Patent Application
20160130365
Methods are provided for prognosing, diagnosing, and treating cancers associated with ADAM8, for reducing or preventing metastasis of established tumors, and for identifying drugs that target ADAM8.
Cancer metastasis results from a multistep process that selects for invasive tumor cells capable of escaping from the primary site and colonizing distant organs. Early in the tumorigenesis process, the hypoxic stress that characterizes the low tissue oxygen microenvironment of a solid tumor can stimulate tumor-induced angiogenesis, which in turn supplies nutrients and oxygen necessary to prevent tumor dormancy and support tumor growth, as well as provides routes for tumor cell dissemination (Harris et al. Nat Rev Cancer 2:38-47, 2002; Majmundar et al. Mol Cell 40:294-309, 2010). Secretion of proangiogenic mediators by tumor cells, most notably vascular endothelial growth factor A (VEGF-A), promotes endothelial cell migration and proliferation (Adams and Alitalo Nat Rev Mol Cell Biol 8:464-478, 2007). The resulting neovascularization of the surrounding stroma facilitates the spreading of tumor cells into the blood stream. The process involves adherence to and transmigration through the endothelium, in particular via processes involving integrins (Avraamides et al. Nat Rev Cancer 8:604-617, 2008; Mierke J Biophys 2008:183516, 2008). Circulating tumor cells (CTCs) can be measured in the peripheral blood even before the tumor is clinically detectable (Eyles et al. J Clin Invest 120:2030-2039, 2010). The concentration of CTCs in breast cancer patients has been associated with high risk for disease recurrence and poor outcome (Cristofanilli et al. N Engl J Med 351:781-791, 2004). Moss et al. shows a method for predicting cancer disease status using assays of urine samples with a fluorescent substrate to detect metalloproteinase (MMP) activity of a large number of MMP family members, including ADAM8 and ADAM12 (WO 2012/088105 A2 published Jun. 28, 2012).
More than 500,000 women worldwide die yearly of metastatic breast disease despite recent advances in the treatment of cancer according to reports from the World Health Organization. Identifying new therapeutic targets to effectively block breast cancer dissemination is crucial to improve the overall survival of patients at high risk. In particular, women with triple-negative breast cancer (TNBC) whose tumors are characterized as negative for estrogen receptor, progesterone receptor, and HER2 (also known as HER2/neu) lack targeted therapy (Boyle Ann Oncol 23 Suppl 6:vi7-12, 2012).
In various embodiments, a method of treating a cancer or a metastasis in a subject, in which cells of the cancer or the metastasis over express ADAM8 protein compared to normal cells includes the steps of: administering to the subject a composition having a therapeutically effective amount of at least one antibody or antibody fragment that binds to at least one extracellular portion (ectodomain) of ADAM8 protein and inhibits at least one activity of ADAM8; and, monitoring effect of the composition on at least one symptom selected from the group consisting of: growth or angiogenesis of a primary or metastatic tumor, presence of circulating tumor cells, and appearance of a new metastasis. In certain embodiments, prior to administering, the antibody is produced by immunizing with an antigen including an amino acid sequence of an ADAM8 protein domain selected from the group consisting of: a metalloproteinase domain (MP), a disintegrin domain (DI), a cysteine-rich domain (CRD), and an EGF-like domain (ELD). In an aspect of an embodiment of the method, the at least one antibody or antibody fragment is selected from a library of antibodies produced in a humanized mammal.
In an embodiment of the method, the cancer is selected from breast, brain, lung, prostate, skin, ovarian, colorectal, bone, renal, head and neck, esophageal, gastric, liver, bladder, cervical, uterine, thyroid, testicular, and pancreatic cancers. In certain embodiments, the breast cancer has at least one characteristic selected from the group consisting of: estrogen receptor negative, progesterone receptor negative, HER2 negative, triple-negative inflammatory, triple-negative, basal-like, invasive, ductal, ductal carcinoma in situ (DCIS), lobular, HER2 positive, and mixed breast cancer. In certain embodiments, the method includes treating the cancer or metastasis by observing a decrease in size of an established tumor or a decrease in size or frequency of metastases.
In various embodiments, the antibody is obtained by immunizing with a peptide from an ADAM8 protein having an amino acid sequence selected from any of: SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; and SEQ ID NO: 9. Alternatively, the antibody is obtained by immunizing with a peptide including an amino acid sequence from ADAM8 protein amino acid residues 158 to 653 corresponding to the ectodomain. In certain embodiments, the method further includes monitoring by measuring a decrease of a symptom of the cancer or metastasis progression. In certain embodiments, the symptom is selected from the group consisting of: size of primary tumor; growth of primary tumor; number of metastases; size of metastases; number of circulating tumor cells in blood; vascularization in tissue adjacent to the tumor or metastases; amount of ADAM8 in body fluids; amount of VEGF-A in body fluids; and, weight loss of the subject.
The sequence listing material in computer readable form ASCII text file (18 kilobytes) created Nov. 12, 2015 entitled “34724-163_SequenceListing_11132015”, containing sequence listings numbers 1-9, has been electronically filed herewith and is incorporated by reference herein in its entirety.
The ADAM8 activity in various embodiments is selected from the group consisting of: ADAM8 metalloproteinase enzyme activity; release of VEGF-A; promotion of angiogenesis; and extravasation or intravasation of metastatic cancer cells through vascular endothelium; and activation of β1-integrin.
An embodiment of the invention provides a method of diagnosing or prognosing a cancer condition in a patient to select a treatment for the patient, the method including analyzing amount of ADAM8 protein or expression of ADAM8 protein in the patient or a patient tumor in comparison to a normal sample from a normal subject or from an unaffected tissue from the patient, in which an increase of ADAM8 amount in the patient in comparison to the normal sample or from the unaffected tissue is an indicium of presence of the ADAM8-associated cancer; and, selecting a course of treatment for the patient, such that the indicium of the cancer that is ADAM8-associated determines selecting the treatment having at least one therapeutic agent that inhibits an ADAM8 activity or reduces expression of ADAM8. In an embodiment of the invention, the therapeutic agent down modulates or eliminates ADAM8 expression.
In various embodiments, the method further includes administering to the patient a therapeutic agent which is a therapeutically effective amount of at least one antibody or antibody fragment that binds to an extracellular portion of the ADAM8 protein. The at least one antibody or antibody fragment in certain embodiments is two or more antibodies or antibody fragments.
In various embodiments of the method, the step of analyzing further includes measuring ADAM8 expression using at least one technique selected from the group consisting of immunohistochemistry (IHC), mass spectrometry, enzyme-linked immunosorbant (ELISA), immunocytochemistry, immunofluorescence, flow cytometry, quantification of ADAM8-associated miRNA, and western blotting.
In various embodiments, the condition diagnosed is selected from the group of cancers consisting of breast, brain, lung, prostate, skin, colorectal, ovarian, bone, renal, head and neck, esophageal, gastric, liver, bladder, cervical, testicular, uterine, thyroid, and pancreatic cancers. The method includes breast cancer that has at least one characteristic selected from the group consisting of estrogen receptor negative, progesterone receptor negative, HER2 negative, triple-negative, triple-negative inflammatory, basal-like, invasive, ductal, ductal carcinoma in situ (DCIS), lobular, HER2 positive, and mixed breast cancer. In various embodiments, the cancer is metastatic. Alternatively, the method diagnoses or prognoses cancer that is a primary tumor absent metastasis.
In various embodiments, the method further includes selecting the at least one antibody or antibody fragment that binds at least one extracellular domain selected from a group consisting of a cysteine-rich (CRD), a metalloproteinase (MP), a disintegrin (DI), and an epidermal growth factor-like (ELD) domains for administration to the patient.
In certain embodiments, the antibody or antibody fragment binds to and/or inhibits the activity of a plurality of ectodomains, for example, the metalloproteinase domain and the disintegrin domain. In an embodiment of the method, the antibody or antibody fragment is recombinantly produced.
In an embodiment of the method, the antibody or antibody fragment is at least one of IgG, IgM, and IgA.
In various embodiments of the method, the antibody or antibody fragment is monoclonal. Alternatively, the antibody or antibody fragment is polyclonal.
In various embodiments, at least one of the antibody, the antibody fragment, or a binding region of the antibody or antibody fragment is selected from the group of: a single chain antibody (scFv), a recombinant camelid heavy-chain-only antibody (VHH), a shark heavy-chain-only antibody (VNAR), a microprotein, a darpin, an anticalin, an adnectin, an aptamer, an Fv, a Fab, a Fab′, and a F(ab′)2.
In various alternative embodiments of the invention, the antibody or antibody fragment is an immunoconjugate. In an embodiment of the method, the immunoconjugate antibody or antibody fragment is bound to at least one of: a chemotherapeutic agent, a photosensitizing agent, a cytotoxic natural product, a phytotoxin, a cytotoxic antibiotic, a bacterial toxin, a fungal toxin, a bioactive protein, a radioisotype, an enzyme, and one or more enzymes that activates an inert pre-cytotoxic agent.
In various embodiments of the method, the antibody or antibody fragment includes a first antibody or antibody fragment that binds to a disintegrin domain (DI) and a second antibody or antibody fragment that binds to a metalloproteinase domain (MP). Alternatively, the at least one antibody or antibody fragment includes a first antibody or antibody fragment that binds to a cysteine-rich domain (CRD) and a second antibody or antibody fragment that binds to an EGF-like domain (ELD).
In various embodiments, the method after administering further includes measuring a decrease of at least one symptom selected from the group consisting of: primary tumor size, number of metastatic tumors, size of metastatic tumors, formation of new metastatic lesions, angiogenesis, and concentration of circulating tumor cells.
An aspect of the invention provides a method of treating a cancer or a metastasis in a subject, in which the cells of the cancer or the metastasis over express ADAM8 compared to normal cells, the method including administering to the subject a composition having a therapeutically effective amount of at least one antibody or antibody fragment that binds to at least one extracellular portion (ectodomain) of ADAM8 protein and inhibits at least one activity of ADAM8; and monitoring effect of the composition on at least one symptom selected from the group consisting of: growth or angiogenesis of a primary or metastatic tumor, presence of circulating tumor cells, and appearance of a new metastasis. The antibody or antibody fragment binds to at least one ectodomain, for example, the antibody or antibody fragment binds to two ectodomains.
In various embodiments, the method includes prior to administering producing the antibody by immunizing with an antigen including an amino acid sequence of an ADAM8 protein domain selected from at least one: an EGF-like domain (ELD); a cysteine-rich domain (CRD); a disintegrin domain (DI); and a metalloproteinase domain (MD). Alternatively, the antibody is selected from a library of antibodies produced in a humanized mammal. In various embodiments, the at least one antibody or antibody fragment is selected from a yeast display library, Bacillus spore display library, or a phage display library by testing for binding to an ADAM8 protein ectodomain. The antibody or antibody fragment binds to at least one ectodomain, for example, binds to the disintegrin domain (DI), the metalloproteinase domain (MP), or both.
