WO2025106979A9

WO2025106979A9 - Extracellular grp78 inhibitors for the removal of treatment resistant cancers

Info

Publication number: WO2025106979A9
Application number: PCT/US2024/056398
Authority: WO
Inventors: Donald J. Davidson
Original assignee: Creative Biotherapeutics LLC
Current assignee: Creative Biotherapeutics LLC
Priority date: 2023-11-16
Filing date: 2024-11-18
Publication date: 2025-07-03
Anticipated expiration: 2026-05-16
Also published as: WO2025106979A2; WO2025106979A3

Abstract

The invention provides methods of treatment of cancer, specifically drug-resistant, immune resistant, and/or metastasized cancer. The invention also provides compositions comprising GRP78 antagonists for the treatment of cancer.

Description

EXTRACELLULAR GRP78 INHIBITORS

FOR THE REMOVAL OF TREATMENT RESISTANT CANCERS

Field of the Invention

The present invention relates to the treatment of cancer.

Background

Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Cancer progression impairs the normal biological process of healthy cells which achieved by the invasion of nearby tissues and metastasize to distant tissues.

Established cancer treatments include surgery, radiation therapy, and chemotherapy. Recent cancer therapy breakthroughs have introduced antibody drug conjugates, bispecific antibodies, radiolabeled ligands, and CAR T-cells for cancer treatment, offering the possibility of durable cancer treatment responses. However, resistance to anticancer treatment remains a complex process, limiting the impact of both established and new treatments on some of the most aggressive forms of cancer. As a result, despite new breakthroughs, chemotherapy remains the most often used method of treating cancer.

Unfortunately, although a design of new chemotherapy agents is growing quickly, effective chemotherapy agents have not been discovered against many advanced stages of cancer, including invasive and metastatic cell lines. Chemoresistance and immune suppression are the main reasons for failure for many therapies leading to tumor recurrence. In immune suppression, the tumor and tumor microenvironment result in “exhausted” immune cells unable to kill tumor cells. In chemoresistance, tumor cells no longer respond to chemotherapy, a major cause of a more aggressive, resistant and metastatic type of tumor. It is estimated that 90% of chemotherapies failures and resulting cancer deaths are due to the invasion and metastasis of drug-resistant cancer.

Summary of the Invention

The invention provides methods for treatment of cancer. In certain aspects, the invention provides the methods of treatment of treatment resistant metastatic breast cancer, brain cancer, or ovarian cancer. Advantageously, methods of the invention provide for the treatment of previous drug and immune resistant cancers. The methods of the invention recognize that Glucose- Regulated Protein 78 (hereinafter GRP78) antagonists are useful in treatment of cancer. Exemplary GRP78 antagonists for use in the methods of the invention are provided in U.S. Patent No. 10,905,750 and U.S. Patent No. 12,060,410, which are herein incorporated by reference in their entirety.

Treatment resistant cancer treatment

Accordingly, the present invention provides methods for treating treatment resistant cancer. The methods provide to a subject having a treatment resistant cancer a composition comprising a GRP78 antagonist. The GRP78 antagonist may comprise a binding domain selected from the group consisting of a plasminogen kringle 5 fragment; a plasminogen kringle 5 fragment attached to immunoglobulin; a ROR1 kringle fragment; a ROR1 kringle fragment attached to an immunoglobulin; a ROR2 kringle fragment; and a ROR2 kringle fragment attached to an immunoglobulin.

The cancer may be metastatic breast cancer, brain cancer, lung cancer or ovarian cancer. The treatment resistant cancer may be targeted drug resistant, chemotherapy resistant, or immune resistant.

The subject may have previously been administered an anti -cancer therapy.

The GRP78 antagonist may be provided to a subject that has already been administered a chemotherapy or a subject to-be administered a chemotherapy. For example, the chemotherapy may comprise use of a drug-pump substrate, including e.g. Doxorubicin or Taxol.

For example, in aspects of the invention, the plasminogen kringle 5 fragment may comprise an amino acid sequence of SEQ ID NO:1. The plasminogen kringle 5 fragment may be attached to immunoglobulin and may comprise 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.

The ROR1 kringle fragment may comprise an amino acid sequence of SEQ ID NO: 19. The R0R1 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.

The R0R2 kringle fragment may comprise the amino acid sequence of SEQ ID NO:33.

The R0R2 kringle fragment may be attached to immunoglobulin and may be 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.

Cancer stem cell prevention

Aspects of the present invention provide methods for the prevention of cancer stem cell (CSC) expansion, understood to be a cause of cancer drug resistance - including chemoresistance and immune suppression, through the use of GRP78 antagonists.

Specifically, the present invention benefits from the insight that, although chemoresistance is a complex process, a subset of about 1% of cells in any given tumor may be cancer stem cells (CSCs). CSCs may be responsible, in part, for a shift to more aggressive, resistant, immune suppressive and metastatic tumors, for example in subjects administered chemotherapies, playing roles in tumor initiation, progression, metastasis, drug resistance and cancer relapse. Chemotherapy may increase the density of CSCs in tumors by selecting for drug resistant CSCs (intrinsic resistance) and/or inducing the conversion of “normal” tumor cells to a more cancer stem cell phenotype (acquired resistance).

The present invention also benefits from the insight that the antigen cell surface protein CD 133 in tumors has been associated with tumor recurrence, chemoresistance and metastasis. CD 133 expression on tumor cells may significantly increase cell proliferation and chemoresistance. CD133 expression may be detected on normal regenerating tissues, cancer stem cells from brain, ovary, liver, prostate, pancreas, kidneys, and colon. By the present invention it was discovered the GRP78 antagonists of the invention prevent the expression and proliferation of CSCs, including CD133 expressing CDCs.

Accordingly, the present invention provides methods for the prevention of cancer stem cell (CSC) expansion that comprise providing to a subject having or at risk of expressing CSCs a composition comprising a GRP78 antagonist. The GRP78 antagonist may comprise 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.

Advantageously, the method may prevent the expression of CSCs expressing CD133. For example, the subject may have been previously administered a chemotherapy. The chemotherapy may have resulted in the expression or proliferation of CSCs, including CSCs expression CD133. Alternatively, because methods of the invention may prevent the formation of CSCs, the GRP78 antagonist may be provided to a subject to-be administered a chemotherapy. For example, the chemotherapy may comprise use of a drug-pump substrate, including Doxorubicin or Taxol. Further advantageously, the methods of the invention may result in synergistic treatment of cancer together with the chemotherapy and or targeted therapy.

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.

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 NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID N0:9, 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.

The ROR1 kringle fragment may comprise an amino acid sequence of SEQ ID NO: 19. The R0R1 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

The R0R2 kringle fragment may be SEQ ID NO:33 and combinations thereof.

The R0R2 kringle fragment attached to immunoglobulin may be 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 N0:41, SEQ ID NO:42, SEQ ID NO43, SEQ ID NO:44, SEQ ID NO:45, and combinations thereof.

The present invention also benefits from the insight that the embryonic protein, Cripto (Cripto-1) may be present and essential for chemoresistance, immune suppression and CSC formation in cancer cells. Cripto may be a marker for chemotherapy induced CSC expansion. In human tumors including breast, lung, renal, ovarian pancreatic, colorectal, prostate, gastric, bladder, esophageal, melanoma and glioblastoma, Cripto may be overexpressed compared to normal tissues. Overexpression of Cripto may lead to signaling through oncogenic WNT, AKT and NOTCH pathways. Cripto is also an obligate TGF-P co-receptor for NODAL and GDF1/3, that may be implicated in both cancer and sternness.

Accordingly, in aspects of the invention, the GRP78 antagonist binds the N-terminal GRP78 domain of extracellular GRP78, thereby preventing GRP78 binding to Cripto.

In aspects of the invention, the subject may be or have been identified as expressing CSCs, by any known means (e.g. a biopsy followed by an immune assay and/or sequencing) The subject may be or have been identified as expressing CSC expressing CD 133.

The subject may be afflicted with any cancer associated with CSC formation, for example the subject may be afflicted with one or more of a lung cancer, renal cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, gastric cancer, bladder cancer, esophageal cancer, DIPG, melanoma, or glioblastoma CBT300

CBT300 is a fusion protein containing a Glucose Regulated Protein 78 (GRP78) binding peptide with an Fc domain (for example, human IgGl).

In certain aspects, the invention recognizes that CBT300 was designed based on the discovery that drug resistant recurrent cancer cells up-regulate a survival pathway that results in the expression of extracellular Glucose-Regulated Protein 78 (ecGRP78) in the tumor microenvironment (TME).

This TME ecGRP78 binds to cancer and immune cell surfaces, which induces a cascade of events to increase drug resistance, immune suppression, and cancer stem cell (CSC) formation. CBT300 targets surface bound ecGRP78 that has been found on breast, lung, ovarian, prostate, melanoma, multiple myeloma, colon, pediatric and adult brain tumors. The data provided herein demonstrates that inhibition of ecGRP78 can a) induce apoptosis of drug resistant tumor cells, b) eliminate drug and immune resistance showing synergistic effects with chemotherapy and immunotherapy, c) decrease the amount of chemotherapy about 90% in combination with CBT300. ecGRP78 is found on many types of tumor cell surfaces but not on normal cell surfaces. In fact, cell surface bound ecGRP78 is important for many aspects of cancer development, including cell survival, proliferation, chemoresistance, angiogenesis, metastasis formation, immune suppression, and stem cell formation. Increased ecGRP78 expression in metastatic breast cancer, Glioblastoma and multiple myeloma patients was significantly associated with late-stage, increased distant metastasis, increased aggressiveness, shorter disease-free survival, and decreased overall survival. In studies to help understand how ecGRP78 causes tumor progression and drug and immune resistance, a novel ecGRP78 binding transmembrane protein was discovered. This GRP78 binding protein on TNBC and brain cancer cells is called Receptor Tyrosine Kinase Orphan Receptor- 1 (ROR1). Using the GRP78 binding domain from ROR1 and a human Fc domain, a biologic fusion protein was created that is a potent and specific ecGRP78 inhibitor called CBT300. As data provided herein demonstrates, CBT300 elimination of csGRP78 destabilizes and removes oncofetal proteins ROR1, and Cripto, and checkpoint protein PD-L1, from tumor cell surfaces resulting in reversal of chemoresistance, reduction in immune suppression, inhibition of stem cell phenotype and increased tumor cell apoptosis. The results with CBT300 demonstrate, by in vitro and in vivo experiments, that GRP78 antagonists are effective on several drug resistant cancers. Thus, the use of GRP78 antagonists have demonstrated the potential to provide a major advance in the treatment of drug resistant cancers either alone or in combination with lower doses of chemotherapy. Exploiting a novel mechanism of action with a non-toxic, efficacious and cost- effective biologic therapy that has shown increased survival in recurrent cancers will bring new hope for these patients.

