Ala Glu Phe Val Gin Gin Lys Asn Phe Leu Val Glu Glu

180 185 190

Glu Gin Arg Gin Leu Gin Glu Leu Glu Lys Asp Glu Arg Glu Gin Leu

195 200 205

Arg lie Leu Gly Glu Lys Glu Ala Lys Leu Ala Gin Gin Ser Gin Ala

210 215 220 Leu Gin Glu Leu lie Ser Glu Leu Asp Arg Arg Cys His Ser Ser Ala

225 230 235 240

Leu Glu Leu Leu Gin Glu Val lie lie Val Leu Glu Arg Ser Glu Ser

245 250 255

Trp Asn Leu Lys Asp Leu Asp IIe Thr Ser Pro Glu Leu Arg Ser Val

260 265 270

Cys His Val Pro Gly Leu Lys Lys Met Leu Arg Thr Cys Ala Val His

275 280 285 lie Thr Leu Asp Pro Asp Thr Ala Asn Pro Trp Leu IIe Leu Ser Glu

290 295 300 Asp Arg Arg Gin Val Arg Leu Gly Asp Thr Gin Gin Ser lie Pro Gly

305 310 315 320

Asn Glu Glu Arg Phe Asp Ser Tyr Pro Met Val Leu Gly Ala Gin His

325 330 335

Phe His Ser Gly Lys His Tyr Trp Glu Val Asp Val Thr Gly Lys Glu

340 345 350

Ala Trp Asp Leu Gly Val Cys Arg Asp Ser Val Arg Arg Lys Gly His

355 360 365

Phe Leu Leu Ser Ser Lys Ser Gly Phe Trp Thr IIe Trp Leu Trp Asn

370 375 380 Lys Gin Lys Tyr Glu Ala Gly Thr Tyr Pro Gin Thr Pro Leu His Leu

385 390 395 400

Gin Val Pro Pro Cys Gin Val Gly lie Phe Leu Asp Tyr Glu Ala Gly

405 410 415 Met Val Ser Phe Tyr Asn IIe Thr Asp His Gly Ser Leu IIe Tyr Ser

420 425 430

Phe Ser Glu Cys Ala Phe Thr Gly Pro Leu Arg Pro Phe Phe Ser Pro 435 440 445

Gly Phe Asn Asp Gly Gly Lys Asn Thr Ala Pro Leu Thr Leu Cys Pro

450 455 460

Leu Asn IIe Gly Ser Gin Gly Ser Thr Asp Tyr

465 470 475

<210> 3 <211> 475

<212> PRT

<213> Homo sapiens

<222> 1 ... 177 COPZ1 <400> 3

Met Glu Ala Leu lie Leu Glu Pro Ser Leu Tyr Thr Val Lys Ala IIe

1 5 10 15

Leu IIe Leu Asp Asn Asp Gly Asp Arg Leu Phe Ala Lys Tyr Tyr Asp

20 25 30

Asp Thr Tyr Pro Ser Val Lys Glu Gin Lys Ala Phe Glu Lys Asn IIe

35 40 45 Phe Asn Lys Thr His Arg Thr Asp Ser Glu lie Ala Leu Leu Glu Gly

50 55 60

Leu Thr Val Val Tyr Lys Ser Ser lie Asp Leu Tyr Phe Tyr Val IIe

65 70 75 80

Gly Ser Ser Tyr Glu Asn Glu Leu Met Leu Met Ala Val Leu Asn Cys

85 90 95

Leu Phe Asp Ser Leu Ser Gin Met Leu Arg Lys Asn Val Glu Lys Arg

100 105 110

Ala Leu Leu Glu Asn Met Glu Gly Leu Phe Leu Ala Val Asp Glu lie

115 120 125 Val Asp Gly Gly Val lie Leu Glu Ser Asp Pro Gin Gin Val Val His 130 135 140