In various embodiments, the method of screening a library of compounds to identify a candidate therapeutic agent to treat a cancer includes contacting a first sample having extracellular ADAM8 protein with at least one compound, and contacting a second sample having extracellular ADAM8 protein with a control lacking the compound and otherwise identical; analyzing amounts of an ADAM8-associated activity in the first and second samples, in which reduced activity in the first sample compared to the second sample identifies the compound as the candidate therapeutic agent; and, testing the compound for at least one of chemical, structural, and biological functional properties.
In an embodiment of the method, the screening further includes testing the compound for ADAM8 specificity to confirm that the compound is not cross-reactive against at least one protein selected from the group consisting of: ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, and ADAM33. In certain embodiments, the first and second ADAM8 samples are extracts or purified proteins in vitro. In an embodiment of the method the activity in the first sample that is reduced is selected from the group consisting of ADAM8 metalloproteinase enzyme activity, release of VEGF-A, promotion of angiogenesis, extravasation of metastatic cancer cells through vascular endothelium, intravasation or extravasation of metastatic cancer cells through vascular endothelium, extravasation of metastatic cancer cells through vascular endothelium, and activation of β1-integrin.
An aspect of the invention provides a pharmaceutical composition that includes a therapeutically effective amount of at least one antibody, antibody fragment, or compound that binds specifically to at least one of the group: ADAM8, a fragment of ADAM8, and a short nucleic acid expressing a modulator for ADAM8, and the antibody or antibody fragment inhibits at least one activity of ADAM8 or reducing expression of ADAM8. In an embodiment, the antibody or antibody fragment specifically binds or inhibits an ADAM8 domain or a portion of at least one domain of ADAM8 selected from the group: metalloproteinase domain, a disintegrin domain, a cysteine-rich domain, and an EGF-like domain. An embodiment provides the composition formulated for systemic delivery to a subject. An embodiment of the route of delivery is an injection. In an embodiment, the pharmaceutical composition is delivered to the subject that is a human. Alternatively, the subject is a high value animal.
In various embodiments, the pharmaceutical composition further includes at least one therapeutic agent selected from the group consisting of: a growth factor, an anti-inflammatory agent, an ADAM8 knockdown, and an inhibitor of ADAM8 synthesis. In an embodiment, the ADAM8 knockdown composition is at least one of the group: short interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA) inhibitor of ADAM8 synthesis. In an aspect of the invention, the antibody or antibody fragment inhibits an ADAM8-associated miRNA.
In various embodiments, the vasopressor agent of the pharmaceutical composition is at least one selected from the group consisting of: nitric oxide, calcium channel blockers, collagenase inhibitors, topical steroids, matrix metalloproteinase inhibitors, ascorbates, angiotensin II, angiotensin III, calreticulin, tetracyclines, fibronectin, collagen, thrombospondin, transforming growth factors, (TGF), keratinocyte growth factors (KGF), fibroblast growth factor (FGF), insulin-like growth factors (IGF), IGF binding proteins, epidermal growth factors (EGF), platelet derived growth factor (PDGF), neu differentiation factor, hepatocyte growth factor, vascular endothelial growth factor, heparin-binding EGF, von Willebrand Factor-C, heparin, heparin sulfates, and hyaluronic acid.
In various embodiments, the pharmaceutical composition further includes at least one from the group consisting of an anti-coagulant agent, anti-tumor agent, anti-viral agent, anti-bacterial agent, anti-myobacterial agent, anti-fungal agent, anti-proliferative agent, and anti-apoptotic agent.
An embodiment of the composition is formulated in a unit dosage.
In various embodiments, the antibody or antibody fragment of the pharmaceutical composition is monoclonal. Alternatively, the antibody or antibody fragment is polyclonal. In various embodiments, the antibody, antibody fragment, or compound is provided in a dosage effective to decrease at least one symptom selected from the group consisting of: growth or angiogenesis of a primary or metastatic tumor, presence of circulating tumor cells, and appearance of new metastases. In an embodiment of the composition, the antibody or antibody fragment is recombinantly produced.
In various embodiments of the pharmaceutical composition, the antibody, antibody fragment, or at least one binding region of the antibody or antibody fragment are selected from the group of: a single chain antibody (scFv), a recombinant camelid heavy-chain-only antibody (VHH), a shark heavy-chain-only antibody (VNAR), a microprotein, a darpin, an anticalin, and adnectin, an aptamer, an Fv, a Fab, a Fab′, and a F(ab′)2.
In various embodiments, the antibody or antibody fragment is provided in a dosage effective to decrease a symptom of a cancer or metastasis progression. For example, the symptom is selected from the group consisting of: size of primary tumor, growth of primary tumor, number of metastases, size of metastases; number of circulating tumor cells in blood, vascularization in tissue adjacent to the tumor or metastases, amount of ADAM8 in body fluids, amount of VEGF-A in body fluids, and weight loss of the subject.
In various embodiments, the therapeutically effective amount is formulated in a dosage within the range of about 0.1 mg to about 500 mg. In certain embodiments, the dosage is selected from about 0.1 mg, about 1 mg, about 5 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, and about 500 mg.
In various embodiments, the unit dose is calculated from the weight of a subject in the range of about 0.01 mg/kg to about 200 mg/kg. For example, a unit dose is calculated from the weight of a subject in the range of about 0.1 mg/kg to about 10 mg/kg, or about 0.5 mg/kg to about 50 mg/kg, or about 1 mg/kg to about 100 mg/kg, or about 1.5 mg/kg to about 200 mg/kg, or about 0.5 mg/kg to about 1.5 mg/kg, or about 0.01 mg/kg to about 0.5 mg/kg. In various embodiments, the composition is administered on at least one regimen selected from the group consisting of: daily, weekly, biweekly, and alternating weeks
In certain embodiments, the at least one antibody or antibody fragment is an immunoconjugate. In certain embodiments, the immunoconjugate antibody or antibody fragment is bound to at least one selected from the group of a chemotherapeutic agent, a photosensitizing agent, a cytotoxic natural product, a phytotoxins, a cytotoxic antibiotic, a bacterial toxin, a fungal toxin, a bioactive protein, a radioisotype, an enzyme, and a combination of enzymes that activate an inert pre-cytotoxic agent.
In various embodiments, a method of formulating a pharmaceutical composition for treating a cancer includes diagnosing by analyzing amount of ADAM8 protein or expression of ADAM8 protein in a patient or in a patient tumor in comparison to a normal sample from a normal subject or from an unaffected tissue from the patient, suxh that an increase of ADAM8 amount in the patient in comparison to the normal sample or the unaffected tissue is an indicium of presence of an ADAM8-associated cancer; and, formulating the composition to contain a therapeutically effective amount of an antibody or an antibody fragment that binds to an extracellular portion (ectodomain) of ADAM8 protein and inhibits at least one activity of ADAM8.
In certain embodiments, the therapeutically effective amount is formulated in a dosage selected from the group consisting of: about 1 mg, about 5 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, and about 500 mg. For example, the therapeutically effective amount is formulated in a unit dose. In certain embodiments, the method according to claim 64, wherein the unit dose is calculated from the weight of the patient, wherein the dose/weight of the patient is a range from about 0.01 mg/kg to about 200 mg/kg.
In an aspect of the method, the ectodomain of ADAM8 is at least one selected from the group of ectodomains consisting of: a metalloproteinase domain (MP), a disintegrin domain (DI), a cysteine-rich domain (CRD), and an EGF-like domain (ELD). In certain embodiments, the composition comprises a plurality of antibodies or antibody fragments, a first antibody or antibody fragment that binds to a first ectodomain and a second antibody or antibody fragment that binds to a second ectodomain. In an aspect of the method, the first ectodomain and the second ectodomain are the metalloproteinase domain (MP) and the disintegrin domain (DI). In certain embodiments, the antibody or antibody fragment binds to and inhibits at least two ectodomain activities. In an aspect of the method, the antibody or antibody fragment binds to and inhibits the metalloproteinase (MP) domain and the disintegrin domain (DI). In certain embodiments, the antibody or antibody fragment binds to at least one ectodomain and inhibits the metalloproteinase domain (MP) and the disintegrin domain (DI).
ADAM8 belongs to a large family of proteins consisting of more than 30 members. Synthesized as a proform, ADAM8 can dimerize or multimerize and autocatalytically clip off its prodomain, leaving an active membrane-anchored metalloproteinase (
An aspect of the invention provides a composition, for example, an antibody or peptide mimetic directed against the ADAM8 ectodomain region that further inhibits growth and/or spreading of cancer cells in patients with breast tumors that express ADAM8. In particular, we claim that an antibody against a region spanning part or all of the cysteine-rich/EGF-like domains, or the metalloproteinase/disintegrin domains will be effective. The term “against” as used herein refers to antibody specificity, such that an antibody against a polypeptide has affinity to recognize and bind to that peptide. Furthermore, as approximately half of breast cancer-derived metastases displayed ADAM8 expression, ADAM8 or downstream signaling molecules are useful as biomarkers for disease progression. Identifying new therapeutic targets to effectively block breast cancer dissemination is crucial to improve the overall survival of patients at high risk, e.g., women with triple-negative breast tumors. Since ADAM8 is expressed also in other human cancers, including brain, lung, prostate, skin, colorectal, ovarian, bone, renal, head and neck, esophageal, gastric, liver, bladder, cervical, testicular, uterine, thyroid, and pancreatic cancers, inhibitors of ADAM8 should also be effective in the treatment of patients with these types of tumors. Further, ADAM8 or downstream signaling molecules can be used as biomarkers for diagnosis of these cancers.
ADAM8 is a transmembrane protein that belongs to the ADAM (a disintegrin and metalloproteinase) family and mediates cell adhesion, cell migration, and proteolysis of a variety of substrates, including cytokine receptors or ligands, cell adhesion molecules and extracellular matrix components (Koller et al. Curr Pharm Des 15:2272-2281, 2009). Synthesized as a proform, ADAM8 can dimerize or multimerize and autocatalytically clip off its prodomain, leaving an active membrane-anchored metalloproteinase (
ADAM8 was found to be non-essential under physiological conditions, as evidenced by the normal development and lack of pathological defects in ADAM8 deficient mice (Kelly et al. Dev Dyn 232:221-231, 2005), despite its expression in a variety of immune cells (Richens et al. Immunobiology 212:29-38, 2007 and Yoshiyama et al. Genomics 41:56-62, 1997). ADAM8 expression was detected under several pathological conditions characterized by inflammation and extracellular matrix remodeling, including cancer (Koller et al. Curr Pharm Des 15:2272-2281, 2009). Using microarray analysis, ADAM8 was identified as a target of a multistep pathway downstream of NF-κB RelB, which is known to promote an aggressive phenotype in breast cancer (Wang et al. Nat Cell Biol 9:470-478, 2007). Analysis herein of publically available microarray databases indicated that ADAM8 is expressed in aggressive breast cancers, including TNBC, and correlated with a poor patient outcome (
Data herein show that ADAM8 is required to maintain the aggressive phenotype of TNBC cells in three dimensional (3D) culture, and promotes angiogenesis, spread of CTCs, and metastatic dissemination in orthotopic breast and cardiac mouse models. The term “orthotopic” as used herein conventionally refers to a disease model affecting the same tissue and location as in the original disease.
