GRP78 antagonists for alzheimer's treatment

Abstract

The present invention provides methods of treating a disorder associated with protein aggregation by providing to a subject having or at risk of having a GRP78-mediated protein aggregation disorder a composition comprising a GRP78 antagonist.

Description

[0001] GRP78 ANTAGONISTS FOR ALZHEIMER’S TREATMENT

[0002] Field of the Invention

[0003] The present invention relates to the treatment of Alzheimer’s disease using GRP78 antagonists.

[0004] Background

[0005] Alzheimer's Disease (AD) is the most common cause of dementia, with 6.7 million adults currently diagnosed. AD is currently incurable and the 5th leading cause of disease related death for the over 62 million US adults that are over the age of 65. From 2000-2019, while the rate of mortality for all ages declined for HIV (-7.3%), Stroke (-10.5%) and Heart Disease (-5.2%), the rate of death related to Alzheimer’s Disease (AD) rose dramatically by 145.2% during the same time period.

[0006] The disease is characterized pathologically by the accumulation of extracellular 0-amyloid (“A0”) plaques and intracellular neurofibrillary tangles in the brain. Diagnosis is made through the clinical assessment of the neurologic and neuropsychiatric signs and symptoms of AD and the exclusion of other causes of dementia. AD is commonly classified into mild, moderate and severe stages by a brief cognitive screening examination, the Mini-Mental State Examination (“MMSE”). Currently, there are no treatments for AD that can halt, reverse, or even stop the neurodegenerative processes associated with the disease. Current approved treatments available are very limited with cholinesterase inhibitors (EXELON), and Glutamate regulators (NAMENDA) offering temporary symptom improvement of memory loss and cognitive function, though they do not halt the disease progression.

[0007] As such approved drugs like Donepezil, and Rivastigmine are drugs used to treat symptoms that can boost the activity of chemicals in the brain to compensate for declines in cognitive and memory, but they cannot stop its progression. For example, anti-amyloid monoclonal antibodies have shown mixed results and are associated with non-trivial risks and treatment burdens.

[0008] Recently, researchers have been rethinking the Amyloid reduction theory for delaying Alzheimer’s Disease. Anti- Amyloid FDA approved antibody therapies, Aducanumab, Lecanemab and soon to be approved Donanemab have come under intense scrutiny by experts not because they fail to reduce Amyloid-beta plaques, but due to risks of amyloid-related imaging abnormalities (ARIAs) that can produce brain swelling or bleeding, with the FDA recently halting the approval process for the monoclonal antibody treatment (Donanemab)

[0009] More recent commentary has found that the effect of anti-Amyloid antibodies “is tiny” with 3 main limitations: 1) there is little evidence that amyloid reduction correlates with clinical outcome, 2) clinical efficacy of anti-amyloid drugs may be explained by functional unblinding and 3) anti-amyloid therapies have no effect on tau aggregation burden.

[0010] While, between 1995 and 2021, pharmaceutical companies have spent an estimated $42.5 billion on AD, only 6 drug candidates out of 235 have reached commercialization and an amazing 117 have been discontinued, equating to a 95% failure rate at the end of 2021. Thus, the critical need for effective therapies at all stages of neurodegenerative disease remains a priority with the US population projected to grow for citizens over the age of 65 from 62 to 74 million by the year 2030. As the at-risk population grows, the disease related mortality risk escalates with those over the age of 85 to 78% with Alzheimer’s compared to 30% for non-Alzheimer’s.

[0011] Summary of the Invention

[0012] The present invention provides novel GRP78 antagonists that prevent or dissolve the formation of protein aggregates, understood to be instrumental in neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s Disease.

[0013] Without being bound by a mechanism of action, the invention recognizes for the first time that misfolded and aggregated proteins in the pathogenesis of neurodegenerative diseases like as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Amyotrophic Lateral Sclerosis (ALS), may be mediated by folding in the endoplasmic reticulum centering around GRP78. For example, extra cellular GRP78 may be essential for tau fibril aggregation which is a key underpinning of Alzheimer’s Disease. Extra cellular GRP78 binds tightly to central nervous system monomers and alpha-synuclein, increasing alpha- sy nuclein and tau fibril formation, leading to tau fibril killing of human brain microglia and neurons in seconds.

[0014] Accordingly, aspects of the invention provide methods of treating a disorder associated with protein aggregation by providing to a subject having or at risk of having a GRP78-mediated protein aggregation disorder a composition comprising a GRP78 antagonist. The GRP78 antagonist comprises a binding domain selected from the group consisting of a plasminogen kringlc 5 fragment, a plasminogen kringlc 5 fragment attached to immunoglobulin, a R0R1 kringlc fragment, a R0R1 kringle fragment attached to an immunoglobulin, a R0R2 kringle fragment; and a ROR2 kringle fragment attached to an immunoglobulin.