Arg Val Ala Leu Arg Gly Glu Asp Val Pro Leu Thr Glu Gin Thr Val

145 150 155 160

Ser Gin Val Leu Gin Ser Ala Lys Glu Gin lie Lys Trp Ser Leu Leu

165 170 175

Arg

<210> 4

<211> 531

<212> PRT

<213> Homo sapiens <400> 4

Met Asp Gly IIe Val Pro Asp IIe Ala Val Gly Thr Lys Arg Gly Ser

1 5 10 15

Asp Glu Leu Phe Ser Thr Cys Val Thr Asn Gly Pro Phe lie Met Ser

20 25 30 Ser Asn Ser Ala Ser Ala Ala Asn Gly Asn Asp Ser Lys Lys Phe Lys

35 40 45

Gly Asp Ser Arg Ser Ala Gly Val Pro Ser Arg Val IIe His lie Arg

50 55 60

Lys Leu Pro IIe Asp Val Thr Glu Gly Glu Val IIe Ser Leu Gly Leu 65 70 75 80

Pro Phe Gly Lys Val Thr Asn Leu Leu Met Leu Lys Gly Lys Asn Gin

85 90 95

Ala Phe lie Glu Met Asn Thr Glu Glu Ala Ala Asn Thr Met Val Asn 100 105 110

Tyr Tyr Thr Ser Val Thr Pro Val Leu Arg Gly Gin Pro lie Tyr lie

115 120 125

Gin Phe Ser Asn His Lys Glu Leu Lys Thr Asp Ser Ser Pro Asn Gin

130 135 140

Ala Arg Ala Gin Ala Ala Leu Gin Ala Val Asn Ser Val Gin Ser Gly

145 150 155 160

Asn Leu Ala Leu Ala Ala Ser Ala Ala Ala Val Asp Ala Gly Met Ala

165 170 175 Met Ala Gly Gin Ser Pro Val Leu Arg lie lie Val Glu Asn Leu Phe

180 185 190

Tyr Pro Val Thr Leu Asp Val Leu His Gin lie Phe Ser Lys Phe Gly

195 200 205

Thr Val Leu Lys IIe lie Thr Phe Thr Lys Asn Asn Gin Phe Gin Ala

210 215 220

Leu Leu Gin Tyr Ala Asp Pro Val Ser Ala Gin His Ala Lys Leu Ser

225 230 235 240

Leu Asp Gly Gin Asn IIe Tyr Asn Ala Cys Cys Thr Leu Arg IIe Asp

245 250 255 Phe Ser Lys Leu Thr Ser Leu Asn Val Lys Tyr Asn Asn Asp Lys Ser

260 265 270

Arg Asp Tyr Thr Arg Pro Asp Leu Pro Ser Gly Asp Ser Gin Pro Ser

275 280 285

Leu Asp Gin Thr Met Ala Ala Ala Phe Gly Leu Ser Val Pro Asn Val 290 295 300

His Gly Ala Leu Ala Pro Leu Ala lie Pro Ser Ala Ala Ala Ala Ala

305 310 315 320

Ala Ala Ala Gly Arg IIe Ala IIe Pro Gly Leu Ala Gly Ala Gly Asn

325 330 335

Ser Val Leu Leu Val Ser Asn Leu Asn Pro Glu Arg Val Thr Pro Gin

340 345 350

Ser Leu Phe lie Leu Phe Gly Val Tyr Gly Asp Val Gin Arg Val Lys

355 360 365 lie Leu Phe Asn Lys Lys Glu Asn Ala Leu Val Gin Met Ala Asp Gly

370 375 380

Asn Gin Ala Gin Leu Ala Met Ser His Leu Asn Gly His Lys Leu His

385 390 395 400

Gly Lys Pro IIe Arg lie Thr Leu Ser Lys His Gin Asn Val Gin Leu

405 410 415

Pro Arg Glu Gly Gin Glu Asp Gin Gly Leu Thr Lys Asp Tyr Gly Asn

420 425 430

Ser Pro Leu His Arg Phe Lys Lys Pro Gly Ser Lys Asn Phe Gin Asn

435 440 445 lie Phe Pro Pro Ser Ala Thr Leu His Leu Ser Asn lie Pro Pro Ser

450 455 460

Val Ser Glu Glu Asp Leu Lys Val Leu Phe Ser Ser Asn Gly Gly Val

465 470 475 480

Val Lys Gly Phe Lys Phe Phe Gin Lys Asp Arg Lys Met Ala Leu IIe 485 490 495

Gin Met Gly Ser Val Glu Glu Ala Val Gin Ala Leu lie Asp Leu His

500 505 510

Asn His Asp Leu Gly Glu Asn His His Leu Arg Val Ser Phe Ser Lys

515 520 525

Ser Thr IIe

530

<210> 5

<211> 290 <212> PRT

<213> Homo sapiens

<400> 5 Met Arg lie Phe Ala Val Phe IIe Phe Met Thr Tyr Trp His Leu Leu

1 5 10 15

Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

Gly Ser Asn Met Thr IIe Glu Cys Lys Phe Pro Val Glu Lys Gin Leu

35 40 45

Asp Leu Ala Ala Leu IIe Val Tyr Trp Glu Met Glu Asp Lys Asn lie

50 55 60 lie Gin Phe Val His Gly Glu Glu Asp Leu Lys Val Gin His Ser Ser

65 70 75 80 Tyr Arg Gin Arg Ala Arg Leu Leu Lys Asp Gin Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Gin IIe Thr Asp Val Lys Leu Gin Asp Ala Gly Val Tyr

100 105 110 Arg Cys Met IIe Ser Tyr Gly Gly Ala Asp Tyr Lys Arg IIe Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys IIe Asn Gin Arg lie Leu Val Val

