Patent Application
20190091227
Cyclin-dependent kinase 4 (CDK4) and the closely related cyclin-dependent kinase 6 (CDK6) are regulators of mammalian mitosis, acting to promote the start of DNA synthesis in preparation for cell division. Several selective inhibitors of CDKs 4 and 6 (“CDK4/6” inhibitors) are at various stages of development, with one, palbociclib, being currently approved in the United States for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer in combination with an endocrine based therapy—either letrozole or fulvestrant. These combinations have been very effective, and are fast becoming the standard of care for such patients.
Breast cancer remains one of the most common and deadly cancers in the United States, with an expected 232,670 newly diagnosed cases and 40,000 related deaths in 2014 alone. Among these, the largest molecular diagnostic subgroup (˜70%) comprises patients with hormone receptor positive (ER+/PR+; ER+/PR−; or ER−/PR+) and HER2 negative (IHC<3+ and not FISH positive) disease. For patients with metastatic disease, the reported median survival ranges from 18-36 months. Metastatic breast cancer is not considered curable. Significant improvements in survival have been obtained, however, coincident with the introduction of improved systemic therapies.
The general therapeutic goal for all patients with ER/PR positive metastatic breast cancer is to prolong survival and improve quality of life. This is accomplished by surgical intervention, where feasible, and medication. Typically endocrine (anti-hormonal) medications are used initially, and maintained until resistance arises. These are preferred because they are effective and relatively non-toxic and their use avoids the toxicities of chemotherapy-based regimens. Current National Comprehensive Cancer Network (NCCN) treatment guidelines state: “systemic treatment of breast cancer recurrence or stage IV disease prolongs survival and enhances quality of life but is not curative. Therefore, treatments associated with minimal toxicity are preferred.”
There are several first line endocrine therapy options for ER/PR positive breast cancer patients with metastatic disease. The term “first-line” is used in this context to indicate the first line of therapy following the appearance of metastatic disease, even if patients have previously been treated in the pre-metastatic setting. In the metastatic setting, either fulvestrant or an aromatase inhibitor (AI, e.g., letrozole) is generally preferred as single agents for first-line therapy. Fulvestrant is a selective estrogen receptor down-regulator (SERD) and is indicated for the treatment of hormone receptor positive metastatic breast cancer in postmenopausal patients with disease progression following anti-estrogen therapy. Aromatase inhibitors block a key step in the synthesis of estrogen.
For postmenopausal patients with metastatic breast cancer who are endocrine therapy-naïve, have progressed >12 months after the end of adjuvant therapy, or who present with de novo metastatic breast cancer, treatment options include an AI plus palbociclib or single-agent therapy using fulvestrant or an AI.
Palbociclib (formerly PD 0332991) is an inhibitor of cyclin-dependent kinases 4 and 6 (CDK 4/6). Palbociclib plus letrozole received US Food and Drug Administration (FDA) accelerated approval as first-line therapy for the treatment of metastatic ER-positive human epidermal growth factor receptor 2 (HER2)-negative breast cancer in 2015. Treatment with letrozole plus palbociclib resulted in a statistically significant increase in progression free survival (PFS) in the combination arm. Overall survival appeared favorable of the combination arm as well, but did not reach statistical significance. There remain limited second-line and beyond endocrine therapy options in the metastatic setting with the AI, exemestane, being one of the more commonly used agents, and the SERD, fulvestrant, being another established option.
Innate and acquired resistance to endocrine therapies pose significant therapeutic challenges in this context, as only those patients who have continuous sensitivity to endocrine therapy experience long-term survival with a reasonably good quality of life. Unfortunately, many patients develop resistance to endocrine therapy, sometimes resulting in very short treatment durations, accelerating the need to initiate cytotoxic chemotherapy.
Like other kinase inhibitors used to treat cancer, the effective use of CDK4/6 inhibitors is limited by resistance—in some cases pre-existing and in most cases developing after a time on treatment. Thus a need exists for low-toxicity methods for treating patients who are resistant to CDK4/6 inhibitor treatment.
The present disclosure addresses the need for non-toxic therapies that prevent or abrogate the resistance that develops to endocrine and CDK inhibitory therapies and provides additional benefits.
Provided herein are compositions and methods for treating ER+, HER2− HRG+ breast cancer (e.g., metastatic ER+, HER2− HRG+ breast cancer) in a human patient, comprising administering to the patient an anti-ErbB3 antibody (e.g., seribantumab), a CDK4/6 inhibitor (e.g., palbociclib, abemaciclib, or ribociclib), and an endocrine based therapy (e.g., letrozole or fulvestrant) according to a particular clinical dosage regimen (i.e., at a particular dose amount and according to a specific dosing schedule).
An exemplary anti-ErbB3 antibody is seribantumab (also known as “MM-121” or “Ab #6”) or antigen binding fragments and variants thereof. In one embodiment, the anti-ErbB3 antibody comprises the heavy and light chain CDRs or variable regions of seribantumab. In one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of seribantumab having the sequence set forth in SEQ ID NO: 10 and the CDR1, CDR2 and CDR3 domains of the VL region of seribantumab having the sequence set forth in SEQ ID NO: 12.
In another embodiment, the antibody comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. In another embodiment, the antibody comprises VH and/or VL regions having the amino acid sequences set forth in SEQ ID NO: 10 and SEQ ID NO: 12, respectively. In another embodiment, the anti-ErbB3 antibody comprises VH and/or VL regions encoded by the nucleic acid sequences set forth in SEQ ID NOs: 9 and 11, respectively. In another embodiment, the anti-ErbB3 antibody comprises heavy and/or light chains having the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
In another embodiment, an antibody is used that competes for binding with and/or binds to the same epitope on human ErbB3 as the above-mentioned antibodies. In a particular embodiment, the epitope comprises residues 92-104 of human ErbB3 (SEQ ID NO: 14). In another embodiment, the epitope includes amino acid residues within positions 92-104 of human ErbB3 (SEQ ID NO: 14). In another embodiment, the antibody competes with seribantumab for binding to human ErbB3 and has at least 90% variable region amino acid sequence identity with the above-mentioned anti-ErbB3 antibodies (e.g., at least about 90%, 95% or 99% variable region identity with SEQ ID NO: 10 and SEQ ID NO: 12).