Using the orthotopic models herein, ADAM8 was found to induce both vascular remodeling by the release of soluble pro-angiogenic factors, including VEGF-A (vascular endothelial growth factor A), and adhesion and transmigration through the endothelium following β1-integrin activation. Overall, these findings indicate that the transmembrane ADAM8 protein constitutes a therapeutic target for treatment of aggressive breast tumors.
Results herein from analyzing samples obtained from publically available cancer microarray datasets, show that ADAM8 mRNA levels were significantly elevated in breast cancer in comparison to normal breast tissue, and more specifically in aggressive subtypes including estrogen receptor-negative breast tumors. Furthermore, immunohistochemistical analysis of a cohort of patients with triple-negative breast tumors (negative for estrogen receptor, progesterone receptor, and HER2) revealed that 34% ( 17/50) were positive for ADAM8 protein, and that adjacent normal tissues were negative. Importantly, 48% ( 27/56) of all breast tumor metastases in the samples were positive for ADAM8. Consistently high ADAM8 mRNA levels were observed to be significantly correlated with poor metastasis relapse-free survival.
ADAM8 was tested herein within breast cancer cells in culture, and data show that this protein plays an essential role in migratory and invasive properties of cancer cells. In particular, ADAM8 promoted formation of invasive branching colonies and anchorage-independent growth in 3D assays of TNBC cells. An orthotopic xenograft mouse model was used to show that tumors derived from MDA-MB-231 TNBC cells in which ADAM8 was knocked down by a specific shRNA, failed to grow beyond a palpable size. In contrast, tumors derived from shControl MDA-MB-231 cells grew to an average size of 1 cm3 over the time course. ADAM8 was observed to be induced in breast cancer cells cultured under hypoxic conditions. High ADAM8 expression in vivo was detected around necrotic areas within tumors derived from shControl MDA-MB-231 cells but not in those from shADAM8 cells. shADAM8 tumors were unable to induce angiogenesis and hence remained dormant. Endothelial vessel density was assessed by immunohistochemical staining for the angiogenic marker CD31 in both tumor groups. CD31 staining revealed that vessel density (angiogenesis) was significantly decreased at the peritumoral area of shADAM8 compared to the shControl MDA-MB-231 cell-derived tumors. All animals injected with shControl cells were positive for brain macrometastases. In contrast, only one mouse had a small metastasis in the shADAM8 group.
To determine the mechanism by which ADAM8 induces angiogenesis, the effect of conditioned medium obtained from MDA-MB-231 cells on ability of Human Umbilical Vein Endothelial Cells (HUVECs) to form vessel-like structures (tube formation) was tested. Conditioned media from shControl MDA-MB-231 cells poorly promoted tube formation. Conditioned media from shADAM8 MDA-MB-231 did not promote tube formation. Furthermore, release of Vascular Endothelial Growth Factor (VEGF)-A and four other angiogenesis mediators was observed to be reduced in the conditioned medium obtained from shADAM8 MDA-MB-231 cells compared to shControl cells using an angiogenesis protein array. Further, the metalloproteinase domain of ADAM8 was found to be required for the release of VEGF-A.
Binding of primary tumor cells and then circulating tumor cells (CTCs) to an endothelial layer followed by transmigration of the CTCs through the blood vessel wall (intravasation and extravasation, respectively) are critical steps in metastasis. ADAM8 knockdown was observed to reduce binding of MDA-MB-231 cells to HUVECs in culture, i.e., shControl cells were found to adhere much more efficiently than shADAM8 cells to a confluent HUVEC monolayer. Furthermore, in a transendothelial cell migration (TEM) assay, shADAM8 cells displayed a profoundly decreased ability to migrate through a confluent HUVEC monolayer in comparison to shControl cells. Thus, ADAM8 promotes tumor cell adhesion onto an endothelial layer, as is found in a vessel wall, and migration through the vessel wall.
The role of ADAM8 involvement in the modulation of β1-integrin activation, a critical step in adhesion of cancer cells to the endothelium, was investigated. Pre-treatment of shControl cells with an antagonist antibody targeting β1-integrin greatly reduced the ability of the cells to bind to HUVECs. Monoclonal antibodies targeting either the cysteine-rich/EGF-like or the metalloproteinase/disintegrin extracellular domains of ADAM8, caused inhibition of adhesion of shControl cells onto HUVECs essentially to the same extent as the β1-integrin antibody, and similarly prevented transmigration of the cells in a TEM assay.
Examples herein show that mice with shADAM8 tumors had fewer CTCs compared to those with shControl tumors, as revealed by analysis of blood samples. The result is consistent with a role for ADAM8 in adhesion and migration of cancer cells from the primary tumor site through induction of angiogenesis and interactions with the endothelium. This effect was observed even at early time points after injection of tumor cells into the mouse mammary fat pad when tumors from both groups were barely palpable and did not show any significant difference in size.
To validate ADAM8 as a therapeutic target for TNBC, the Mab1031 ADAM8 antibody targeting the MP/DI domains was selected for use in an orthotopic model.
Mice were treated with either 0.5 mg/kg of Mab1031 antibody (n=9) or with this dose of a control isotype-matched IgG2B (n=8) by i.p. injection twice weekly from the day of implantation of shCtrl-3 cells. Anti-ADAM8 treatment was observed to result in a significantly decreased primary tumor burden by about 50% (
To test potential use of this ADAM8 antibody in a more clinically relevant system, a tumor resection model was used (
Mice were injected in the MFP with MDA-MB-231 shCtrl-3 cells, and palpable tumors were observed 12 days later. Treatment was initiated with 1.5 mg/kg of Mab1031 anti-ADAM8 antibody or isotype control IgG2B. Additional doses of antibody were administered on day 15 and on day 17, at which point tumors reached a volume of about 0.15 cm3 and tumor resection was performed (
In the control group, metastases to the brain and lungs were seen in almost all of the subjects (8/8 and 7/8), respectively. In contrast in the anti-ADAM8-treated group, only 2/9 and 1/9 mice showed metastases in the brains and lungs, respectively. Thus, treatment with an anti-ADAM8 MP/DI antibody resulted in a profound reduction in frequency of metastases to each of the brain and lungs compared to the frequency of metastases in the isotype control antibody treated group.
To test the role of ADAM8 in the ability of breast cancer cells to colonize typical distant metastasis sites, MDA-MB-231-derived shCtrl-3 and shA8-20 cells, which stably express Luciferase, were injected in the left ventricle of female NOD/SCID mice (n=4/group). Total tumor burden was monitored weekly by Luciferase activity measurement using the Xenogen Biophotonic Imaging system. Organ and bone metastases were assessed at sacrifice using this method.
Cardiac injection of MDA-MB-231-derived shA8-20 cells resulted in a significantly reduced total tumor burden after 2 and 3 weeks compared to injection of shCtrl-3 cells (
Since both the Mab10311 (CRD/ELD) and the Mab1031 (MP/DI) antibodies reduced amount of transendothelial cell migration to about the same extent (
The Mab10311 antibody was observed to have inhibited the MP activity about 40%, a lesser extent than Mab1031, 70%. (
After three weeks, no significant change in primary tumor burden was observed as a result of treatment with anti-ADAM8 Mab10311 (
A portion of the invention herein has been published in a paper entitled, “ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis”, EMBO Molecular Medicine 6(2): 278-294, 2014, published online Dec. 27, 2013, by co-authors Mathilde Romagnoli, Nora D. Mineva, Michael Polmear, Catharina Conrad, Srimathi Srinivasan, Delphine Loussouarn, Sophie Barillé-Nion, Irene Georgakoudi, Áine Dagg, Enda W. McDermott, Michael J. Duffy, Patricia M. McGowan, Uwe Schlomann, Maddy Parsons, Joerg W. Bartsch, and Gail E. Sonenshein, and is hereby incorporated herein by reference in its entirety.
The invention having now been fully described is exemplified by the following examples and claims, which are exemplary and are not to be construed as further limiting. All references cited are hereby incorporated herein by reference.
The present invention relates to compositions, methods, and kits for prognosing, diagnosing and treating ADAM8-related cancer conditions. The term “antibody” as used herein includes whole antibodies and any antigen binding fragment (i.e., “antigen-binding portion”) or single chains of these. A naturally occurring “antibody” is a glycoprotein including at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or is a camelid antibody having a single chain.
As used herein, an antibody that “specifically binds” a target protein or cell is intended to refer to an antibody that binds to a domain or portion of the target having an amino acid sequence. For example, the antibody has a KD of 5×10−9 M or less, 2×10−9 M or less, or 1×10−10 M or less. For example, the antibody is monoclonal. Alternatively, the antibody is polyclonal. The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for the target or for a particular epitope of target. The antibody is an IgM, IgE, IgG such as IgG1 or IgG2b, and IgA.
Also useful for systems, method, and kits herein is an antibody that is recombinantly produced. The term “recombinant human antibody”, as used herein, includes all anti-ADAM8 or control antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from human genes expressed in an animal (e.g., mouse, pig, goat, rabbit, sheep, primate, monkey, dog, and high value animals). Mammalian host cells for expressing the recombinant anti-ADAM8 antibodies used in the methods herein include Chinese Hamster Ovary (CHO cells) including dhfr-CHO cells and NSO myeloma cells, (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980) used with a DHFR selectable marker (e.g., R. J. Kaufman et al. Mol. Biol. 159:601-621, 1982), COS cells, and SP2 cells. Analysis was performed with the GS gene expression system shown in WO 87/04462, WO 89/01036, and EP 338,841.
To produce antibodies, expression vectors encoding anti-ADAM8 or control antibody genes are introduced into mammalian, yeast, or bacterial host cells, and the host cells are cultured for a period of time sufficient for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Antibodies are recovered from the culture medium using standard protein purification methods.
Standard assays to evaluate the binding ability of the antibodies toward any of the various ADAM8 target species ectodomain antigens are known in the art, including for example, ELISAs, western blots, and RIPAs. The binding kinetics (e.g., binding affinity) of the antibodies are assessed by standard assays known in the art, such as by Biacore analysis.
General methodologies for antibody production, including criteria to be considered when choosing an animal or a cell line for the production of antisera, are described in Harlow et al., Antibodies, Cold Spring Harbor Laboratory, pp. 93-117, 1988. For example, an animal of suitable size such as a mouse, pig, goat, rabbit, sheep, primate, monkey, dog, camel, or high value animal, is immunized by administration of an amount of immunogen, such as the intact protein or a portion thereof containing an epitope which is an amino acid sequence from a human ADAM8 ectodomain, effective to produce an immune response. In certain embodiments, the animal is subcutaneously injected in the back with 100 micrograms to 100 milligrams of antigen, dependent on the size of the animal, followed three weeks later with an intraperitoneal injection of 100 micrograms to 100 milligrams of immunogen dependent on the size of the animal with adjuvant, for example Freund's complete adjuvant. Additional i.p. injections every two weeks with adjuvant, for example Freund's incomplete adjuvant, are administered until a suitable titer of antibody in the animal's blood is achieved. Exemplary titers include a titer of at least about 1:5000 or a titer of 1:100,000 or more, i.e., the dilution having a detectable activity. The antibodies are purified, for example, by affinity purification on columns containing the virulence factor or a portion thereof.
