US20140227252A1 - Methods and Monitoring of Treatment with a DLL4 Antagonist - Google Patents

Methods and Monitoring of Treatment with a DLL4 Antagonist Download PDF

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US20140227252A1
US20140227252A1 US14/068,890 US201314068890A US2014227252A1 US 20140227252 A1 US20140227252 A1 US 20140227252A1 US 201314068890 A US201314068890 A US 201314068890A US 2014227252 A1 US2014227252 A1 US 2014227252A1
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antibody
bnp
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Steven Eugene Benner
Robert Joseph Stagg
Jakob Dupont
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Oncomed Pharmaceuticals Inc
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Oncomed Pharmaceuticals Inc
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Assigned to ONCOMED PHARMACEUTICALS, INC. reassignment ONCOMED PHARMACEUTICALS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BENNER, STEVEN EUGENE, DUPONT, JAKOB, STAGG, ROBERT JOSEPH
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Priority to US15/070,373 priority patent/US20160334423A1/en
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    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin
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    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to the field of treating diseases with a DLL4 antagonist. More particularly, the invention provides methods for treating cancer comprising administering a DLL4 antagonist, either alone or in combination with other anti-cancer agents, and monitoring for cardiovascular side effects and/or toxicity.
  • Cancer is one of the leading causes of death in the developed world, with over one million people diagnosed with cancer and 500,000 deaths per year in the United States alone. Overall it is estimated that more than 1 in 3 people will develop some form of cancer during their lifetime. There are more than 200 different types of cancer, four of which—breast, lung, colorectal, and prostate—account for almost half of all new cases (Siegel et al., 2011 , CA: A Cancer J. Clin. 61:212-236).
  • the Notch pathway is involved in multiple aspects of vascular development including proliferation, migration, smooth muscle differentiation, angiogenesis, and arterial-venous differentiation (Iso et al., 2003, Arterioscler. Thromb. Vasc. Biol., 23:543).
  • the Notch receptor ligand DLL4 (Delta-like ligand 4) is an important component of the Notch pathway and plays a role in angiogenesis.
  • Blocking DLL4 signaling has been shown to reduce tumor growth by multiple different mechanisms (Ridgway et al., 2006 , Nature, 444:1083-87; Noguera-Troise et al., Nature, 444:1032-37; Hoey et al., 2009 , Cell Stem Cell, 5:168-77).
  • DLL4 blocking antibodies have been reported to result in endothelial cell proliferation and the development of blood vessels, however, these blood vessels lack a functional lumen. This dysangiogenic effect has been reported to block tumor growth by promoting the development of only non-functional blood vessels (Ridgway et al.
  • DLL4 blocking antibodies have been shown to inhibit tumor growth by reducing the proliferation of tumor cells and reducing cancer stem cell frequency. Although the mechanism behind the reduction of cancer stem cells or CSCs is unknown, it is hypothesized that DLL4 is required for the self-renewal of CSCs and maintains these cells in an undifferentiated state (Hoey et al., 2009. Cell Stem Cell, 5:168-77).
  • blockade of DLL4 signaling by anti-human DLL4 antibodies can result in endothelial hypertrophy and the creation of non-functional microvessels. Consequently, even in the presence of tumor angiogenic factors, blockade of DLL4 signaling, through administration of anti-human DLL4 antibodies, can result in dysangiogenesis which inhibits the ability of the tumor to induce the functional blood vessel formation needed to support growth of the tumor.
  • Chemotherapy is a well-established therapeutic approach for numerous cancers, but its efficacy can be limited by side effects and/or toxicity.
  • targeted therapies such as the anti-ErbB2 receptor (HER2) antibody trastuzumab (HERCEPTIN), tyrosine kinase inhibitors imatinib (GLEEVEC), dasatinib (SPRYCEL), nilotibib (TASIGNA), sunitinib (SUTENT), sorafenib (NEXAVAR), the anti-VEGF antibody bevacizumab (AVASTIN), and anti-angiogenesis drugs sunitinib (SUTENT) and sorafenib (NEXAVAR), are known to cause, or are likely to cause, side effects and/or toxicity in subjects who take them.
  • HER2 receptor HER2 receptor
  • GLEEVEC tyrosine kinase inhibitors imatinib
  • SPRYCEL dasatinib
  • TASIGNA
  • bevacizumab, sunitinib, and sorfenib are known to cause hypertension in about one-third of patients who take them.
  • anti-DLL4 antibodies may have side effects and/or toxicity in some subjects.
  • anti-DLL4 antibodies can cause hypertension in some patients. This was surprising, since anti-DLL4 antibodies have been reported to inhibit tumor angiogenesis by promoting dysangiogensis, a mechanism different than that of traditional anti-angiogenic treatments.
  • the present invention provides improved methods for treating diseases comprising administering to a subject a therapeutically effective amount of a DLL4 antagonist.
  • the invention provides methods of screening for, detecting, identifying, monitoring, reducing, preventing, and/or attenuating a cardiovascular side effect and/or toxicity related to treatment with a DLL4 antagonist.
  • the methods comprise determining the level of a natriuretic peptide in a sample from a patient who has received, is receiving, will receive, or is being considered for initial or further treatment with a DLL4 antagonist, including but not limited to an anti-DLL4 antibody.
  • the invention provides methods of selecting a subject for treatment with a DLL4 antagonist, comprising: obtaining a biological sample from the subject, determining the level of a biomarker in the sample, and selecting the subject for treatment with the DLL4 antagonist if the level of the biomarker is below a predetermined level.
  • the biomarker is a natriuretic peptide.
  • the method of selecting a subject for treatment with a DLL4 antagonist comprises: obtaining a biological sample from the subject, determining the level of a natriuretic peptide in the sample, and selecting the subject for treatment with the DLL4 antagonist if the level of the natriuretic peptide is below a predetermined level.
  • the biological sample is blood, serum, or plasma.
  • the natriuretic peptide is B-type natriuretic peptide (BNP).
  • BNP B-type natriuretic peptide
  • the predetermined level is about 300 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 200 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 250 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 200 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 150 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 100 pg/ml or less in a blood, serum, or plasma sample.
  • the invention provides methods of identifying a subject as eligible for treatment with a DLL4 antagonist, comprising: obtaining a biological sample from the subject, determining the level of a biomarker in the sample, and identifying the subject as eligible for treatment with the DLL4 antagonist if the level of the biomarker is below a predetermined level.
  • the biomarker is a natriuretic peptide.
  • the method of identifying a subject as eligible for treatment with a DLL4 antagonist comprises: obtaining a biological sample from the subject, determining the level of a natriuretic peptide in the sample, and identifying the subject as eligible for treatment with the DLL4 antagonist if the level of the natriuretic peptide is below a predetermined level.
  • the natriuretic peptide is BNP.
  • the biological sample is blood, serum, or plasma.
  • the predetermined level is about 300 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 200 pg/ml or less in a blood, serum, or plasma sample.
  • the predetermined level is about 250 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 200 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 150 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 100 pg/ml or less in a blood, serum, or plasma sample.
  • the invention provides methods of monitoring a subject receiving treatment with a DLL4 antagonist for the development of cardiovascular side effects and/or toxicity, comprising: obtaining a biological sample from the subject receiving treatment, determining the level of a biomarker in the sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein an increase in the level of the biomarker indicates development of cardiovascular side effects and/or toxicity.
  • the biomarker is a natriuretic peptide.
  • the method of monitoring a subject receiving treatment with a DLL4 antagonist for the development of cardiovascular side effects and/or toxicity comprises: obtaining a biological sample from the subject receiving treatment, determining the level of a natriuretic peptide in the sample, and comparing the level of the natriuretic peptide in the sample to a predetermined level of the natriuretic peptide, wherein an increase in the level of the natriuretic peptide indicates development of cardiovascular side effects and/or toxicity.
  • the natriuretic peptide is BNP.
  • the invention provides methods of detecting the development of cardiovascular side effects and/or toxicity in a subject receiving treatment with a DLL4 antagonist, comprising: obtaining a biological sample from the subject receiving treatment, determining the level of a biomarker in the sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein an increase in the level of the biomarker indicates development of cardiovascular side effects and/or toxicity.
  • the biomarker is a natriuretic peptide.