In certain aspects, the invention provides methods of treatment of cancer in mammals. In certain embodiments, the invention provides methods of treatment of cancer in humans or animals. In certain embodiments, the invention provides methods of treatment of cancer in humans. In certain embodiments, the invention provides methods of treatment of cancer in animals.

In certain aspects, the invention provides methods of treatment of metastatic breast cancer, ovarian cancer, or brain cancer, 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.

In certain aspects, the invention provides methods of treatment of brain cancer, wherein the method comprises administration of composition comprising a therapeutically effective amount of a GRP78 antagonist or a pharmaceutically acceptable salt thereof. The brain cancer may be pediatric or adult brain cancer.

In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 1 mg/kg to about 100 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 1 mg/kg to about 50 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 1 mg/kg to about 25 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 1 mg/kg to about 10 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 10 mg/kg to about 100 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 10 mg/kg to about 50 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 10 mg/kg to about 25 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 1 mg/kg to about 5 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 5 mg/kg to about 10 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 5 mg/kg to about 25 mg/kg. In certain embodiments, the GRP78 antagonist is CBT300 or a pharmaceutically acceptable salt thereof.

In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 1 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 2 mg/kg. In certain embodiments, the method comprises administration of GRP78 or a pharmaceutically acceptable salt thereof at a dose of about 3 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 4 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 5 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 6 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 7 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 7.5 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 8 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 9 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 10 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 11 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 12 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 13 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 14 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 15 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 16 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 17 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 18 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 19 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 20 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 21 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 22 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 23 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 24 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 25 mg/kg. In certain embodiments, the GRP78 antagonist is CBT300 or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises from about 10 mg to about 1000 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises from about 100 mg to about 1000 mg of GRP78 antagonist or a pharmaceutically acceptable salt. In certain embodiments, the composition comprises from about 200 mg to about 1000 mg GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises from about 400 mg to about 1000 mg GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises from about 500 mg to about 1000 mg GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises from about 600 mg to about 1000 mg GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises from about 1 mg to about 100 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises from about 1 mg to about 10 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises from about 5 mg to about 10 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 100 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 200 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 300 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 400 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 500 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 600 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 700 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 800 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 900 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 1000 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the GRP78 antagonist is CBT300 or a pharmaceutically acceptable salt thereof. 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.

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.

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 are 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 are 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.

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.

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 NON, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NON, 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.

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 ROR2 kringle fragment attached to immunoglobulin, wherein the ROR2 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.

Brief Description of the Figures

FIG. 1 provides data pertaining to binding of extracellular GRP78.

FIG. 2 provides data pertaining to drug resistance and its correlation with extracellular GRP78.

FIG. 3 provides flow cytometry data demonstrating the activity of a GRP78 inhibitor of the invention. FIG. 4 provides the data demonstrating that a GRP78 inhibitor of the invention eliminates extracellular GRP78 in BT549 cells and reverses drug resistance. (Figure 6 write up should be on FIG. 4 and FIG.4 write should be on FIG. 6.)

FIG. 5 provides data demonstrating that a GRP78 inhibitor of the invention reverses drug resistance in patient-derived TNBC spheroids.

FIG. 6 provides the data demonstrating the impact of a GRP78 inhibitor of the invention on extracellular GRP78 and drug resistance, provides the data demonstrating the impact of a GRP78 inhibitor of the invention on extracellular GRP78 and drug resistance.

FIG. 7 provides the data demonstrating the inhibition of TNBC tumors upon administration of a GRP78 inhibitor of the invention in mice.

FIG. 8 provides data demonstrating that a GRP78 inhibitor of the invention coadministered with doxorubicin regresses tumors.

FIG. 9 provides a schematic for the role of GRP78 in tumor progression and the role of CBT300 in tumor apoptosis.

FIG. 10 provides stability data for pharmaceutical compositions comprising a GRP78 inhibitor of the invention.

FIG. 11 provides a schematic for the role of GRP78 in tumor promotion by cell surface GRP78.

FIG. 12 is a schematic illustration of human GRP78 domains and where known GRP78 inhibitors bind.

FIGS. 13A-C show that inhibition of GRP78 results in glioma regressions and extended survival.

FIGS. 14A-B show that a GRP78 inhibitor of the invention inhibits U87 GMB Tumor growth.

FIG. 15 is a table of pediatric High-Grade Glioma and diffuse midline glioma cells. FIGS. 16A-F show that patient derived DIPG 2D cell proliferation and 3D spheroids are inhibited by a GRP78 inhibitor of the invention.

FIG. 17 shows a GRP78 inhibitor of the invention inhibition of sphere formation in human primary glioblastoma stem cells (GSCs).

FIG. 18A-C provide data pertaining to drug resistance in ovarian cancer and its correlation with extracellular GRP78.

FIG. 19A shows a study design for identifying GRP78 bound proteins.

FIG. 19B shows an image from an electrophoresis assay identifying GRP78 bound proteins.

FIG. 19C shows a graph of GRP78 binding to R0R1, Cripto, PD-L1, or CD44.

FIG. 20A shows a tissue array study with ovarian tumor cores and normal ovary cores.

FIG. 20B is a graph of fold increase expression of GRP78, R0R1, Cripto, and PD-L1 in HGSC tissue compared to normal ovary tissue.

FIG 20C are images from normal ovarian tissue and High Grade Serious ovarian Carcinoma (HGCS) tissue stained for DNA, ecGRP78, R0R1 or Cripto, or PD-L1.

FIG. 21A shows an image from an electrophoresis assay of a GRP78 inhibitor of the invention.

FIG. 21B shows SEC-HPLC analysis of a GRP78 inhibitor of the invention.

FIG. 22 shows a graph of ovarian cancer cell viability following a GRP78 inhibitor of the invention dosing.

FIG. 23A-I show flow cytometry analysis of R0R1, PD-L1, and Cripto expression from ovarian cancer cells treated with a GRP78 inhibitor of the invention.

FIG. 24A shows graphs of live and dead ovarian cancer tumor cells after dosing with a GRP78 inhibitor of the invention. FIG. 24B shows graphs from control conditions for a cytotoxic T-lymphocyte (CTL) killing assay with a GRP78 inhibitor of the invention.

FIG. 25 shows a graph of T cell killing of 0VCAR3 cells with and without a GRP78 inhibitor of the invention.

FIG. 26A-D how cell viability graphs of multiple cancers expressing GRP78 following treatment.

FIG. 27A shows brain cancer and normal brain derived core tissue sample slides.

FIG. 27B-C shows immunohistochemistry of core tissues stained with DAPI, GRP78, PD- L1 or Cripto.

FIG. 27D-E show bar graphs of core tissues stained with DAPI, GRP78, PD-L1 or Cripto.

FIG. 28A shows raw data of flow cytometry and a histogram analysis of human BT549 triple negative breast cancer cells following incubation with GRP78 and CBT300.

FIG. 28B shows a bar graph of the average percent of BT549 cells positive for proteins GRP78, R0R1, Cripto- 1, and PD-L1

FIG. 29A-D show graphs of breast cancer cell viability and surface GRP78 with CBT300 treatment.

FIG. 30 shows measurements of cancer cell line growth in spheroids treated with Doxorubicin, Taxol, Cisplatin and CBT300.

FIG. 31A-D show graphs of spheroid sizes following incubation with CBT300.

FIG. 32 shows a mouse syngeneic triple negative breast cancer 4T1 model treated with mouse anti-PD-1, CBT300, doxorubicin and a combination of doxorubicin and CBT300.

FIG. 33A shows mouse syngeneic orthotopic 4T1 tumor volumes with various treatments.

FIG. 33B shows pictures of actual 4T1 tumors from each of the treatment groups. FIG 34 displays immunohistochemistry analysis of 4T1 treated tumors for DNA (DAPI), surface GRP78 (anti-GRP78-FITC), CD8 T-cells (anti-CD8-PE) and CD133 stem cells (anti- CD133-PE).

FIG. 35A-C show graphs of normalized expression of tumor GRP78, T-cells and stem cells in tumor section.

Detailed Description

The present invention is directed to methods of treatment of cancer by administration of compositions comprising GRP78 antagonists. Specifically, the methods of the invention are directed to the methods of treatment of metastatic breast cancer. Methods of the invention are also directed to the methods of treatment of brain cancer, including pediatric and adult brain cancers. Methods of invention area also directed to methods of treatment of ovarian cancer.

Receptor tyrosine kinase orphan receptors (RORs)

Receptor tyrosine kinase orphan receptors (RORs) play a crucial role in tumor growth and progression. Receptor tyrosine kinase orphan receptors (RORs) are transmembrane tyrosine kinase peptides that belong to a family of orphan receptor kinases in mammals that consist of two members R0R1 and R0R2. R0R1 consists of (1) an Ig-like domain (Ig), (2) a frizzled domain (FZD), (3) a kringle domain (KRD), (4) a transmembrane, and (5) a TKD-like tyrosine kinase domain. The highest expression of R0R1 in tissues that are not in a tumor microenvironment, is during early embryonic development and that this expression of R0R1 drops strongly around day 16, with only very low expression levels observed in adult tissues. Mice with homozygous disruption in the R0R1 gene die within 24 hours after birth from respiratory defects.

In a tumor microenvironment, R0R1 can be found on the leukemia cells of patients with chronic lymphocytic leukemia, and either R0R1 or R0R2 is expressed by neoplastic cells of a variety of different cancers including glioblastoma. Cancer-cell expression of R0R1 has been associated with enhanced cancer-cell migration, epithelial mesenchymal transition, increased associated risk for relapse and metastasis, and unfavorable prognosis. More recently, R0R1 has been identified on ovarian cancer stem cells, which have enhanced capacity for migration/spheroid formation in vitro and engraftment/metastasis in vivo. R0R1 may function as a receptor for Wnt5a that induces noncanonical Wnt signaling which then potentially leads to enhanced tumor-cell growth, directional migration, and/or tissue-cell polarity during organogenesis. R0R1 is a receptor for tumor expressed soluble GRP78 on activated endothelial cells, stressed tumor cells and immature dendritic cells. The binding to GRP78 to the kringle domain on R0R1 leads to a cascade of signaling that can induce angiogenesis, chemo-resistance and dendritic cell tolerance.

GRP78

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 (FIG. 9). For example, patients with glioblastoma exhibit extreme immunosuppression, both systemically as well as within the tumor microenvironment. We now know that 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 chemoresistance and proliferation and suggest that GRP78 is a soluble immunomodulatory molecule.

To understand how GRP78 binding can lead to dendritic cell tolerance and tumor chemoresistance, pull down experiments to identify the surface binding protein were conducted. We have discovered that soluble 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 does not lead to activation of ROR-1.