[0015] The protein aggregation disorder may be any protein aggregation disorder mediated by GRP78, for example, Tau protein associated disorders or an alpha- sy nuclein (aSyn) associated disorders. The disorder may be Alzheimer’s disease, Parkinson’s disease, or Amyotrophic Lateral Sclerosis (ALS).

[0016] The fragment binding GRP78 may be described by the specific sequences disclosed herein and in U.S. Patent No. 10,905,750, which is incorporated herein by reference in its entirety.

[0017] For example, in aspects of the invention, the plasminogen kringle 5 fragment comprises an amino acid sequence of SEQ ID NO:1. The plasminogen kringle 5 fragment attached to immunoglobulin comprises an amino acid sequence from the group consisting of SEQ ID NO:2, SEQ ID NOG, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NOG, SEQ ID NOTO, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18.

[0018] The ROR1 kringle fragment may comprise an amino acid sequence of SEQ ID NO: 19.

[0019] The ROR1 kringle fragment attached to immunoglobulin may comprise an amino acid sequence from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and combinations thereof.

[0020] The ROR2 kringle fragment may be SEQ ID NO:33 and combinations thereof.

[0021] The ROR2 kringle fragment attached to immunoglobulin may be selected from the group consisting of SEQ ID NO:34, SEQ ID NOGS, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and combinations thereof. Advantageously, the GRP78 antagonist may bind to the N-terminal GRP78 domain of extracellular GRP78 and prevents GRP78 mediated aggregation of proteins.

[0022] In certain aspects, the invention provides methods of treatment of protein aggregation disorders, wherein the method comprises administration of composition comprising a therapeutically effective amount of a GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition is administered parenterally. In certain embodiments, the GRP78 antagonist is CBT300. In certain embodiments, the composition is administered as an intravenous infusion. In certain embodiments, the composition is administered as a subcutaneous injection. In certain embodiments, the composition is administered once a month. In certain embodiments, the composition is administered once every four weeks. In certain embodiments, the composition is administered once every three weeks. In certain embodiments, the composition is administered once every two weeks. In certain embodiments, the composition is administered once every week.

[0023] In certain aspects, the invention further provides pharmaceutical compositions comprising GRP78. In certain embodiments, the invention further provides that the pharmaceutical composition comprising GRP78 antagonist comprises a buffer. The buffers used in the pharmaceutical compositions of the invention could be any pharmaceutically acceptable buffers. In certain embodiments, the buffer is phosphate buffered saline (PBS). In certain embodiments, the buffer is a citrate buffer. In certain embodiments, the buffer is an acetate buffer.

[0024] In certain embodiments, the pH of the pharmaceutical composition is from about 6 to about 8. In certain embodiments, the pH of the pharmaceutical composition is from about 6.5 to about 7.5. In certain embodiments, the pH of the pharmaceutical composition is from about 7 to about 7.4. In certain embodiments, the pH of the pharmaceutical composition is about 7.2. In certain embodiments, the pH of the pharmaceutical composition is 7.2.

[0025] The present invention also provides compositions, medicaments, and uses of compositions comprising a GRP78 antagonist for use in treating disorders associated with protein aggregation by providing to a subject having or at risk of having a GRP78-mediated protein aggregation disorder the composition of the invention.

[0026] Brief Description of the Drawings

[0027] FIG. 1 shows a graph of absorbance and CBT300 concentration in the present of Tau fibril and monomers.

[0028] FIG. 2A show immunohistochemistry (IHC) of wells with pre-formed fibrils following incubation with a control, CBT300, and GRP78.

[0029] FIG. 2B-C show graphs of Tau tangle aggregation size and number / area of tau tangle aggregation following incubation with a control, CBT300, and GRP78.

[0030] FIG. 3A shows immunofluorescence Tau fibrils in human microglia cells following incubation with a control, CBT300, and extracellular GRP78.

[0031] FIG. 3B shows a graph of internalization of Tau aggregative in human microglia cells following incubation with a control, CBT300, and extracellular GRP78.

[0032] FIG. 4 is a graph of cell death in HMC3 microglia after incubation with PBS (control), CBT300, or extracellular GRP78.

[0033] Detailed Description

[0034] The present invention provides methods of treating a disorder associated with protein aggregation by providing to a subject having or at risk of having a GRP78-mediated protein aggregation disorder a composition comprising a GRP78 antagonist.