  • the method of detecting the development of a cardiovascular side effect and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprises: obtaining a biological sample from the subject receiving treatment, determining the level of a natriuretic peptide in the sample, and comparing the level of the natriuretic peptide in the sample to a predetermined level of the natriuretic peptide, wherein an increase in the level of the natriuretic peptide indicates development of a cardiovascular side effect and/or toxicity.
  • the natriuretic peptide is BNP.
  • the invention provides methods for identifying cardiovascular side effects and/or toxicity in a subject receiving treatment with a DLL4 antagonist, comprising: obtaining a biological sample from the subject receiving treatment, determining the level of a biomarker in the sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein if the level of the biomarker in the sample is higher than the predetermined level of the biomarker then a cardiovascular side effect and/or toxicity is indicated.
  • the biomarker is a natriuretic peptide.
  • the method for identifying cardiovascular side effects and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprises: obtaining a biological sample from the subject receiving treatment, determining the level of a natriuretic peptide in the sample, and comparing the level of the natriuretic peptide in the sample to a predetermined level of the natriuretic peptide, wherein if the level of the natriuretic peptide in the sample is higher than the predetermined level of the natriuretic peptide then a cardiovascular side effect and/or toxicity is indicated.
  • the natriuretic peptide is BNP.
  • the invention provides methods for monitoring cardiovascular side effects and/or toxicity in a subject receiving treatment with a DLL4 antagonist, comprising: obtaining a biological sample from the subject receiving treatment, determining the level of a biomarker in the sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein if the level of the biomarker in the sample is higher than the predetermined level of the biomarker then a cardiovascular side effect and/or toxicity is indicated.
  • the biomarker is a natriuretic peptide.
  • the method for monitoring cardiovascular side effects and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprises: obtaining a biological sample from the subject receiving treatment, determining the level of a natriuretic peptide in the sample, and comparing the level of the natriuretic peptide in the sample to a predetermined level of the natriuretic peptide, wherein if the level of the natriuretic peptide in the sample is higher than the predetermined level of the natriuretic peptide then a cardiovascular side effect and/or toxicity is indicated.
  • the natriuretic peptide is BNP.
  • the biological sample is blood, serum, or plasma.
  • the predetermined level is about 300 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 200 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 250 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 200 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 150 pg/ml or less in a blood, serum, or plasma sample.
  • the predetermined level is about 100 pg/ml or less in a blood, serum, or plasma sample.
  • the predetermined level of a biomarker e.g., natriuretic peptide or BNP
  • the predetermined level of a biomarker is the amount of the biomarker in a sample obtained at an earlier date.
  • the predetermined level of a biomarker e.g., natriuretic peptide or BNP
  • the predetermined level of a biomarker is a normal reference level.
  • the predetermined level for BNP is about 100 pg/ml or less in blood, serum, or plasma.
  • a biological sample is obtained approximately every week, every 2 weeks, every 3 weeks, or every 4 weeks.
  • the subject is administered a therapeutically effective amount of a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker.
  • a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker.
  • the natriuretic peptide is BNP and the predetermined level is about 100 pg/ml.
  • the subject is administered a therapeutically effective amount of an inhibitor and/or a ⁇ -blocker.
  • the natriuretic peptide is BNP and the predetermined level is about 200 pg/ml. In some embodiments of the methods described herein, wherein the natriuretic peptide level in the sample is above a predetermined level for any one sample, the subject is administered a therapeutically effective amount of an ACE inhibitor and/or a ⁇ -blocker and the DLL4 antagonist is withheld. In some embodiments, the natriuretic peptide is BNP and the predetermined level is about 300 pg/ml.
  • natriuretic peptide level decreases to below about 200 pg/ml after administration of the ACE inhibitor and/or a ⁇ -blocker, then administration of the DLL4 antagonist is resumed. In some embodiments, if the natriuretic peptide level decreases to below about 300 pg/ml after administration of the ACE inhibitor and/or a ⁇ -blocker, then administration of the DLL4 antagonist is resumed.
  • the invention provides methods of reducing cardiovascular side effects and/or toxicity in a subject receiving treatment with a DLL4 antagonist, comprising: obtaining a biological sample from the subject receiving treatment, determining the level of a natriuretic peptide in the sample, comparing the level of the natriuretic peptide in the sample to a predetermined level of the natriuretic peptide, and administering to the subject a therapeutically effective amount of a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker if the level of the natriuretic peptide in the sample is higher than the predetermined level of the natriuretic peptide.
  • a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker
  • the invention provides methods of preventing or attenuating the development of cardiovascular side effects and/or toxicity in a subject receiving treatment with a DLL4 antagonist, comprising: obtaining a biological sample from the subject prior to treatment with the DLL4 antagonist, determining the level of a natriuretic peptide in the sample, comparing the level of the natriuretic peptide in the sample to a predetermined level of the natriuretic peptide, administering to the subject a therapeutically effective amount of a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker, and administering to the subject the DLL4 antagonist.
  • a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker
  • the invention provides methods of ameliorating cardiovascular side effects and/or toxicity in a subject administered a DLL4 antagonist, comprising: determining the level of a natriuretic peptide in the sample, and administering to the subject a therapeutically effective amount of a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker.
  • a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker.
  • the natriuretic peptide is BNP.
  • the invention provides methods of screening a subject for the risk of cardiovascular side effects and/or toxicity from treatment with a DLL4 antagonist, comprising: obtaining a biological sample from the subject prior to treatment with the DLL4 antagonist, determining the level of a natriuretic peptide in the sample, and comparing the level of the natriuretic peptide in the sample to a predetermined level of the natriuretic peptide, wherein if the level of the natriuretic peptide in the sample is higher than the predetermined level then the subject is at risk for cardiovascular side effects and/or toxicity.
  • the subject is administered a therapeutically effective amount of a therapeutic agent directed to the cardiovascular side effect and/or toxicity prior to treatment with the DLL4 antagonist.
  • the DLL4 antagonist specifically binds human DLL4.
  • the DLL4 antagonist is an antibody that specifically binds the extracellular domain of human DLL4.
  • the DLL4 antagonist specifically binds an epitope within amino acids 27-217 of the extracellular domain of human DLL4 (SEQ ID NO:17).
  • the DLL4 antagonist binds an epitope comprising amino acids 66-73 (QAVVSPGP, SEQ ID NO: 18) of human DLL4.
  • the DLL4 antagonist binds an epitope comprising amino acids 139-146 (LISKIAIQ, SEQ ID NO:19) of human DLL4.
  • the DLL4 antagonist binds an epitope comprising amino acids 66-73 (QAVVSPGP, SEQ ID NO: 18) and amino acids 139-146 (LISKIAIQ, SEQ ID NO:19) of human DLL4. In some embodiments, the DLL4 antagonist binds human DLL4 with a dissociation constant (K D ) of about 10 nM to about 0.1 nM.
  • K D dissociation constant
  • the DLL4 antagonist is an anti-DLL4 antibody.
  • the DLL4 antagonist is an antibody comprising a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:1), a heavy chain CDR2 comprising YISSYNGATNYNQKFKG (SEQ ID NO:3), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:5), and a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:6), a light chain CDR2 comprising AASNQGS (SEQ ID NO:7), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:8).
  • the DLL4 antagonist is an antibody comprising a heavy chain variable region comprising the amino acids of SEQ ID NO: 10. In certain embodiments, the DLL4 antagonist is an antibody which further comprises a light chain variable region comprising the amino acids of SEQ ID NO: 12. In certain embodiments, the DLL4 antagonist comprises the same heavy and light chain amino acid sequences as an antibody encoded by a plasmid deposited with ATCC having deposit no. PTA-8425 or PTA-8427. In certain embodiments, the DLL4 antagonist comprises the heavy chain CDR amino acid sequences and the light chain CDR amino acid sequences that are contained in the 21M18 antibody produced by the hybridoma deposited with ATCC having deposit no. PTA-8670.
  • the DLL4 antagonist is encoded by the plasmid having ATCC deposit no. PTA-8425 which was deposited with American Type Culture Collection (ATCC), at 10801 University Boulevard, Manassas, Va. 20110, under the conditions of the Budapest Treaty on May 10, 2007.
  • the DLL4 antagonist is encoded by the plasmid having ATCC deposit no. PTA-8427 which was deposited with American Type Culture Collection (ATCC), at 10801 University Boulevard, Manassas, Va., 20110, under the conditions of the Budapest Treaty on May 10, 2007.