GRP78 antagonists

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 anti angiogenic 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 anti angiogenic 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.

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.

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 NOG, SEQ ID NO:4, SEQ ID NOG, SEQ ID NOG, 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.

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.

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 . 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.

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 NO: 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.

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.

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 IDNO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ IDNO:41, SEQ IDNO: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. 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 N0:71, 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.

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.

In certain aspects, the invention provides methods of treatment of metastatic breast cancer, 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.

In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 1 mg/kg to about 100 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 1 mg/kg to about 50 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 1 mg/kg to about 25 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 1 mg/kg to about 10 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 10 mg/kg to about 100 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 10 mg/kg to about 50 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 10 mg/kg to about 25 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 1 mg/kg to about 5 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 5 mg/kg to about 10 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 5 mg/kg to about 25 mg/kg. In certain embodiments, the GRP78 antagonist is CBT300 or a pharmaceutically acceptable salt thereof. The term “about,” when used in the context of describing the dose of the GRP78 antagonist or a pharmaceutically acceptable salt thereof, means ± 5% of the recited dose. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 1 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 2 mg/kg. In certain embodiments, the method comprises administration of GRP78 or a pharmaceutically acceptable salt thereof at a dose of about 3 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 4 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 5 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 6 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 7 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 7.5 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 8 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 9 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 10 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 11 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 12 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 12 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 13 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 14 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 15 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 16 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 17 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 18 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 19 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 20 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 21 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 22 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 23 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 24 mg/kg. In certain embodiments, the method comprises administration of GRP78 antagonist or a pharmaceutically acceptable salt thereof at a dose of about 25 mg/kg. In certain embodiments, the GRP78 antagonist is CBT300 or a pharmaceutically acceptable salt thereof.

In certain embodiments, the composition comprises from about 10 mg to about 1000 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises from about 100 mg to about 1000 mg of GRP78 antagonist or a pharmaceutically acceptable salt. In certain embodiments, the composition comprises from about 200 mg to about 1000 mg GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises from about 400 mg to about 1000 mg GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises from about 500 mg to about 1000 mg GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises from about 600 mg to about 1000 mg GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises from about 1 mg to about 100 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises from about 1 mg to about 10 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises from about 5 mg to about 10 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 100 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 200 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 300 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 400 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 500 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 600 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 700 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 800 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 900 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises about 1000 mg of GRP78 antagonist or a pharmaceutically acceptable salt thereof. The term “about,” when used in the context of describing the quantity of the GRP78 antagonist or a pharmaceutically acceptable salt thereof in a composition, means ± 5% of the recited quantity of GRP78 antagonist in the composition. In certain embodiments, the GRP78 antagonist is CBT300 or a pharmaceutically acceptable salt thereof.

This TME ecGRP78 binds to cancer and immune cell surfaces, which induces a cascade of events to increase drug resistance, immune suppression, and cancer stem cell (CSC) formation. CBT300 targets surface bound ecGRP78 that has been found on breast, lung, ovarian, prostate, melanoma, multiple myeloma, colon, pediatric and adult brain tumors. The data provided herein demonstrates that inhibition of ecGRP78 can a) induce apoptosis of drug resistant tumor cells, b) eliminate drug and immune resistance showing synergistic effects with chemotherapy and immunotherapy, c) decrease the amount of chemotherapy about 90% in combination with CBT300. Recent publications show that ecGRP78 is found on many types of tumor cell surfaces but not on normal cell surfaces. In fact, cell surface bound ecGRP78 is important for many aspects of cancer development, including cell survival, proliferation, chemoresistance, angiogenesis, metastasis formation, immune suppression, and stem cell formation. Recently, it has been shown that increased ecGRP78 expression in metastatic breast cancer, Glioblastoma and multiple myeloma patients was significantly associated with late-stage, increased distant metastasis, increased aggressiveness, shorter disease-free survival, and decreased overall survival. In studies to help understand how ecGRP78 causes tumor progression and drug and immune resistance, a novel ecGRP78 binding transmembrane protein was discovered. This protein binds on TNBC and brain cancer cells called Receptor Tyrosine Kinase Orphan Receptor-1 (R0R1). Using the GRP78 binding domain from R0R1 and a human Fc domain, a biologic fusion protein was created that is a potent and specific ecGRP78 inhibitor called CBT300. As data provided herein demonstrates, CBT300 elimination of csGRP78 destabilizes and removes oncofetal proteins R0R1, and Cripto, and checkpoint protein PD-L1, from tumor cell surfaces resulting in reversal of chemoresistance, reduction in immune suppression, inhibition of stem cell phenotype and increased tumor cell apoptosis. The results with CBT300 demonstrate, by in vitro and in vivo experiments, that GRP78 antagonists are effective on several drug resistant cancers. Thus, the use of GRP78 antagonists have demonstrated the potential to provide a major advance in the treatment of drug resistant cancers either alone or in combination with lower doses of chemotherapy. Exploiting a novel mechanism of action with a non-toxic, efficacious and cost-effective biologic therapy that has shown increased survival in recurrent cancers will bring new hope for these patients.

GRP78 compositions

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.

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.

The data for stability for the compositions is provided in FIG. 10. The data provided in FIG. 10 was measured using a binding assay. The data demonstrate that CBT300 is stable when compared to an historic standard for GRP78 binding when stored at -18°C and -4°C for at least 6 months. The binding affinity values compared to standard CBT300 Lot A, as measured in Kd values, are within acceptable assay 95% confidence interval of -15% to 25%. The room temperature (26°C) stored CBT300’s Kd values are acceptable for 3 months but show a large deviation at the 4^th month of -54.85% change. Although the 37°C stored CBT300 displays an acceptable value for Kd of 20.4 nM at 4 months, the Maximum Saturation Absorbance shows a greater than 15% decrease in maximum absorbance indicating that a significant percent of CBT300 is degraded when stored at 37°C for more than 3 months. This indicates that at higher concentrations (> 10 nM) of CBT300 there could be some aggregation or association of the fusion protein when stored at room temperature or at 37°C for more than 3 months. However, when stored at -18°C or 4°C CBT300 is stable for at least 6 months. 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.

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 IDNO: 12, SEQ IDNO: 13, SEQ IDNO: 14, SEQ IDNO: 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.

In certain embodiments, the GRP78 antagonist is a ROR2 kringle fragment attached to immunoglobulin, wherein the ROR2 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 IDNO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ IDNO:42, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, and combinations thereof. Cancer stem cells (CSCs)

Cancer stem cells (CSCs) are cancer cells, found within tumors or hematological cancers, that possess characteristics associated with normal stem cells, including the ability to give rise to all cell types found in a particular cancer sample. CSCs are tumorigenic (tumor-forming) and may generate tumors through the stem cell processes of self-renewal and differentiation into multiple cell types. As CSCs form a small proportion of the tumor, many drugs may not act specifically on stem cells, with conventional chemotherapies being limited to eliminating differentiated or differentiating cells that form the bulk of the tumor. A population of CSCs, even following chemotherapy, may persist and then gave rise to it diverse cancer cell types, resulting in relapse.

Unfortunately, CSCs are inherently more resistant to chemotherapeutic agents. Without being limited to a mechanism of action, this may be due to (i) limitations in physically contacting CSCs with large concentrations of anti-cancer drugs due to their location within solid tumors, (ii) the expression of a variety of transmembrane proteins (such as MDR1 and BCRP), that pump drugs out of the cytoplasm, (iii) slower division of CSCs (as with adult stem cells) resulting in resistant to chemotherapeutic agents that target rapidly replicating cells via damaging DNA or inhibiting mitosis, (iv) upregulation of DNA damage repair proteins, and (v) overactivation of anti- apoptotic signaling pathways.

As a result, today chemotherapy drugs alone are considered to be ineffective in destroying cancer cells. Additional treatment targeting the removing of CSCs, once they have developed, in addition to cancerous somatic cells must be used to prevent this. However, CSC targeting interventions once CSC cell types have formed, for example in response to chemotherapy, have been limited.

CD 133

CD133 antigen, also known as prominin-1, is a membrane glycoprotein in humans. CD133 antigen is a member of the pentaspan transmembrane family, which localize to cellular protrusions. The protein consists of five transmembrane segments, with the first and second segments and the third and fourth segments connected by intracellular loops while the second and third as well as fourth and fifth transmembrane segments are connected by extracellular loops. The precise function of CD 133 is unknown, however it has been thought to act as an organizer of cell membrane topology, as with other members of the pentaspan family. CD133 is normally expressed in hematopoietic stem cells, endothelial progenitor cells, glioblastoma, neuronal and glial stem cells, various pediatric brain tumors, as well as adult kidney, mammary glands, trachea, salivary glands, uterus, placenta, digestive tract, and testes.

More recently, however, CD 133 has been characterized as a biomarker for cancer stem cells (CSCs). Without being bound by a mechanism of action, it is thought that CD133 interacts with the Wnt/p-catenin and PI3K-Akt signaling pathways, and may be able upregulate the expression of the FLICE-like inhibitory protein (FLIP), inhibiting apoptosis and resulting in proliferation. In addition, CD 133 can increase angiogenesis by activating the Wnt signaling pathway and increasing the expression of vascular endothelial growth factor-A (VEGF-A) and interleukin-8.

CD 133 -positive populations have shown the ability to propagate tumors when injected into immune-compromised mice, including brain tumors. CD133+ melanoma cells may play a critical role in melanoma recurrence. For solid cancers, CD 133 has been reported as a cancer stem cell marker for brain tumors, prostate cancer, colon cancer, lung cancer, and hepatic cancer.

CD 133 and GRP78

Drug resistant, recurrent cancers up regulate a survival pathway that results in the expression of glucose-regulated protein 78 (GRP78) on tumor cell surfaces and in the tumor microenvironment (TME). The extracellular GRP78 binds cancer and immune cells, which induces a cascade of events to increase cell growth, drug resistance, immune suppression.

Normally, GRP78 is an endoplasmic reticulum (ER) resident protein that exists in the ER to facilitate the folding of proteins and targeting misfolded proteins for ER-associated degradation. However, in tumor cells during stress, like hypoxia and low glucose, GRP78 is released from the ER, translocated to the cell surface, and secreted into the TME. This cell surface form of GRP78 has been shown to be more highly expressed in breast, lung, brain, leukemia, renal, melanoma, pancreatic and ovarian tumors compared to normal tissues. Cell surface GRP78 expression in these cancers correlates with stage, drug and immune resistance, metastases, and aggressiveness. Recently, it has been shown that increased cell surface GRP78 expression in Triple Negative Breast Cancer (TNBC) patients was significantly associated with later stage, increased distant metastasis, increased aggressiveness, shorter disease-free survival, and decreased overall survival.