[0035] GRP78

[0036] GRP78 mostly resides in the endoplasmic reticulum (ER), where it functions in protein folding and assembly, targeting misfolded protein for degradation, ER Ca2+-binding and controlling the activation of trans-membrane ER stress sensors. GRP78 is a member of the 70 kilodalton heat shock protein (HSP70) family, and it’s up-regulation is part of the general cellular defense mechanism of stressed cells that is referred to as the unfolded protein response. The expression of GRP78 and other members of the unfolded protein response in tumors has led to a significant scientific interest in targeting members of the unfolded protein response in cancer. Overexpression of GRP78 in many different cancers has established that GRP78 contributes to tumor growth and confers drug resistance to cancer cells. Accordingly soluble GRP78 and cell surface bound GRP78 are possible biomarkers and therapeutic targets for many cancers including glioblastoma. GRP78 has also been found to be released at times of cellular stress and has been shown to have extracellular properties that are anti-inflammatory or favor the resolution of inflammation. GRP78 produced by the tumor cells is believed to interfere with adaptive immune responses of antigen-presenting cells (APCs). Soluble GRP78 is believed to bind to transmembrane RORs on APCs which induces of self-tolerance of APCs which helps explain how tumors can remain invisible from immune surveillance and become drug resistant. For example, patients with glioblastoma exhibit extreme immunosuppression, both systemically as well as within the tumor microenvironment. There are many factors produced by the tumor and tolerogenic immune cells that can induce immune tolerance and tumor resistance that are not inhibited by current therapies. For example, tumor derived soluble GRP78 can bind to immature dendritic cells (DCs) and regulate their maturation to produce a tolerogenic phenotype by upregulating IL- 10, B7H1, B7H3, and B7H4 expression and down regulating maturity marker expression of CD86 leading to a tolerogenic phenotype that is stable with LPS stimulation. This immunosuppressive DC phenotype is stable upon lipopolysaccharide (LPS) stimulation. GRP78- treated dendritic cells also reduce T-cell proliferation and induce T-cell apoptosis. Finally, increased generation of T-regs from GRP78 treated myeloid antigen presenting cells were observed in vitro and ex vivo. This data shows that GRP78 surface binding on tumor cells leads to chemo-resistance and proliferation and suggest that GRP78 is a soluble immunomodulatory molecule.

[0037] It has also been discovered that GRP78 binds to a cell- surface receptor orphan tyrosine kinase receptor-1 (ROR-1) on dendritic, glioma, and endothelial cells. GRP78 antagonists reverse this tolerogenic and resistant phenotype. The data demonstrates that GRP78 binds to the kringle domain of ROR-1 and ROR-2 leading to ROR signaling through several different non-canonical pathways. This ROR signaling in dendritic cells induces a tolerogenic phenotype and a resistant phenotype in tumor cells. Wnt5a has been shown to be a second ligand for ROR-1 and binds to the frizzled domain, which leads to increased migration and proliferation of leukemia cells. Blocking the GRP78 binding to ROR-1, the Wnt5a binding docs not lead to activation of ROR-1.

[0038] GRP78 antagonists

[0039] The preferred GRP78 antagonists of the invention comprise anti- angiogenic kringle fragment peptides from mammalian plasminogen, R0R1, and or R0R2. These kringle fragment peptides may be in the form of free kringle fragments peptides, or in a form fused onto immunoglobulin, or in a form modified with various linking agents that are designed to bind to blood or tissue peptides when introduced into the blood stream of a patient. These kringle fragments fused to immunoglobulin compound and the modified kringle fragments realize extended in vivo half-life times as compared to their corresponding non-modified kringle fragment peptides. These modified kringle fragment peptides include succimidyl or maleimido reactive linking groups which can then subsequently react with amino groups, hydroxyl groups and / or thiol groups of blood or tissue peptides to form the more stable biologically active components. The present invention also includes a method for treating a patient in need of antiangiogenesis therapy comprising administering these kringle containing antiangiogenic peptides to the patient. The present invention also includes compositions for treating a patient in need of anti-angiogenesis therapy comprising a compound containing at least one of these kringle containing antiangiogenic peptides with a pharmaceutically acceptable excipient and / or optionally sustained release compounds to form a therapeutic composition. The preferred GRP78 antagonists are provided in U.S. Patent No. 10,905,750 and U.S. Patent Publication No. 2021 / 0324047, which are herein incorporated by reference in their entirety.

[0040] In particular, the present invention provides the three broadly defined different types of GRP78 antagonists that specifically inhibit surface bound GRP78 binding and receptor signaling. These broadly defined different grouping of GRP78 antagonists include (1) plasminogen kringle 5 fragment fusion compounds, (2) ROR1 kringle derivative compounds, and (3) ROR2 kringle derivative compounds.