An exemplary CDK4/6 inhibitor is palbociclib. In another embodiment, the CDK4/6 inhibitor is abemaciclib. In another embodiment, the CDK4/6 inhibitor is ribociclib.
An exemplary endocrine based therapy is letrozole or fulvestrant.
Accordingly, in one aspect, methods of treating a human patient with a ER+, HER2− breast cancer are provided, the methods comprising administering to the patient an anti-ErbB3 antibody (e.g., seribantumab), a CDK4/6 inhibitor (e.g., palbociclib), and an endocrine based therapy (e.g., letrozole or fulvestrant).
In another aspect, methods of treating a human patient with a ER+, HER2− breast cancer are provided, the method comprising administering to the patient an anti-ErbB3 antibody (e.g., seribantumab) and an endocrine-based therapy (e.g., letrozole or fulvestrant). In one embodiment, the method does not comprise administration of a CDK4/6 inhibitor (e.g., palbociclib, abemaciclib, or ribociclib). In another embodiment, the method comprises administering to the patient an anti-ErbB3 antibody (e.g., seribantumab) and fulvestrant. In another embodiment, the method comprises administering to the patient an anti-ErbB3 antibody (e.g., seribantumab) and letrozole.
In one embodiment, no more than three other antineoplastic agents (e.g., CDK4/6 inhibitors and/or endocrine based therapies) are administered in combination with seribantumab within a treatment cycle. In another embodiment, no more than two other antineoplastic agents are administered in combination with seribantumab within a treatment cycle. In another embodiment, no more than one other antineoplastic agent is administered in combination with seribantumab within a treatment cycle.
In one embodiment, a treatment cycle is 21 days. In another embodiment, the treatment comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 cycles. Treatment is continued for any suitable period of time (e.g., until a complete response (CR) has been achieved). In one embodiment, the treatment is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, or 11 months. In another embodiment, the treatment is administered for at least one year. In another embodiment, the treatment is administered for at least two years.
The therapeutic agents described herein (e.g., seribantumab, palbociclib, letrozole, and fulvestrant) can be administered to a patient by any suitable means. In one embodiment, seribantumab is formulated for intravenous administration. In one embodiment, palbociclib is formulated for oral administration (e.g., as a capsule or tablet). In one embodiment, letrozole is formulated for oral administration (e.g., as a capsule or tablet). In one embodiment, fulvestrant is formulated as a sterile solution for intramuscular injection.
In one embodiment, the dose of the anti-ErbB3 antibody (e.g., seribantumab), the CDK4/6 inhibitor (e.g., palbociclib) and the endocrine based therapy (e.g., letrozole or fulvestrant) is a dose that is fixed irrespective of the weight of the patient. For example, seribantumab may be administered at a fixed dose of 3 g without regard to the patient's weight. Palbociclib may be administered at a fixed dose of a 125 mg capsule without regard to the patient's weight. Letrozole may be administered at a fixed dose of a 2.5 mg without regard to the patient's weight. Fulvestrant may be administered at a fixed dose of 500 mg without regard to the patient's weight. In certain embodiments, dosage regimens are adjusted to provide the optimum desired response (e.g., an effective response).
In one aspect, palbociclib, letrozole, and seribantumab are administered in combination according to a particular dosage regimen. In one embodiment, a 125 mg palbociclib capsule is administered orally once daily for 21 consecutive days, followed by 7 days off treatment for a 28 day cycle. In this embodiment, 2.5 mg of letrozole is given once daily continuously throughout the 28 day cycle. In this embodiment, seribantumab is administered at a dose of 3 g every two weeks by IV infusion throughout the cycle.
In another aspect, palbociclib, fulvestrant, and seribantumab are administered in combination according to a particular dosage regimen. In this embodiment, a 125 mg palbociclib capsule is administered orally once daily for 21 consecutive days, followed by 7 days off treatment for a 28 day cycle. In this embodiment, fulvestrant is administered at a dose of 500 mg on days 1, 15, 29, and once monthly or once every 28 days thereafter. In this embodiment, seribantumab is administered at a dose of 3 g every two weeks by IV infusion throughout the cycle.
Accordingly, in one aspect, methods of treating a human patient with a ER+, HER2− breast cancer are provided, the methods comprising administering to the patient:
In another aspect, methods of treating a patient who has been previously treated with palbociclib and a hormonal therapy, and whose cancer has progressed on this treatment, are provided, the method comprising concurrently administering to the patient:
In another aspect, methods of treating a patient with ER/PR+, HER2− breast cancer expressing HRG as measured by RNA in-situ hybridization (RNA-ISH) are provided. In one embodiment, the breast cancer is locally advanced or metastatic breast cancer. In another embodiment, the method comprises a 28-day cycle, wherein:
In one embodiment, the method comprises at least one subsequent treatment cycle. In another embodiment, fulvestrant is administered only on day 1 of each subsequent treatment cycle.
In another aspect, the method of treating a patient with ER/PR+, HER2− breast cancer expressing HRG as measured by RNA in-situ hybridization (RNA-ISH) comprises a 28-day cycle, wherein:
In one embodiment, a heregulin RNA in situ hybridization (RNA-ISH) score of 1+ of higher has been measured in a biological sample from the patient prior to treatment.
The efficacy of the treatment methods provided herein can be assessed using any suitable means. In one embodiment, the treatment produces at least one therapeutic effect selected from the group consisting of reduction in size of a tumor, reduction in metastasis, complete remission, partial remission, stable disease, increase in overall response rate, or a pathologic complete response. In one embodiment, the treatment results in the patient exhibiting stable disease, a partial response, or a complete response.
Further provided are kits that include an anti-ErbB3 antibody, such as seribantumab, a CDK4/6 inhibitor, such as palbociclib, and an endocrine based therapy, such as letrozole or fulvestrant. In one embodiment, the kit comprises: (a) a dose of seribantumab, (b) a dose of palbociclib, (c) a dose of letrozole or fulvestrant, and (d) instructions for using letrozole or fulvestrant in combination with seribantumab and palbociclib, in the methods described herein. In another embodiment, the kit comprises: (a) a dose of seribantumab, (b) a dose of letrozole or fulvestrant, and (c) instructions for using letrozole or fulvestrant in combination with seribantumab, in the methods described herein.