  • the DLL4 antagonist is the antibody produced by the hybridoma having ATCC deposit no.
  • the DLL4 antagonist is a humanized version of the antibody produced by the hybridoma having ATCC deposit no. PTA-8670.
  • the DLL4 antagonist competes for specific binding to human DLL4 with an antibody encoded by the plasmid deposited with ATCC having deposit no. PTA-8425 or PTA-8427.
  • the subject has cancer.
  • the cancer is selected from the group consisting of: lung cancer, pancreatic cancer, breast cancer, colon cancer, colorectal cancer, melanoma, gastrointestinal cancer, gastric cancer, renal cancer, ovarian cancer, liver cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, glioma, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, and head and neck cancer.
  • the subject is treated with the DLL4 antagonist in combination with one or more additional anti-cancer agents.
  • the present invention encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members.
  • the present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.
  • FIG. 1 The table is a summary of some of the subjects enrolled in the Phase 1b clinical trial for treatment of NSCLC with OMP-21M18 in combination with carboplatin and pemetrexed.
  • FIG. 2 Percent change in target tumor lesions in subjects enrolled in the Phase 1b clinical trial for treatment of NSCLC with OMP-21M18 in combination with carboplatin and pemetrexed.
  • FIG. 3 Percent change in target tumor lesions in subjects enrolled in the Phase 1b clinical trial for treatment of pancreatic cancer with OMP-21M18 in combination with gemcitabine.
  • the present invention relates to treating diseases with a DLL4 antagonist. More particularly, the invention provides methods for treating cancer comprising administering a DLL4 antagonist, either alone or in combination with other anti-cancer agents, and monitoring for cardiovascular side effects and/or toxicity, including those related to the DLL4 antagonist.
  • the anti-DLL4 antibody OMP-21M18 was administered to subjects in a Phase 1 single agent dose escalation trial.
  • the data from this early trial, as well as results from animal studies suggested that administration of a DLL4 antagonist such as an anti-DLL4 antibody may result in cardiovascular side effects and/or toxicity in certain patients.
  • the study showed that increased BNP levels may be an early indicator that a patient being treated with a DLL4 antagonist is at risk of developing cardiotoxicity, allowing for intervention with cardioprotective medications.
  • antagonists refer to any molecule that partially or fully blocks, inhibits, reduces or neutralizes a biological activity of a target and/or signaling pathway (e.g., Notch signaling).
  • a target and/or signaling pathway e.g., Notch signaling
  • antagonists refers to a molecule that partially or fully blocks, inhibits, neutralizes, or interferes with the biological activities of a DLL4 protein.
  • Suitable antagonist molecules specifically include, but are not limited to, antagonist DLL4 antibodies or antibody fragments.
  • modulation and “modulate” as used herein refer to a change or an alteration in a biological activity. Modulation includes, but is not limited to, stimulating or inhibiting an activity. Modulation may be an increase or a decrease in activity (e.g., a decrease in Notch signaling), a change in binding characteristics, or any other change in the biological, functional, or immunological properties associated with the activity of a protein, pathway, or other biological point of interest.
  • antibody refers to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing, through at least one antigen recognition site within the variable region of the immunoglobulin molecule.
  • the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments), single chain Fv (scFv) antibodies, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site as long as the antibodies exhibit the desired biological activity.
  • antibody fragments such as Fab, Fab′, F(ab′)2, and Fv fragments
  • scFv single chain Fv
  • multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site as long as the antibodies exhibit the desired biological activity.
  • An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • the different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations.
  • Antibodies can be naked or conjugated to other molecules, including but not limited to, toxins and radioisotopes.
  • antibody fragment refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody.
  • antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments.
  • Antibody fragment as used herein comprises an antigen-binding site or epitope-binding site.
  • variable region of an antibody refers to the variable region of the antibody light chain, or the variable region of the antibody heavy chain, either alone or in combination.
  • the variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs), also known as “hypervariable regions”.
  • FR framework regions
  • CDRs complementarity determining regions
  • the CDRs in each chain are held together in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding sites of the antibody.
  • CDRs There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al., 1991 , Sequences of Proteins of Immunological Interest, 5 th Edition, National Institutes of Health, Bethesda Md.), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., 1997 , J. Mol. Biol., 273:927-948). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.
  • the term “monoclonal antibody” as used herein refers to a homogenous antibody population involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies that typically include a mixture of different antibodies directed against different antigenic determinants.
  • the term “monoclonal antibody” encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab′, F(ab′)2, Fv), single chain (scFv) antibodies, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site (antigen-binding site).
  • “monoclonal antibody” refers to such antibodies made by any number of techniques, including but not limited to, hybridoma production, phage selection, recombinant expression, and transgenic animals.
  • humanized antibody refers to forms of antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences.
  • humanized antibodies are human immunoglobulins in which residues of the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and/or binding capability (Jones et al., 1986 , Nature, 321:522-525; Riechmann et al., 1988 , Nature, 332:323-327; Verhoeyen et al. 1988 , Science, 239:1534-1536).
  • a non-human species e.g., mouse, rat, rabbit, or hamster
  • the Fv framework region residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and/or binding capability.
  • the humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or binding capability.
  • the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDRs that correspond to the non-human immunoglobulin whereas all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region or domain
  • human antibody refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any of the techniques known in the art. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • chimeric antibody refers to an antibody wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species.
  • the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and/or binding capability, while the constant regions are homologous to the sequences in antibodies derived from another species (e.g., human).
  • affinity-matured antibody refers to an antibody with one or more alterations in one or more CDRs thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alterations(s).
  • Preferred affinity-matured antibodies will have nanomolar or even picomolar affinities for the target antigen.
  • Affinity-matured antibodies are produced by procedures known in the art. For example, Marks et al. 1992 , Bio/Technology 10:779-783, describes affinity maturation by VH and VL domain shuffling.
  • Random mutagenesis of CDR and/or framework residues is described by Barbas et al., 1994 , PNAS, 91:3809-3813; Schier et al., 1995 , Gene, 169:147-155; Yelton et al., 1995 , J. Immunol. 155:1994-2004; Jackson et al., 1995 , J. Immunol., 154:3310-9; and Hawkins et al., 1992 , J. Mol. Biol., 226:889-896.
  • epitopes and “antigenic determinant” are used interchangeably herein and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody.
  • the antigen is a polypeptide
  • epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein.
  • Epitopes formed from contiguous amino acids also referred to as linear epitopes
  • epitopes formed by tertiary folding also referred to as conformational epitopes
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
  • selectively binds or “specifically binds” mean that a binding agent or an antibody reacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including unrelated or related proteins.
  • specifically binds means, for instance, that an antibody binds a protein with a K D of about 0.1 mM or less, but more usually less than about 1 ⁇ M.
  • “specifically binds” means that an antibody binds a target at times with a K) of at least about 0.1 ⁇ M or less, at other times at least about 0.01 ⁇ M or less, and at other times at least about 1 nM or less. Because of the sequence identity between homologous proteins in different species, specific binding can include an antibody that recognizes a protein in more than one species (e.g., human DLL4 and mouse DLL4). Likewise, because of homology within certain regions of polypeptide sequences of different proteins, specific binding can include an antibody (or other polypeptide or binding agent) that recognizes more than one protein.
  • an antibody or binding moiety that specifically binds a first target may or may not specifically bind a second target.
  • “specific binding” does not necessarily require (although it can include) exclusive binding, i.e. binding to a single target.
  • an antibody may, in certain embodiments, specifically bind more than one target.
  • multiple targets may be bound by the same antigen-binding site on the antibody.
  • an antibody may, in certain instances, comprise two identical antigen-binding sites, each of which specifically binds the same epitope on two or more proteins.
  • an antibody may be bispecific or multispecific and comprise at least two antigen-binding sites with differing specificities.
  • a bispecific antibody may comprise one antigen-binding site that recognizes an epitope on one protein and further comprise a second, different antigen-binding site that recognizes a different epitope on a second protein.
  • reference to binding means specific binding.
  • polypeptide and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids
  • the polypeptides of this invention may be based upon antibodies, in certain embodiments, the polypeptides can occur as single chains or associated chains (e.g., dimers).