130 135 140 Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gin Ala Glu Gly Tyr

145 150 155 160

Pro Lys Ala Glu Val lie Trp Thr Ser Ser Asp His Gin Val Leu Ser

165 170 175

Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn

180 185 190

Val Thr Ser Thr Leu Arg lie Asn Thr Thr Thr Asn Glu lie Phe Tyr

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220 Val IIe Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His

225 230 235 240

Leu Val lie Leu Gly Ala IIe Leu Leu Cys Leu Gly Val Ala Leu Thr

245 250 255

Phe IIe Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys 260 265 270

Gly lie Gin Asp Thr Asn Ser Lys Lys Gin Ser Asp Thr His Leu Glu

275 280 285

Glu Thr

290

<210> 6

<211> 288 <212> PRT

<213> Homo sapiens

<400> 6 Met Gin lie Pro Gin Ala Pro Trp Pro Val Val Trp Ala Val Leu Gin

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gin Thr Asp Lys Leu Ala

65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gin Pro Gly Gin Asp Cys Arg Phe Arg

85 90 95

Val Thr Gin Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala lie Ser Leu

115 120 125

Ala Pro Lys Ala Gin lie Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gin Phe Gin Thr Leu Val Val Gly Val Val Gly Gly

165 170 175

Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val IIe Cys

180 185 190

Ser Arg Ala Ala Arg Gly Thr IIe Gly Ala Arg Arg Thr Gly Gin Pro

195 200 205

Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly

210 215 220 Glu Leu Asp Phe Gin Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro

225 230 235 240

Cys Val Pro Glu Gin Thr Glu Tyr Ala Thr lie Val Phe Pro Ser Gly

245 250 255

Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270

Ser Ala Gin Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu

275 280 285

<210> 7 <211> 1255

<212> PRT

<213> Homo sapiens

<400> 7

Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu

1 5 10 15

Pro Pro Gly Ala Ala Ser Thr Gin Val Cys Thr Gly Thr Asp Met Lys

20 25 30 Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His

35 40 45

Leu Tyr Gin Gly Cys Gin Val Val Gin Gly Asn Leu Glu Leu Thr Tyr

50 55 60

Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gin Asp IIe Gin Glu Val 65 70 75 80