The technique of in vitro immunization of human lymphocytes is used to generate monoclonal antibodies. Techniques for in vitro immunization of human lymphocytes are well known to those skilled in the art (See, e.g., Inai et al. Histochemistry 99(5):335 362, 1993; Mulder et al. Hum. Immunol. 36(3):186 192, 1993; Harada et al. J. Oral Pathol. Med. 22(4):145 152, 1993; Stauber, et al. J. Immunol. Methods 161(2):157 168, 1993; Venkateswaran, et al. Hybridoma 11(6) 729 739, 1992). These techniques are used to produce antigen-reactive monoclonal antibodies, including antigen-specific IgG, IgA, and IgM monoclonal antibodies.
In various embodiments of the pharmaceutical composition, at least one of the anti-ADAM8 antibody or at least one binding region of the antibody is selected from the group of: a single chain antibody (scFv), a recombinant camelid heavy-chain-only antibody (VHH), a shark heavy-chain-only antibody (VNAR), a microprotein, a darpin, an anticalin, an adnectin, an aptamer, an Fv, a Fab, a Fab′, and a F(ab′)2.
Peptide libraries used to select an antibody that specifically binds to an ADAM8 protein ectodomain and can be constructed to be displayed on any of various vectors having viable units which are easily manipulated. For example, viable units include any of a phage display library, a Bacillus spore display library, an E. coli cell display library, and a yeast display library. A vector is used to transfect the viable unit with a gene encoding a protein of interest inserted into a gene expressed on the outside of the unit causing the protein of interest to be displayed on the outside of the phage, yeast cell, or Bacillus spore (Marks et al. Journal of Molecular Biology 222(3): 581-597, 5 Dec. 1991; Boder et al. Nature Biotechnology 15, 553-557, 1 Jun. 1997; Isticato et al. J. Bacteriol. 183(21): 6294-6301, 2001). In the Examples herein, the gene codes for an extracellular portion (ectodomain) of ADAM8 protein or for a protein that inhibits an ADAM8 activity.
An aspect of the present invention provides pharmaceutical compositions that include at least one of an antibody, antibody chain, or antibody fragment specific for at least part of a target protein which is ADAM8 protein or a fragment thereof, or at least one of a short nucleic acid expressing a modulator for the protein, for treating an ADAM8-associated cancer disorder by negatively modulating the amount of ADAM8 expression or activity of the ADAM8 protein or pathway. For example, the modulator is an anti-ADAM8 antibody linked to a toxin. In various embodiments, the pharmaceutical composition is compounded for systemic delivery to a subject, for example the composition is formulated as an injection. The composition in another embodiment is formulated as a systemic formulation for administration to the patient and may be compounded for delivery to affected tissues. In related embodiments, the pharmaceutical composition is formulated sufficiently pure for administration to a human subject, e.g., to the vascular system or endothelial system of a human patient in need of treatment for cancer.
In various embodiments, the compositions herein optionally further include one or more additional therapeutic agents, for example, known or new chemotherapeutic agents. In certain embodiments, the additional therapeutic agent or agents are selected from the group consisting of growth factors, anti-inflammatory agents, vasopressor agents including but not limited to nitric oxide and calcium channel blockers, collagenase inhibitors, topical steroids, matrix metalloproteinase inhibitors, ascorbates, angiotensin II, angiotensin III, calreticulin, tetracyclines, fibronectin, collagen, thrombospondin, transforming growth factors (TGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), epidermal growth factor (EGF), platelet derived growth factor (PDGF), neu differentiation factor (NDF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), heparin-binding EGF (HBEGF), thrombospondins, von Willebrand Factor-C, heparin and heparin sulfates, and hyaluronic acid.
In various embodiments, a plurality of therapeutic agents are included in the pharmaceutical composition to treat the same, a concurrent or a related symptom, condition or disease. In various embodiments, the therapeutic agent is a drug that may include without limitation anti-coagulant, anti-tumor, anti-viral, anti-bacterial, anti-mycobacterial, anti-fungal, anti-proliferative or anti-apoptotic agents. In particular, cancer patients frequently present with a bacterial or viral infection due to immune insufficiency, and comorbidity rates are elevated from bacterial pneumonia. Drugs to treat comorbid diagnoses such as bacterial infections that are included in the compositions of the invention are well known in the art. See for example, Goodman & Gilman's The Pharmacological Basis of Therapeutics, 13th Ed., Brenton, L. et al., eds., McGraw-Hill, 2010, the content of which is herein incorporated by reference in its entirety. For example, in certain embodiments, the therapeutic agent is azithromycin, clarithromycin, erythromycin, doxycycline, gemifloxacin, levofloxacin, moxifloxacin, cefaclor, cefadroxil, cefprozil, cefuroxime, cephalexin, amoxicillin, amoxicillin with clavulanate, ampicillin, piperacillin, ticarcillin with clavulanate, rifamycin, isoniazid, and vancomycin.
An antibody or antibody fragment in various embodiments is an anti-ADAM8 that binds to an ADAM8 ectodomain and is conjugated to an agent so that growth of a target tumor cell is inhibited or the target tumor cell is killed (U.S. Pat. No. 7,226,596). Antibodies are conjugated to anticancer agents, such as cytotoxic agents, chemotherapeutic agents, toxin, or a radioactive isotope, i.e., a radioconjugate to form an immunoconjugate (U.S. patent application publication number 2008/0175842). A variety of radionuclides are covalently bound or complexed as chelates to form radioconjugated antibodies (U.S. Pat. No. 7,226,596), e.g., 212Bi, 131I, 131In, 90Y and 186Re. (Ibid.). Toxins for immunoconjugates include diphtheria A chain, nonbinding active fragments of diphtheria toxin, cholera toxin, botulinus toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, saporin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. (U.S. patent application publication number 2008/0175842). Small molecule toxins for conjugating to anti-ADAM8 antibodies include calicheamicins (U.S. Pat. No. 5,053,394), maytansinoids (U.S. Pat. No. 5,208,020), palytoxin, and CC1065 (U.S. Pat. No. 7,226,596).
Bifunctional protein coupling agents are used to make antibody/cytotoxic agent immunoconjugates, e.g., N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds, such as 1,5-difluoro-2,4-dinitrobenzene (U.S. patent application publication number 2008/0175842). For example, preparation of a ricin immunotoxin is described in Vitetta et al. Science 238:1098 (1987) (Ibid.).
Two conjugated antibodies have been approved for use in the clinic. For example, T-DM1 (trastuzumab emtansine, the first antibody-drug conjugate) was approved February 2014 for patients with metastatic breast cancer that overexpresses HER2. T-DM1 is an immunoconjugate of trastuzumab with DM1, a chemotherapy drug.
As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences, 18th Ed.; Gennaro, Mack Publishing, Easton, Pa. (1995) provides various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Examples of materials which serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as glucose and sucrose; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, preservatives and antioxidants can also be present in the composition, the choice of agents and non-irritating concentrations to be determined according to the judgment of the formulator.
Methods provided herein involve contacting cancer or pre-cancerous or tumor cells or tissues with a pharmaceutical composition that modulates or inhibits ADAM8 amount or activity, for example, administering to a subject having the cancer or cells or tissue a therapeutically effective amount of a pharmaceutical composition having as an active agent at least one of an antibody specific for ADAM8, or a nucleic acid modulator (inhibitor) of expression of the ADAM8 protein or modulator of transcription of mRNA to ADAM8 protein, to the subject in need thereof, in such amounts and for such time as is necessary to achieve the desired result which is reduction of or preventing indicia of the cancer-related condition.
The compositions, according to the method of the present invention, may be administered using any amount and by any route of administration effective for treating the cancer-related disorder. Thus, the expression “amount effective for treating a cancer-related disease or condition”, as used herein, refers to a sufficient amount of the composition to beneficially prevent or ameliorate the symptoms of the disease or condition.
The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, e.g., liver function, cancer progression, and/or intermediate or advanced stage of macular degeneration; age, weight and gender of the patient; diet, time and frequency of administration; route of administration; drug combinations; reaction sensitivities; and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered hourly, twice hourly, every three to four hours, daily, twice daily, every three to four days, every week, or once every two weeks depending on half-life and clearance rate of the particular composition.
The active agents of the anti-ADAM8 compositions of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression “dosage unit form” as used herein refers to a physically discrete unit of active agent appropriate for the patient to be treated. The total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. For any active agent, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, as provided herein, usually mice, but also potentially from pigs, goats, rabbits, sheep, primates, monkeys, dogs, camels, or high value animals. The cell-based, animal, and in vivo models provided herein are also used to achieve a desirable concentration and total dosing range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active agent that ameliorates the symptoms or condition or prevents progression of the disease or condition. Therapeutic efficacy and toxicity of active agents are determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use.
The daily dosage of the products may be varied over a wide range, such as from 0.1 mg to 500 mg per adult human per day. For intravenous or local intra-tumoral administration, the compositions are provided for example in the form of a solution containing at least about 0.001 mg, about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.5 mg, about 1 mg, about 5 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 250 mg, or about 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
A unit dose typically contains from about 0.001 mg to about 500 mg of the active ingredient, preferably from about 0.1 mg to about 100 mg of active ingredient, more preferably from about 1.0 mg to about 10 mg of active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.01 mg/kg to about 200 mg/kg of body weight. For example, the range is from about 0.01 to 100 mg/kg of body weight per day, or from about 0.5 mg/kg to 50 mg/kg of body weight per day, or from about 1.0 mg/kg to about 25 mg/kg of body weight per day. The compositions may be administered on a regimen of, for example, one to four or more times per day or per week. A unit dose may be divided for example, administered in two or more divided doses.
Administration of a source of inhibitor of expression of the ADAM8 protein includes administration of a dose of a viral vector or a nucleic acid vector encoding the inhibitor of ADAM8 expression. For example the dose contains at least about 50, 100, 500, 1000, or at least about 5000 vector particles per cell to be treated. Alternatively, the dose of a viral vector or a nucleic acid vector is at least about 104 to about 105; about 105 to about 106; 106 to about 107; 107 to about 108; about 108 to about 109; about 109 to about 1010; or at least about 1010 to about 1011. The dose effective for treating a specific number of cells is calculated from the area in need of treatment by methods known to one of skill in the art.