[0041] This first type of GRP78 antagonists that are disclosed in the present application are the plasminogen kringle five fragment fusion compound includes various peptide fragments of K5 (SEQ ID NO: 189) fused to immunoglobulin herein abbreviated as the K5-frag-Fc fusion peptides. These K5-frag-Fc fusion peptides of the first type of GRP78 antagonists include but not limited to those selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, and SEQ ID NO: 18, as provided in U.S. Patent No. 10,905,750, which is incorporated herein by reference in its entirety.

[0042] The second type of GRP78 antagonists that are disclosed in the present application are the ROR1 kringle derivatives that include the kringle active zone component itself (SEQ ID NO: 19) abbreviated as Krl or specifically the Krl(l-93 Active Zone) fragment; Krl active zone fragment peptides here abbreviated as Krl -frag peptides; Krl active zone fragment peptides fusion complexes herein abbreviated as Krl-frag-Fc; and the modified active zone fragments of Krl herein abbreviated as mod-Krl-frag peptides.

[0043] The Krl-frag-Fc fusion peptides of the second type of GRP78 antagonists include but not limited to those selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, as provided in U.S. Patent No. 10,905,750, which is incorporated herein by reference in its entirety .

[0044] The Krl -frag peptides of the second type of GRP78 antagonists include but not limited to those selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61, as provided in U.S. Patent No. 10,905,750, which is incorporated herein by reference in its entirety.

[0045] The mod-Krl-frag peptides of the second type of GRP78 antagonists include but not limited to those selected from the group consisting of SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NG:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO:131, SEQ ID NO:186, SEQ ID NO: 187, and SEQ ID NO:188, as provided in U.S. Patent No. 10,905,750, which is incorporated herein by reference in its entirety.

[0046] The third type of GRP78 antagonists that are disclosed in the present application are the ROR2 kringle derivatives that include SEQ ID NO:33 which is the abbreviated as Kr2 or specifically abbreviated the Kr2(l-85 Active Zone) fragment; Kr2 active zone fragment fusion peptides herein abbreviated as the Kr2-frag-Fc fusion peptides; active zone fragments of Kr2 herein abbreviated as Kr2-frag peptides; and the modified active zone fragments of Kr2 herein abbreviated as mod-Kr2-frag peptides.

[0047] The Kr2-Fc fusion peptides of the third type of GRP78 antagonists include but not limited to those selected from the group consisting of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45, as provided in U.S. Patent No. 10,905,750, which is incorporated herein by reference in its entirety.

[0048] The Kr2-frag peptides of the third type of GRP78 antagonists include but not limited to those selected from the group consisting of SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77, as provided in U.S. Patent No. 10,905,750, which is incorporated herein by reference in its entirety.

[0049] The mod-Kr2-frag peptides of the third type of GRP78 antagonists include but not limited to those selected from the group consisting of SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO: 140, SEQ ID NO: 141 , SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO: 184, and SEQ ID NO: 185, as provided in U.S. Patent No. 10,905,750, which is incorporated herein by reference in its entirety.

[0050] GRP78 compositions

[0051] In certain aspects, the invention further provides pharmaceutical compositions comprising GRP78. In certain embodiments, the invention further provides that the pharmaceutical composition comprising GRP78 antagonist comprises a buffer. The buffers used in the pharmaceutical compositions of the invention could be any pharmaceutically acceptable buffers. In certain embodiments, the buffer is phosphate buffered saline (PBS). In certain embodiments, the buffer is a citrate buffer. In certain embodiments, the buffer is an acetate buffer.

[0052] In certain embodiments, the pH of the pharmaceutical composition is from about 6 to about 8. In certain embodiments, the pH of the pharmaceutical composition is from about 6.5 to about 7.5. In certain embodiments, the pH of the pharmaceutical composition is from about 7 to about 7.4. In certain embodiments, the pH of the pharmaceutical composition is about 7.2. In certain embodiments, the pH of the pharmaceutical composition is 7.2. The term “about,” when used in the context of describing the pH of the compositions of the invention, means ± 0.2.

[0053] In certain embodiments, the aqueous solubility of the GRP78 antagonists in an aqueous buffer is 160 mg / mL. In certain embodiments, the compositions of the invention comprising GRP78 antagonists are shelf-stable when stored at 4 °C. In certain embodiments, the compositions of the invention comprising GRP78 antagonists arc shelf- stable when stored at -20 °C. In certain embodiments, the compositions of the invention comprising GRP78 antagonists are stable for at least 6 months when stored at 4 °C or at -20 °C. In certain embodiments, the compositions of the invention comprising GRP78 antagonists are stable for at least 12 months when stored at 4 °C or at -20 °C. In certain embodiments, the compositions of the invention comprising GRP78 antagonists arc stable at 26 °C and / or 37 °C for 3 months. In certain embodiments, the compositions of the invention comprising GRP78 antagonists are stable at 26 °C and / or 37 °C for more than 3 months.