As used herein, the term “subject” or “patient” is a human patient (e.g., a patient having ER+, HER2− HRG+ metastatic breast cancer).
As used herein, the term “estrogen receptor positive” (ER+) refers to tumors (e.g., carcinomas), typically breast tumors, in which the tumor cells score positive (i.e., using conventional histopathology methods) for estrogen receptor (ER). According to recommendations provided by the College of American Pathologists (CAP) and the American Society of Clinical Oncology (ASCO), a tumor is ER+ if at least 1% of the tumor cells tested (e.g., by immunohistochemistry) score ER positive.
The terms “ErbB2,” “HER2,” and “HER2 receptor,” as used interchangeably herein, refer to the protein product of the human neu oncogene, also referred to as the ErbB2 oncogene or the HER2 oncogene. According to guidelines provided by CAP and the ASCO, a tumor designated HER2 negative (HER2-) is a tumor in which an immunoassay such as immunohistochemistry (IHC) test shows no staining or membrane staining in <30% of tumor cells. For one such assay, marketed as HERCEPTEST®, a score of 0 or 1+ is considered HER2 negative, a score of 2+ is considered equivocal—requiring further testing by fluorescence in-situ hybridization (FISH) for definitive characterization, and a score of 3+ is considered HER2 positive. Therefore a patient with a biopsy scoring 0 or 1+ by HERCEPTEST, or 2+ by HERCEPTEST and negative by FISH is considered HER2 negative, while a patient scoring 3+ by HERCEPTEST or 2+ by HERCEPTEST and FISH positive is deemed HER2 positive.
As used herein, “HRG” indicates any and all isotypes of heregulin (neuregulin-1, “NRG”), a set of naturally occurring ligands of ErbB3. HRG expression can be evaluated, for example, using a RNA in situ-hybridization (ISH)-based assay, e.g., according to the protocol described in Example 1 of U.S. Ser. No. 14/965,301; WO 2015/100459, which is expressly incorporated herein by reference. In one embodiment, the RNA-ISH is read out via a chromogenic signal. In a particular embodiment, the probes used to detect HRG by RNA-ISH hybridize specifically to a nucleic acid that comprises nucleotides 442-2977 of the nucleotide sequence set forth in GenBank accession number NM-013956 (SEQ ID NO:13). In certain embodiments the probes hybridize specifically to RNAs encoding each of the HRG isoforms α, β1, β1b, β1c, β1d, β2, β2b, β3, β3b, γ, γ2, γ3, ndf43, ndf34b, and GGF2. In another embodiment, the HRG score is determined by RT-PCR using probes specific for HRG.
The terms “ErbB3” and “HER3,” as used interchangeably herein, refer to human ErbB3 protein, as described in U.S. Pat. No. 5,480,968. The human ErbB3 protein sequence is shown in SEQ ID NO:4 of U.S. Pat. No. 5,480,968, wherein the first 19 amino acids (aas) correspond to the leader sequence that is cleaved from the mature protein. ErbB3 is a member of the ErbB family of receptors, other members of which include ErbB1 (EGFR), ErbB2 (HER2/Neu) and ErbB4. ErbB3 itself lacks tyrosine kinase activity, but is itself phosphorylated upon dimerization of ErbB3 with another ErbB family receptor, e.g., ErbB1 (EGFR), ErbB2 and ErbB4, which are receptor tyrosine kinases. Ligands for the ErbB family receptors include heregulin (HRG), betacellulin (BTC), epidermal growth factor (EGF), heparin-binding epidermal growth factor (HB-EGF), transforming growth factor alpha (TGF-α), amphiregulin (AR), epigen (EPG) and epiregulin (EPR). The amino acid sequence of human ErbB3 is provided at Genbank Accession No. NP_001973.2 (receptor tyrosine-protein kinase erbB-3 isoform 1 precursor) and is assigned Gene ID: 2065.
As used herein, the term “ErbB3 inhibitor” is intended to include therapeutic agents that inhibit, downmodulate, suppress or downregulate activity of ErbB3. The term is intended to include chemical compounds, such as small molecule inhibitors, and biologic agents, such as antibodies, interfering RNA (shRNA, siRNA), soluble receptors and the like. An exemplary ErbB3 inhibitor is an anti-ErbB3 antibody, such as seribantumab.
As used herein, the term “agent,” refers to an active molecule, e.g., a therapeutic protein, e.g., a drug.
As used herein, “effective treatment” refers to treatment producing a beneficial effect, e.g., amelioration of at least one symptom of a disease or disorder. A beneficial effect can take the form of an improvement over baseline, i.e., an improvement over a measurement or observation made prior to initiation of therapy according to the method. Effective treatment may refer to alleviation of at least one symptom of cancer.
As used herein, the term “effective amount” refers to an amount of an agent that provides the desired biological, therapeutic, and/or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, and/or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount can be administered in one or more administrations.
As used herein, the term “administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a formulation of the molecules disclosed herein) into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease, or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.
As used herein, the terms “fixed dose”, “flat dose” and “flat-fixed dose” are used interchangeably and refer to a dose that is administered to a patient without regard for the weight or body surface area (BSA) of the patient. The fixed or flat dose is therefore not provided as a mg/kg dose, but rather as an absolute amount of the agent.
The terms “treat,” “treating,” and “treatment,” as used herein, refer to therapeutic or preventative measures described herein. The methods of “treatment” employ administration to a subject, the combination disclosed herein in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
As used herein, adjunctive or combined administration (coadministration) includes simultaneous administration of the agents in the same or different dosage form, or separate administration of the agents (e.g., sequential administration). For example, the agents can be formulated for separate administration and administered concurrently or sequentially. Such concurrent or sequential administration preferably results in the agents being simultaneously present in treated patients.