The present invention identifies and recognizes GRP78 binding to protein Cripto, associated with cell sternness, immune suppression, drug resistance and aggressiveness in cancer. Without being bound by a mechanism of action, it is now shown that GRP78 antagonists bind GRP78 at a similar location as R0R1, Cripto, and PD-L1. CBT300 may eliminate cell surface expression of GRP78 causing a destabilization of Cripto surface expression on many types of cancer cells and prevention of the expression of CSCs, including CD133 CSCs. This may lead to a reversal of drug resistance, immune suppression, cancer stem cell formation and increased tumor cell apoptosis in vitro and in vivo.

Experimental Examples

Breast Cancer and GRP78

Metastatic breast cancer impacts a significant amount of patients worldwide. There are no presently available effective therapies for treatment of metastatic breast cancer:

• Recurrent metastatic breast cancer (MBC) is incurable.

• MBC accounts for over 40,000 deaths each year in the US for over 30 years.

• Nearly 90% of those deaths are due to drug resistant recurrent disease.

• About 45% of MBC deaths can be attributed to an aggressive subtype called metastatic triple negative breast cancer (TNBC).

• Despite MBC’s initial response to chemotherapy, it often recurs in more than 40% of stage I-III and 80% of stage TV patients (2).

• After recurrence, the 5-year survival is 12%.

• African-American women have a 40% greater incidence of death from MBC despite a lower risk of diagnosis.

The invention further recognizes that: • CBT300 is a fusion protein containing a Glucose Regulated Protein 78 (GRP78). binding peptide with an Fc domain.

• CBT300 inhibits a unique a tumor specific extracellular surface bound survival factor, ecGRP78.

• ecGRP78 has been found on 95% of MBC and 93% of TNBC tumors.

• Surface bound ecGRP78 is important for MBC development including proliferation, chemoresistance, angiogenesis, metastasis formation, immune suppression and stem cell formation.

• Increased surface bound ecGRP78 expression in MBC patients correlates with later stage, distant metastasis, aggressiveness, shorter disease-free survival and decreases overall survival.

• CBT300 eliminates surface bound ecGRP78 resulting in the a) reduction of drug resistance, b) lessening of immune suppression, c) reversal of stem cell expansion, d) and induction of tumor cell apoptosis.

As discussed in detail below, the data provided in the application demonstrates that GRP78 antagonists, including CBT300, are efficacious in treatment of cancer. Specifically, the data provided herein demonstrates that CBT300 is efficacious in inhibiting metastatic breast cancer tumors in mice. The data provided herein demonstrates that:

• Extracellular GRP78 binds and stabilizes R0R1, Cripto and PD-L1 on Triple Negative Breast cancer cells.

• Extracellular surface bound GRP78 induces chemoresistance 5-20-fold.

• CBT300 inhibits extracellular and surface bound GRP78 to reverses drug and immune resistance and induce apoptosis.

• CBT300 regresses triple negative breast cancer spheroids alone and in combination with chemotherapy.

• CBT300 with Doxorubicin Regresses TNBC Tumors by 68% and Extends Survival.

The data provided in FIG. 1 demonstrates that extracellular GRP78 binds to ROR-1, PD- L1 and Cripto on MBC Cell Surfaces. The details of the data provided in FIG. 1 are provided here. Left: Extracellular GRP78 binds to R0R1, PD-L1 and Cripto-1 on TNBC (BT549) cells. A) BT549 cell membrane proteins were solubilized using a non-denaturing membrane isolation kit. The membrane lysates were precleared with IgG-his agarose-Ni beads. Then GRP78-his was mixed with membrane lysates and bound proteins were precipitated with agarose-Ni beads. Bound proteins were eluted with elution buffer (pH 2.0) and analyzed by SDS-PAGE. Lane 1 : Molecular Weight Standards, Lane 2: Surface protein pull down with GRP78-His. Protein bands were identified by mass spectrometry analysis. ROR1 (top band), PD-L1 (middle band) and Cripto-1 (bottom band) Right: Extracellular GRP78 binds to ROR1, PD-L1 and Cripto-1 with similar affinity but weaker to CD44. Bound GRP78-HRP on 96 well plates with either ROR-l-ECD, PD- Ll-ECD, Cripto-1 -ECD or CD44-ECD bound was detected with TMB reagent. All assays were an average of 4 wells.

The data provided in FIG. 2 demonstrates that surface bound extracellular GRP78 (ecGRP78) induces drug resistance on multiple cancer cells. Specifically, the data provided in FIG. 2 demonstrates that the ecGRP78 induces drug resistance on BT549 metastatic breast cancer cells, U118 glioblastoma cells, SW626 ovarian cancer proliferation, and A549 lung cancer cells. The details of the experiments and/or data provided in FIG. 2 are provided here. Tumor cells were plated in 96 well plates overnight at 3000 cells per well. After cell attachment, media was removed and fresh media with 5 pg/ml of extracellular GRP78 was added to half of the wells. Doxorubicin was also added at the indicated concentrations and incubated at 37C, 5% CO2 for 5 days. Cell counting kit 8 was added per manufactures protocol and the number of live cells was measured from Absorbance at 450 nm. Each point is an average of 3 wells.

The data provided in FIG. 3 demonstrates that CBT300 significantly reduces csGRP78, ROR1, Cripto, and PD-L1 on BT549 Cells. The details of the experiments and/or data provided in FIG. 3 are provided here. A) BT549 cells in full media were incubated for three days +/- sGRP78 (5 ug/ml) and CBT300(20 nM). Cells were then washed and stained with PE-labeled mouse antihuman monoclonal antibodies against either GRP78, ROR1, Cripto-1 or PD-L1. FACs analysis was performed on a Guava PCA. B) Average percent of BT549 cells (n-3) positive for indicated proteins as indicated by FACs. Average of 3 independent analyses. **** p < 0.001, ***p<0.01, **p<0.05. The data provided in FIG. 4 demonstrates that CBT300 eliminates ecGRP78 from BT549 cells and reverses drug resistance. The details of the experiments and/or data provided in FIG. 4 are provided here. Left: BT549 cells were plated on slide chambers with ecGRP78(5 ug/ml) in full media overnight. CBT300 (100 nM) was added with Doxorubicin (100 uM) for 48 hours. Cells were washed, fixed (not permeabilized) and surface GRP78 was detected. Anti-GRP78-FITC and DAPI to mark the nucleus. Right: BT549 cells (5000 per well) were added to wells with exGRP78 and attached. Doxorubicin with and without CBT300 was added. After 4 days live cells were detected. Each point is a replicate of 3.

The data provided in FIG. 5 demonstrates that CBT300 reverses drug resistance and regresses patient-derived TNBC spheroids. The details of the experiments and/or data provided in FIG. 5 are provided here. A) BT549, HS578 or HCC1937 TNBC cells (10,000) were added to U- shaped low attachment 96 well plates. The spheroids were let grow for 5-days then Doxorubicin, Taxol, CBT300 or Cisplatin with or without CBT300 were added to the TNBC spheres for 7 days. The wells were performed in quadruplicate. B-E) Spheroid sizes (n = 3-10) were measured from above pictures with image.

The data provided in FIG. 6 demonstrates that CBT300 eliminates ecGRP78 from BT549 cells and reverses drug resistance. The details of the experiments and/or data provided in FIG. 6 are provided here. A-B) MBC cell lines and mouse triple negative mammary cell lines were plated in 96 well plates at 10,000 cells per well. The cells were let attach overnight and therapies were added the following day. Live cells were determined by CCK-8 assay after 5 days from adding drug. Each point is an average of 4 wells. C) Immunofluorescence staining of normal breast and invasive breast carcinoma cancer tissue. The tissue Micro-Array from human patients with invasive breast cancer and normal breast tissue was stained for ROR1 with anti-RORl-PE(red) and GRP78 with anti-GRP78-FITC (green) and DNA nucleus with DAPI.

The data provided in FIG. 7 demonstrates that CBT300 inhibits TNBC cancer tumor growth and metastasis in mice. The details of the experiments and/or data provided in FIG. 7 are provided here. A) Tumor growth of PBS Control, CBT300, and Cisplatin treated 4T1 triple negative breast cancer cells orthotopically implanted in mice 10 days earlier (Day -10). Treatments were started on Day 1 when tumors reached -100 mm3. Each group started with 10 mice. *p<0.05 B) Individual tumors in mice measured on day 22. **p=0.01, ***p<0.001 C) Survival of mice with 4T1 triple negative breast cancer orthotopic tumors. Mice were removed from study when the tumors reached 1.6 cc tumor volume or lost more than 20% body weight. D) Number of lung metastasis counted on day 22 or when mice were removed from study.

The data provided in FIG. 8 demonstrates that CBT300 administered with doxorubicin regresses TNBC tumors by 65% and extends survival. The details of the experiments and/or data provided in FIG. 8 are provided here. Tumor growth curves of PBS, CBT300, mouse Keytruda (mPD-1) Doxorubicin and CBT300 with Doxorubicin treated 4T1 tumors orthotopically implanted in mice 10 days earlier (Day -10). Treatments were started on day 1 when tumors reached -100 mm3. Each group started with 8 mice. CBT300 in combination with Doxorubicin showed significant tumor growth inhibition compared to SOC mouse Keytruda or Doxorubicin alone. B) Survival curve of 4T1 TNBC treated mice up to day 24. CBT300 in combination with Doxorubicin showed 100% survival at day 24 C) Mice tumor sizes were measured and compared at day 18 before any of the mice were removed from study. D) Individual 4T1 tumors at day 24 were excised from mice are compared with Control, Doxorubicin+CBT300, Doxorubicin.

Brain Cancer and GRP78

Study design and summary

Brain cancer is the leading cause of disease-related death in children and adolescents under the age of 20 and is the 6th leading cause of disease-related death in adults. Despite aggressive treatments that include maximal surgical resection, stereotactic radiosurgery [SRS], whole-brain radiation therapy [WBRT], chemotherapy, molecularly targeted therapeutics, and immunotherapies, the recurrence rate for most types of brain cancer is nearly 100%. These recurrences are many times drug resistant, which leads to the dismal 2-year median survival rate for children and adults with high grade gliomas like Glioblastoma Multiforme (GBM). For childhood brain cancers like Diffuse Intrinsic Pontine Glioma (DIPG), and Diffuse Midline Gliomas (DMG-H3.3K27M) the median survival rate is less than 1 year.