[0054] CBT300 has shown a long half-life around 11 days with a bioavailability around 60-70%. CBT300 has also shown to exhibit no acute or observable toxicitics whether dosed SC or intravenously (IV) up to doses of 400 mg / kg daily in mice.

[0055] In certain embodiments, the compositions of the invention comprising GRP78 antagonists comprise GRP78 antagonist at a concentration of from about 10 mg / mL to about 500 mg / mL. In certain embodiments, the compositions of the invention comprising GRP78 antagonists comprise GRP78 antagonist at a concentration of from about 50 mg / mL to about 200 mg / mL. In certain embodiments, the compositions of the invention comprising GRP78 antagonists comprise GRP78 antagonist at a concentration of about 50 mg / mL. In certain embodiments, the compositions of the invention comprising GRP78 antagonists comprise GRP78 antagonist at a concentration of about 100 mg / mL. In certain embodiments, the compositions of the invention comprising GRP78 antagonists comprise GRP78 antagonist at a concentration of about 150 mg / mL.

[0056] In certain embodiments, the GRP78 antagonist is a is a plasminogen kringle 5 fragment attached to immunoglobulin, wherein the plasminogen kringle 5 fragment attached to immunoglobulin is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and combinations thereof, as provided in U.S. Patent No. 10,905,750, which is incorporated herein by reference in its entirety.

[0057] In certain embodiments, the GRP78 antagonist is a ROR1 kringle fragment attached to immunoglobulin, wherein the ROR1 kringle fragment attached to immunoglobulin is selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and combinations thereof, as provided in U.S. Patent No. 10,905,750, which is incorporated herein by reference in its entirety. In certain embodiments, the GRP78 antagonist is a R0R2 kringle fragment attached to immunoglobulin, wherein the R0R2 kringle fragment attached to immunoglobulin is selected from the group consisting of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and combinations thereof.

[0058] Tau proteins

[0059] Tau proteins (abbreviated from tubulin associated unit) are proteins that form a group of six highly soluble protein isoforms produced by alternative splicing from the gene MAPT (microtubule-associated protein tau). Tau proteins promote stability of microtubules in axons and are abundant in the neurons of the central nervous system (CNS).

[0060] Hyperphosphorylation of the tau protein can result in the self-assembly of tangles of paired helical filaments and straight filaments, which are thought to be involved in the pathogenesis of Alzheimer's disease. All of the six tau isoforms are present in an often hyperphosphorylated state in paired helical filaments in the Alzheimer's disease brain. In AD, pathological tau aggregation spreads progressively throughout the brain, possibly along existing neural networks.

[0061] The common minimal connection between Alzheimer's Disease and all the tauopathies is the aggregation state of tau. Under all these diseased conditions, monomeric tau is known to be converted into polymeric ordered fibrils. Neurofibrillary tangles (NFTs), which are comprised of fibrillar tau aggregates, are a neuropathological hallmark of tauopathies. Applicants have discovered that spreading of tau pathology in the brain may be caused by a form of tau aggregate released from a “donor” cell entering a second “recipient” cell, and inducing further misfolding and aggregation of tau in the recipient cell via direct protein-protein contact. The specific form of tau aggregate which facilitates this cell-to-cell spread of tau aggregates is referred to as “tau seeds” and the activity may be referred to herein as “seeding activity”, since this form of tau aggregate seeds or nucleates tau aggregation in the cell it enters (i.e. the “recipient cell”).

[0062] Tau can exist in both a monomeric form and in different aggregated forms. As used herein, the term “tau aggregate” refers to a molecular complex that comprises two or more tau monomers. Without wishing to be bound by theory, a tau aggregate may comprise a nearly unlimited number of monomers bound together. For example, a tau aggregate may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more tau monomers. Alternatively, a tau aggregate may comprise 20, 30, 40, 50, 60, 70, 80, 90, 100 or more tau monomers. A tau aggregate may also comprise 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more tau monomers. The terms “fibrillar tau aggregate” and “tau fibril” refer to forms of tau aggregates, and these terms are used interchangeably herein. A fibrillar tau aggregate is a polymeric, ordered fiber comprising tau. Tau fibrils are generally not soluble, but shorter assemblies, or oligomers, can be soluble. Tau aggregate also refers to soluble tau oligomers and protofibrils, which may act as intermediates during tau aggregation. Also included in the definition of tau aggregate is the term “tau seed”, which refers to a tau aggregate that is capable of nucleating or “seeding” intracellular tau aggregation when internalized by a cell, or when exposed to monomeric tau in vitro. Tau seeding activity may be assessed in a cellular tau aggregation assay as described herein.