Anti-ErB3 antibodies (or VH/VL domains derived therefrom) suitable for use in the invention can be generated using methods well known in the art. Alternatively, art recognized anti-ErbB3 antibodies can be used, for example, AV-203 (as described in U.S. Pat. No. 8,481,687), GSK2849330 (as described in U.S. Pat. No. 9,085,622), KTN3379 (as described in U.S. Pat. No. 9,220,775), duligotuzumab (as described in U.S. Pat. No. 8,597,652), elgemtumab (as described in U.S. Pat. No. 8,735,551), futuximab (as described in WO2008/104183), lumretuzumab (as described in U.S. Pat. No. 8,859,737), and patritumab (as described in U.S. Pat. No. 7,705,130). Antibodies that compete with any of these art-recognized antibodies for binding to ErbB3 also can be used.
An exemplary anti-ErbB3 antibody is seribantumab (also known as “MM-121” or “Ab #6”) or antigen binding fragments and variants thereof. Seribantumab is a human monoclonal anti-ErbB3 IgG2 (see, e.g., U.S. Pat. Nos. 7,846,440; 8,691,771 and 8,961,966; 8,895,001, U.S. Patent Publication Nos., 20110027291, 20140127238, 20140134170, and 20140248280), as well as international publication Nos. WO/2013/023043, WO/2013/138371, WO/2012/103341, and U.S. Provisional Patent Application Ser. No. 62/090,780, the teachings of which are expressly incorporated herein by reference).
In one embodiment, the anti-ErbB3 antibody comprises the heavy and light chain CDRs or variable regions of seribantumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of seribantumab having the sequence set forth in SEQ ID NO: 10 and the CDR1, CDR2 and CDR3 domains of the VL region of seribantumab having the sequence set forth in SEQ ID NO: 12. In another embodiment, the antibody comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. In another embodiment, the antibody comprises VH and/or VL regions having the amino acid sequences set forth in SEQ ID NO: 10 and SEQ ID NO: 12, respectively. In another embodiment, the anti-ErbB3 antibody comprises VH and/or VL regions encoded by the nucleic acid sequences set forth in SEQ ID NOs: 9 and 11, respectively. In another embodiment, the anti-ErbB3 antibody comprises heavy and/or light chains having the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. In another embodiment, an antibody is used that competes for binding with and/or binds to the same epitope on human ErbB3 as the above-mentioned antibodies. In a particular embodiment, the epitope comprises residues 92-104 of human ErbB3 (SEQ ID NO: 14). In another embodiment, the antibody competes with seribantumab for binding to human ErbB3 and has at least 90% variable region amino acid sequence identity with the above-mentioned anti-ErbB3 antibodies (see, e.g., U.S. Pat. No. 7,846,440 and US Patent Publication No. 20100266584).
Art recognized CDK4/6 inhibitors can be used. An exemplary CDK4/6 inhibitor is palbociclib. Palbociclib (codenamed PD-0332991, trade name IBRANCE) is a drug for the treatment of ER-positive and HER2-negative breast cancer. It is a selective inhibitor of the cyclin-dependent kinases CDK4 and CDK6. IBRANCE capsules for oral administration contain 125 mg, 100 mg, or 75 mg of palbociclib, a kinase inhibitor. The molecular formula for palbociclib is C24H29N7O2. The molecular weight is 447.54 daltons. The chemical name is 6-acetyl-8-cyclopentyl-5-methyl-2-{[5-(piperazin-1-yl)pyridin-2-yl]amino}pyrido[2,3-d]pyrimidin-7(8H)-one, and its structural formula is:
Palbociclib is a yellow to orange powder with pKa of 7.4 (the secondary piperazine nitrogen) and 3.9 (the pyridine nitrogen). At or below pH 4, palbociclib behaves as a high-solubility compound. Above pH 4, the solubility of the drug substance reduces significantly. Palbociclib contains the following inactive ingredients: Microcrystalline cellulose, lactose monohydrate, sodium starch glycolate, colloidal silicon dioxide, magnesium stearate, and hard gelatin capsule shells. The light orange, light orange/caramel and caramel opaque capsule shells contain gelatin, red iron oxide, yellow iron oxide, and titanium dioxide; and the printing ink contains shellac, titanium dioxide, ammonium hydroxide, propylene glycol and simethicone.
The recommended dose of palbociclib is a 125 mg capsule taken orally once daily for 21 consecutive days followed by 7 days off treatment to comprise a complete cycle of 28 days. IBRANCE should be taken with food.
When coadministered with palbociclib, the recommended dose of letrozole is 2.5 mg taken once daily continuously throughout the 28-day cycle.
When coadministered with palbociclib, the recommended dose of fulvestrant is 500 mg administered on Days 1, 15, 29, and once monthly thereafter.
Another exemplary CDK4/6 inhibitor is abemaciclib. Abemaciclib (codenamed LY2835219; trade name IBRANCE) is an investigational drug for various types of cancer. It is an orally selective inhibitor of the cyclin-dependent kinases CDK4 and CDK6. The molecular formula for abemaciclib is C27H32F2N8. The molecular weight is 506.61 daltons. The chemical name is 2-pyrimidinamine, N-(5-((4-ethyl-1-piperazinyl)methyl)-2-pyridinyl)-5-fluoro-4-(4-fluoro-2-methyl-1-(1-methylethyl)-1H-benzimidazol-6-yl), and its structural formula is:
Another exemplary CDK4/6 inhibitor is ribociclib. Ribociclib (codenamed LEE011; trade name KISQUALI) is a drug for the treatment of various cancers, including hormone receptor-positive and HER2-negative advanced or metastatic breast cancer. It is an orally available, highly selective inhibitor of the cyclin-dependent kinases CDK4 and CDK6. The molecular formula for Ribociclib is C23H30N8O. The molecular weight is 434.55 daltons. The chemical name is 7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide, and its structural formula is:
KISQALI tablets are recommended to be are taken daily with or without food. Recommended starting dose: 600 mg orally (three 200 mg tablets) taken once daily with or without food for 21 consecutive days followed by 7 days off treatment.
Art recognized endocrine based therapies can be used. Exemplary endocrine based therapies include non-steroidal aromatase inhibitors (e.g., letrozole, anostrozole) and selective estrogen receptor degraders (e.g., fulvestrant, brilanestrant, elacestrant).