  • polynucleotide and “nucleic acid” are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • the percent identity may be measured using sequence comparison software or algorithms or by visual inspection.
  • Various algorithms and software that may be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof.
  • two nucleic acids or polypeptides of the invention are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%. 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the sequences that is at least about 10, at least about 20, at least about 40-60 residues, at least about 60-80 residues in length or any integral value therebetween.
  • identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a nucleotide sequence.
  • a “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e
  • substitution of a phenylalanine for a tyrosine is a conservative substitution.
  • conservative substitutions in the sequences of the polypeptides and antibodies of the invention do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the antigen(s), i.e., the one or more RSPO protein(s) to which the polypeptide or antibody binds.
  • Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well-known in the art.
  • vector means a construct, which is capable of delivering, and usually expressing, one or more gene(s) or sequence(s) of interest in a host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.
  • a polypeptide, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature.
  • Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature.
  • a polypeptide, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.
  • substantially pure refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.
  • cancer and “cancerous” as used herein refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth.
  • examples of cancer include, but are not limited to, carcinoma, blastoma, sarcoma, and hematologic cancers such as lymphoma and leukemia.
  • tumor and “neoplasm” as used herein refer to any mass of tissue that results from excessive cell growth or proliferation, either benign (non-cancerous) or malignant (cancerous) including pre-cancerous lesions.
  • metalastasis refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location.
  • a “metastatic” or “metastasizing” cell is one that loses adhesive contacts with neighboring cells and migrates (e.g., via the bloodstream or lymph) from the primary site of disease to invade neighboring body structures.
  • cancer stem cell and “CSC” and “tumor stem cell” and “tumor initiating cell” are used interchangeably herein and refer to cells from a cancer or tumor that: (1) have extensive proliferative capacity; 2) are capable of asymmetric cell division to generate one or more types of differentiated cell progeny wherein the differentiated cells have reduced proliferative or developmental potential; and (3) are capable of symmetric cell divisions for self-renewal or self-maintenance.
  • CSC cancer stem cell
  • tumor stem cell undergo self-renewal versus differentiation in a chaotic manner to form tumors with abnormal cell types that can change over time as mutations occur.
  • cancer cell and “tumor cell” refer to the total population of cells derived from a cancer or tumor or pre-cancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the cancer cell population, and tumorigenic stem cells (cancer stem cells).
  • cancer stem cells tumorigenic stem cells
  • tumorigenic refers to the functional features of a cancer stem cell including the properties of self-renewal (giving rise to additional tumorigenic cancer stem cells) and proliferation to generate all other tumor cells (giving rise to differentiated and thus non-tumorigenic tumor cells).
  • tumorigenicity refers to the ability of a random sample of cells from the tumor to form palpable tumors upon serial transplantation into immunocompromised hosts (e.g., mice).
  • subject refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment.
  • subject and patient are used interchangeably herein in reference to a human subject.
  • pharmaceutically acceptable refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.
  • pharmaceutically acceptable excipient, carrier or adjuvant refers to an excipient, carrier or adjuvant that can be administered to a subject, together with at least one binding agent (e.g., an antibody) of the present disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic effect.
  • binding agent e.g., an antibody
  • an effective amount or “therapeutically effective amount” or “therapeutic effect” refer to an amount of a binding agent, an antibody, polypeptide, polynucleotide, small organic molecule, or other drug effective to “treat” a disease or disorder in a subject.
  • the therapeutically effective amount of a drug has a therapeutic effect and as such can reduce the number of cancer cells; decrease tumorigenicity, tumorigenic frequency, or tumorigenic capacity; reduce the number or frequency of cancer stem cells: reduce the tumor size; reduce the cancer cell population; inhibit or stop cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibit and stop tumor or cancer cell metastasis; inhibit and stop tumor or cancer cell growth; relieve to some extent one or more of the symptoms associated with the cancer; reduce morbidity and mortality; improve quality of life; or a combination of such effects.
  • the agent for example an antibody, prevents growth and/or kills existing cancer cells, it can be referred to as cytostatic and/or cytotoxic.
  • treating or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to both i) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder.
  • prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder.
  • a subject is successfully “treated” according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including the spread of cancer cells into soft tissue and bone; inhibition of or an absence of tumor or cancer cell metastasis; inhibition or an absence of cancer growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity: reduction in the number or frequency of cancer stem cells; or some combination of effects.
  • reference to “about” or “approximately” a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to “about X” includes description of “X”.
  • the DLL4 antagonist specifically binds the extracellular domain of human DLL4.
  • the DLL4 antagonist is an antibody.
  • the DLL4 antagonist or antibody specifically binds an epitope within amino acids 27-217 of the extracellular domain of human DLL4 (SEQ ID NO: 17).
  • the DLL4 antagonist or antibody specifically binds an epitope formed by a combination of the N-terminal region of human DLL4 (SEQ ID NO:14) and the DSL region of human DLL4 (SEQ ID NO:15).
  • the DLL4 antagonist or antibody specifically binds within the N-terminal region of human DLL4 (SEQ ID NO: 14).
  • the DLL4 antagonist or antibody binds an epitope comprising amino acids 66-73 (QAVVSPGP; SEQ ID NO: 18) of human DLL4. In some embodiments, the DLL4 antagonist or antibody binds an epitope comprising amino acids 139-146 (LISKIAIQ; SEQ ID NO:19) of human DLL4. In some embodiments, the DLL4 antagonist or antibody binds an epitope comprising amino acids 66-73 (QAWSPGP; SEQ ID NO: 18) and amino acids 139-146 (LISKIAIQ; SEQ ID NO: 19) of human DLL4.
  • the DLL4 antagonist (e.g., an antibody) binds to human DLL4 with a dissociation constant (K D ) of about 1 ⁇ M or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, or about 1 nM or less. In certain embodiments, the DLL4 antagonist or antibody binds to human DLL4 with a K D of about 40 nM or less, about 20 nM or less, about 10 nM or less, or about 1 nM or less. In certain embodiments, the DLL4 antagonist binds to human DLL4 with a K D of about 1 nM.
  • K D dissociation constant
  • the DLL4 antagonist binds to human DLL4 with a K D of about 0.8 nM. In certain embodiments, the DLL4 antagonist binds to human DLL4 with a K D of about 0.6 nM. In certain embodiments, the DLL4 antagonist binds to human DLL4 with a K D of about 0.5 nM. In certain embodiments, the DLL4 antagonist binds to human DLL4 with a K D of about 0.4 nM. In some embodiments, the K D is measured by surface plasmon resonance.
  • the dissociation constant of the antagonist or antibody to DLL4 is the dissociation constant determined using a DLL4 fusion protein comprising a DLL4 extracellular domain (e.g., a DLL4 ECD-Fc fusion protein) immobilized on a Biacore chip.
  • a DLL4 fusion protein comprising a DLL4 extracellular domain (e.g., a DLL4 ECD-Fc fusion protein) immobilized on a Biacore chip.
  • the DLL4 antagonist binds to DLL4 with a half maximal effective concentration (EC 50 ) of about 1 ⁇ M or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 100 nM or less, or about 1 nM or less.
  • the DLL4 antagonist or antibody binds to human DLL4 with an EC 50 of about 40 nM or less, about 20 nM or less, about 10 nM or less, or about 1 nM or less.
  • the DLL4 antagonist is a polypeptide. In certain embodiments, the DLL4 antagonist or polypeptide is an antibody. In certain embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In certain embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a bispecific antibody. In certain embodiments, the antibody is a humanized antibody. In certain embodiments, the antibody is a human antibody. In certain embodiments, the antibody is an antibody fragment comprising an antigen-binding site.
  • the DLL4 antagonists (e.g., antibodies) of the present invention can be assayed for specific binding by any method known in the art.
  • the immunoassays which can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Biacore analysis, FACS analysis, immunofluorescence, immunocytochemistry, Western blot analysis, radioimmunoassay, ELISA, “sandwich” immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitin reaction, immunodiffusion assay, agglutination assay, complement-fixation assay, immunoradiometric assay, fluorescent immunoassay, and protein A immunoassay.
  • the specific binding of a DLL4 antagonist (e.g., an antibody) to human DLL4 may be determined using ELISA.