[0063] Alpha-synuclein (aSyn)

[0064] Alpha-synuclein (aSyn) is a protein that, in humans, is encoded by the SNCA gene. Alpha- synuclein is a neuronal protein that regulates synaptic vesicle trafficking and subsequent neurotransmitter release. aSyn is abundant in the brain, found mainly in the axon terminals of presynaptic neurons.

[0065] The human alpha-synuclein protein is made of 140 amino acids. An alpha-synuclein fragment, known as the non-amyloid beta (non-Abeta) component of Alzheimer's disease amyloid, originally found in an amyloid-enriched fraction, was shown to be a fragment of its precursor protein, NACP.

[0066] In Parkinson's disease and other synucleinopathies, insoluble forms of alpha-synuclein accumulate as inclusions in Lewy bodies. Familial Parkinson's disease is associated with mutations in the -synuclcin (SNCA) gene. In the process of seeded nucleation, alpha-synuclein acquires a cross-sheet structure similar to other amyloids.

[0067] Numerous reports have shown that a-syn is deeply involved in the pathophysiology of AD. Elevated a-syn levels in AD could facilitate A0 oligomerization, tau phosphorylation, activation of kinases, dissociations of tan and tubulin, and tau aggregation. Furthermore, the association of a-syn with genetic factors may accelerate AD pathology.

[0068] Experimental examples

[0069] GRP78 inhibitor of the invention were examined for the first time in inhibiting Tau protein deposits that are associated with nerve and microglia cell breakdown. Tau protein neurofibrillary tangles are stringy aggregates composed largely of the tau protein and extracellular GRP78 and are associated with cognitive loss. These tangle aggregates have been shown to be within, rather than between, nerve and microglia cells and their presence strongly correlates with nerve and microglia cell damage leading to memory loss and reduced mental processing.

[0070] It was shown by the present invention that inhibition of extracellular GRP78 with the novel inhibitors CBT200 and CBT300 significantly reduces p-tau aggregate size, number, and toxicity.

[0071] GRP78 and AD disease progression

[0072] Currently, there are several studies that show up regulation of GRP78 and not inhibition of extracellular GRP78 will delay AD progression in mice. These studies show that healthy neurons and microglia in AD patients have an increased amount of GRP78 expression compared to the AD diseased neurons and microglia. Moreover, there are pre-clinical AD therapies that up regulate GRP78 gene expression leading to an increase in neuron and microglia survival in mice, which significantly delays AD disease progression.

[0073] However, the data below demonstrates that although the addition of extracellular GRP78 alone reduced p-tau aggregate size, it did not reduce the number of aggregates or eliminate p-tau fibril toxicity in microglia cells. Without being limited by a mechanism, this may be the result of extracellular’ GRP78 “glue” that keeps tau and other types of tangles aggregated and this GRP78 “glue” remains important for internalization and toxicity of aggregates.

[0074] Without being bound by a mechanism of action, the present data, for the first time, supports the mechanism that extracellular’ GRP78 increases the formation of aggregated “tangles” from tau, and alpha-synuclein in the brain microenvironment. The accumulation of these smaller protein tangles (p-tau and alpha-synuclein) intracellularly generate Endoplasmic Reticulum (ER) stress, followed by the activation of the unfolded protein response, which up regulates GRP78 and other stress proteins. This adaptive response starts to alleviate the non-folded protein “tangles” overload inside cells by degradation and secretion, thus preserving neuronal and microglia viability. However, if this stress persists, the ER stress-mediated cell death program (ERAD) then leads to neuronal and microglia loss.

[0075] Accordingly, although it may be detrimental to inhibit intracellular GRP78, up regulation of GRP78 (for example, as shown in cancer cells), leads to the secretion of extracellular GRP78. This is also observed in PD / AD patients’ blood and CNS fluid where extracellular GRP78 is higher than in normal patients.

[0076] The data below demonstrates that blocking extracellular GRP78 binding to tau and alpha- synuclein tangles, dissolves the tangles allowing for microglia degradation of the tangles and for survival of neurons and microglia.

[0077] CBT300 binding to Tau fibrils

[0078] Tau pre-formed fibrils and Tau monomers were tested for the presence of GRP78 to confirm the role of GRP78 in fibril / tangle formation, p-tau fibrils were pre-formed with the addition of stress proteins including GRP78. p-Tau monomers and p-Tau pre-formed fibrils were bound to a tissue coated 96 well plate overnight. The plates were washed and blocked and then CBT300-HRP was added to the wells at various concentrations and measured for binding.

[0079] FIG. 1 shows a graph of absorbance and CBT300 concentration in the present of Tau fibril and monomers.