An exemplary endocrine based therapy is letrozole. Letrozole (trade name FEMARA) is a nonsteroidal aromatase inhibitor (inhibitor of estrogen synthesis). Letrozole inhibits the aromatase enzyme by competitively binding to the heme of the cytochrome P450 subunit of the enzyme, resulting in a reduction of estrogen biosynthesis in all tissues. It is chemically described as 4,4′-(1H-1,2,4-Triazol-1-ylmethylene)dibenzonitrile, and its structural formula is
Letrozole is a white to yellowish crystalline powder, practically odorless, freely soluble in dichloromethane, slightly soluble in ethanol, and practically insoluble in water. It has a molecular weight of 285.31, empirical formula C17H11N5, and a melting range of 184° C.−185° C.
FEMARA (letrozole tablets) is available as 2.5 mg tablets for oral administration. Letrozole contains the following inactive Ingredients: colloidal silicon dioxide, ferric oxide, hydroxypropyl methylcellulose, lactose monohydrate, magnesium stearate, maize starch, microcrystalline cellulose, polyethylene glycol, sodium starch glycolate, talc, and titanium dioxide.
The recommended dose of FEMARA (letrozole tablets) is one 2.5 mg tablet administered once a day, without regard to meals.
Another exemplary endocrine based therapy is anastrozole (trade name ARIMIDEX). ARIMIDEX (anastrozole) is an orally available aromatase inhibitor which competively blocks the conversion of androgens to estrogens in peripheral (extra-gonadal) tissues. The chemical name is a,a,a′,a′-Tetramethyl-5-(1H-1,2,4-triazol-1-ylmethyl)-1,3-benzenediacetonitrile. The molecular formula is C17H19N5 and its structural formula is:
Anastrozole is freely soluble in methanol, acetone, ethanol, and tetrahydrofuran, and very soluble in acetonitrile. Each tablet contains as inactive ingredients: lactose, magnesium stearate, hydroxypropylmethylcellulose, polyethylene glycol, povidone, sodium starch glycolate, and titanium dioxide. ARIMIDEX is available as 1 mg tablets for oral administration and the recommended dose of ARIMIDEX is one tablet daily.
Another exemplary endocrine based therapy is fulvestrant (trade name FASLODEX). FASLODEX (fulvestrant) Injection for intramuscular administration is an estrogen receptor antagonist. The chemical name is 7-alpha-[9-(4,4,5,5,5-penta fluoropentylsulphinyl) nonyl]estra-1,3,5-(10)-triene-3,17beta-diol. The molecular formula is C32H47F5O3S and its structural formula is:
Fulvestrant is a white powder with a molecular weight of 606.77. The solution for injection is a clear, colorless to yellow, viscous liquid. Each injection contains as inactive ingredients: 10% w/v Alcohol, USP, 10% w/v Benzyl Alcohol, NF, and 15% w/v Benzyl Benzoate, USP, as co-solvents, and made up to 100% w/v with Castor Oil, USP as a co-solvent and release rate modifier.
The recommended dose of FASLODEX is 500 mg and should be administered intramuscularly into the buttocks slowly (1-2 minutes per injection) as two 5 mL injections, one in each buttock, on days 1, 15, 29 and once monthly thereafter. A dose of 250 mg is recommended in patients with moderate hepatic impairment to be administered intramuscularly into the buttock slowly (1-2 minutes) as one 5 mL injection on days 1, 15, 29 and once monthly thereafter.
Another exemplary endocrine based therapy is brilanestrant (Code names: GDC-0810, ARN-810, RG-6046, RO-7056118). Brilanestrant is an investigational drug for the treatment of metastatic estrogen receptor-positive breast cancer. It is a non-steroidal combined selective estrogen receptor modulator (SERM) and selective estrogen receptor degrader (SERD). The chemical name is (2E)-3-{4-[(1E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl]phenyl}prop-2-enoic acid. The molecular formula is C26H20ClFN2O2 and its structural formula is:
Brilanestrant is orally available and does not need to be administered by intramuscular injection.
Another exemplary endocrine based therapy is elacestrant (Code names: RAD-1901, ER-306323). Elacestrant is an investigational drug for the treatment of estrogen receptor-positive breast cancer, endometrial cancer, and kidney cancer. It is a non-steroidal combined selective estrogen receptor modulator (SERM) and selective estrogen receptor degrader (SERD). The chemical name is (6R)-6-{2-[ethyl({4-[2-(ethylamino)ethyl]phenyl}methyl)amino]-4-methoxyphenyl}-5,6,7,8-tetrahydronaphthalen-2-ol. The molecular formula is C30H38N2O2 and its structural formula is:
Elacestrant is orally available and does not need to be administered by intramuscular injection.
Provided herein are compositions and methods for treating ER+, HER2− breast cancer (e.g., metastatic ER+, HER2− breast cancer) in a human patient, comprising administering to the patient an anti-ErbB3 antibody (e.g., seribantumab or istiratumab), a CDK4/6 inhibitor (e.g., palbociclib, abemaciclib, or ribociclib), and an endocrine based therapy (e.g., letrozole or fulvestrant) according to a particular clinical dosage regimen (i.e., at a particular dose amount and according to a specific dosing schedule). Also provided herein, are composition and methods for treating ER+, HER2− breast cancer (e.g., metastatic ER+, HER2− breast cancer) in a human patient, comprising administering to the patient an anti-ErbB3 antibody (e.g., seribantumab or istiratumab), and an endocrine based therapy (e.g., letrozole or fulvestrant) according to a particular clinical dosage regimen (i.e., at a particular dose amount and according to a specific dosing schedule)
Treatment outcomes can be evaluated using standard measures for tumor response. Target lesion (tumor) responses to therapy are classified as:
Non-target lesion responses to therapy are classified as:
In another embodiment, the patient so treated experiences tumor shrinkage and/or decrease in growth rate, i.e., suppression of tumor growth. In another embodiment, tumor cell proliferation is reduced or inhibited. Alternately, one or more of the following can indicate a beneficial response to treatment: the number of cancer cells can be reduced; tumor size can be reduced; cancer cell infiltration into peripheral organs can be inhibited, retarded, slowed, or stopped; tumor metastasis can be slowed or inhibited; tumor growth can be inhibited; recurrence of tumor can be prevented or delayed; one or more of the symptoms associated with cancer can be relieved to some extent. Other indications of a favorable response include reduction in the quantity and/or size of measurable tumor lesions or of non-target lesions.