It has been discovered that recurrent drug resistant brain cancers up-regulate a survival pathway resulting in the expression and secretion of (extracellular) Glucose-Regulated Protein 78 (ecGRP78) in the tumor microenvironment (TME) FIG. 11 provides a schematic for the role of GRP78 in tumor promotion by cell surface. GRP78 leading to stabilization of R0R1 , PD-L1 , and binding of NODAL to Activin/Cripto- 1/GRP78. This leads to drug resistance and immune suppression resulting in tumor promotion. Tumor suppression by elimination csGRP78 with CBT300 results in the reduced expression of R0R1, PD-L1, Cripto-1, and drug pumps. This reduced expression plus the increase in Smad 2/3 signaling results in reduction in drug resistance, immune suppression and tumor growth.

It has further been discovered that ecGRP78 is important for drug and immune resistance as well as glioma stem cell formation. Extracellular GRP78 stabilizes essential oncofetal proteins, R0R1 and Cripto-1, along with the checkpoint protein, PD-L1, on tumor and immune cell surfaces inducing a cascade of events to increase drug resistance, immune suppression, and cancer stem cell (CSC) formation.

GRP78 inhibitors of the invention specifically block the N-terminal domain of ecGRP78 from binding to and stabilizing these pro-tumorigenic proteins on the tumor surface without disrupting normal cell function. CBT300 targets ecGRP78 that has been specifically found on the surface of breast, lung, ovarian, prostate, melanoma, multiple myeloma, colon, pediatric and adult brain tumors and not on normal cells.

By the present invention, it has been discovered that inhibition of ecGRP78 can a) induce apoptosis of drug resistant tumor cells, b) eliminate drug and immune resistance showing synergistic effects with chemotherapy and immunotherapy, c) decrease the amount of chemotherapy needed, thus lowering the toxic side effects. From initial in vivo human HGG tumor results, mice treated with only ecGRP78 inhibitors of the present invention displayed an increase in pathological complete responses of over 50% with no observable toxicity. These treated mice also showed a significant increase in median overall survival of over 42 days compared to 30 days for controls. This 12-day increase in survival in mice correlates to an estimated 1 1/2 years in human patients. A therapeutic alternative that is effective at targeting recurrent brain tumors and metastasis leading to a significant increase in overall survival that is non-toxic would prove to be paradigm altering, as shown by the present invention.

Surprisingly, GRP78 inhibitors of the invention bind to the N-terminal domain of GRP78 and specifically eliminate extracellular cell surface GRP78 that is found only on tumor cells and not on normal cells. This specificity to inhibit only extracellular cell surface GRP78 greatly reduces the dose limiting toxicities observed with other GRP78 inhibitors and eradicates glioma brain tumors in 60% of mice leading to complete responses verified by pathological exam and significantly increase survival.

The present invention demonstrates that CBT300 eliminates GRP78 from cancer cells including High-Grade Glioma (HGG) cells, resulting in decreased drug resistance, lessened immune suppression, reduced stem cell formation and increased apoptosis without normal cell interruption.

FIG. 12 is a schematic illustration of human GRP78 domains and where known GRP78 inhibitors bind. Inhibitors IT- 139, AR- 12, EGCG, and PAT-SM6 were tested in phase I clinical studies. (SBD: substrate binding domain, KDEL: 4 residue C-terminal peptide)

The pharmacokinetic characteristics of CBT300 in mice dosed IV showed a low volume of distribution (V = 0.21) with a half-life in mice around 21 hours. Subcutaneous(sc) dosing of CBT300 showed a half-life around 11 days with a bioavailability around 60-70%. CBT300 has also shown no observable toxicities dosed sc or IV at doses of 400 mg/kg daily in mice. CBT300 is stable at 4C and -20C for over 12 months.

By the present invention it was shown that CBT300 reverses drug resistance and reduces immune suppression in adult and pediatric HGG cells. Unlike other GRP78 inhibitors, CBT300 binds and eliminates extracellular cell surface GRP78 on HGG cells. Reducing cell surface GRP78, which then destabilizes and greatly reduces the expression of three major HGG cell surface survival proteins ROR1, Cripto-1 and PD-L1.

By the present invention it has was also shown that CBT300 inhibits HGG tumor growth alone and is synergistic with chemotherapy to enhance HGG tumor inhibition. The inhibition and removal of extracellular cell surface GRP78 on HGG cells leads to a decrease in chemoresistance and immune suppression in vitro. Extracellular cell surface GRP78’s inhibition with CBT300 alone reduced DIPG tumor growth in neurosphere assays by an average of about 70%. In combination therapy, CBT300 with Doxorubicin or Temozolomide demonstrated about a 90% reduction in HGG neurosphere growth. N-terminal GRP78 inhibitor, CBT200, crosses the Blood Tumor Barrier and significantly inhibits HGG tumor growth leading to pathological complete responses and increased overall survival. The in vivo human adult HGG tumor results with GRP78 inhibitor, CBT200, showed that treated mice displayed an increase in pathological complete responses of over 60% with no observable toxicity. These treated mice also showed a significant increase in median overall survival.

Study protocol and preliminary data

The novel extracellular factor called Glucose Regulated Protein 78 (ecGRP78), is highly up regulated under the stressed Tumor Microenvironment (TME). GRP78 is normally only an endoplasmic reticulum (ER) resident protein that facilitates the folding of proteins and targeting misfolded proteins for ER-associated degradation. However, in cells during stress, GRP78 is released from the ER and secreted into the TME. This extracellular form of GRP78 (ecGRP78) has been shown to be highly expressed in brain (GBM) cancers. The increased ecGRP78 expression in brain tumors correlates with later stage, increased resistance, and more metastases. Inhibition of surface bound ecGRP78 on GBM cells and brain endothelial cells by GRP78 inhibitors lead to tumor cell apoptosis and inhibition of angiogenesis. When U87 GBM cells were transduced to express GRP78 inhibitors and implanted in the forebrain of nude mice, glioma growth was significantly suppressed and long-term survival of mice past 120 days promoted. The GRP78 inhibitors in immune competent mice significantly recruited CD3 T-cells including, CD4 and NKT cells. This recruitment led to a >300 day extended survival in mice with GRP78 inhibitor expressing tumor cell.

The present design was established to determine if systemic dosing of the GRP78 inhibitor would be effective at inhibiting human D54 brain tumor xenografts in mice like the GRP78 inhibitor transduced tumor cell lines. The results showed that systemic delivery of the GRP78 inhibitor significantly increased survival with increases in complete responses.

FIGS. 13A through 13C show that GRP78 inhibition results in glioma regressions and extended survival.

FIG. 13A shows regression of orthotopic D54 glioma tumors by treatment with systemic presence of the GRP78 inhibitor. 0.5 x 10⁵ D54 human glioma cells were stereo tactically injected into the forebrain of nude mice. After 13 days of tumor growth, osmotic minipumps were implanted with GRP78 inhibitor or vehicle (PBS) in the subcutaneous flank and replaced every 14 days. T1 -weighted MRI analysis of D54 gliomas occurred on Days 28, 42, and 65 The MRI analysis of D54 gliomas on Day 38, 42 and 65 show a complete eradication of the brain tumor mass in mouse #79 (top) and mouse #39 (middle). Mouse #65 showed slower D54 tumor growth with low dose GRP78 inhibitor but still had measurable brain tumor at the end of the study. The 30% complete response mice demonstrated no observable tumors by MRI analysis. The low dose GRP78 inhibitor group slowed tumor growth but did not show complete regression of the tumors.

FIG. 13B is a graph showing survival of orthotopic adult D54 glioma tumors by treatment with the GRP78 inhibitor. Kaplan-Meier survival plot with log-ranking testing was used to effect pairwise comparisons between the control and the GRP78 inhibitor treated groups. The survival curve shows an increase in survival for the GRP78 inhibitor treated mice and that 30% of the mice survived for at least 30 days longer than the control mice.

FIG. 13C is immunohistochemistry analysis of a tumor from the control group and from the low dose GRP78 inhibitor (1 mg/kg) group at day 65. Vascular density of the tumor tissue was measured by CD31 staining. Hypoxic regions were measured by HlF-la staining and cell death was measured by tunnel staining. Tumor tissue stained with anti-CD31 (blood vessel marker), antiHypoxia Inducible Factor 1 alpha (hypoxic areas) and Tunnel stain (dead cells) clearly demonstrate that the GRP78 inhibitor drastically reduces brain tumor vasculature which then increases the hypoxic areas in the tumor resulting in tumor and endothelial cell death. It was concluded that inhibition of extracellular/surface bound GRP78 by the GRP78 inhibitor is a potent method to treat brain tumors and that the GRP78 inhibitor can cross the blood tumor barrier (BTB).

FIGS. 14A and 14B show that CBT200 inhibits U87 GMB Tumor growth. CBT200 showed significant regressions with complete responses (MRI) in about 65% (10/15) of mice with established D54 GBM tumors and an average 73% survival at the end of the study compared to 27% of control mice. A histopathological report of the mice brain tumors from the control and CBT200 groups found that mice from the treated group had “no abnormalities detected” in the brain tissue whereas the tumor tissue from the control group showed “extensive neoplastic involvement; tumor not well circumscribed; cell highly pleomorphic; numerous mitotic figures and scattered foci of necrosis”. This indicated that CBT200 can cross the BTB and completely eradicate GBM tumors.

To determine how extracellular and surface bound GRP78 induces pediatric and adult glioma cell sternness, drug resistance and immune suppression ecGRP78 pull down experiments were performed with a pediatric GBM stem cell line.

To determine if ecGRP78 induces drug resistance on DMG (DIPG) stem cells, 5 ug/ml ecGRP78 (circulating concentration in GBM patients) were added to DIPG-13 cells and inhibition with Doxorubicin was tested.

FIG. 15 is a table of pediatric High-Grade Glioma and diffuse midline glioma cells.

FIGS. 16A through 16F show that patient derived DIPG 2D cell proliferation and 3D spheroids are inhibited by CBT300.

FIG. 16A shows pediatric GBM and DIPG cells (5,000) plated in 96 well plates and let attach overnight. CBT300 was added to the well and 96 well plates were incubated for 5 days. Live cell count was measured with CCK8 reagent. CBT300 showed nM efficacy with all GBM and DIPG cells tested. It was shown that ecGRP78 increases cell resistance to Doxorubicin by about 10-fold, which is inhibited by low doses of CBT300.

To determine if CBT300 could block tumor cell growth, we used 2D proliferation and 3D Patient Derived (PDX) spheroid assays.

FIG. 16B shows the results of the 2D proliferation assay. Patient Derived (PDX) Stem Cells, DIPG-48 (10,000), were added to U-bottom low binding plates to form spheroids. Spheroids were dosed twice with treatments on Day 1 and Day 19. Pictures were taken on days indicated with quadruplicate wells and measured by Image! Average tumor sphere volumes were calculated and graphed in prism. , CBT300 potently inhibited 2 PDX pediatric GBM cell lines and 4 DIPG cell lines proliferation in the nanomolar range.