As formulated with an appropriate pharmaceutically acceptable carrier in a desired dosage, the anti-ADAM8 antibody pharmaceutical composition or ADAM8 expression modulator provided herein is administered to humans and other mammals for example topically for skin tumors (such as by powders, ointments, creams, or drops), orally for cancers of the mouth and tongue, rectally, mucosally, sublingually, parenterally, intracisternally, intravaginally, intraperitoneally, intravenously, subcutaneously, bucally, sublingually, ocularly, or intranasally, depending on preventive or therapeutic objectives and the severity and nature of the cancer-related disorder or condition.
Injections of anti-ADAM8 antibodies include intravenous injection or intra-ocular injection into the tumor directly, for example, for esophageal, breast, brain, head and neck, and prostate tumors.
Liquid dosage forms for example for intravenous, ocular, mucosal, or other administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to at least one active agent, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents; solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan; and mixtures thereof. Besides inert diluents, the ocular, oral, or other systemically-delivered compositions can also include adjuvants such as wetting agents, and emulsifying and suspending agents.
Dosage forms for topical or transdermal administration of an inventive pharmaceutical composition include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active agent is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. For example, ocular or cutaneous routes of administration are achieved with aqueous drops, a mist, an emulsion, or a cream. Administration may be therapeutic or prophylactic. The invention includes implantation devices, surgical devices, or products which contain disclosed compositions (e.g., gauze bandages or strips), and methods of making or using such devices or products. These devices may be coated with, impregnated with, bonded to or otherwise treated with a composition as described herein.
Transdermal patches have the added advantage of providing controlled delivery of the active ingredients to the eye and body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
Injectable preparations of anti-ADAM8 antibody or other ADAM8 modulators, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono-glycerides or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, by irradiation, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. In order to prolong the effect of an active agent, slowing absorption of the agent from subcutaneous or intratumoral injection is often desirable. Delayed absorption of a parenterally administered active agent may be accomplished by dissolving or suspending the agent in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the agent in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of active agent to polymer and the nature of the particular polymer employed, the rate of active agent release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the agent in liposomes or microemulsions which are compatible with body tissues.
Compositions for treating rectal or vaginal termors are preferably suppositories which can be prepared by mixing the active anti-ADAM8 agent(s) of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active agent(s).
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active agent is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol, and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof.
Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active agent(s) may be admixed with at least one inert diluent such as sucrose or starch. Such dosage forms may also include, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also include buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active agent(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.
ADAM8 protein is expressed in most primary, non-small cell lung cancer tumors, particularly in advanced-stage lung ademocarcinomas, and in patient serum (Ishikawa et al. Clin Cancer Research 10(24):8363-70, 2004). ADAM8 is a marker for diagnosis of lung cancer (Ibid.). Ectopic ADAM8 expression increases the migratory phenotype of fibroblasts and COS cells in culture (Ibid.).
ADAM8 mRNA levels are higher in pancreatic cancer than in the normal pancreas or in chronic pancreatitis (Valkoskaya et al. Journal of Cellular and Molecular Medicine 11:1162-1174, 2007; Valkoskaya Exp Oncol 30 (2):129-132, 2008). Overexpression of ADAM8 correlates with poor patient survival. Invasiveness is reduced by ADAM8 knockdown in ASPC-1 cells in culture using siRNAs. In Valkoskaya 2007 and Valkoskaya et al. 2008, hypoxia induced ADAM8 level in approximately half of the cell lines examined in culture.
Zhang et al. observed higher ADAM8 levels in liver tissue in patients with hepatocellular carcinoma (HCC) compared to liver tissue from healthy individuals (Zhang et al. Tumor Biol. 33(6):2167-72, 2012; Zhang et al. Surgeon 11(2):67-71, 2013). Patients with ADAM8-positive tumors have shorter post-operative survival time that those with ADAM8-negative tumors (Ibid.).
In patients with gliomas, ADAM8 expression correlates with clinicopathological features and prognosis (He et al. Med Oncol. 29(3):2032-7, 2012). ADAM8 expression has been shown by IHC and western blot analysis to be overexpressed in human gliomas and is associated with poor clinical outcome (Ibid.).
In medulloblastoma tissues compared to normal cerebellum tissues, ADAM8 mRNA and protein are highly expressed (Zhang et al. J Neurol Sci. 323(1-2):46-51, 2012). In brain cancer, high ADAM8 protein levels correlate with advanced metastatic stage and aggressive histopathological type (Ibid.).
mRNA expression of ADAM8 protein increases as a function of tumor status in kidney cancer, and compared to noncancerous kidney tissues (Roemer et al. J Urol. 172 (6 Pt 1):2162-6, 2004; Roemer et al. Oncol Rep. 11(2):529-36, 2004).
An embodiment of the invention provides a method of treating a primary cancer or metastasis in a subject, in which the cancer or metastasis expresses ADAM8, the method including administering to the subject a composition having a therapeutically effective amount of an antibody or antibody fragment that binds to at least part of an extracellular portion (ectodomain) of ADAM8 protein and inhibits at least one activity of ADAM8, and a pharmaceutically acceptable carrier.
The method generally uses an anti-ADAM8 antibody that binds to the ADAM8 ectodomain, produced having specific affinity to an antigen having an amino acid sequence of at least one ADAM8 protein ectodomain selected from at least one of a cysteine-rich domain (CRD), a disintegrin domain (DI), a metalloproteinase domain (MP), and an EGF-like domain (ELD). The immunizing antigen is at least four to seven amino acids in length, and may be as long as the entire ectodomain having length of several hundred amino acids.
In general, the method is appropriate to any cancer, for example, the cancer is selected from breast, brain, lung, prostate, skin, colorectal, ovarian, bone, renal, head and neck, esophageal, gastric, liver, bladder, cervical, testicular, uterine, thyroid, and pancreatic cancers. The method includes in various embodiments, a cancer that is an established tumor. In the embodiment of breast cancer, the method includes breast cancer that is triple-negative or that is estrogen receptor negative. Embodiments of the method include the cancer which is a primary tumor, for example, the tumor is non-metastatic. Alternatively the method includes a cancer that is metastatic. In various embodiments, the subject is a human; alternatively the subject is a high value animal or is a research animal.
The antibody in various embodiments is obtained by immunizing or selecting from a yeast or phage or other type of display library with a peptide of an ADAM8 protein having an amino acid sequence selected from sequences as shown in any of: SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; and SEQ ID NO: 9 as a target. For example, the antibody is obtained by immunizing with a peptide including an amino acid sequence within residues 158 to 653 corresponding to the ectodomain of ADAM8, or selecting for a member of the library that binds to the ectodomain or ectodomain fragment.
The method in an embodiment further involves analyzing a decrease in at least one symptom of cancer or metastasis progression. In various alternative embodiments the symptom is at least one selected from: decreased size of primary tumor; cessation of growth of primary tumor; decreased number of metastases; decreased size of metastases; absence of new metastases; decreased number of circulating tumor cells in blood; decreased vascularization in tissue at the periphery of or within the tumor or metastases; decreased amount of ADAM8 in body fluids; decreased amount of VEGF-A in body fluids; and, weight loss of the subject.
An embodiment of the invention provides a method of diagnosing or prognosing a presence of a cancer disease condition in a patient and selecting a course of treatment for the patient, the method including: analyzing amount of expression of ADAM8 protein in the patient or patient tumor; measuring the amount in a normal subject or in an unaffected tissue of the cancer patient, such that an increase in expression in the cancer of the patient in comparison to the normal subject or the normal tissue of the patient is an indicium of the cancer; and, prognosing a course of treatment for the patient, such that the indicium of ADAM8-related cancer guides selecting at least one therapeutic agent that inhibits or reduces expression or activity of ADAM8.
An embodiment of the method further involves administering to the patient a therapeutically effective amount of an antibody or antibody fragment that binds to an extracellular portion (ectodomain) of ADAM8 protein and inhibits the activity of ADAM8.
The activity of ADAM8 in various alternative embodiments of prognosing the course of treatment is at least one of: ADAM8 metalloproteinase enzyme activity; release of VEGF-A; promotion of angiogenesis; extravasation or intravasation of cancer cells through vascular endothelium; and β1-integrin activation.
An embodiment of the invention provides also a method of screening a library of compounds to identify a candidate therapeutic agent to treat a cancer, the method including: contacting a first ADAM8 sample with at least one compound, and contacting a second ADAM8 sample with a control lacking the compound and otherwise identical; analyzing amounts of ADAM8 or an ADAM8 activity in the first and second samples, such that a reduced amount or reduced activity in the first sample compared to the second sample identifies the compound as the candidate therapeutic agent; testing the compound for at least one of chemical structural and biological functional properties; and, testing the compound for ADAM8 specificity by determining if there is cross-reactivity with closely-related ADAM family members: ADAMS, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, and ADAM33. The specific compound inhibits ADAM8 and does not inhibit closely related ADAM family members. In various embodiments, the compound is selected from an antibody fragment library or a peptide library.
In an embodiment of this method the first and second ADAM8 samples are located on cells in culture or in vivo, i.e., the samples are cells in culture and the ADAM8 is located on the cells.
In an alternative embodiment of this method the first and second ADAM8 samples are cell extracts or are purified proteins in vitro.
In various alternative embodiments of this method the activity is selected from: ADAM8 metalloproteinase enzyme activity; release of VEGF-A; promotion of angiogenesis; and extravasation or intravasation of cancer cells through vascular endothelium; and β1-integrin activation.
Human TNBC cell lines (MDA-MB-231, Hs578T, MDA-MB-468, BT549), non-tumoral mammary epithelial cell line MCF-10A, and umbilical vein endothelial cell line HUVEC were purchased from the American Type Culture Collection (ATCC) and maintained in medium recommended by ATCC. Inflammatory breast cancer cell line SUM149 was maintained as described in Forozan et al. Br J Cancer 81(8): 1328-1334, 1999. MDA-MB-231-derived LM1 and LM2 cells were isolated in vivo because of the cells' ability to metastasize to the lung (Minn et al. Nature 436:518-524, 2005). Stable clones of ADAM8 shRNA (shA8) and Control shRNA (shCtrl) MDA-MB-231 cells, expressing GFP, were established using transfection of either ADAM8 shRNA or scramble control vector (Origene), respectively, using LTX Lipofectamine (Invitrogen), selection with 1 μg/ml puromycin (Sigma), and limiting dilution. MDA-MB-231 clones were authenticated using short tandem repeat analysis (Genetica DNA Laboratories), which demonstrated 100% identity with the MDA-MB-231 cell line of ATCC. For culture in 3D suspension conditions, single-cell suspensions in complete media were plated at 1.25×104 cells per well in ultra low-attachment 6-well plates (Costar) and incubated for 48 h. For culture in hypoxic conditions, cells were incubated in 1% oxygen for 6 or 24 h in 6-well plates.
Transient RNAi-mediated ADAM8 knockdown was performed with the following short interfering RNAs (siRNAs; QIAGEN):
AllStar negative control siRNA (QIAGEN) was used in each example as a non-silencing control siRNA (siCtrl). siRNAs (10 nM) were introduced into cells using Lipofectamine RNAi Max Transfection Reagent (Invitrogen) by reverse transfection according to the manufacturer's protocol. Transfected cells were used 24 h later in functional assays.