[0080] The results confirm that CBT300 bound to pre-formed fibrils 100-fold tighter than to the p-tau monomers. Extracellular GRP78 inhibitors of the invention (CBT300, CBT200) were shown to bind extracellular GRP78 on the pre-formed tau and alpha- synuclein fibrils 100-fold tighter than monomers. Unlike, monoclonal antibody drugs to amyloid-beta, tau and alpha-synuclein “tangles,” CBT300 and CBT200 bind 100-fold tighter to pre-formed tangles than to monomers. Without being bound to a mechanism of action, this may be due to the extracellular GRP78 bound in the forming aggregates.

[0081] Extracellular GRP78 and CBT300 binding to Tau fibrils Extracellular GRP78 inhibition using CBT300 was analyzed against high concentrations of extracellular GRP78 (1 uM), to assess whether inhibition or over-expression would lead to a decrease in p-tau preformed fibril aggregation size and numbers. Tau fibrils were plated in low binding 96 well plates over. Either PBS pH 7.2 as a control, CBT300 (330 nm,) or extracellular CRTP78 (1|1M) were added to the plates and incubated for 24 hours at 37C.

[0082] FIG. 2A show immunohistochemistry (IHC) of wells with pre-formed fibrils following incubation with a control, CBT300, and GRP78.

[0083] FIG. 2B-C show graphs of Tau tangle aggregation size and number / area of tau tangle aggregation following incubation with a control, CBT300, and GRP78. A total of 8 wells and an average of 4-5 measurements per well at each condition were recorded with *p<0.05 ****p<0.0001.

[0084] CBT300, dissolved preformed p-tau aggregates by 96% and reduced the number of tangle aggregates by over 98%. This demonstrated that GRP78 inhibitors specific to extracellular GRP78 can reduce the number and shrink the size of p-tau aggregates significantly.

[0085] Although 1 uM extracellular GRP78 alone shrunk the size of the p-tau aggregates, it did not reduce the numbers of aggregates. From IHC staining of Tau aggregates, CBT300 significantly (p<0.0001) reduced the amount of extracellular GRP78 and aggregated Tau protein in the treated wells, whereas extracellular GRP78 did not reduce the amount of protein aggregation or number of aggregates.

[0086] Tau pre-formed fibril aggregation was shown to be significantly inhibited by CBT300. Preformed tau fibrils or tangles formed much larger aggregates with added extracellular GRP78 at concentrations shown to exist in AD patients’ cerebral spinal fluid within 24 hours. With the addition of extracellular GRP78 inhibitors, it was shown that these tangle aggregates were significantly dissolved.

[0087] In contrast, Tau tangle aggregation size but not tangle numbers, were inhibited by high concentrations of extracellular GRP78. Thus, high concentrations of extracellular GRP78 may reduce the Tau tangle aggregation size but also significantly increases the number of Tau tangles. Tau aggregation and internationalization in Human microglia cells

[0088] Tau preformed fibrils were incubated with Thioflavin T to analyze aggregation of fibrils in HMC3 human brain microglia cells to determine if cells could internalize and degrade aggregates.

[0089] Thioflavin stained Tau aggregates treated with either PBS (control), CBT300 (330 nM), or extracellular GRP78 (1 uM) were incubated for 24 hours at 37C and then added to wells with HMC3 human microglia cells to determine the amount of internalization of Tau aggregates.

[0090] FIG. 3A shows immunofluorescence Tau fibrils in human microglia cells following incubation with a control, CBT300, and extracellular GRP78. The columns show duplicate wells of HMC3 treated as listed above each column. After 24 hours the cells were carefully washed, and immunofluorescence was recorded.

[0091] FIG. 3B shows a graph of internalization of Tau aggregative in human microglia cells following incubation with a control, CBT300, and extracellular GRP78.

[0092] With CBT300 treatment, the amount of tau aggregates internalized was very minimal with a 93% (p<0.0003) reduction in internalized tau aggregates. Although the GRP78 (1 uM) treated tau aggregates were smaller in size, the number of aggregates were higher than in the CBT300 treated wells and the amount of internalized tau aggregates was much higher, similar to the PBS control. This shows that in the presence of the control and extracellular GRP78, tau aggregates were highly internalized but not degraded at 24 hours. The data also indicates that CBT300 reduces tau fibril aggregation leading to less tau aggregate internalization into microglia cells.

[0093] Human brain microglia survival

[0094] CBT300, extracellular GRP78 and PBS (control) treated tau fibril aggregates were added to human brain microglia cells and cell survival was measured their survival after 24 hours, to ascertain how these treated tau fibril aggregates affect human microglia cell survival.