Studies have shown that patient derived stem cell 3D spheroids can replicate elements of the TME and predict clinical efficacy with a high degree of accuracy. FIGS. 16C, 16D, 16E, and 16F show that CBT300 regresses and inhibits DIPG spheroid growth alone and in combination with chemotherapy (Doxorubicin, Temozolomide) and can significantly regress all DIPG patient derived spheroids. Patient Derived stem cells from 5 difference PDX DIPG tumors were grown as listed above and treatments were added after spheres were formed and classified as Day 1. Percent inhibition was calculated from measured tumor volumes and wells were repeated in quadruplicate. Results demonstrate 95-100% spheroid regression with either CBT300 alone or in combination with doxorubicin in 3 out of the 5 DIPG stem cell spheroids. With two other stem cell spheroids, DIPG-38, DIPG-48, CBT300 alone shows between 65%-75% inhibition.

CBT300 also removed cell surface GRP78 leading to the destabilization and cell surface removal of Cripto, PD-L1, and ROR1, as determined by flow cytometry and IHC analysis. These changes also resulted in decreased ABCB1 drug pump expression.

CBT300 was also shown to inhibit adult human glioma stem cell neurosphere formation.

FIG. 17 shows CBT300 inhibition of sphere formation in human primary GSCs. 827 human primary GSCs were plated on 24-well non-tissue culture treated plastic in neural basal media with 15 ng/ml EGF and 15 ng/ml bFGF and allowed to form spheres. The following day wells were treated with 550 nM Drug A (“Dead” CBT300) or 550 nM Drug B (“Active CBT300). At days 3, 5, and 7, entire wells were imaged with the Leica Inverted microscope and spheres (containing 5 or more cells) counted. Statistical analysis at day 7; t-test, p value =0.039. As shown, CBT300 significantly inhibits neurosphere formation by about 25% on day 7 or about 52% when compared to day 0. drug A was linear “dead” CBT300 (E. coli produced) and Drug B was correctly folded CBT300 (CHO produced). The linear “dead” CBT300 had some activity and still had effects on neurosphere formation. Dead CBT300 showed much weaker binding to GRP78.

Ovarian cancer and GRP78

Recurrent drug-resistant metastatic ovarian cancer is incurable. 57% of ovarian cancer patients are diagnosed with metastatic disease, with recurrent ovarian cancer accounting for over 206,000 deaths worldwide in 2022. After recurrence, the 5-year survival rate for a patient is only 31%. Current therapies are not effective against metastatic drug-resistant ovarian cancer. By the present invention, it was discovered that GRP78 inhibitors of the invention were potent anti-tumor and anti -angiogenic therapies in recurrent ovarian cancers. Tumor cell surface GRP78 inhibition results in showed reduction of drug resistance, b) lessening of immune suppression, c) reversal of stem cell formation, d) and induction of tumor cell apoptosis.

Notably, without being bound by a mechanism of action, surface bound GRP78 binds to R0R1, Cripto and PD-L1, which are highly expressed on Ovarian Tumors. CBT300 was shown to be a potent cell surface GRP78 inhibitor that showed anti-angiogenic and anti-tumor. The fusion protein that contains a kringle domain from R0R1 and a human IgG Fc domain.

Ovarian tumor Mouse survival following GRP 78 inhibition

MA148 human ovarian tumor cells (2 x 10⁶) were injected into the subcutaneous flank of 6-8 weeks old female athymic nude mice (n=10). After 2 days, mice were then injected intramuscularly with AAV-GFP or AAV-CBT300 (5xl0⁹) viral particles. Tumor growth was determined by weekly measurements. *p<0.05, **p<0.001.

Extracellular GRP78 mediated resistance in ovarian cancer

5 Day tumor cell viability assays were conducted with and without extracellular GRP78 (5 ug/ml) and with Doxorubicin. Tumor cells were plated in 96 well plates overnight at 3000 cells per well. After cell attachment, media was removed and fresh media with 5 mg/ml of extracellular GRP78 was added to half of the wells. Doxorubicin was also added at the indicated concentrations and incubated at 37C, 5% CO2 for 5 days. Cell counting kit 8 was added per manufactures protocol and the number of live cells was measured from Absorbance at 450 nm. Each point is an average of 3 wells.

FIG. 19A-C show graphs of ovarian cancer drug resistance correlated with extracellular GRP78.

Viability was measured by CCK8 WST-1 assay. Surface bound extracellular GRP78 (ecGRP78) induced drug resistance in multiple cancer cell types. Extracellular surface bound GRP78 induced chemoresistance in multiple types of ovarian cancer cells by 7-17-fold. Specifically, ecGRP78 induced drug resistance and proliferation of SW626 (7x), Ca-0V3 (14x), and PA-1 ovarian cancer (17x).

Extracellular GRP78 binds and stabilizes R0R1, Cripto and PD-L1 on cancer cells.

Identification of GRP78 bound membrane proteins

Tumor cell membrane proteins were biotinylated and solubilized using a non-denaturing membrane isolation kit. Tumor cell membranes were pulled down and mixed with GRP78-His and separated but Ni-magnetic bead separation. GRP78 bound proteins were eluted and analyzed by SDS-PAGE and mass spectrometry analysis.

FIG. 20A shows a study design for identifying GRP78 bound proteins.

FIG. 20B shows an image from an electrophoresis assay identifying GRP78 bound proteins. R0R1, PD-L1, and Cripto were identified as GRP78 bound.

FIG. 20C shows a graph of GRP78 ELISA binding to R0R1, Cripto, PD-L1, or CD44. All assays were an average of 3-4 wells. Extracellular GRP78 bound to each of R0R1, Cripto, PD- Ll.

Co-localization of GRP78 to identified membrane proteins in high grade serious ovarian carcinoma

A tissue array was prepared to analyze GRP78 localization in ovarian cancer.

FIG. 21A shows a tissue array study with ovarian tumor cores and normal ovary cores. A3-A4 and A7-A8 are High Grade Serious ovarian Carcinoma (HGCS) cores. C1-C8 show adjacent normal tissue cores.

FIG. 21B is a graph of fold increase expression of GRP78, R0R1, Cripto, and PD-L1 in HGSC tissue compared to normal ovary tissue. Surface GRP78 and Cripto showed an 80-fold increase in HGSC tissues. R0R1 and PD-L1 showed a 20-fold increase in HGSC tissues.

FIG 21C are images from normal ovarian tissue and High Grade Serious ovarian Carcinoma (HGCS) tissue stained for DNA, ecGRP78, R0R1 or Cripto, or PD-L1. Human normal and Ovarian tumor tissue microarray cores were stained for DNA (D API- blue), ecGRP78 (FITC-green), R0R1 (PE-red), Cripto (PE-red) or PD-L1 (PE-red). GRP78 was colocalized with R0R1, Cripto, and PD-L1 in HGSC tissue but not in normal adjacent ovary cells.

Surface bound GRP78, R0R1, Cripto and PD-L1 were highly expressed on high grade epithelial patient tumors but not expressed on normal or adjacent ovarian tissue.

CBT300, novel fusion protein inhibits ovarian cancer cell survival

FIG. 22A shows an image from an electrophoresis assay of CBT300 reduced (lane 1) and non-reduced (lane 2).

FIG. 22B shows SEC-HPLC analysis of CBT300. Purified CBT300 shows >99% purity of HPLC-mass spectrometry analysis.

Live OVCAR-3, CaOV-3, PA-1, and SW626 ovarian cancer cells were assayed with varying CBT-300 doses for 5 days to analyze ovarian cell viability and expression of the membrane proteins identified in HGSCs.

FIG. 23 shows a graph of ovarian cancer cell viability following CBT-300 dosing. CBT- 300 reduced ovarian cancer cell survival across OVCAR-3, CaOV-3, PA-1, and SW626 ovarian cancer cell lines in a dose dependent manner.

FIG. 24A-I show flow cytometry analysis of R0R1 , PD-L1, and Cripto expression from OVCAR-3 ovarian cancer cells treated with CBT300. Extracellular GRP78 increased expression of R0R1, PD-L1- and Cripto in ovarian cancer cells. Treatment with CBT300 not only reduced R0R1, PD-L1, and Cripto expression to pre-ecGRP78 levels but resulted in reduction of cell marker expression overall (R0R1, -33.1%; PD-L1, -24.1%, Cripto, -9%).

CBT300 induces apoptosis ovarian cancer cells in 2D and 3D assays. CBT300 also inhibited extracellular and surface bound GRP78 to reverse drug and immune resistance and induce tumor regression. CBT300 mediates T-cell killing of ovarian cancer cells

0VCAR3 cells with/and without CBT300 were mixed and incubated at 37C, 5% CO2. After 72 hours, the 0VCAR3 cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) dye and mixed with activated T-cells at various ratios for 4 hours. Dead cells were stained with 7-AAD and analyzed by flow cytometry.

FIG. 25A shows graphs of live and dead ovarian cancer tumor cells after dosing with CBT300. CBT300 in the presence of activated T-cells resulted in T-cell mediated ovarian tumor cell death across effector cell to tumor cell ratios and CBT300 doses.

As a control, for the cytotoxic T-lymphocyte (CTL) killing assay, (i) activated T-cells and (ii) 0VCAR3 cells with 2 nM CBT300 were assayed for 72 hrs. (iii) CFSE labeled 0VCAR3 cells alone and (iv) 0VCAR3 cells with 100 nM CBT300 were assayed for 3 days.

FIG. 25B shows graphs from control conditions for a cytotoxic T-lymphocyte (CTL) killing assay with CBT300. Activated T cells alone resulted in equivalent cell death to OVCAR cells alone. CBT300 in the absence of activated T-cells resulted in tumor cell death in a dose dependent manner.

FIG. 26 shows a graph of T cell killing of OVCAR3 cells with and without CBT300. CBT300, at 2nM doses, resulted in greater than 70% ovarian cancer cell death across T-cell :tumor cell ratios.

CBT300 increased CD3 T-cell killing of ovarian cancer cells and reduces exhaustion.

Cancer Stem Cell (CSC) prevention

By the present invention, it is shown that CBT300’s elimination of cell surface GRP78 destabilizes and removes oncofetal proteins ROR1, and Cripto-1, and checkpoint protein PD-L1, and stem cell marker CD133 from tumor cell surfaces resulting in reversal of chemoresistance, reduction in immune suppression, inhibition of stem cell phenotype and increased tumor cell apoptosis. By the present invention it was shown that GRP78 antagonists bind tightly to GRP78 with Kd’s in the low nM range on the N-terminal domain of GRP78 at a similar location as R0R1 , Cripto, and PD-L1. By the present invention, the only known GRP78 inhibitors were developed that bind to the N-terminal domain of GRP78 to block R0R1, Cripto and PD-L1 tumor cell surface expression, thereby being effective on rare CSCs, including CD 133 expressing CSCs.