The target sequence of the ADAM8 shRNA used in this study was
WCEs from cells in culture or frozen homogenized tumor tissue were prepared using radio-immunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors (Pierce), and further supplemented with EDTA and phenanthroline to inhibit the autocatalytic activity of ADAM8. Samples (25 μg) were analyzed by immunoblotting as described in Romagnoli et al. Cancer Res 72:6268-6278, 2012. Antibodies specific for ADAM8 were purchased from Millipore (AB19017) and LSBio (B4068). Antibody specific for VEGF-A was purchased from R&D Systems (AF293NA). Antibodies specific for β-Actin (AC-15) and β-tubulin (TUB 2.1) were purchased from Sigma. HIF-1α antibody (NB 100-105) was purchased from Novus Biologicals. Antibodies against HA-tag (sc-805) and Lamin B (sc-6217) were purchased from Santa Cruz Biotechnology. For western blot analysis of released VEGF-A, serum-free medium incubated with cells for 16 h was collected, centrifuged at 4,000 rpm for 10 min to remove cell debris and was concentrated using centrifugal 10K filters (Amicon Ultra-15). Volumes of the conditioned medium corresponding to 2.5×105 cells were analyzed by immunoblotting using a VEGF-A antibody (R&D Systems, MAB293).
As a measure of cellular metabolism and therefore cell growth, ATP levels were assessed in 3×103 MDA-MB-231 cells using an ATPlite luminescence ATP detection assay system (Perkin Elmer), as described in Mineva et al. J Cell Physiol; 220(3):593-9, 2009. Average ATP levels for triplicate samples are presented relative to control condition set to 1 (Mean±S.D. from three independent examples).
For soft agar assays, 5×104 cells in a mix of 0.4% Bacto Agar (BD Biosciences) in complete media were plated in triplicate on 6-well dishes pre-coated with a 1:1 mix of 2×DMEM medium supplemented with 10% FBS and 1.6% Bacto Agar. Cells were fed three times per week with complete medium. After 8-12 days, cells were stained overnight with 0.2 mg/ml iodonitrotetrazolium chloride (Sigma) and photographed at 10× magnification. Colonies with diameters of approximately 20 μm or greater were counted using ImageJ software (NIH). Matrigel outgrowth assays were carried out as in Wang et al. Nat Cell Biol 9:470-478, 2007, using 5×103 cells plated in duplicate on 24-well plates. Cultures were incubated for 4-7 days and photographed at 20× magnification.
Migration and invasion assays were performed in triplicate using polycarbonate filter Transwells (Costar) with 8-μm-diameter pores, without precoating or precoated with growth factor-reduced Matrigel (BD Biosciences, 356231), respectively. Transendothelial migration assays were performed as described herein with Transwells precoated with HUVECs that had been allowed to grow until a confluent monolayer was achieved. Suspensions of 1×105 tumor cells were layered in the upper compartments of the Transwell and incubated at 37° C. After 24 h, number of cells that migrated or invaded to the lower side of the filter were evaluated by crystal violet staining and OD570 nm determination. The control condition (siCtrl, shCtrl, or isotype antibody control) was set to 100% and the mean±S.D. from three independent replicated assays is given.
Tube formation assays were performed as described (Kim et al. Mol Cancer Res 5:321-329, 2007). Briefly, 2×104 HUVEC cells were plated, in triplicate, on glass chambers coated with growth factor reduced Matrigel. Cells were incubated in 200 μl EBM-2 Basal Medium supplemented with 1% FBS and concentrated conditioned medium from shCtrl and shA8 MDA-MB-231 clones (volume corresponding to 5×105 tumor cells) or conditioned media in the absence of tumor cells. Following incubation for 13 h, three random fields per well were photographed (40× magnification). Incomplete networks were excluded and numbers of closed networks of vessel-like tubes counted in three fields (n=9). The average percentage relative to control samples is presented ±S.D. from a representative of three independent replicated assays.
Endothelial cell adhesion assays were performed as described in Price et al. Br J Cancer 74:1762-1766, 1996. Briefly, 1×105 HUVEC cells were plated, in duplicate, in 48-well plates. After 24 h, 5×104 shCtrl or shA8 MDA-MB-231 cells stained with NucBlue (Invitrogen) were incubated for 15 min at 37° C. in 300 μl of EBM-2 Basal Medium supplemented with 1% FBS on top of the confluent HUVEC monolayer or in empty wells (without HUVECs). Unattached MDA-MB-231 cells were washed three times with PBS and attached NucBlue-positive cells were counted in three random fields per well (n=6). The average percentage relative to control samples is presented ±S.D. from three independent examples. For inhibition examples with antibodies, tumor cells and/or HUVECs were pre-treated with 10 μg/ml of β1-integrin antibody (BD Biosciences, 552828), 20 μg/ml of antibody specific for an ADAM8 ectodomain (R&D Systems, MAB10311 and Mab1031), or control isotypes (rat or mouse, respectively) for 30 min at room temperature and then washed twice with phosphate buffered saline (PBS) before being used in the endothelial cell adhesion assay.
Secreted levels of 55 angiogenesis related proteins were analyzed in the conditioned medium from shControl (shCtrl-3 and -5) and shADAM8 (shA8-17 and -20) MDA-MB-231 clones using the Proteome Profiler™ Human Angiogenesis antibody array (R&D Systems, ARY007). Cells incubated with 2% fetal bovine serum (FBS) conditioned medium for 24 h were centrifuged at 4,000 rpm for 10 min to remove cell debris and a volume of medium corresponding to 2.5×105 cells was analyzed by the antibody array according to the manufacturer's protocol. The signal intensity of the detected proteins (two spots for each protein) was measured using ImageJ, the background signal subtracted, and the average intensity (mean±S.D) for the two clones was calculated.
Six-week-old female nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (Jackson Laboratory) were implanted with 2.5×106 cells in 50 μL 50% Matrigel (BD Biosciences, CB-40230A) solution (1:1 dilution of Matrigel with DMEM medium) in the fourth inguinal mammary fat pad. Primary tumor growth was monitored by caliper measurement twice a week. Tumor volumes were calculated as (length×width2)/2. Mice were sacrificed after 3-4 weeks when tumors derived from shCtrl MDA-MB-231 cells had reached a volume of about 1 cm3. Mice were photographed, tumors were dissected, photographed, weighed and fixed in 10% Neutral Buffered Formalin for histological and IHC analyses. For metalloproteinase/disintegrin antibody treatment examples, control isotype IgG2B (n=8) or antibody for ADAM8 (anti-A8 mAb; n=9; Mab004 and Mab1031 respectively, R&D Systems) was injected i.p. twice weekly at the dosage of 0.5 mg/kg from the day of shCtrl-3 cell implantation in the MFP.
For the cysteine-rich/EGF-like antibody treatment examples, control isotype IgG1 (n=7) or anti-A8 Ab (n=7; Mab002 and Mab10311, R&D Systems) was injected i.p. twice weekly at 1.5 mg/kg from the day of shCtrl-3 cell implantation in the MFP.
Alternatively, treatment with doses of 1.5 mg/kg was performed using a dosage regiment of administering treatment after the primary tumors became palpable and continuing with an injection on the day of resection when the tumors reached a volume of about 0.15 cm3 and twice weekly for the following five weeks. For metastasis detection, brains and lungs were removed by dissection, stored in cold PBS and examined for GFP expression using a fluorescent dissecting microscope (Leica MZ FLIII).
Six-week-old female nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (Jackson Laboratory) were implanted with 0.5×106 cells in 100 μL PBS using an ultrasound guided injection with a 27½-gauge needle into the left ventricle. Immediately following cardiac injection, systemic vascular dissemination of the tumor cells was analyzed by detection of Luciferase activity using the Xenogen Biophotonic Imaging system. Total tumor burden was monitored weekly as described herein. Mice were sacrificed three weeks after initial cell injection. Organs and bones were harvested and specific Luciferase activity detected as described herein.
Blood was collected at sacrifice either by cardiac puncture (about 500 μl; day 33), or by drawing from mice using the submandibular blood collection method (about 100 μl) at time points 7, 14 and 21 days after inoculation of tumor cells into the MFP. To evaluate level of autofluorescence, blood was collected also from control NOD/SCID mice that were not injected with any tumor cells. Blood samples were collected in heparin (100 USP units/sample), and were diluted and processed the same day for detection of GFP-positive CTCs using a confocal flow cytometry system at a flow rate of 4.5 μl per min, as described in Greiner et al. Cytometry A 79:874-883, 2011. Enumerated cells were identified with an algorithm as described in Hwu et al. J Biomed Opt 16:040501, 2011 based on the combined detection of peaks appearing simultaneously in a fluorescence channel optimized for GFP detection and in at least one of the channels detecting scattering at either 405 or 488 nm.
Diluted whole blood samples were flowed in microfluidic devices, each having five single 50 μm (width) by 70 μm (height) and 10 mm (length) channels. To flow cells in a microfluidic channel for light scattering and fluorescence emission measurements, the outlet tubing was placed into eppendorf tubes and the cell suspension was drawn by a syringe pump (Harvard Apparatus) at a flowrate of 6 μl min−1. Operating at a constant pressure, a 6 μl min−1 set flowrate calibrated for water yielded an actual value of 4.5 μl min−1 due to the increased viscosity of blood. Channels were pre-wetted by flowing PBS, and the blood sample flow was allowed to stabilize prior to data acquisition. Of the initial approximately 100 μl of whole blood collected, 34±17, 55±17, and 57±9 μl, respectively, were analyzed for non-tumor bearing, shControl, and shADAM8 mice, respectively. At the final 33 day time point, 100 μl of blood was analyzed.
Counts positive for GFP fluorescence were detected in the 500 to 590 nm “green” channel following excitation with 488 nm light and were time-correlated with 405 and 488 nm scattering values of sufficient intensity. The incident 633 nm light interrogated cells for red autofluorescence, which was detected in the 650-690 nm channel and analyzed separately from green correlated counts. Within the volume analyzed, leukocytes, and CTCs constituted approximately 0.5% of the acquired backscattered and fluorescent light data stream. Using a 25 kHz sampling frequency, the lower 99.5% of the intensity values defined the mean and standard deviation of the background signal. Individual peaks were demarcated by 6 to 7 data points and the maximum value defined the intensity used to determine whether the count contained sufficiently intense scattering and fluorescence values for CTC identification. Within a Matlab program, the intensity threshold was defined based on the number of standard deviations above the mean of the background signal. The number of standard deviations was observed to be, respectively, 4, 4, 7, and 5 for the 405, 488, 650-690, and 500-590 nm channels, respectively. Of the non-tumor bearing samples assayed, a concentration of mean±S.D. μl−1 green autofluorescent cells were detected and subtracted from the detected counts in tumor-bearing samples, yielding the reported CTC concentration values.