Gin Gly Tyr Val Leu lie Ala His Asn Gin Val Arg Gin Val Pro Leu

85 90 95

Gin Arg Leu Arg lie Val Arg Gly Thr Gin Leu Phe Glu Asp Asn Tyr

100 105 110 Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro

115 120 125

Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gin Leu Arg Ser

130 135 140

Leu Thr Glu lie Leu Lys Gly Gly Val Leu lie Gin Arg Asn Pro Gin

145 150 155 160

Leu Cys Tyr Gin Asp Thr IIe Leu Trp Lys Asp IIe Phe His Lys Asn

165 170 175 Asn Gin Leu Ala Leu Thr Leu lie Asp Thr Asn Arg Ser Arg Ala Cys

180 185 190

His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser

195 200 205

Ser Glu Asp Cys Gin Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys

210 215 220

Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gin Cys

225 230 235 240

Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu

245 250 255 His Phe Asn His Ser Gly lie Cys Glu Leu His Cys Pro Ala Leu Val

260 265 270

Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg

275 280 285

Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu 290 295 300

Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gin

305 310 315 320

Glu Val Thr Ala Glu Asp Gly Thr Gin Arg Cys Glu Lys Cys Ser Lys

325 330 335

Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu

340 345 350

Val Arg Ala Val Thr Ser Ala Asn lie Gin Glu Phe Ala Gly Cys Lys

355 360 365 Lys lie Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp

370 375 380

Pro Ala Ser Asn Thr Ala Pro Leu Gin Pro Glu Gin Leu Gin Val Phe

385 390 395 400

Glu Thr Leu Glu Glu IIe Thr Gly Tyr Leu Tyr IIe Ser Ala Trp Pro

405 410 415

Asp Ser Leu Pro Asp Leu Ser Val Phe Gin Asn Leu Gin Val IIe Arg

420 425 430

Gly Arg IIe Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gin Gly Leu

435 440 445 Gly lie Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly

450 455 460

Leu Ala Leu IIe His His Asn Thr His Leu Cys Phe Val His Thr Val

465 470 475 480

Pro Trp Asp Gin Leu Phe Arg Asn Pro His Gin Ala Leu Leu His Thr 485 490 495

Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His

500 505 510

Gin Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gin Cys

515 520 525

Val Asn Cys Ser Gin Phe Leu Arg Gly Gin Glu Cys Val Glu Glu Cys

530 535 540

Arg Val Leu Gin Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys

545 550 555 560 Leu Pro Cys His Pro Glu Cys Gin Pro Gin Asn Gly Ser Val Thr Cys

565 570 575

Phe Gly Pro Glu Ala Asp Gin Cys Val Ala Cys Ala His Tyr Lys Asp

580 585 590

Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu

595 600 605

Ser Tyr Met Pro lie Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gin

610 615 620

Pro Cys Pro IIe Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys

625 630 635 640 Gly Cys Pro Ala Glu Gin Arg Ala Ser Pro Leu Thr Ser lie IIe Ser

645 650 655

Ala Val Val Gly lie Leu Leu Val Val Val Leu Gly Val Val Phe Gly

660 665 670 lie Leu IIe Lys Arg Arg Gin Gin Lys IIe Arg Lys Tyr Thr Met Arg 675 680 685

Arg Leu Leu Gin Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly

690 695 700

Ala Met Pro Asn Gin Ala Gin Met Arg lie Leu Lys Glu Thr Glu Leu 705 710 715 720

Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys

725 730 735

Gly IIe Trp IIe Pro Asp Gly Glu Asn Val Lys IIe Pro Val Ala IIe

740 745 750 Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu lie Leu

755 760 765

Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg

770 775 780

Leu Leu Gly IIe Cys Leu Thr Ser Thr Val Gin Leu Val Thr Gin Leu 785 790 795 800

Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg

805 810 815

Leu Gly Ser Gin Asp Leu Leu Asn Trp Cys Met Gin lie Ala Lys Gly

820 825 830 Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala

835 840 845

Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys lie Thr Asp Phe

850 855 860

Gly Leu Ala Arg Leu Leu Asp IIe Asp Glu Thr Glu Tyr His Ala Asp 865 870 875 880

Gly Gly Lys Val Pro IIe Lys Trp Met Ala Leu Glu Ser lie Leu Arg

885 890 895

Arg Arg Phe Thr His Gin Ser Asp Val Trp Ser Tyr Gly Val Thr Val

900 905 910

Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly lie Pro Ala

915 920 925

Arg Glu lie Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gin Pro

930 935 940 Pro IIe Cys Thr lie Asp Val Tyr Met lie Met Val Lys Cys Trp Met

945 950 955 960 lie Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe

965 970 975

Ser Arg Met Ala Arg Asp Pro Gin Arg Phe Val Val IIe Gin Asn Glu

980 985 990

Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu

995 1000 1005

Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr

1010 1015 1020 Leu Val Pro Gin Gin Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly

1025 1030 1035

Ala Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg

1040 1045 1050

Ser Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu 1055 1060 1065

Glu Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser

1070 1075 1080

Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu

1085 1090 1095

Gin Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gin Arg Tyr Ser

1100 1105 1110

Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val

1115 1120 1125 Ala Pro Leu Thr Cys Ser Pro Gin Pro Glu Tyr Val Asn Gin Pro

1130 1135 1140

Asp Val Arg Pro Gin Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro

1145 1150 1155

Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu

1160 1165 1170

Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly

1175 1180 1185

Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gin Gly Gly Ala

1190 1195 1200 Ala Pro Gin Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp

1205 1210 1215

Asn Leu Tyr Tyr Trp Asp Gin Asp Pro Pro Glu Arg Gly Ala Pro

1220 1225 1230

Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr 1235 1240 1245

Leu Gly Leu Asp Val Pro Val

1250 1255 <210> 8

<211> 1210

<212> PRT

<213> Homo sapiens <400> 8

Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala

1 5 10 15

Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gin

20 25 30

Gly Thr Ser Asn Lys Leu Thr Gin Leu Gly Thr Phe Glu Asp His Phe

35 40 45

Leu Ser Leu Gin Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn

50 55 60 Leu Glu lie Thr Tyr Val Gin Arg Asn Tyr Asp Leu Ser Phe Leu Lys

65 70 75 80

Thr lie Gin Glu Val Ala Gly Tyr Val Leu lie Ala Leu Asn Thr Val

85 90 95

Glu Arg lie Pro Leu Glu Asn Leu Gin IIe IIe Arg Gly Asn Met Tyr 100 105 110

Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn

115 120 125

Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gin Glu lie Leu

130 135 140

His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu

145 150 155 160

Ser lie Gin Trp Arg Asp lie Val Ser Ser Asp Phe Leu Ser Asn Met

165 170 175 Ser Met Asp Phe Gin Asn His Leu Gly Ser Cys Gin Lys Cys Asp Pro

180 185 190

Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gin

195 200 205 Lys Leu Thr Lys IIe lie Cys Ala Gin Gin Cys Ser Gly Arg Cys Arg

210 215 220

Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gin Cys Ala Ala Gly Cys

225 230 235 240

Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp

245 250 255

Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro

260 265 270

Thr Thr Tyr Gin Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly

275 280 285 Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His

290 295 300

Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu

305 310 315 320 Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val

325 330 335

Cys Asn Gly lie Gly lie Gly Glu Phe Lys Asp Ser Leu Ser lie Asn

340 345 350 Ala Thr Asn IIe Lys His Phe Lys Asn Cys Thr Ser IIe Ser Gly Asp

355 360 365

Leu His lie Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr

370 375 380

Pro Pro Leu Asp Pro Gin Glu Leu Asp IIe Leu Lys Thr Val Lys Glu 385 390 395 400

IIe Thr Gly Phe Leu Leu lie Gin Ala Trp Pro Glu Asn Arg Thr Asp

405 410 415

Leu His Ala Phe Glu Asn Leu Glu lie IIe Arg Gly Arg Thr Lys Gin

420 425 430 His Gly Gin Phe Ser Leu Ala Val Val Ser Leu Asn lie Thr Ser Leu

435 440 445

Gly Leu Arg Ser Leu Lys Glu lie Ser Asp Gly Asp Val lie IIe Ser

450 455 460

Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr IIe Asn Trp Lys Lys Leu 465 470 475 480

Phe Gly Thr Ser Gly Gin Lys Thr Lys IIe lie Ser Asn Arg Gly Glu

485 490 495

Asn Ser Cys Lys Ala Thr Gly Gin Val Cys His Ala Leu Cys Ser Pro

500 505 510

Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn

515 520 525

Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly

530 535 540 Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys lie Gin Cys His Pro

545 550 555 560

Glu Cys Leu Pro Gin Ala Met Asn lie Thr Cys Thr Gly Arg Gly Pro

565 570 575

Asp Asn Cys IIe Gin Cys Ala His Tyr IIe Asp Gly Pro His Cys Val

580 585 590

Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp

595 600 605

Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys

610 615 620 Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly

625 630 635 640

Pro Lys lie Pro Ser lie Ala Thr Gly Met Val Gly Ala Leu Leu Leu

645 650 655

Leu Leu Val Val Ala Leu Gly IIe Gly Leu Phe Met Arg Arg Arg His 660 665 670 lie Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gin Glu Arg Glu Leu

675 680 685

Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gin Ala Leu Leu

690 695 700

Arg IIe Leu Lys Glu Thr Glu Phe Lys Lys IIe Lys Val Leu Gly Ser

705 710 715 720

Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp lie Pro Glu Gly Glu

725 730 735 Lys Val Lys lie Pro Val Ala IIe Lys Glu Leu Arg Glu Ala Thr Ser

740 745 750

Pro Lys Ala Asn Lys Glu IIe Leu Asp Glu Ala Tyr Val Met Ala Ser

755 760 765

Val Asp Asn Pro His Val Cys Arg Leu Leu Gly IIe Cys Leu Thr Ser

770 775 780

Thr Val Gin Leu lie Thr Gin Leu Met Pro Phe Gly Cys Leu Leu Asp

785 790 795 800

Tyr Val Arg Glu His Lys Asp Asn IIe Gly Ser Gin Tyr Leu Leu Asn

805 810 815 Trp Cys Val Gin IIe Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg

820 825 830

Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro

835 840 845

Gin His Val Lys IIe Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala 850 855 860

Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro IIe Lys Trp

865 870 875 880

Met Ala Leu Glu Ser lie Leu His Arg IIe Tyr Thr His Gin Ser Asp

885 890 895

Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser

900 905 910

Lys Pro Tyr Asp Gly lie Pro Ala Ser Glu lie Ser Ser lie Leu Glu

915 920 925 Lys Gly Glu Arg Leu Pro Gin Pro Pro IIe Cys Thr IIe Asp Val Tyr

930 935 940

Met IIe Met Val Lys Cys Trp Met lie Asp Ala Asp Ser Arg Pro Lys

945 950 955 960

Phe Arg Glu Leu IIe IIe Glu Phe Ser Lys Met Ala Arg Asp Pro Gin

965 970 975

Arg Tyr Leu Val lie Gin Gly Asp Glu Arg Met His Leu Pro Ser Pro

980 985 990

Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp

995 1000 1005 Asp Val Val Asp Ala Asp Glu Tyr Leu IIe Pro Gin Gin Gly Phe

1010 1015 1020

Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu

1025 1030 1035

Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys IIe Asp Arg Asn 1040 1045 1050