Preliminary work shows that CBT300’s inhibition of cell surface GRP78 potently (IC50s HnM to 250nM) inhibits in vitro proliferation of breast, lung, ovarian, colon, and adult brain cancer cell lines as well as patient derived adult GBM and pediatric glioma stem cells. 3D tumor spheroids are more representative of therapy effectiveness in patients, with data showing that CBT300 significantly regresses triple negative breast cancer, lung cancer, ovarian cancer, brain cancer including patient derived GBM and DIPG stem cell spheroids and is synergistic with chemotherapies, Taxol, and Doxorubicin in TNBC spheroids.

By the present invention, it was discovered that CBT300 specifically inhibits CSCs, including CD133 expressing CSCs.

Extracellular, surface bound GRP78 induces drug resistance when bound to tumor cells.

FIG. 27A-D show cell viability graphs of multiple cancers expressing GRP78 following treatment.

Tumor cells were plated in 96 well plates overnight at 3000 cells per well. The next day, after cell attachment, the media was removed and fresh media with 5 ug/ml of soluble GRP78 was added to half of the wells. Doxorubicin was also added at the indicated concentrations and incubated at 37C, 5% CO2 for 5 days. Cell counting kit 8 was added per manufactures protocol and the number of live cells was measured from Absorbance at 450 nM. Each point is an average of 3 wells.

As shown, cell surface bound GRP78 from extracellular GRP78 at 5 ug/ml, induced Doxorubicin resistance in TNBC cells (BT549), ovarian cancer cells (SW626), glioblastoma cells (Ul i 8), and lung cancer cells (A549). To rule out Doxorubicin binding directly to GRP78, Doxorubicin binding to a GRP78 bound plate was tested. No binding was found between Doxorubicin and GRP78. However, GRP78 binding to tumor cell surfaces, resistance to doxorubicin, as measured by IC50 values, was increased 5 times to 20 times in a 5-day proliferation assay. The data suggests that soluble GRP78 from the tumor microenvironment can bind to tumor cells to induce drug resistance several fold.

Cell surface GRP78 co-localizeswith Cripto andPD-Ll on late-stage patient glioblastoma (GBM) tissues

Two brain tissue microarrays with patient core tissue samples of late stage, heavily treated glioblastoma and normal tissues were stained for tumor cell markers

FIG. 28A shows brain cancer and normal brain derived core tissue sample slides.

FIG. 28B-C shows immunohistochemistry of core tissues stained with DAPI, GRP78, PD- L1 or Cripto. The first microarray was stained with DAPI (blue-DNA), and antibodies to cell surface GRP78 (green-FITC), and PD-L1 (red-PE). The last column is an overlay of all three stains to show co-localization. Cores B6 (stage 4 GBM) and C5 (normal cerebrum) are pictured. A second microarray was stained with DAPI (blue-DNA), and antibodies to cell surface GRP78 (green- FITC), Cripto (red-PE). The last column is an overlay of all three stains to show co-localization. Cores B2 (stage 4 GBM) and C6 (normal cerebrum) are pictured.

FIG. 28D-E show bar graphs of core tissues stained with DAPI, GRP78, PD-L1 or Cripto. Each bar is an average of 6 images.

As shown, cell surface GRP78 co-localizes with Cripto and PD-L1 on late-stage patient GBM tissues but not on normal brain cerebrum tissues.

GRP 78 inhibition with CBT300 eliminates cell surface expression of GRP78, ROR1, Cripto-1 andPD-Ll on BT549 cells

BT549 TNBC cells were grown in full media (10% FBS) plus GRP78 (5 ug/ml) with and without CBT300 to determine if the expression of surface bound GRP78 changed with CBT300 treatment.

FIG. 29A shows raw data and a histogram analysis of flow cytometry analysis of human BT549 triple negative breast cancer cells following incubation with GRP78 and CBT300. Human Metastatic Triple Negative Breast Cancer Cells, BT549, in full media were incubated for three days +/- extracellular GRP78 (5 ug/ml) and CBT300 (2 nM). Cells were then washed and stained with PE-labeled mouse anti -human monoclonal antibodies against either GRP78, R0R1, Cripto-1 or PD-L1. FACs analysis was performed on a Guava PCA. Cells were not permeabilized. B) Histogram analysis of flow cytometry data

FIG. 29B shows a bar graph of the average percent of BT549 cells (n-3) positive for proteins GRP78, R0R1, Cripto-1, and PD -LI. Average of 3 independent analyses. **** p < 0.001, ***p<0.01, **p<0.5, ns = not significant. As shown, if the GRP78 N-terminal binding domain was blocked with CBT300, the expression of surface expressed ROR1, PD-L1 and Cripto-1 were reduced by >76% for ROR1, >75% for PD-L1 and >90% for Cripto-1

A concentration of 5 ug/ml for GRP78 was because it has been shown that extracellular GRP78 circulates in cancer patients at this concentration. The addition of extracellular GRP78 increased surface bound GRP78 on BT549 cells from about 29% of the cells to 85% of cells or an increase of almost 3-fold. Cells that were incubated with CBT300 had an almost complete elimination of surface bound GRP78. The data suggest that by blocking the N-terminal domain of GRP78 with CBT300, surface-bound GRP78 is removed. Since ROR1 and Cripto-1 also bind to the N-terminal domain of GRP78, it is proposed that CBT300 should reduce the surface expression of ROR1, PD-L1, and Cripto-1 on BT549 cell surfaces.

CBT300 inhibits proliferation and reduces chemoresistance of human metastatic breast cancer (MBC) cells.

Whether the significant decrease in PD-L1, Cripto-1 and ROR1 cell surface expression had functional effects on MBC cells was studied.

To determine if CBT300 could inhibit growth of human and mouse metastatic breast cancer (MBC) cells, we tested TNBC cell lines, BT549(triple negative-TN), BT474(ER+, PR+, Her2+) T47D(ER+, PR+, Her2-), HS578.T(TN), and mouse mammary TN cancer cell lines, 4T1 and EMT-6, with CBT300.

FIG. 30A-D shows viability of attached cancer cells treated with Doxorubicin, and or CBT300. Various human and mouse breast cancer cells were plated in 96 well plates at 5000 cells per well and allowed to attach overnight. GRP78 (5 ug/ml) was added to all wells except for the last 3 columns. Doxorubicin with and without CBT300 was also added to wells. After 4 days, live BT549 cells were detected with CCD8 reagent. Each point is a replicate of 3. BT549 cells were plated on slide chambers with GRP78 (5 mcg/ml) in full media overnight. CBT300 was added to 2 chambers and Doxorubicin was added to all wells. Cells were incubated for another 24 hours and stained for GRP78 (polyclonal to C-Terminal FITC green), and DAPI blue for DNA nucleus. Doxorubicin naturally fluoresces red.

To determine if CBT300 could inhibit growth of human and mouse MBC cells, TNBC cell lines, BT549 (triple negative-TN), BT474(ER+, PR+, Her2+) T47D(ER+, PR+, Her2-), HS578.T(TN), and mouse mammary TN cancer cell lines, 4T1 and EMT-6 cell lines were tested with CBT300. All cells were tested in full media supplemented with 5 ug/ml GRP78 and showed potent proliferation inhibition with CBT300. Mouse and human GRP78 have a 99.4% homology and show similar binding constants to CBT300.

As shown, CBT300 potently (nanomolar range) inhibited the proliferation of 4 human MBC cell lines and 2 mouse mammary TN cell lines. This data suggests that GRP78 inhibition with CBT300 can potently inhibit proliferation of chemo resistant MBC cells.

FIG 30C-D demonstrates that CBT300 eliminates ecGRP78 from BT549 cells and reverses drug resistance. The details of the experiments and/or data provided in FIG. 30 are provided here. BT549 cells were plated on slide chambers with ecGRP78(5 ug/ml) in full media overnight. CBT300 (100 nM) was added with Doxorubicin (100 uM) for 48 hours. Cells were washed, fixed (not permeabilized) and surface GRP78 was detected. Anti-GRP78-FITC and DAPI to mark the nucleus. BT549 cells (5000 per well) were added to wells with exGRP78 and attached. Doxorubicin with and without CBT300 was added. After 4 days live cells were detected. Each point is a replicate of 3.

ROR1 knockdown may also reduce ABCB1 (P-Glycoprotein) protein expression by repressing MAPK/ERK/AKT activity, resulting in the restoration of MDA-MB-231 TNBC cells sensitivity to doxorubicin. Whether CBT300’s inhibition of GRP78 could lead to an increased sensitivity of BT549 cells to doxorubicin was tested. BT549 cells were plated in 96 well plates and in chamber slides overnight to let cells attach. Then CBT300, at a concentration of 1 nM was added to the cells with either Doxorubicin at 100 nM or a dose response curve of Doxorubicin (96 well plates) and grown for 3 days. After 3 days, cells were washed and stained for surface bound GRP78 in green, DNA in stained in blue and internalized Doxorubicin is red with natural fluorescence.

As shown, there was a high amount of surface GRP78 on the BT549 cells and no Doxorubicin (red) inside the cells without CBT300 added. CBT300 eliminated surface bound GRP78 (green), allowing high concentrations of Doxorubicin to be internalized in the tumor cells. These cells all died within a day. The amount of cell killing with CBT300 and Doxorubicin was analyzed by determining the number of live cells with CCK-8 reagent. It was shown that that surface bound GRP78 increased BT549 cells’ resistance to Doxorubicin by about 20-fold. In contrast, the addition of CBT300 to BT549 TNBC cells increases their sensitivity to Doxorubicin killing by about 400-fold. It was also demonstrated that CBT300 alone is a potent inhibitor of BT549 cell growth with an IC50 at 13 nM. It was further shown that with the addition of CBT300 the amount of Doxorubicin needed for similar cell killing compared to Doxorubicin alone is about 400 times less.

CBT300 regresses Metastatic Breast Cancer spheroid formation and growth.

FIG. 31 shows measurements of cancer cell line growth in spheroids treated with Doxorubicin, Taxol, Cisplatin and CBT300.

To determine whether CBT300 could reduce drug resistance and induce tumor cell apoptosis in a more clinical representative model, 3D spheroid or mammosphere assay was conducted.

Studies have shown that 3D cultures (spheroids) can replicate elements of the tumor environment, such as hypoxia, necrosis, cell adhesion, and growth much better than 2D assays. In this assay, BT549, HS578T and HCCC1937 triple negative breast cancer cells (10,000) were added to each well of a 96 well low-attachment plate in stem cell media (no serum). Spheroids were grown for 5 days before CBT300, and/or Doxorubicin or Taxol or Cisplatin were added. Seven days later, microscope pictures were taken of spheres and recorded. Spheroid size was measured with the Image J program.