Also provided herein are kits which include an anti-ErbB3 antibody (e.g., seribantumab or istiratumab), a CDK4/6 inhibitor (e.g., palbociclib, abemaciclib, or ribociclib) and an endocrine based therapy (e.g., letrozole or fulvestrant), in a therapeutically effective amount adapted for use in the preceding methods. In another embodiment, the kits include an anti-ErbB3 antibody (e.g., seribantumab or istiratumab) and an endocrine based therapy (e.g., letrozole or fulvestrant), in a therapeutically effective amount adapted for use in the preceding methods. The kits optionally also can include instructions, e.g., comprising administration schedules, to allow a practitioner (e.g., a physician, nurse, or patient) to administer the therapeutic agents contained therein to a patient having cancer. The kit also can include a syringe. Instruments or devices necessary for administering the pharmaceutical composition(s) also may be included in the kits.
In one embodiment, the present invention provides a kit comprising: (a) a dose of seribantumab or istiratumab, (b) a dose of palbociclib, (c) a dose of letrozole or fulvestrant, and (d) instructions for using letrozole or fulvestrant in combination with seribantumab or istiratumab and palbociclib, in the methods described herein. In another embodiment, the kit comprises (a) a dose of seribantumab or istiratumab, (b) a dose of letrozole or fulvestrant, and (c) instructions for using letrozole or fulvestrant in combination with seribantumab or istiratumab, in the methods described herein.
The following examples are merely illustrative and should not be construed as limiting the scope of this disclosure in any way as many variations and equivalents will become apparent to those skilled in the art upon reading the present disclosure.
The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.
MCF-7, T47D, ZR75-1 and HCC-1428 and were obtained from the American Type Culture Collection (“ATCC” Rockville, Md., USA). All cells were cultured in RPMI1640 medium supplemented with 10% v/v Heat inactivated FBS, 5% v/v L-glutamine and 5% v/v penicillin-streptomycin solution. All culture reagents were from Gibco unless otherwise stated. When hormone-free conditions were required, cells were cultured in phenol red free RPMI1640 medium supplemented with 10% v/v charcoal stripped, heat inactivated FBS, 5% v/v L-glutamine and 5% v/v penicillin-streptomycin solution. To ensure low growth factor conditions for growth factor stimulation assays, cells were cultured in low serum conditions such as 3% v/v heat inactivated FBS or 1% v/v heat inactivated FBS with normal supplementation of other media components. All cell lines were cultured at 37° C. in a humid atmosphere with 95% air, 5% CO2. The identities of all cells used were verified by microsatellite analyses at ATCC. Recombinant heregulin (HRGβ1) was from R&D Systems (396-HB). Cell Titer-Glo assay reagents were from Promega. Estradiol (E8875) and fulvestrant (14409) were from Sigma-Aldrich. Tamoxifen (S1972), palbociclib (S1579), abemaciclib (S7158) and ribociclib (S7440) were all from SelleckChem.
Cells were seeded in duplicate or triplicate at 1500 to 5000 cells per well in 96 well plates, Opaque-walled 96-well plates with clear bottom: 96-well, Black/Clear, Flat bottom with lid, FALCON, 353219 in reduced serum conditions at either 3% v/v FBS or 1% v/v FBS. The day after plating recombinant HRGβ1 was added to yield a final concentration of 10 nM. Control wells received media without HRGβ1. Plates were then incubated for the indicated time period of 4 to 6 days at 37° C. in a humid atmosphere with 95% air, 5% CO2.
For studies including HRGβ1 and the ErbB3 targeting mAb, seribantumab (MM-121) or CDK inhibitors such as palbociclib, abemaciclib or ribociclib, cells were seeded in duplicate or triplicate at 1500 to 5000 cells per well in 96 well plates, Opaque-walled 96-well plates with clear bottom: Nano-Culture plates, MS pattern, low binding, SCIVAX Life Science NCP-LS96-10 in reduced serum conditions at either 3% v/v FBS or 1% v/v FBS. The day after plating recombinant HRGβ1 was added to yield a final concentration of 10 nM. Control wells received media without HRGβ1. Where indicated seribantumab was added to achieve a final concentration of 1 μM or over a dilution series to achieve 10 dilutions per plate to achieve a dose-response curve with 2 controls wells per row. Where indicated CDK inhibitors were added at 10 μM and dilutions were performed to generate a dose-response curve with 2 controls wells per row. Plates were then incubated for the indicated time period of 4 to 6 days at 37° C. in a humid atmosphere with 95% air, 5% CO2. Growth inhibition was calculated as a function of the relative inhibition or proliferation of cells treated with either growth factor or antagonists to cells treated with diluent only, over the same time period.
For studies including HRGβ1 and the ErbB3 targeting mAb, seribantumab (MM-121) or CDK inhibitors such as palbociclib, abemaciclib or ribociclib cells were seeded in duplicate or triplicate at 1500 to 5000 cells per well in 96 well plates, Opaque-walled 96-well plates with clear bottom: Nano-Culture plates, MS pattern, low binding, SCIVAX Life Science NCP-LS96-10 in reduced serum conditions at either 3% v/v FBS or 1% v/v FBS. The day after plating recombinant HRGβ1 was added to yield a final concentration of 10 nM. Control wells received media without HRGβ1. Where indicated, seribantumab was added to achieve a final concentration of 1 μM or over a dilution series to achieve 10 dilutions per plate to achieve a dose-response curve with 2 controls wells per row. Where indicated CDK inhibitors were added at 10 μM and dilutions were performed to generate a dose-response curve with 2 controls wells per row. Plates were then incubated for the indicated time period of 4 to 6 days at 37° C. in a humid atmosphere with 95% air, 5% CO2.