HEK-293 cells were plated at 5×105 cells/ml in P60 dishes. After 24 h, cultures were co-transfected with 1 μg of plasmid DNA encoding C-terminal HA-tagged CD23 (CD23 membrane isoform b, purchased from Addgene), and 1 μg vector DNA expressing either ADAM8 full-length or remnant form, or empty vector (EV) pCDNA3.1 Version B DNA. The medium was replaced after 6 h with culture medium without FBS. After 16 h, the serum-free media was harvested and centrifuged to remove cell debris, and cells were trypsinized and counted. The supernatants were concentrated using Amicon-ultra 4 centrifugal filter units (Millipore), and volumes corresponding to equal cell numbers were assessed for cleaved CD23 using Western blotting. WCEs from lysed cells were assessed for ADAM8 levels.
Paraffin-embedded tissues from Formalin-fixed tumors were selected (see Table 1 and
Specimens were cut in 3-μm-thick sections and stained with H&E. Alternatively, sections were processed for immunohistochemistry. Sections were deparaffinized in xylene and rehydrated through an alcohol gradient, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Samples were steamed before incubation for antigen retrieval with citrate buffer (pH 6.0). Slides were incubated with a primary antibody targeting ADAM8 (1:150, B4068, LSBio) on an automated immunostainer using a labelled streptavidin-biotin method (Dako) followed by 3,3′-diaminobenzidine chromogen detection. Immunostained slides were counterstained with hematoxylin. Negative controls were included in each run by omitting the primary antibody. ADAM8 immunoreactivity in carcinomatous cells was scored by a pathologist using a five-tiered semiquantitative system (0: no staining; 1+: focal weak staining<10%; 2+: 10-25%; 3+: 25-50%; 4+: >50%). Non-carcinomatous cells were excluded from scoring.
Primary antibodies targeting ADAM8 (1:150, B4068, LSBio), CD31 (1:50, ab28364, Abeam) and the active conformation of β1-integrin (1:100, B2861, LSBio) were used in the same protocol described as herein for patient specimens. For evaluation of vessel density, two slides/tumor were cut 200 μm apart and analyzed by immunostaining for CD31. Slides were examined at low magnification and three areas with the greatest numbers of vessels (hot spots) at the tumor periphery were identified for each tumor slice. These were photographed at 40× magnification, the numbers of vessels/hotspot counted and the average value of vessel density/tumor plotted (n=6/tumor).
Cancer datasets were downloaded from ONCOMINE™ (Compendia Bioscience, www.oncomine.com), as described in Romagnoli et al., 2012. The rank for ADAM8 across the 14 analyses and its P-value for the comparison of ADAM8 expression between breast carcinoma and normal breast, were obtained directly from the ONCOMINE™ website. A comprehensive gene expression microarray data set of 295 primary breast tumors (obtained from Rosetta Inpharmatics) prepared by van de Vijver and colleagues (van de Vijver et al. N Engl J Med 347:1999-2009, 2002), was used to compare ADAM8 mRNA levels across the different molecular breast cancer subtypes and to assess correlation of this protein with clinicopathological variables and patient outcome. Univariate relationships between ADAM8 mRNA log ratio data and patient outcome were assessed using the Log-rank test (RR, CI). A RR (relative risk) of 1 indicates that the risk of relapse or mortality is the same in both groups, while a RR of more than 1 indicates an increased risk. Confidence intervals (CIs) were calculated for the RR, which was then tested for statistical significance using the Wald test. Data were assigned arbitrary cut-off points. Data shown are representative of results obtained using the 75th percentile. Results are presented using Kaplan-Meier curves. P-values less than 0.05 (P<0.05) were regarded as being statistically significant. Data were analyzed using PASW statistics version 18.0 (SPSS Inc.). The Pearson's pairwise correlation plot showing the correlation between ADAM8 and CD31 (PECAM1) expression was obtained using breast tumor microarray datasets from the bc-GenExMiner Database (http://bcgenex.centregauducheau.fr). All other analyses were performed using a two-tailed Student's t-test for samples with equal variance, in which P-values less than 0.05 were considered statistically significant.
Analysis of the ONCOMINE™ database was used to compare the copy number of ADAM8 DNA in breast cancer samples with that of normal breast tissue. No copy number gain was found among samples from invasive ductal and lobular carcinoma (n=14), invasive ductal (n=647), invasive lobular (n=71), mixed lobular and ductal (n=9), or mucinous (n=9) cancerous tissues as compared to normal breast tissue (n=111). In fact, a decrease in copy number was observed for invasive ductal tissue compared to normal breast tissue, and for mucinous compared to normal breast tissue, as is typical of deletions that arise during cancer.
The ONCOMINE™ microarray database was used to assess ADAM8 mRNA levels in breast cancer. ADAM8 levels were observed significantly overexpressed in invasive breast carcinoma samples from different origins (ductal, lobular, mixed), in comparison to normal breast tissues (
IHC analysis was performed to assess ADAM8 expression directly in breast tumors (
IHC analysis of 56 human breast cancer-derived metastases revealed that 48.2% ( 27/56) were positive for ADAM8 expression (
These findings demonstrate that ADAM8 is aberrantly expressed in a large number of breast cancers, in particular in patients with TNBC, and expression of ADAM8 is associated with a worse prognosis. Thus, a role of ADAM8 was observed in TNBC, cancers which are characterized by high rates of recurrence and dissemination to distant organs and have fewer effective targeted therapies (Boyle, 2012).
ADAM8 is synthesized as a 120 kD proform, which multimerizes and autocatalytically clips off its prodomain, leaving a proteolytically active 90 kD membrane-anchored metalloproteinase, which can be further processed to a 60 kD remnant form (
Using western blot analysis, high ADAM8 proform expression was found in all TNBC cell lines tested compared to the non-tumoral mammary epithelial cell line MCF-10A when grown in adherent conditions (
To test the role of ADAM8 on the aggressive phenotype of MDA-MB-231 and Hs578T cells in vitro, the effects of ADAM8 knockdown were measured on growth in soft agar, and migration and invasion through Matrigel, assays that are performed in 3D-culture conditions.
Decreased ADAM8 expression led to reduced abilities of both cell lines to grow under anchorage-independent conditions (
Thus, presence of ADAM8 was observed to be required for the maintenance of a migratory and invasive phenotype in TNBC cells in culture.
The effects of stable ADAM8 knockdown in vivo was tested using a mouse orthotopic xenograft model. Clones of MDA-MB-231 cells expressing a specific ADAM8 shRNA (shA8-17 and shA8-20) or Control shRNA (shCtrl-3 and shCtrl-5) were isolated. The Control shRNA clones expressed levels of ADAM8 comparable to the parental cells. Effective knockdown of ADAM8 expression by shADAM8 was verified by western blot (
The two shADAM8 MDA-MB-231 clones showed significantly reduced ability both to migrate and to invade through Matrigel compared to the shControl clones (
To test the effects of ADAM8 knockdown on tumor growth and metastasis, 2.5×106 MDA-MB-231 shCtrl-3 and shA8-20 cells were injected into the fourth inguinal mammary fat pad of female NOD/SCID mice (n=seven per group). Tumor growth at the orthotopic site was recorded by caliper measurements twice weekly. Between 13-16 days after cell implantation, mice from the shControl group started to develop mammary tumors that progressed with a high growth rate (
CTCs were detected based on stable GFP expression in the MDA-MB-231 clones, using flow cytometry on freshly collected blood following termination of the time course. A profound decrease in the numbers of CTCs was observed with ADAM8 knockdown (12.8±7.0 vs 0.0±2.0 per μl of blood;
To test whether knockdown of ADAM8 affects metastasis, isolated brains were examined from six animals per group under a fluorescent dissecting microscope for the presence of GFP-positive metastases. All of the animals injected with shControl cells were observed to be positive for brain metastases (
When solid tumors reach a few millimeters in diameter, nutrients and oxygen become insufficient and hypoxic stress is induced (Majmundar et al. Mol Cell 40:294-309, 2010). This leads to cell death by necrosis as well as to the release of factors that promote endothelial cell recruitment and angiogenesis that are needed to support continued tumor growth (Bergers et al. Nat Rev Cancer 3:401-410, 2003; Naumov et al. Cell Cycle 5:1779-1787, 2006). ADAM8 levels have been shown to be induced under hypoxia in pancreatic cancer cells (Valkovskaya et al. J Cell Mol Med 11:1162-1174, 2007). Thus, the effects of growth under reduced oxygen on ADAM8 expression in MDA-MB-231 and Hs578T breast cancer cells were measured.
Incubation of these cells under conditions of 1% of oxygen was observed to have led to substantial increases in ADAM8 protein levels (
To determine whether Hs578T cells behaved similarly under hypoxic conditions as compared to MDA-MB-231 cell lines, MDA-MB-231, Hs578T, shCtrl-3, and shA8-20 cells were incubated for 8 h under hypoxic conditions and nuclear extracts were subjected to Western blotting analysis for HIF-1α and Lamin B. All lanes were from the same gel, but two different times of exposure were used for the Hs578T cells (5 minutes) and MDA-MB-231-derived lines (30 minutes), as indicated by the vertical line. Therefore, HIF-la is induced in various breast cancer cell lines under hypoxia (
Consistently strong ADAM8 staining was detected around the necrotic areas in the shCtrl-3 tumors, and was absent in tumors derived from shA8-20 cells (
To determine if expression levels of VEGF-A mRNA are altered by ADAM8 knockdown. RNA isolated from MDA-MB-231 shCtrl-3 or shA8-20 cells was assessed for VEGF-A expression by reverse transcription (RT)-PCR. Samples were normalized based on GAPDH expression levels. Results show that VEGF-A mRNA levels are unchanged in stable ADAM8 knockdown cells (
To assess angiogenesis IHC staining of the tumors for the endothelial cell marker CD31 was performed. A significant decrease in the number of peritumoral vessels was measured in shA8-20 vs shCtrl-3 tumors (
To determine the mechanism whereby ADAM8 induces angiogenesis, the ability of conditioned media from MDA-MB-231 clones to stimulate the formation of vessel-like structures (tube formation) by human umbilical vein endothelial cells (HUVECs) on Matrigel was assessed (
To identify pro-angiogenic factors released by ADAM8, conditioned media from the two shControl and two shADAM8 MDA-MB-231 cell clones were analyzed using a Human Angiogenesis antibody array, which can detect the expression of 55 angiogenesis related proteins. Expression levels of the detected proteins were quantified and those that were significantly downregulated by more than an average of two-fold in the shADAM8 clones are presented in
To determine whether the remnant form of ADAM8 can induce VEGF-A, MDA-MB-231-derived shCtrl-3 or shA8-20 clones were transfected with the vectors expressing ADAMS remnant form (Remnant) or with an empty vector (EV) DNA (
To test ectopic expression of full-length ADAM8 and remnant form in stable ADAM8 knockdown cells, MDA-MB-231-derived shCtrl-3 or shA8-20 clones were transfected with the indicated vectors expressing ADAM8, its remnant form (Remnant) or empty vector (EV) DNA for 24 h. Then, the medium was replaced by serum-free medium and incubated for 16 h. Supernatants and cells were collected. Cells were lysed and WCEs were assessed by Western blotting for ADAM8 expression. VEGF-A released into the media was measured (
CTCs can be measured prior to formation of a detectable primary tumor (Eyles et al. J Clin Invest 120:2030-2039, 2010). Thus, the role of ADAM8 in the appearance of these early CTCs was investigated using a second set of mice (n=four/group;
Binding of primary tumor cells and then CTCs to an endothelial layer followed by transmigration through the blood vessel wall (intravasation and extravasation, respectively) are critical steps in metastasis (Scott et al. Nat Rev Cancer 12:445-446, 2012; Steeg Nat Med 12:895-904, 2006). Thus, the effects of ADAM8 on binding of MDA-MB-231 cells to HUVECs were determined. shControl and shADAM8 cells were added to tissue culture plates in the presence or absence of a HUVEC monolayer. The two shControl clones were observed to adhere much more efficiently to a confluent HUVEC monolayer than the two shADAM8 clones, and adhesion on plastic was not affected by ADAM8 expression (
The MDA-MB-231-derived shCtrl-3 clone and the total MDA-MB-231 population were transfected with either Control siRNA (siCtrl) or two specific ADAM8 siRNAs (siA8) for 72 h. Cancer cell adhesion to (
WCEs from HUVECs and MDA-MB-231 cells, as a positive control for ADAM8 expression, were subjected to western blotting for ADAM8 (LSBio antibody) and tubulin (
Activation of β1-integrin in shCtrl-3 cells was observed to be required for adhesion to HUVECs, as judged by reduced endothelial cell-binding in vitro following addition of an antagonist β1-integrin antibody (
To validate ADAM8 as a therapeutic target for TNBC, the Mab1031 ADAM8 antibody was selected for use in an orthotopic model.