Gly Leu Gin Ser Cys Pro IIe Lys Glu Asp Ser Phe Leu Gin Arg

1055 1060 1065

Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser IIe Asp

1070 1075 1080

Asp Thr Phe Leu Pro Val Pro Glu Tyr IIe Asn Gin Ser Val Pro

1085 1090 1095

Lys Arg Pro Ala Gly Ser Val Gin Asn Pro Val Tyr His Asn Gin

1100 1105 1110 Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gin Asp Pro

1115 1120 1125

His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gin

1130 1135 1140

Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala

1145 1150 1155

Gin Lys Gly Ser His Gin lie Ser Leu Asp Asn Pro Asp Tyr Gin

1160 1165 1170

Gin Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly IIe Phe Lys

1175 1180 1185 Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gin

1190 1195 1200

Ser Ser Glu Phe IIe Gly Ala

1205 1210 <210> 9

<211> 142

<212> PRT

<213> Homo sapiens

<400> 9

Met Gly Ala Pro Thr Leu Pro Pro Ala Trp Gin Pro Phe Leu Lys Asp

1 5 10 15 His Arg lie Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Cys Ala

20 25 30

Cys Thr Pro Glu Arg Met Ala Glu Ala Gly Phe lie His Cys Pro Thr

35 40 45

Glu Asn Glu Pro Asp Leu Ala Gin Cys Phe Phe Cys Phe Lys Glu Leu

50 55 60

Glu Gly Trp Glu Pro Asp Asp Asp Pro IIe Glu Glu His Lys Lys His

65 70 75 80

Ser Ser Gly Cys Ala Phe Leu Ser Val Lys Lys Gin Phe Glu Glu Leu

85 90 95 Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys Asn Lys

100 105 110 lie Ala Lys Glu Thr Asn Asn Lys Lys Lys Glu Phe Glu Glu Thr Ala

115 120 125

Lys Lys Val Arg Arg Ala lie Glu Gin Leu Ala Ala Met Asp 130 135 140

<210 10

<211> 435 <212> PRT

<213> Homo sapiens

<400> 10 Met Glu Met Glu Lys Glu Phe Glu Gin lie Asp Lys Ser Gly Ser Trp

1 5 10 15

Ala Ala IIe Tyr Gin Asp lie Arg His Glu Ala Ser Asp Phe Pro Cys

20 25 30

Arg Val Ala Lys Leu Pro Lys Asn Lys Asn Arg Asn Arg Tyr Arg Asp

35 40 45

Val Ser Pro Phe Asp His Ser Arg lie Lys Leu His Gin Glu Asp Asn

50 55 60

Asp Tyr lie Asn Ala Ser Leu IIe Lys Met Glu Glu Ala Gin Arg Ser

65 70 75 80 Tyr lie Leu Thr Gin Gly Pro Leu Pro Asn Thr Cys Gly His Phe Trp

85 90 95

Glu Met Val Trp Glu Gin Lys Ser Arg Gly Val Val Met Leu Asn Arg

100 105 110

Val Met Glu Lys Gly Ser Leu Lys Cys Ala Gin Tyr Trp Pro Gin Lys 115 120 125

Glu Glu Lys Glu Met lie Phe Glu Asp Thr Asn Leu Lys Leu Thr Leu

130 135 140 lie Ser Glu Asp IIe Lys Ser Tyr Tyr Thr Val Arg Gin Leu Glu Leu 145 150 155 160

Glu Asn Leu Thr Thr Gin Glu Thr Arg Glu lie Leu His Phe His Tyr

165 170 175

Thr Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser Phe Leu

180 185 190 Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu Ser Pro Glu His