Specifically, BT549, HS578 or HCC1937 TNBC cells (10,000) were added to U-shaped low attachment 96 well plates. The spheroids were let grow for 5 days then Doxorubicin, Taxol, CBT300 or Cisplatin with or without CBT300 were added to the TNBC spheres for 7 days. The wells were performed in quadruplicate.

FIG. 32A-D show measurements of spheroid sizes (n = 3-10). ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

As shown, CBT300 significantly regressed spheroids alone and in combination with Doxorubicin and Taxol which are both drug pump substrates. Cisplatin did not show a synergistic effect with CBT300. Without being limited by a mechanism of action, and in agreement with the literature, Cisplatin is not a substrate for known drug pumps. The spheroids formed with BT549, HS578T and HCC1937 were smaller when dosed with CBT300 alone. The BT549, Hs578T and HCC1937 spheroids were eliminated with combination of CBT300 and chemotherapy at doses 1/10 the suggested effective dose. Doxorubicin alone displayed weaker regression and actual increased spheroid size demonstrating that CBT300 can reverse drug resistance for improved regression of breast cancer cell spheroids. Because this spheroid assay has been shown to be highly predictive of clinical efficacy of test drugs, the data suggests significant tumor regressions in MBC clinical studies with CBT300.

GRP 78 antagonists inhibit tumor growth in a triple negative mouse mammary syngeneic orthotopic model.

To investigate the effects of GRP78 inhibition with CBT300 in vivo, a mouse derived triple negative mammary tumor cell line (4T1) orthotopically implanted in the right mammary fat pad of syngeneic mice was used. The 4T1 model, derived from BALB/c mice, is aggressive, immune suppressive, and drug resistant. This cell line was chosen because it has been shown to express both Cripto and ROR1 and is sensitive to CBT300 in vitro.

FIG. 33 shows a mouse syngeneic triple negative breast cancer 4T1 model treated with mouse anti-PD-1, CBT300, doxorubicin and a combination of doxorubicin and CBT300. Tumor growth curves of 4T1 tumors orthotopically implanted in mice (Day -10). Treatments were started when tumors reached -100 mm3. n=8. Mice tumor sizes were measured and compared at day 18 before control mice had to be removed because of tumor size. Individual 4T1 tumors at days 19-24 were excised from mice. Tumor sizes recorded and frozen for sectioning.

FIG. 34A shows mouse syngeneic orthotopic 4T1 tumor volumes with various treatments.

FIG. 34B shows pictures of actual 4T1 tumors from each of the treatment groups.

BALB/c female mice (8 weeks) were injected orthotopically in the mammary fat pad with 1 x 10⁶ tumor cells (in a 0.05 mL cell suspension). Around 10 days after tumor cell implantation (Day 1 of the study) animals were sorted into groups (n=8 per group) with individual tumor volumes between 63 to 88 mm³ per group. Mice were dosed with blinded samples of Doxorubicin, anti-mPD-Ll, CBT300 and a combination of Doxorubicin and CBT300.

As shown, CBT300 in combination with Doxorubicin displayed a strong antitumor response with a 68% tumor regression. The data again demonstrated the potent antitumor efficacy of CBT300 for triple-negative breast cancer (TNBC) and its ability to reverse drug resistance.

GRP 78 antagonists reduce Crip to and cell surface GRP 78 causing the elimination of CD133 cancer stem cells and an influx of CD8 T-cells.

In the 4T1 syngeneic orthotopic mouse tumor model, tumors were collected and sectioned from each group. The tumor sections were stained for DNA (DAPI), surface GRP78 (anti-GRP78- FITC), CD8 T-cells (anti-CD8-PE) and CD133 stem cells (anti-CD133-PE).

FIG 35 displays immunohistochemistry analysis of 4T1 treated tumors.

FIG 36A-C show graphs of immunohistochemistry analysis of 4T1 treated tumors.

4T1 tumors were orthotopically implanted in mice (Day -10) and treated, n=8. Tumors were excised on day 24 from each group and sectioned for staining. Sections were stained with DAPI (DNA-blue), anti-ecGRP78 (FITC-green), anit-CD8 (PE-red) or CD133 (anti-CD133-PE). Normalized expression of tumor GRP78, T-cells and stem cells in tumor sections are graphed. GRP78, CD8 and CD 133 % area analyses were divided by % tumor area (DAPI) for each group. Average of 3 sections. **p<0.01, ***p<0.001, ****p<0.0001. As shown, the control tumor sections had low expression of surface GRP78 whereas the Doxorubicin treatment significantly increased the tumor cell surface GRP78 about 15-fold. The Doxorubicin treatment alone slightly increased CD8 T-cell infiltration (~2-fold) into the 4T1 tumors but massively increased the amount of stem cells remaining in the 4T1 tumor tissue. The result validated that chemotherapy “selects or switches” to a more drug resistant, immune suppressive and metastatic cancer stem cell phenotype. However, when CBT300 was added to the doxorubicin chemotherapy, the expression of surface GRP78 and CD133 positive cancer stem cells was almost non-existent and the number of infiltrated CD8 T-cells increases over 5-fold. The results suggest that GRP78 antagonists’ elimination of tumor cell surface GRP78 destabilizes and removes Cripto and PD-L1 resulting in the “switching” of the cancer stem cells back to a more normal, chemo and immune sensitive type of tumor cell, thereby leading to significant tumor regression.

Discussion

Drug resistant recurrent cancer cells up-regulate a survival pathway that results in the expression of extracellular Glucose-Regulated Protein 78 (ecGRP78) in the tumor microenvironment (TME). This TME ecGRP78 binds to cancer and immune cell surfaces, which induces a cascade of events to increase drug resistance, immune suppression.

By the present invention, it has been discovered the GRP78 expression may result in cancer stem cell (CSC) formation. CBT300 targets surface bound ecGRP78 that has been found on breast, lung, ovarian, prostate, melanoma, multiple myeloma, colon, pediatric and adult brain tumors. It is now shown for the first time that inhibition of ecGRP78 can induce apoptosis of drug resistant tumor cells, eliminate drug and immune resistance showing synergistic effects with chemotherapy and immunotherapy, and may decrease the amount of chemotherapy by about 90% in combination with CBT300. ecGRP78 has been found on many types of tumor cell surfaces but not on normal cell surfaces. In fact, cell surface bound ecGRP78 is important for many aspects of cancer development, including cell survival, proliferation, chemoresistance, angiogenesis, metastasis formation, immune suppression, and stem cell formation. It has been shown that increased ecGRP78 expression in metastatic breast cancer, glioblastoma and multiple myeloma patients was significantly associated with later stage, increased distant metastasis, increased aggressiveness, shorter disease-free survival, and decreased overall survival.

By the presenting invention, CBT300’s elimination of csGRP78 was shown to destabilize and remove oncofetal proteins R0R1 and Cripto, and checkpoint protein PD-L1 from tumor cell surfaces resulting in reversal of chemoresistance, reduction in immune suppression, inhibition of stem cell phenotype (CD133) and increased tumor cell apoptosis. The results with CBT300 demonstrate proof of concept data in vitro and in vivo for novel ecGRP78 inhibitors on several drug resistant cancers demonstrating the potential to provide a major advance in the treatment of drug resistant cancers either alone or in combination with lower doses of chemotherapy. Exploiting a novel mechanism of action with a non-toxic, efficacious and cost-effective biologic therapy that has shown increased survival in recurrent cancers.

Incorporation by Reference

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.

Equivalents

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

What is claimed is:

1. A method for treating a treatment resistant cancer, wherein the method comprises administration of 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 cancer is metastatic breast cancer, brain cancer, or ovarian cancer.

3. The method of claim 1, wherein the treatment resistance cancer is drug resistant, chemotherapy resistant, or immune resistant.

4. The method of claim 1, wherein the subject has previously been treatment with an anti-cancer therapy.

5. The method of claim 1, wherein the subject has previously been administered a chemotherapy.

6. The method of claim 1, wherein the GRP78 antagonist comprises a plasminogen kringle 5 fragment comprising an amino acid sequence of SEQ ID NO: 1.

7. The method of claim 6, wherein the plasminogen kringle 5 fragment is attached to an immunoglobulin and comprises an amino acid sequence 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 N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID N0: 13, SEQ ID NO: 14, SEQ ID NO : 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18.

8. The method of claim 1, wherein the GRP78 antagonist comprises a R0R1 kringle fragment comprises an amino acid sequence of SEQ ID NO: 19.

9. The method of claim 8, wherein the R0R1 kringle fragment is attached to immunoglobulin and 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.

10. The method of claim 1, wherein the GRP78 antagonist comprises a ROR2 kringle fragment comprising the amino acid sequence of SEQ ID NO:33.

11. The method of claim 10, wherein the ROR2 kringle fragment is attached to immunoglobulin and 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.

12. A method for the prevention of cancer stem cell (CSC) expression, the method comprising: providing to a subject having or at risk of expressing CSCs 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 ROR1 kringle fragment; a ROR1 kringle fragment attached to an immunoglobulin; a ROR2 kringle fragment; and a ROR2 kringle fragment attached to an immunoglobulin.

13. The method of claim 12, wherein the method prevents the expression of CSCs expressing CD133.

14. The method of claim 12, wherein the subject has been previously administered a chemotherapy.

15. The method of claim 12, wherein the plasminogen kringle 5 fragment comprises an amino acid sequence of SEQ ID NO: 1.

16. The method of claim 12, 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 NON, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID N0:9, 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.

17. The method of claim 12, wherein the ROR1 kringle fragment comprises an amino acid sequence of SEQ ID NO: 19.

18. The method of claim 12, wherein the ROR1 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.

19. The method of claim 12, wherein the ROR2 kringle fragment is SEQ ID NO:33 and combinations thereof.

20. The method of claim 12, wherein the ROR2 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.

21. The method of claim 12, wherein the GRP78 antagonist binds to the N-terminal GRP78 domain of extracellular GRP78, thereby preventing GRP78 binding to Cripto.

22. The method of claim 12, wherein the subject has been identified as expressing CSCs.

23. The method of claim 13, wherein the subject has been identified as expressing CSC expressing CD133.

24. The method of claim 24, wherein the subject is afflicted with one or more of a brain cancer, lung cancer, renal cancer, ovarian cancer, pancreatic cancer, liver cancer, colorectal cancer, prostate cancer, gastric cancer, bladder cancer, esophageal cancer, melanoma, or glioblastoma.

26. The method of claim 15, wherein the chemotherapy comprises a drug pump substrate.

27. The method of claim 26, wherein the method results in synergistic treatment of cancer together with the chemotherapy.