To measure proliferation, Cell Titer-Glo (CTG) assays were performed as per manufacturer's instructions. Specifically, reagent-1 and reagent-2 were equilibrated to room temperature at which point reagent-1 was added to reagent-2 and mixed by vortex. Test plates containing cells were equilibrated to room temperature for 30 minutes at which point an equal volume of CTG reagent was added to each well of the test plate, typically 100 μl to give a final volume of 200 μl per well. Each plate was then sealed with a foil plate sealer and mixed on an orbital shaker for 10 minutes to lyse cells and release cellular ATP. Following mixing plates were incubated at room temperature for 15 minutes to stabilize the luminescent signal. Data was collected by measuring relative luminescence on a Synergy H1 plate reader using the luminescence program. Growth was calculated and expressed as a relative value to the unstimulated control wells on each individual plate. Growth inhibition was calculated as a function of the relative inhibition of cells treated with antagonists to the growth of cells treated with diluent only, over the same time period. Data was then plotted using GraphPad Prism software.
ErbB3 is a member of the human epidermal growth factor receptor (ErbB or HER) family which is comprised of four receptors (ErbB1-4). A defining feature of the ErbB network is that two members of the family, ErbB2 and ErbB3, are non-autonomous. ErbB2 lacks the capacity to interact with a growth-factor ligand, whereas the kinase activity of ErbB3 is defective. Heregulin (HRG), the ErbB3 ligand, has been identified as a potent driver of proliferation and enhanced survival. HRG expression leads to a distinct tumor cell phenotype characterized by an inability to respond to the effects of numerous Standard of Care (SOC) therapies, including chemotherapies, anti-hormonal agents and other targeted therapeutics.
In surveys of HRG expression, HRG+ cells are present in approximately 50% of the cases of most solid tumor types. It is hypothesized that these HRG+ cells are protected from the effects of SOC therapy and continue to proliferate even in the presence of SOC, resulting in limited clinical benefit. In this model, if HRG activity is blocked, HRG+ cells become susceptible to SOC, resulting in enhanced clinical benefit. Seribantumab is a fully human anti-ErbB3 monoclonal antibody designed to block HRG activity by inhibiting the binding of HRG to ErbB3. In the presence of seribantumab, HRG+ tumor cells are predicted to be able to respond to co-administered SOC therapy.
For hormone receptor positive (HR+) breast cancer, hormone deprivation strategies have proven clinical benefit in the adjuvant and metastatic settings. Unfortunately, clinical benefit from these therapies can be short-lived in some patients. Optimal clinical management of these patients requires a comprehensive molecular understanding of the drivers of rapid clinical progression. It has been shown that HRG mRNA expression measured in tumor samples defines a subgroup of patients who derive only limited clinical benefit from SOC when compared to patients whose tumors do not express HRG. This was observed in a previously published Phase 2 clinical study with exemestane, and preclinically with multiple classes of anti-hormonal agents, including letrozole and fulvestrant—treatments that currently represent the mainstay of treatment options for HR+, HER2 negative (HER2−) advanced breast cancer.
The data supports the hypothesis that phenotypically distinct HRG+ cells in breast cancer models persist despite treatment with SOC and various novel classes of therapy. Moreover, the data suggests that addition of seribantumab to these other therapies is important for sustained treatment responses. Continued expansion of HRG+ cells could be the key to rapid clinical progression in breast cancer patients treated with SOC therapy. These findings support the development of seribantumab in combination with anti-hormonal agents in a Phase 3 clinical trial in HR+, HER2− advanced breast cancer.
HRG expression in breast cancer cells can contribute to cancer progression and resistance to therapies by activating HER3 signaling. To elaborate on this finding, the prevalence of HRG mRNA in the TCGA public data base and by directly measuring HRG mRNA in 197 ER-positive, HER2-negative breast cancer tumors using a clinically relevant HRG RNA-ISH assay was examined. Both the TCGA database and the patient samples were found to have a prevalence of 45% for HRG mRNA (
ER+, HER2− breast cancers cell lines with HRG for 6 days were stimulated and proliferation was measured in vitro. Results (
Fulvestrant is classified a “SERD”, selective estrogen receptor degrader and is widely used to treat patients with advanced ER+ breast cancers. SERDs antagonize hormone binding to the receptor and promote degradation of receptor protein, thereby having a dual mechanism of action (MOA) to inhibit hormone receptor signaling and cancer cell growth. As shown in
ER+, HER2− breast cancer cells were treated with CDK4/6 inhibitors in the absence or presence of HRG with or without the addition of seribantumab, followed by measurement of proliferation using the CTG assay (
MCF7 cells were initially treated with various combinations of 1) palbociclib or abemaciclib or ribociclib, 2) HRG, 3) fulvestrant and 4) seribantumab, and proliferation was measured by CTG assay. When MCF7 cells were treated with the combination of a CDK4/6 inhibitor plus fulvestrant (50 nM), the degree of inhibition of proliferation was greater than the activity of the CDK4/6 inhibitor alone (
A patient with ER+, HER2− metastatic breast cancer is treated with one palbociclib 125 mg capsule taken orally once daily for 21 consecutive days, followed by 7 days off treatment to comprise a complete cycle of 28 days. The patient is concurrently treated with letrozole, 2.5 mg taken once daily continuously throughout the 28-day cycle, or with fulvestrant at a dose of 500 mg administered on days 1, 15, 29, and once monthly thereafter. The patient is also concurrently treated with seribantumab at a dose of 3 g every two weeks by IV infusion. Such treatment results in a beneficial result, e.g., stable disease, a partial response, or a complete response.
A patient with ER+, HER2− metastatic breast cancer who has been previously treated with palbociclib and either letrozole or fulvestrant and has become resistant to this treatment is treated with one palbociclib 125 mg capsule taken orally once daily for 21 consecutive days followed by 7 days off treatment to comprise a complete cycle of 28 days. The patient is concurrently treated with either letrozole (if the patient had been previously treated with fulvestrant) or fulvestrant (if the patient had been previously treated with letrozole). Letrozole is administered at a dose of 2.5 mg taken once daily continuously throughout the 28-day cycle, or fulvestrant is administered at a dose of 500 mg administered on days 1, 15, 29, and once monthly thereafter. The patient is also concurrently treated with seribantumab at a dose of 3 g every two weeks by IV infusion. Such treatment results in a beneficial result, e.g., stable disease, a partial response, or a complete response.