195 200 205

Gly Pro Val Val Val His Cys Ser Ala Gly lie Gly Arg Ser Gly Thr

210 215 220

Phe Cys Leu Ala Asp Thr Cys Leu Leu Leu Met Asp Lys Arg Lys Asp 225 230 235 240

Pro Ser Ser Val Asp IIe Lys Lys Val Leu Leu Glu Met Arg Lys Phe

245 250 255

Arg Met Gly Leu IIe Gin Thr Ala Asp Gin Leu Arg Phe Ser Tyr Leu

260 265 270 Ala Val IIe Glu Gly Ala Lys Phe IIe Met Gly Asp Ser Ser Val Gin

275 280 285

Asp Gin Trp Lys Glu Leu Ser His Glu Asp Leu Glu Pro Pro Pro Glu

290 295 300

His IIe Pro Pro Pro Pro Arg Pro Pro Lys Arg IIe Leu Glu Pro His 305 310 315 320

Asn Gly Lys Cys Arg Glu Phe Phe Pro Asn His Gin Trp Val Lys Glu

323 330 333

Glu Thr Gin Glu Asp Lys Asp Cys Pro lie Lys Glu Glu Lys Gly Ser

340 345 350

Pro Leu Asn Ala Ala Pro Tyr Gly IIe Glu Ser Met Ser Gin Asp Thr

355 360 365

Glu Val Arg Ser Arg Val Val Gly Gly Ser Leu Arg Gly Ala Gin Ala

370 375 380 Ala Ser Pro Ala Lys Gly Glu Pro Ser Leu Pro Glu Lys Asp Glu Asp

385 390 395 400

His Ala Leu Ser Tyr Trp Lys Pro Phe Leu Val Asn Met Cys Val Ala

405 410 415

Thr Val Leu Thr Ala Gly Ala Tyr Leu Cys Tyr Arg Phe Leu Phe Asn

420 425 430

Ser Asn Thr

435

<210> 11 <211> 232

<212> PRT

<213> Homo sapiens

<400> 11 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu

1 5 10 15

Tyr Leu His His Ala Lys Trp Ser Gin Ala Ala Pro Met Ala Glu Gly

20 25 30

Gly Gly Gin Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gin

35 40 45

Arg Ser Tyr Cys His Pro IIe Glu Thr Leu Val Asp lie Phe Gin Glu

50 55 60 Tyr Pro Asp Glu lie Glu Tyr IIe Phe Lys Pro Ser Cys Val Pro Leu

65 70 75 80

Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro

85 90 95

Thr Glu Glu Ser Asn lie Thr Met Gin lie Met Arg lie Lys Pro His

100 105 110

Gin Gly Gin His lie Gly Glu Met Ser Phe Leu Gin His Asn Lys Cys

115 120 125

Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gin Glu Lys Lys Ser Val

130 135 140 Arg Gly Lys Gly Lys Gly Gin Lys Arg Lys Arg Lys Lys Ser Arg Tyr

145 150 155 160

Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys Leu Met Pro Trp

165 170 175

Ser Leu Pro Gly Pro His Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys 180 185 190