  • An ELISA assay comprises preparing DLL4 antigen, coating wells of a 96 well microtiter plate with antigen, adding to the wells the DLL4 antagonist or antibody conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase), incubating for a period of time and detecting the presence of the binding agent or antibody.
  • an enzymatic substrate e.g., horseradish peroxidase or alkaline phosphatase
  • the DLL4 antagonist or antibody is not conjugated to a detectable compound, but instead a second conjugated antibody that recognizes the DLL4 antagonist or antibody is added to the well.
  • the DLL4 antagonist or antibody can be coated on the well, antigen is added to the coated well and then a second antibody conjugated to a detectable compound is added.
  • ELISAs e.g., Ausubel et al., Editors, 1994-present, Current Protocols in Molecular Biology , John Wiley & Sons, Inc., New York, N.Y.
  • the specific binding of a DLL4 antagonist (e.g., an antibody) to human DLL4 may be determined using FACS.
  • a FACS screening assay may comprise generating a cDNA construct that expresses an antigen as a fusion protein, transfecting the construct into cells, expressing the antigen on the surface of the cells, mixing the DLL4 antagonist with the transfected cells, and incubating for a period of time.
  • the cells bound by the DLL4 antagonist may be identified by using a secondary antibody conjugated to a detectable compound (e.g., PE-conjugated anti-Fc antibody) and a flow cytometer.
  • a detectable compound e.g., PE-conjugated anti-Fc antibody
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3 H or 125 I), or fragment or variant thereof, with the antibody of interest in the presence of increasing amounts of unlabeled antigen followed by the detection of the antibody bound to the labeled antigen.
  • labeled antigen e.g., 3 H or 125 I
  • the affinity of the antibody for the antigen and the on-off rates can be determined from the data by Scatchard plot analysis.
  • Biacore kinetic analysis is used to determine the binding affinities and on-off rates of antagonists or antibodies that bind DLL4.
  • Biacore kinetic analysis comprises analyzing the binding and dissociation of antibodies from antigens (e.g., DLL4 proteins) that have been immobilized on the surface of a Biacore chip.
  • Biacore kinetic analyses can be used to study binding of different antibodies in qualitative epitope competition binding assays.
  • the DLL4 antagonists are polyclonal antibodies.
  • Polyclonal antibodies can be prepared by any known method. Polyclonal antibodies are prepared by immunizing an animal (e.g., a rabbit, rat, mouse, goat, donkey) by multiple subcutaneous or intraperitoneal injections of the relevant antigen (e.g., a purified peptide fragment, full-length recombinant protein, fusion protein, etc.). The antigen can be optionally conjugated to a carrier protein such as keyhole limpet hemocyanin (KLH) or serum albumin.
  • KLH keyhole limpet hemocyanin
  • the antigen (with or without a carrier protein) is diluted in sterile saline and usually combined with an adjuvant (e.g., Complete or Incomplete Freund's Adjuvant) to form a stable emulsion.
  • an adjuvant e.g., Complete or Incomplete Freund's Adjuvant
  • polyclonal antibodies are recovered from blood, ascites, and the like, of the immunized animal.
  • Polyclonal antibodies can be purified from serum or ascites according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and dialysis.
  • the DLL4 antagonists are monoclonal antibodies.
  • Monoclonal antibodies can be prepared using hybridoma methods known to one of skill in the art (see e.g., Kohler and Milstein, 1975 , Nature 256:495). Using the hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized as described above to elicit lymphocytes to produce antibodies that will specifically bind the immunizing antigen. In some embodiments, lymphocytes are immunized in vitro.
  • the immunizing antigen e.g., DLL4 is a human protein or a portion thereof.
  • the immunizing antigen (e.g., DLL4) is a mouse protein or a portion thereof. In some embodiments, the immunizing antigen is an extracellular domain of human DLL4. In some embodiments, the immunizing antigen is an extracellular domain of mouse DLL4. In some embodiments, a mouse is immunized with a human antigen. In some embodiments, a mouse is immunized with a mouse antigen.
  • lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol.
  • the hybridoma cells are selected using specialized media as known in the art and unfused lymphocytes and myeloma cells do not survive the selection process.
  • Hybridomas that produce monoclonal antibodies directed against a target antigen may be identified by a variety of techniques including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (e.g., flow cytometry, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA)).
  • the hybridomas can be propagated either in in vitro culture using standard methods (J. W.
  • the monoclonal antibodies can be purified from the culture medium or ascites fluid according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and dialysis.
  • monoclonal antibodies can be made using recombinant DNA techniques as known to one skilled in the art (see e.g., U.S. Pat. No. 4,816,567).
  • the polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using conventional techniques.
  • the isolated polynucleotides encoding the heavy and light chains are cloned into suitable expression vectors which produce the monoclonal antibodies when transfected into host cells such as E.
  • recombinant monoclonal antibodies, or fragments thereof can be isolated from phage display libraries expressing variable domain regions or CDRs of a desired species (see e.g., McCafferty et al., 1990 , Nature, 348:552-554; Clackson et al., 1991 , Nature, 352:624-628; and Marks et al., 1991 , J. Mol. Biol., 222:581-597).
  • the polynucleotide(s) encoding a monoclonal antibody can be modified, for example, by using recombinant DNA technology to generate alternative antibodies.
  • the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted for those regions of, for example, a human antibody to generate a chimeric antibody or for a non-immunoglobulin polypeptide to generate a fusion antibody.
  • the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody.
  • site-directed or high-density mutagenesis of the variable region can be used to optimize specificity, affinity, and/or other biological characteristics of a monoclonal antibody.
  • site-directed mutagenesis of the CDRs can be used to optimize specificity, affinity, and/or other biological characteristics of a monoclonal antibody.
  • the DLL4 antagonist is a humanized antibody.
  • humanized antibodies are human immunoglobulins in which residues from the complementary determining regions (CDRs) are replaced by residues from CDRs of a non-human species (e.g., mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and/or capability by methods known to one skilled in the art.
  • the Fv framework region residues of a human immunoglobulin are replaced with the corresponding framework region residues from a non-human immunoglobulin that has the desired specificity, affinity, and/or capability.
  • the humanized antibody is further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability.
  • the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all, or substantially all, of the CDRs that correspond to the non-human immunoglobulin whereas all, or substantially all, of the framework regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region or domain
  • such humanized antibodies are used therapeutically because they should be less antigenic and may reduce HAMA (human anti-mouse antibody) responses when administered to a human subject.
  • HAMA human anti-mouse antibody
  • One skilled in the art would be able to obtain a functional humanized antibody with reduced immunogenicity following known techniques (see, e.g., U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761; and 5,693,762).
  • the invention provides an antibody that specifically binds the extracellular domain of human DLL4, wherein the antibody comprises one, two, three, four, five, and/or six of the CDRs of antibodies 21M18, 21M18 H9L2, and/or 21M18 H7L2.
  • Antibodies 21M18 H7L2 and 21M18 H9L2 are humanized versions of the murine 21M18 antibody.
  • Antibody 21M18 H7L2 is also referred to as OMP-21M18 and demcizumab.
  • the invention provides a DLL4 antagonist, wherein the antagonist is a DLL4 antibody that specifically binds an epitope within amino acids 27-217 of the extracellular domain of human DLL4, and wherein the antibody comprises: a heavy chain CDR1 comprising TAYYIH (SEQ ID NO: 1), a heavy chain CDR2 comprising YISCYNGATNYNQKFKG (SEQ ID NO:2), YISSYNGATNYNQKFKG (SEQ ID NO:3), or YTSVYNGATNYNQKFKG (SEQ ID NO:4), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:5).
  • TAYYIH SEQ ID NO: 1
  • a heavy chain CDR2 comprising YISCYNGATNYNQKFKG (SEQ ID NO:2), YISSYNGATNYNQKFKG (SEQ ID NO:3), or YTSVYNGATNYNQKFKG (SEQ ID NO:
  • the antibody further comprises a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:6), a light chain CDR2 comprising AASNQGS (SEQ ID NO:7), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:8).
  • the antibody comprises a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:6), a light chain CDR2 comprising AASNQGS (SEQ ID NO:7), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:8).
  • the DLL4 antibody comprises a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:1), a heavy chain CDR2 comprising YISSYNGATNYNQKFKG (SEQ ID NO:3), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:5); and a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:6), a light chain CDR2 comprising AASNQGS (SEQ ID NO:7), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:8).