The following experiments demonstrate that HER3 inhibitors can block non-canonical CDK2 complex by HRG in the presence of CDK4/6 inhibition by drugs, such as palbociclib, abemaciclib and ribociclib.
MCF7 cells were treated with 10 nM HRG, 100 nM fulvestrant, 100 nM palbociclib, 100 nM abemaciclib or 1 uM of seribantumab either alone or in combination for 20-24 hours as shown in
The implications of these findings are that one of the mechanisms by which HRG inhibits the activity of endocrine therapies (such as fulvestrant) and CDK4/6 inhibitors (such as palbociclib or abemaciclib) may be the non-canonical activation of CDK2 to promote cell cycle transition. These results suggest that seribantumab blocks this activation of CDK2 and therefore restores the activity of standard-of-care therapies, such as fulvestrant and CDK4/6 inhibitors.
Treatment of ER+ HER2− cell lines with multiple receptor tyrosine kinase ligands (RTKL) or estrogen (E2), illustrates that heregulin (HRG) is the most effective RTKL at inhibiting the activity of fulvestrant, palbociclib, or the combination of fulvestrant and palbociclib.
The purpose of this experiment was to determine if the observed effect of HRG on the activity of anti-estrogen therapies (e.g., fulvestrant) and CDK4-6 inhibitors (e.g., palbociclib) or their combinations is specific to HRG or if there is a broader effect that might be mediated by other growth promoting RTK ligands found in various cancers. To test this hypothesis, the ER+ HER2− cell lines MCF7 (
EGF Family Ligands:
Other Ligands:
The data indicate that HRG is the most effective ligand out of all the ligands tested at inhibiting fulvestrant activity, palbociclib activity, and the combination of palbociclib and fulvestrant. These include both EGF family ligands and other ligands such as E2, IGF1 and FGF2.
The objective of this experiment was to determine the effect of HRG on the activity of palbociclib and fulvestrant at the level of cell cycle progression. In addition, this experiment was designed to determine if seribantumab restores the cell cycle inhibitory activity of the individual components or the additive activity of a clinically approved drug combination by blocking the effect of HRG.
MCF7 cells were treated with combinations of palbociclib, fulvestrant, HRG and seribantumab for 24 hours, pulse-labelled with 10 μM EdU for 2 hours, fixed, double stained with Click-iT® EdU Alexa Fluor® 488 and FxCycle™ Violet stain and analyzed by flow cytometry.
The data indicate that HRG can promote S-phase cell cycle progression of ER+ HER2− breast cancer cells (
Finally, since both palbociclib and fulvestrant are commonly used in combination in ER+, HER2− breast cancer patients, the effect of HRG on the activity of the combination and whether seribantumab could restore this activity was tested. Consistent with our previous findings, HRG blocked the cell cycle inhibitory activity of the palbociclib-fulvestrant combination and seribantumab restored this activity (
The purpose of this experiment was to determine if the addition of seribantumab to either fulvestrant or palbociclib or their combination increases efficacy in an orthotopic xenograph model of ER+ HER2− breast cancer.
Female SHO mice (Charles River Laboratories) aged 8-10 weeks were implanted with 17-beta-estradiol pellets (0.72 mg/pellet) and 2 days later, injected with 100 μl of 5×106 MCF7 cells suspended in 50% DPBS/50% matrigel into the abdominal mammary fat pad. Tumor growth was monitored twice a week and tumor volumes were calculated following external caliper measurement according to the formula (tumor size=π/6×[length×width2]). Once the average measured tumor volume had reached approximately 200 mm3, mice were randomized into groups and treatment was administered. Overall, the average starting tumor volume per group was equivalent across all groups. Fulvestrant was dosed at 500 μg per mouse once per week via subcutaneous delivery. Palbociclib was dosed at 25 mg/kg, orally every day for 5 days, Monday through Friday. Seribantumab was dosed a 600 μg per mouse twice per week via intraperitoneal injection.
This data is consistent with the broader in vitro findings that HRG can block the activity of anti-endocrine therapies such as tamoxifen or fulvestrant, CDK4-6 inhibitors (e.g., palbociclib, ribociclib or abemaciclib), and combinations thereof.
The purpose of this experiment was to examine the effect of HRG on the key cell cycle protein RB which is involved in mediating cell cycle progression via CDK4/6 activity. CDK4/6 inhibitors (e.g., palbociclib, ribociclib and abemaciclib) have a mechanism of action that is dependent on the cyclin D-CDK 4/6 complex and Rb protein. CDK4/6 inhibitors cause dephosphorylation the Rb protein, which represses transcription of the E2F gene and thus cell cycle inhibition.
MCF7 cells were cultured as described above. Cells were treated with 10 nM HRG, 50 nM fulvestrant, 40 nM palbociclib, 40 nM abemaciclib or 1 uM of seribantumab either alone or in combination for 20-24 hours as shown in
A model of post-menopausal ER+ breast cancer was used to determine the effect of blocking HRG-mediated ERB3 signaling and/or estrogen-mediated ER activation on tumor growth (
The MCF-7Ca-derived xenograft tumors initially responded to letrozole, but started to develop resistance after approximately 7-8 weeks of treatment (
Such treatment regimens result in a beneficial result, e.g., stable disease, a partial response, or a complete response.
In some instances, the patient meets some or any of the following inclusion criteria:
In some instances, the patient does not meet any of the following exclusion criteria:
This application claims priority to U.S. Provisional Application Nos. 62/308,783 filed Mar. 15, 2016, 62/356,127 filed Jun. 29, 2016 and 62/431,242, filed Dec. 7, 2016. The contents of the aforementioned applications are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/022517 | 3/15/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62431242 | Dec 2016 | US | |
| 62356127 | Jun 2016 | US | |
| 62308783 | Mar 2016 | US |