Mice were treated with either 0.5 mg/kg of Mab1031 antibody (n=9) or a control isotype-matched IgG2B (n=8) by i.p. injection twice weekly from the day of implantation of shCtrl-3 cells. Anti-ADAM8 treatment was observed to have resulted in significantly decreased primary tumor burden by about 50% (
Anti-ADAM8 MP/DI antibody inhibits the metalloproteinase activity of ADAM8. HEK293 cells were co-transfected with vectors expressing CD23, which is a well-established substrate of ADAM8 protease activity, and either ADAM8 or its remnant form or EV DNA. Six hours later, the medium was replaced with serum-free medium. After 16 h, supernatants were collected and volumes corresponding to equal cell numbers were assessed for cleaved CD23 (29 kDa) using Western blotting. Cells were harvested and WCEs were assessed for expression of ADAM8 and tubulin. All lanes were from the same gel, but cut to re-align as indicated by the vertical line (
To test the use of this ADAM8 antibody in a more clinically relevant setting, a tumor resection model was used (
Twelve days after MDA-MB-231 shCtrl-3 cells were implanted into the MFP of mice, palpable tumors were observed, and treatment was initiated with 1.5 mg/kg of anti-ADAM8 MP/DI antibody (Mab1031) or isotype control IgG2B (Mab004). Additional doses of antibody were further administered on day 15 and on day 17, at which point tumors reached a volume of about 0.15 cm3 and tumor resection was performed. Antibody treatment was continued twice weekly for a 5-week period, and then organ metastasis was examined using fluorescent microscopy (
In the isotype-treated control group, metastases to the brain and lungs were seen in 8/8 and 7/8 mice, respectively. In the anti-ADAM8-treated group only 2/9 and 1/9 mice showed metastases in the brain and lungs. Thus, treatment with an anti-ADAM8 antibody resulted in a profound reduction in the frequency of metastases to both the brain and lungs compared to the frequency of metastases in the isotype control antibody treated group.
To test the role of ADAM8 in the ability of breast cancer cells to colonize typical distant metastasis sites, MDA-MB-231-derived shCtrl-3 and shA8-20 cells, which stably express Luciferase, were injected in the left ventricle of female NOD/SCID mice (n=4/group). Total tumor burden was monitored weekly by Luciferase activity measurement using the Xenogen Biophotonic Imaging system. Organ and bone metastases were assessed using this method at sacrifice.
Cardiac injection of MDA-MB-231-derived shA8-20 cells caused a significantly lower total tumor burden after 2 or 3 weeks compared to injection of shCtrl-3 cells (
Since treatment of breast cancer cells with Mab10311 (CRD/ELD) and Mab1031 (MP/DI) antibodies reduced amount of transendothelial cell migration through a HUVEC monolayer to about the same extent (
After three weeks, no significant change in primary tumor burden was seen following the anti-ADAM8 Mab10311 treatment (
Overall, data herein validate ADAM8 as a therapeutic target for TNBC. ADAM8, which is abundantly expressed in aggressive patient breast tumors and metastases, was shown to be essential for tumor growth and dissemination in orthotopic mouse models using knockdown and antibody strategies. Notably, treatment of pre-existing tumors with an antibody targeting the MP/DI domains of ADAM8 profoundly reduced tumor ability to metastasize in a resection model. Two essential novel roles for ADAM8 in tumor progression were identified (
Binding of integrins to cell-surface adhesion molecules and extracellular matrix proteins governs cell adhesion and shape via transduction of intracellular signals (Avraamides et al. Nat Rev Cancer 8:604-617, 2008; Desgrosellier et al. Nat Rev Cancer 10:9-22, 2010). Beta1-integrin was found to mediate adhesion of breast cancer and other tumor cells to quiescent endothelial cells (Price et al. Br J Cancer 74:1762-1766, 1996). Several ADAM proteins, which contain RGD-like integrin-binding motifs in the disintegrin domain, constitute ligands for integrins (Bridges & Bowditch, Curr Pharm Des 11:837-847, 2005). Notably, an antibody targeting of the MP/DI domains of ADAM8 was observed to have greatly reduced the ability of TNBC cells to bind endothelial cells in vitro and to disseminate in mice. In addition, another antibody directed against the ADAM8 CRD/ELD domains was equally effective in vitro, suggesting allosteric hindrance or altered ADAM8 structure may also play a role. This antibody consistently inhibited dissemination in vivo.
Early in the tumorigenesis process, solid tumors stop increasing in mass (tumor dormancy) unless they can overcome hypoxic stress by stimulating angiogenesis, which provides oxygen and nutrients essential for further tumor growth (Bergers et al. Nat Rev Cancer 3:401-410, 2003; Majmundar et al. Mol Cell 40:294-309, 2010; Naumov et al. Cell Cycle 5:1779-1787, 2006). In TNBC cells, hypoxia was observed to have induced expression of ADAM8 proform and active processed form. Mammary tumors of shCtrl cells displayed a substantial increase in ADAM8 protein around necrotic areas, but not in tumors of shA8 animals. Using Transfac analysis, several putative Hypoxia Response Elements were identified in the ADAM8 promoter. The extent of HIF-1α induction and amount of necrosis in tumors derived in shCtrl compared to shA8 cells was similar (
Activation of the VEGFR2 tyrosine kinase receptor by VEGF-A, which promotes endothelial cell proliferation, sprouting, migration, as well as vascular permeability, is a major pathway inducing angiogenesis (Lohela et al. Curr Opin Cell Biol 21:154-165, 2009). High VEGF-A expression has been associated with increased microvessel density, metastasis, and a shorter overall survival in patients with primary invasive breast cancer (Mohammed et al. Br J Cancer 96:1092-1100, 2007). VEGF-A activity is regulated to a large extent in the extracellular space, where cell-surface or extracellular matrix bound VEGF-A reservoirs can be made bioavailable through proteolytic cleavage by various enzymes, including metalloproteinases MMP-3, -7 and -9 (Hawinkels et al. Eur J Cancer 44:1904-1913, 2008; Lee et al. J Invest Dermatol 130:763-773, 2005). TNBCs with higher intratumoral VEGF-A levels compared to non-TNBCs have a shorter time to relapse (Linderholm et al. Ann Oncol 20:1639-1646, 2009). Knockdown of ADAM8 in MDA-MB-231 cells resulted in reduced VEGF-A levels in conditioned media, but did not appear to affect VEGF-A mRNA levels (
Biological roles of ADAM proteins in malignancy identified in Duffy et al. (Duffy et al. Clin Proteomics, 8:9, 2011; Maretzky et al. Nat Commun 2:229, 2011; Duffy et al. Clin Cancer Res 15:1140-1144, 2009). ADAM15 cytoplasmic domain variants have been implicated in mammary carcinoma (Zhong et al. Mol Cancer Res 6:383-394, 2008). An ADAM12 secreted isoform promotes breast tumor metastasis in vivo (Roy et al. J Biol Chem 286:20758-20768, 2011). ADAM10 is upregulated in melanoma metastasis compared to primary melanoma (Lee et al. J Invest Dermatol 130:763-773, 2010). ADAM8 expression was previously correlated with tumor stage or an unfavorable prognosis for patients with esophageal-gastric, lung, pancreatic or brain cancers, and with invasiveness of tumor cells in culture (Baren et al. Br J Cancer 107:143-149, 2012; Koller et al. Curr Pharm Des 15:2272-2281, 2009), although little was explored about its mechanism of action. No evidence of ADAM8 gene amplification in these cancers was reported. Several pathways commonly dysregulated in aggressive malignancies were found to induce ADAM8 mRNA levels, e.g., RelB NF-κB and EGFR (Sriraman et al. Biol Reprod 78:1038-1048, 2008).
In examples herein, ADAM8 expression levels were correlated with a poor prognosis in breast cancer patients and concomitantly with increased numbers of CTCs and metastases in an orthotopic mouse model. CTCs and micrometastases have been recently shown to occur early during the progression of cancers from various origins (melanoma, breast and cervical cancers; Toh et al. Immunol Res 53:229-234, 2012). ADAM8 and ADAM12 activities measured in urine samples to identify breast cancer patients with invasive and metastatic disease (Roy et al. Clin Biochem 44:1434-1439, 2011).
Data herein show that the potential use of monitoring ADAM8 in a non-invasive method as a diagnostic/prognostic biomarker for early cancer detection or potentially to follow disease progression and recurrence, and to treat breast cancer and prevent metastasis is feasible.
This application claims the benefit of U.S. provisional application No. 61/822,738 filed May 13, 2013 and 61/898,830 filed Nov. 1, 2013, and international application PCT/US14/37857 filed May 13, 2014, by inventors Gail E. Sonenshein, Mathilde Romagnoli, and Nora D. Mineva, each of which is hereby incorporated by reference herein in its entirety.
This invention was made with government support under grants CA129129 and ES011624 awarded by the National Institutes of Health and grant W81XWH-10-1-1003 awarded by the U.S. Army. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61898830 | Nov 2013 | US | |
| 61822738 | May 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2014/037857 | May 2014 | US |
| Child | 14940344 | US |