  • the invention provides an antibody that specifically binds an epitope within amino acids 27-217 of the extracellular domain of human DLL4, wherein the antibody comprises a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 1, and/or a light chain variable region having at least 80% sequence identity to SEQ ID NO: 12.
  • the antibody comprises a heavy chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 111.
  • the antibody comprises a light chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:12. In certain embodiments, the antibody comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO:9, and/or a light chain variable region having at least about 95% sequence identity to SEQ ID NO:12. In certain embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO:9, and/or a light chain variable region comprising SEQ ID NO: 12. In certain embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO:9 and a light chain variable region comprising SEQ ID NO:12.
  • the antibody comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO: 10, and/or a light chain variable region having at least about 95% sequence identity to SEQ ID NO: 12. In certain embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 10, and/or a light chain variable region comprising SEQ ID NO: 12. In certain embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 10 and a light chain variable region comprising SEQ ID NO: 12. In certain embodiments, the antibody comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO:11, and/or a light chain variable region having at least about 95% sequence identity to SEQ ID NO: 12.
  • the antibody comprises a heavy chain variable region comprising SEQ ID NO: 11, and/or a light chain variable region comprising SEQ ID NO:12. In certain embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO:11 and a light chain variable region comprising SEQ ID NO:12.
  • the anti-DLL4 antibody is the antibody produced by the hybridoma deposited with ATCC on Sep. 28, 2007 and having ATCC deposit number PTA-8670, also known as murine 21M18.
  • the murine 21M18 antibody is described in detail in U.S. Pat. No. 7,750,124, filed Sep. 28, 2007.
  • the anti-DLL4 antibody is the antibody encoded by the plasmid deposited with ATCC on May 10, 2007, having ATCC deposit number PTA-8425, also known as 21M18 H7L2 and OMP-21M18.
  • the OMP-2M18 antibody is described in detail in U.S. Pat. No. 7,750,124, filed Sep. 28, 2007.
  • the anti-DLL4 antibody OMP-21M18 comprises a heavy chain variable region comprising CDR amino acid sequences CDR1 (SEQ ID NO:1); CDR2 (SEQ ID NO:3); and CDR3 (SEQ ID NO:5); and a light chain variable region comprising CDR amino acid sequences CDR1 (SEQ ID NO:6); CDR2 (SEQ ID NO:7); and CDR3 (SEQ ID NO:8).
  • the OMP-21M18 antibody comprises the heavy chain variable region sequence of SEQ ID NO:10 and the light chain variable region sequence of SEQ ID NO:12.
  • the anti-DLL4 antibody is the antibody encoded by the plasmid deposited with ATCC on May 10, 2007, having ATCC deposit number PTA-8427, also known as 21M18 H9L2.
  • the 21M18 H9L2 antibody is described in detail in U.S. Pat. No. 7,750,124, filed Sep. 28, 2007.
  • the DLL4 antagonist (e.g., an antibody) binds to the same epitope that an antibody comprising the heavy chain variable region comprising SEQ ID NO: 10, and/or a light chain variable region comprising SEQ ID NO: 12 binds. In certain embodiments, the DLL4 antagonist (e.g., an antibody) binds to the same epitope that an antibody comprising the heavy chain variable region comprising SEQ ID NO:9, and/or a light chain variable region comprising SEQ ID NO: 12 binds. In certain embodiments, the DLL4 antagonist (e.g., an antibody) binds to the same epitope that an antibody comprising the heavy chain variable region comprising SEQ ID NO:9, and/or a light chain variable region comprising SEQ ID NO: 12 binds. In certain embodiments, the DLL4 antagonist (e.g., an antibody) binds to the same epitope that an antibody comprising the heavy chain variable region comprising SEQ ID NO:9, and/or a light chain variable region
  • an antibody binds to the same epitope that an antibody comprising the heavy chain variable region comprising SEQ ID NO: 11, and/or a light chain variable region comprising SEQ ID NO: 12 binds.
  • the DLL4 antagonist or antibody binds to the same epitope as murine antibody 21M18.
  • the DLL4 antagonist or antibody binds to the same epitope as humanized antibody 21M18 H7L2 (OMP-21M18).
  • the DLL4 antagonist or antibody binds to the same epitope as humanized antibody 21M18 H9L2.
  • the DLL4 antagonist (e.g. an antibody) competes for specific binding to an extracellular domain of human DLL4 with an antibody, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 10, and/or a light chain variable region comprising SEQ ID NO: 12.
  • the DLL4 antagonist (e.g., an antibody) competes for specific binding to an extracellular domain of human DLL4 with an antibody, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO:9, and/or a light chain variable region comprising SEQ ID NO: 12.
  • the DLL4 antagonist (e.g., an antibody) competes for specific binding to an extracellular domain of human DLL4 with an antibody, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 11, and/or a light chain variable region comprising SEQ ID NO: 12.
  • the DLL4 antagonist competes for specific binding to an extracellular domain of human DLL4 with an antibody encoded by the plasmid deposited with ATCC having deposit no. PTA-8425.
  • the DLL4 antagonist or antibody competes for specific binding to an extracellular domain of human DLL4 with an antibody encoded by the plasmid deposited with ATCC having deposit no. PTA-8427.
  • the DLL4 antagonist or antibody competes for specific binding to an extracellular domain of human DLL4 with an antibody produced by the hybridoma deposited with ATCC having deposit no. PTA-8670. In some embodiments, the DLL4 antagonist or antibody competes for specific binding to an epitope within amino acids 27-217 of the extracellular domain of human DLL4 in a competitive binding assay.
  • Other anti-DLL4 antibodies are known in the art and may be used in the methods described herein.
  • the DLL4 antagonist is a human antibody.
  • Human antibodies can be directly prepared using various techniques known in the art.
  • human antibodies may be generated from immortalized human B lymphocytes immunized in vitro or from lymphocytes isolated from an immunized individual. In either case, cells that produce an antibody directed against a target antigen can be generated and isolated (see, e.g., Cole et al., 1985 , Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, p. 77; Boerner et al., 1991 , J. Immunol., 147:86-95; and U.S. Pat. Nos. 5,750,373; 5,567,610; and 5,229,275).
  • the human antibody can be selected from a phage library, wherein the phage library expresses human antibodies (Vaughan et al., 1996 , Nature Biotechnology, 14:309-314; Sheets et al., 1998 , PNAS, 95:6157-6162; Hoogenboom and Winter, 1991 , J. Mol. Biol., 227:381; Marks et al., 1991 , J. Mol. Biol., 222:581).
  • phage display technology can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable domain gene repertoires from unimmunized donors. Techniques for the generation and use of antibody phage libraries are described in U.S. Pat. Nos.
  • affinity maturation strategies known in the art, including but not limited to, chain shuffling (Marks et al., 1992 , Bio/Technology, 10:779-783) and site-directed mutagenesis, may be employed to generate high affinity human antibodies.
  • human antibodies can be made in transgenic mice that contain human immunoglobulin loci. Upon immunization these mice are capable of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production. This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.
  • the DLL4 antagonist is a bispecific antibody.
  • Bispecific antibodies are capable of specifically recognizing and binding to at least two different epitopes.
  • the different epitopes can either be within the same molecule or on different molecules.
  • the antibodies can specifically recognize and bind a first antigen target, (e.g., DLL4) as well as a second antigen target, such as an effector molecule on a leukocyte (e.g., CD2, CD3, CD28, or B7) or a Fc receptor (e.g., CD64, CD32, or CD16) so as to focus cellular defense mechanisms to the cell expressing the first antigen target.
  • a first antigen target e.g., DLL4
  • a second antigen target such as an effector molecule on a leukocyte (e.g., CD2, CD3, CD28, or B7) or a Fc receptor (e.g., CD64, CD32, or CD16)
  • the antibodies can be used to direct cytotoxic agents to cells which express a particular target antigen, such as DLL4. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. In certain embodiments, the antibodies can be used to affect angiogenesis.
  • the bispecific antibody specifically binds DLL4, as well as a second Notch ligand (e.g., Jagged1 or Jagged2), or at least one Notch receptor selected from the group consisting of Notch 1, Notch2, Notch3, and Notch4. In certain embodiments, the bispecific antibody specifically binds DLL4, as well as VEGF.