[0095] FIG. 4 is a graph of cell death in HMC3 microglia after incubation with PBS (control), CBT300, or extracellular GRP78. After 24 hours thioflavin labeled tau treated aggregated were applied to well containing HMC3 cells. After another 24 hours, live cells were measured by CCK8 assay. Each bar column in an average of 3 wells with *p<0.05 and ****p<0.0001. Only the CBT300 treated tau fibril aggregates showed no killing of human microglia cells. The GRP78 treated fibril aggregates, although smaller, were more numerous and still stained highly with thio flavin indicating highly aggregated tangles. Extracellular GRP78 treated tau aggregates killed 50% of the microglia cells compared to 70% killing with PBS treated aggregates and 0% killing with the CBT300 treated aggregates.

[0096] Despite data showing increased intracellular GRP78 expression leading to delayed AD disease progression in mice, the present data clearly shows that increased extracellular GRP78 may reduce protein aggregation size, but there is a significant increase in aggregate numbers and the effects of these GRP78 treated tau aggregates lead to killing of human brain microglia cells.

[0097] Accordingly, CBT300, but not excess extracellular GRP78, increased microglia survival by over 200%. Tau tangle aggregated treated with CBT300 were smaller, fewer in number and did not induce apoptosis in microglia cells. In fact, there was shown to be a 200% increase in microglia cell survival with CBT300 treatment compared to control and high concentration extracellular GRP78 treatment.

[0098] Conclusion

[0099] The accumulation of misfolded and aggregated proteins play a critical role in the pathogenesis of neurodegenerative diseases like Alzheimer’s (AD), Parkinson’s (PD), Amyotrophic lateral sclerosis (ALS). Extracellular GRP78, critical in the folding of proteins in the endoplasmic reticulum, is a chaperone that is essential for the pathogenesis of these neurodegenerative diseases. Without being bound by a mechanism of action, GRP78 binds to brain endothelial cell receptor called LRP-1, which is a known internalization pathway for proteins. Extracellular GRP78 may be essential in aggregates of p-tau, or alpha-synuclein for internalization and thus apoptotic toxicity of the aggregates.

[0100] It was discovered that extracellular GRP78 stimulates tau fibril aggregation which is a key underpinning of Alzheimer’s Disease. It was also shown by the present invention that extracellular GRP78 binds tightly to fibrils of alpha-synuclein, to significantly increase larger fibril formation, and aggregation. These extracellular GRP78 alpha-synuclein and tau fibrils in vitro killed human brain microglia and neurons in seconds. Following dosing with extracellular GRP78 inhibitors, at least 95% of the tangles were dissolved which then reversed microglia and neuronal cell death. The results show that novel inhibitors of the invention block binding of extracellular GRP78 to fibrils of tau and alpha- synuclein, thus reversing aggregated tau and alpha- sy nuclein fibril formation saving brain microglia without disrupting normal cell function. A therapy that can stop or even reverse phosphorylated-tau and alpha-synuclein aggregations leading to the halting of microglia and neural cell apoptosis with no adverse bleeding or swelling complications provides a major advancement in the treatment of neurodegenerative diseases.

[0101] Incorporation by Reference

[0102] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, publicly accessible databases, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0103] Equivalents

[0104] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

Claims1. A method of treating a disorder associated with protein aggregation, the method comprising: providing to a subject having or at risk of having a GRP78 -mediated protein aggregation disorder a composition comprising a GRP78 antagonist, wherein the GRP78 antagonist comprises a binding domain selected from the group consisting of: a plasminogen kringle 5 fragment; a plasminogen kringle 5 fragment attached to immunoglobulin; a R0R1 kringle fragment; a R0R1 kringle fragment attached to an immunoglobulin; a R0R2 kringle fragment; and a R0R2 kringle fragment attached to an immunoglobulin.

2. The method of claim 1, wherein the protein aggregation disorder is a Tau protein associated disorder or an alpha-synuclein (aSyn) associated disorder.

3. The method of claim 2, wherein the disorder is Alzheimer’s disease, Parkinson’s disease, or Amyotrophic Lateral Sclerosis (ALS).

4. The method of claim 1, wherein the plasminogen kringle 5 fragment comprises an amino acid sequence of SEQ ID NO:1.

5. The method of claim 1, wherein the plasminogen kringle 5 fragment attached to immunoglobulin comprises an amino acid sequence from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18.

6. The method of claim 1, wherein the ROR1 kringle fragment comprises an amino acid sequence of SEQ ID NO: 19.

7. The method of claim 1 , wherein the R0R1 kringle fragment attached to immunoglobulin comprises an amino acid sequence from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and combinations thereof.

8. The method of claim 1, wherein the R0R2 kringle fragment is SEQ ID NO:33 and combinations thereof.

9. The method of claim 1, wherein the R0R2 kringle fragment attached to immunoglobulin is selected from the group consisting of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and combinations thereof.

10. The method of claim 1, wherein the GRP78 antagonist binds to the N-terminal GRP78 domain of extracellular GRP78 and prevents GRP78 mediated aggregation of proteins.

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