His Leu Phe Val Gin Asp Pro Gin Thr Cys Lys Cys Ser Cys Lys Asn

195 200 205

Thr Asp Ser Arg Cys Lys Ala Arg Gin Leu Glu Leu Asn Glu Arg Thr

210 215 220

Cys Arg Cys Asp Lys Pro Arg Arg

225 230 <210> 12

<211> 889

<212> PRT

<213> Homo sapiens <400> 12

Met Glu Glu Gly Ala Pro Arg Gin Pro Gly Pro Ser Gin Trp Pro Pro

1 5 10 15

Glu Asp Glu Lys Glu Val lie Arg Arg Ala lie Gin Lys Glu Leu Lys

20 25 30

IIe Lys Glu Gly Val Glu Asn Leu Arg Arg Val Ala Thr Asp Arg Arg

35 40 45

His Leu Gly His Val Gin Gin Leu Leu Arg Ser Ser Asn Arg Arg Leu

50 55 60 Glu Gin Leu His Gly Glu Leu Arg Glu Leu His Ala Arg lie Leu Leu

65 70 75 80

Pro Gly Pro Gly Pro Gly Pro Ala Glu Pro Val Ala Ser Gly Pro Arg

85 90 95 Pro Trp Ala Glu Gin Leu Arg Ala Arg His Leu Glu Ala Leu Arg Arg

100 105 110

Gin Leu His Val Glu Leu Lys Val Lys Gin Gly Ala Glu Asn Met Thr

115 120 125 His Thr Cys Ala Ser Gly Thr Pro Lys Glu Arg Lys Leu Leu Ala Ala

130 135 140

Ala Gin Gin Met Leu Arg Asp Ser Gin Leu Lys Val Ala Leu Leu Arg

145 150 155 160

Met Lys lie Ser Ser Leu Glu Ala Ser Gly Ser Pro Glu Pro Gly Pro

165 170 175

Glu Leu Leu Ala Glu Glu Leu Gin His Arg Leu His Val Glu Ala Ala

180 185 190

Val Ala Glu Gly Ala Lys Asn Val Val Lys Leu Leu Ser Ser Arg Arg

195 200 205 Thr Gin Asp Arg Lys Ala Leu Ala Glu Ala Gin Ala Gin Leu Gin Glu

210 215 220

Ser Ser Gin Lys Leu Asp Leu Leu Arg Leu Ala Leu Glu Gin Leu Leu

225 230 235 240

Glu Gin Leu Pro Pro Ala His Pro Leu Arg Ser Arg Val Thr Arg Glu 245 250 255

Leu Arg Ala Ala Val Pro Gly Tyr Pro Gin Pro Ser Gly Thr Pro Val

260 265 270

Lys Pro Thr Ala Leu Thr Gly Thr Leu Gin Val Arg Leu Leu Gly Cys

275 280 285

Glu Gin Leu Leu Thr Ala Val Pro Gly Arg Ser Pro Ala Ala Ala Leu

290 295 300

Ala Ser Ser Pro Ser Glu Gly Trp Leu Arg Thr Lys Ala Lys His Gin

305 310 315 320 Arg Gly Arg Gly Glu Leu Ala Ser Glu Val Leu Ala Val Leu Lys Val

325 330 335

Asp Asn Arg Val Val Gly Gin Thr Gly Trp Gly Gin Val Ala Glu Gin

340 345 350

Ser Trp Asp Gin Thr Phe Val lie Pro Leu Glu Arg Ala Arg Glu Leu

355 360 365

Glu IIe Gly Val His Trp Arg Asp Trp Arg Gin Leu Cys Gly Val Ala

370 375 380

Phe Leu Arg Leu Glu Asp Phe Leu Asp Asn Ala Cys His Gin Leu Ser

385 390 395 400 Leu Ser Leu Val Pro Gin Gly Leu Leu Phe Ala Gin Val Thr Phe Cys

405 410 415

Asp Pro Val lie Glu Arg Arg Pro Arg Leu Gin Arg Gin Glu Arg lie

420 425 430

Phe Ser Lys Arg Arg Gly Gin Asp Phe Leu Arg Ala Ser Gin Met Asn 435 440 445

Leu Gly Met Ala Ala Trp Gly Arg Leu Val Met Asn Leu Leu Pro Pro

450 455 460

Cys Ser Ser Pro Ser Thr lie Ser Pro Pro Lys Gly Cys Pro Arg Thr 465 470 475 480

Pro Thr Thr Leu Arg Glu Ala Ser Asp Pro Ala Thr Pro Ser Asn Phe

485 490 495

Leu Pro Lys Lys Thr Pro Leu Gly Glu Glu Met Thr Pro Pro Pro Lys

500 505 510 Pro Pro Arg Leu Tyr Leu Pro Gin Glu Pro Thr Ser Glu Glu Thr Pro

515 520 525

Arg Thr Lys Arg Pro His Met Glu Pro Arg Thr Arg Arg Gly Pro Ser

530 535 540

Pro Pro Ala Ser Pro Thr Arg Lys Pro Pro Arg Leu Gin Asp Phe Arg 545 550 555 560

Cys Leu Ala Val Leu Gly Arg Gly His Phe Gly Lys Val Leu Leu Val

565 570 575

Gin Phe Lys Gly Thr Gly Lys Tyr Tyr Ala lie Lys Ala Leu Lys Lys

580 585 590 Gin Glu Val Leu Ser Arg Asp Glu lie Glu Ser Leu Tyr Cys Glu Lys

595 600 605

Arg lie Leu Glu Ala Val Gly Cys Thr Gly His Pro Phe Leu Leu Ser

610 615 620

Leu Leu Ala Cys Phe Gin Thr Ser Ser His Ala Cys Phe Val Thr Glu 625 630 635 640

Phe Val Pro Gly Gly Asp Leu Met Met Gin IIe His Glu Asp Val Phe

645 650 655

Pro Glu Pro Gin Ala Arg Phe Tyr Val Ala Cys Val Val Leu Gly Leu

660 665 670

Gin Phe Leu His Glu Lys Lys lie lie Tyr Arg Asp Leu Lys Leu Asp

675 680 685

Asn Leu Leu Leu Asp Ala Gin Gly Phe Leu Lys lie Ala Asp Phe Gly

690 695 700 Leu Cys Lys Glu Gly IIe Gly Phe Gly Asp Arg Thr Ser Thr Phe Cys

705 710 715 720

Gly Thr Pro Glu Phe Leu Ala Pro Glu Val Leu Thr Gin Glu Ala Tyr

725 730 735

Thr Arg Ala Val Asp Trp Trp Gly Leu Gly Val Leu Leu Tyr Glu Met

740 745 750

Leu Val Gly Glu Cys Pro Phe Pro Gly Asp Thr Glu Glu Glu Val Phe

755 760 765

Asp Cys IIe Val Asn Met Asp Ala Pro Tyr Pro Gly Phe Leu Ser Val

770 775 780 Gin Gly Leu Glu Phe IIe Gin Lys Leu Leu Gin Lys Cys Pro Glu Lys

785 790 795 800

Arg Leu Gly Ala Gly Glu Gin Asp Ala Glu Glu IIe Lys Val Gin Pro

805 810 815

Phe Phe Arg Thr Thr Asn Trp Gin Ala Leu Leu Ala Arg Thr He Gin 820 825 830

Pro Pro Phe Val Pro Thr Leu Cys Gly Pro Ala Asp Leu Arg Tyr Phe

835 840 845

Glu Gly Glu Phe Thr Gly Leu Pro Pro Ala Leu Thr Pro Pro Ala Pro

850 855 860

His Ser Leu Leu Thr Ala Arg Gin Gin Ala Ala Phe Arg Asp Phe Asp

865 870 875 880

Phe Val Ser Glu Arg Phe Leu Glu Pro

885 [00110] Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure.

Although the disclosure has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art are intended to be within the scope of the disclosure. This application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure come within known customary practice within the art to which the disclosure pertains and may be applied to the essential features herein before set forth.