  • the bispecific antibody is a bispecific antibody disclosed in U.S. patent application Ser. No. 13/625,417, filed on Sep. 24, 2012.
  • the anti-VEGF/DLL4 bispecific antibody is 219R45-MB-21M18, 219R45-MB-21R79, 219R45-MB-21R75, or 219R45-MB-21R83 as disclosed in U.S. patent application Ser. No. 13/625,417, filed on Sep. 24, 2012.
  • Bispecific antibodies can be intact antibodies or antibody fragments. Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be prepared (Tutt et al., 1991 , J. Immunol., 147:60). Thus, in certain embodiments the antibodies to DLL4 are multispecific.
  • the DLL4 antagonists e.g., antibodies or other polypeptides
  • the DLL4 antagonists may be monospecific.
  • each of the one or more antigen-binding sites that an antibody contains is capable of binding (or binds) a homologous epitope on DLL4.
  • the DLL4 antagonist is an antibody fragment.
  • Antibody fragments may have different functions or capabilities than intact antibodies; for example, antibody fragments can have increased tumor penetration.
  • Various techniques are known for the production of antibody fragments including, but not limited to, proteolytic digestion of intact antibodies.
  • antibody fragments include a F(ab′)2 fragment produced by pepsin digestion of an antibody molecule.
  • antibody fragments include a Fab fragment generated by reducing the disulfide bridges of an F(ab′)2 fragment.
  • antibody fragments include a Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent.
  • antibody fragments are produced recombinantly.
  • antibody fragments include Fv or single chain Fv (scFv) fragments.
  • Fab, Fv, and scFv antibody fragments can be expressed in, and secreted from, E. coli or other host cells, allowing for the production of large amounts of these fragments.
  • antibody fragments are isolated from antibody phage libraries as discussed herein. For example, methods can be used for the construction of Fab expression libraries (Huse et al., 1989 , Science, 246:1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for DLL4, or derivatives, fragments, analogs or homologs thereof.
  • antibody fragments are linear antibody fragments.
  • antibody fragments are monospecific or bispecific.
  • the DLL4 antagonist is a scFv.
  • Various techniques can be used for the production of single-chain antibodies specific to DLL4 (see, e.g., U.S. Pat. No. 4,946,778).
  • modified antibodies, or fragments thereof can comprise any type of variable region that provides for the association of the antibody with DLL4.
  • the variable region may be derived from any type of mammal that can be induced to mount a humoral response and generate immunoglobulins against a desired antigen (e.g., DLL4).
  • the variable region of the modified antibodies can be, for example, of human, murine, non-human primate (e.g., cynomolgus monkeys, macaques, etc.) or lepine origin.
  • both the variable and constant regions of the modified immunoglobulins are human.
  • variable regions of compatible antibodies can be engineered or specifically tailored to improve the binding properties or reduce the immunogenicity of the molecule.
  • variable regions useful in the present invention can be humanized or otherwise altered through the inclusion of imported amino acid sequences.
  • variable domains in both the heavy and light chains are altered by at least partial replacement of one or more CDRs and, if necessary, by partial framework region replacement and sequence modification.
  • the CDRs may be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of a different class and preferably from an antibody from a different species. It may not be necessary to replace all of the CDRs with all of the CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another. Rather, it may only be necessary to transfer those residues that are necessary to maintain the activity of the antigen binding site.
  • the modified antibodies of this invention will comprise antibodies (e.g., full-length antibodies or antigen-binding fragments thereof) in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics, such as increased tumor localization, increased tumor penetration, reduced serum half-life or increased serum half-life when compared with an antibody of approximately the same immunogenicity comprising a native or unaltered constant region.
  • the constant region of the modified antibodies comprises a human constant region. Modifications to the constant region include additions, deletions, or substitutions of one or more amino acids in one or more domains.
  • the modified antibodies disclosed herein may comprise alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2 or CH3) and/or to the light chain constant domain (CL).
  • one or more domains are partially or entirely deleted from the constant regions of the modified antibodies.
  • the entire CH2 domain has been removed ( ⁇ CH2 constructs).
  • the omitted constant region domain is replaced by a short amino acid spacer (e.g., 10 aa residues) that provides some of the molecular flexibility typically imparted by the absent constant region.
  • the modified antibodies are engineered to fuse the CH3 domain directly to the hinge region of the antibody.
  • a peptide spacer is inserted between the hinge region and the modified CH2 and/or CH3 domains.
  • constructs may be expressed wherein the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region with a 5-20 amino acid spacer.
  • a spacer may be added to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible.
  • amino acid spacers can, in some cases, prove to be immunogenic and elicit an unwanted immune response against the construct. Accordingly, in certain embodiments, any spacer added to the construct will be relatively non-immunogenic so as to maintain the desired biological qualities of the modified antibodies.
  • the modified antibodies may have only a partial deletion of a constant domain or substitution of a few or even a single amino acid.
  • the mutation of a single amino acid in selected areas of the CH2 domain may be enough to substantially reduce Fc binding and thereby increase tumor localization and/or tumor penetration.
  • Such partial deletions of the constant regions may improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject constant region domain intact.
  • the constant regions of the disclosed antibodies may be modified through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct.
  • the modified antibodies comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as decreasing or increasing effector function or provide for more cytotoxin or carbohydrate attachment sites.
  • the constant region mediates several effector functions. For example, binding of the C1 component of complement to the Fc region of IgG or IgM antibodies (bound to antigen) activates the complement system. Activation of complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and can also be involved in autoimmune hypersensitivity.
  • the Fc region of an antibody can bind to a cell expressing a Fc receptor (FcR).
  • Fc receptors There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors).
  • Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells, release of inflammatory mediators, placental transfer and control of immunoglobulin production.
  • the DLL4 antibodies provide for altered effector functions that, in turn, affect the biological profile of the administered antibody.
  • the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified antibody (e.g., DLL4 antibody) thereby increasing tumor localization and/or penetration.
  • the constant region modifications increase or reduce the serum half-life of the antibody.
  • the constant region is modified to eliminate disulfide linkages or oligosaccharide moieties allowing for enhanced tumor localization and/or penetration.
  • a DLL4 antibody does not have one or more effector functions.
  • the antibody has no antibody-dependent cellular cytoxicity (ADCC) activity and/or no complement-dependent cytoxicity (CDC) activity.
  • ADCC antibody-dependent cellular cytoxicity
  • CDC complement-dependent cytoxicity
  • the antibody does not bind to an Fc receptor and/or complement factors.
  • the antibody has no effector function.
  • the present invention further embraces variants and equivalents which are substantially homologous to the chimeric, humanized, and human antibodies, or antibody fragments thereof, set forth herein. These can contain, for example, conservative substitution mutations, i.e. the substitution of one or more amino acids by similar amino acids.
  • the present invention provides methods for generating an antibody that binds the extracellular domain of human DLL4.
  • the method for generating an antibody that binds DLL4 comprises using hybridoma techniques.
  • the method comprises using an extracellular domain of mouse DLL4 or human DLL4 as an immunizing antigen.
  • the method of generating an antibody that binds DLL4 comprises screening a human phage library.
  • the present invention further provides methods of identifying an antibody that binds to DLL4.
  • the antibody is identified by screening for binding to DLL4 with flow cytometry (FACS).
  • the antibody is screened for binding to human DLL4.
  • the antibody is screened for binding to mouse DLL4.
  • the antibody is identified by screening for inhibition or blocking of DLL4-induced Notch activation.
  • the DLL4 is human DLL4.
  • the Notch is human Notch1, Notch2, Notch3, or Notch4.
  • the antibodies described herein are isolated. In certain embodiments, the antibodies described herein are substantially pure.
  • anti-DLL4 antibodies have been described, for example, in U.S. Pat. No. 7,750,124. Additional anti-DLL4 antibodies are described in, e.g., International Patent Publication Nos. WO 2008/091222 and WO 2008/0793326, and U.S. Patent Application Publication Nos. 2008/0014196, 2008/0175847, 2008/0181899, 2008/0107648, and 2010/0196385.
  • the DLL4 antagonists are polypeptides.
  • the polypeptides can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides that bind an epitope comprising amino acids within the extracellular domain of human DLL4.
  • the polypeptides comprise an antibody or fragment thereof that binds an epitope within the extracellular domain of human DLL4. It will be recognized by those of skill in the art that some amino acid sequences of a polypeptide can be varied without significant effect on the structure or function of the protein.
  • the polypeptides further include variations of the polypeptides which show substantial binding activity to an epitope of the human DLL4 protein.
  • amino acid sequence variations of polypeptides include deletions, insertions, inversions, repeats, and/or type substitutions.
  • the polypeptides and variants thereof can be further modified to contain additional chemical moieties not normally part of the polypeptide.
  • the derivatized moieties can improve the solubility, the biological half-life, or the absorption of the polypeptide.
  • the moieties can also reduce or eliminate any undesirable side effects of the polypeptides and variants. An overview for such chemical moieties can be found in Remington: The Science and Practice of Pharmacy, 21 st Edition, 2005, University of the Sciences in Philadelphia, Pa.
  • the isolated polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing a DNA sequence encoding isolated polypeptide sequences and expressing those sequences in a suitable host.
  • a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest.
  • the sequence can be mutagenized by site-specific mutagenesis to provide functional variants thereof.
  • a DNA sequence encoding a polypeptide of interest may be constructed by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and by selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize a polynucleotide sequence encoding a polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5′ and/or 3′ overhangs for complementary assembly.
  • the polynucleotide sequences encoding a particular polypeptide of interest can be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the polypeptide in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and/or expression of a biologically active polypeptide in a suitable host. As is well-known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.
  • recombinant expression vectors are used to amplify and express DNA encoding DLL4 antagonists such as polypeptides or antibodies, or fragments thereof.
  • recombinant expression vectors can be replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of an anti-DLL4 antibody, or fragment thereof, operatively linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes.
  • a transcriptional unit generally comprises an assembly of (1) a regulatory element or elements having a role in gene expression, for example, transcriptional promoters and/or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences.
  • Regulatory elements can include an operator sequence to control transcription.
  • the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can also be incorporated.
  • DNA regions are “operatively linked” when they are functionally related to each other.
  • DNA for a signal peptide is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation.
  • Structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • recombinant protein is expressed without a leader or transport sequence, it can include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus.
  • Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli , including pCR1, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and other filamentous single-stranded DNA phages.
  • Suitable host cells for expression of a DLL4 antagonist polypeptide or antibody include prokaryotes, yeast, insect, or higher eukaryotic cells under the control of appropriate promoters.
  • Prokaryotes include gram-negative or gram-positive organisms, for example, E. coli or Bacilli.
  • Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems can also be employed.
  • mammalian or insect cell culture systems are used to express recombinant protein. Expression of recombinant proteins in mammalian cells may be preferred because such proteins are generally correctly folded, appropriately modified, and biologically functional.
  • suitable mammalian host cell lines include COS-7 (monkey kidney-derived), L-929 (murine fibroblast-derived), C127 (murine mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast-derived) cell lines, and HEK-293 (human embryonic kidney-derived) cell lines and variants thereof.
  • Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5′ or 3′ flanking non-transcribed sequences, and 5′ or 3′ non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art (see, e.g., Luckow and Summers, 1988 , Bio/Technology, 6:47).
  • the proteins produced by a transformed host can be purified according to any suitable method.
  • Such methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification.
  • Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column.
  • Isolated proteins can be physically characterized using such techniques as proteolysis, high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and x-ray crystallography.
  • supernatants from expression systems which secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix.
  • a suitable purification matrix for example, an anion exchange resin is employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups.
  • the matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification.
  • a cation exchange step is employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups.
  • a hydroxyapatite media is employed, including but not limited to, ceramic hydroxyapatite (CHT).
  • CHT ceramic hydroxyapatite
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • recombinant protein produced in bacterial culture is isolated, for example, by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange, or size exclusion chromatography steps.
  • HPLC is employed for final purification steps.
  • Microbial cells employed in expression of a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
  • Methods known in the art for purifying antibodies and other proteins also include, for example, those described in U.S. Patent Application Pub. Nos. 2008/0312425, 2009/0187005, and U.S. Pat. No. 7,691,980.
  • the DLL4 antagonist is a polypeptide that is not an antibody.
  • a variety of methods for identifying and producing non-antibody polypeptides that bind with high affinity to a protein target are known in the art. See, e.g., Skerra, 2007 , Curr. Opin. Biotechnol., 18:295-304; Hosse et al., 2006 , Protein Science, 15:14-27; Gill et al., 2006 , Curr. Opin. Biotechnol., 17:653-658; Nygren, 2008 , FEBS J. 275:2668-76; and Skerra, 2008 , FEBS J. 275:2677-83.
  • phage display technology may be used to produce and/or identify a DLL4 antagonist polypeptide.
  • the DLL4 antagonist polypeptide comprises a protein scaffold of a type selected from the group consisting of protein A, protein G, a lipocalin, a fibronectin domain, an ankyrin consensus repeat domain, and thioredoxin.
  • the DLL4 antagonists or antibodies can be used in any one of a number of conjugated (e.g., an immunoconjugate or radioconjugate) or non-conjugated forms.
  • the antibodies are used in non-conjugated form to harness the subject's natural defense mechanisms including CDC and/or ADCC to eliminate malignant or cancerous cells.
  • the DLL4 antagonist (e.g., an antibody or polypeptide) is conjugated to a cytotoxic agent.
  • the cytotoxic agent is a chemotherapeutic agent including, but not limited to, methotrexate, adriamicin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents.
  • the cytotoxic agent is a enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria 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, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • diphtheria A chain nonbinding active fragments of diphtheria toxin
  • exotoxin A chain ricin A chain
  • abrin A chain abrin A chain
  • modeccin A chain alpha-sarcin
  • the cytotoxic agent is a radioactive isotope to produce a radioconjugate or a radioconjugated antibody.
  • a radioactive isotope to produce a radioconjugate or a radioconjugated antibody.
  • radionuclides are available for the production of radioconjugated antibodies including, but not limited to, 90 Y, 125 I, 131 I, 123 I, 111 In, 131 In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re, 188 Re and 212 Bi.
  • Conjugates of an antibody and one or more small molecule toxins such as a calicheamicin, maytansine, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, can also be used.
  • Conjugates of an antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as 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).
  • SPDP N-succinimidyl-3-(
  • Heteroconjugate antibodies are also within the scope of the present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune cells to unwanted cells (U.S. Pat. No. 4,676,980). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • the DLL4 antagonist is a non-protein molecule. In certain embodiments, the DLL4 antagonist is a small molecule.
  • a cell-based, luciferase reporter assay utilizing a TCF/Luc reporter vector containing multiple copies of the TCF-binding domain upstream of a firefly luciferase reporter gene may be used to measure DLL4-induced Notch signaling levels in vitro.
  • a cell-based, luciferase reporter assay utilizing a CBF/Luc reporter vector containing multiple copies of the CBF-binding domain upstream of a firefly luciferase reporter gene may be used.
  • the level of Notch activation induced by DLL4 in the presence of a DLL4 antagonist is compared to the level of Notch activation induced by DLL4 in the absence of the DLL4 antagonist.
  • the effect of a DLL4 antagonist on DLL4/Notch signaling can be assessed by measuring the effect of the agent on the expression level of one or more Notch pathway target genes.
  • the present invention provides methods of treating diseases such as cancer with a DLL4 antagonist, while screening for, monitoring, preventing, and/or controlling side effects and/or toxicities, including, but not limited to cardiovascular side effects and/or toxicities associated with the DLL4 antagonist.
  • Side effects and/or toxicities associated with cancer treatment may include, but are not limited to, fatigue, vomiting, nausea, diarrhea, pain, hair loss, neutropenia, anemia, thrombocytopenia, cardiovascular complications, and any combination thereof.
  • Cardiovascular complications e.g., cardiovascular side effects and/or toxicities
  • vascular conditions include but are not limited to, atherosclerosis, hypertension, arterial thrombosis, vasculitis, and deep venous thrombosis/pulmonary embolus.
  • cardiac structural problems and “cardiac dysfunction and heart failure” include but are not limited to, valvular heart disease, pericardial effusion, pericardial constriction, angina, coronary artery disease, cardiomyopathy, myocardial ischemia, myocardial infarction (MI), arrhythmias, myocarditis, left ventricular dysfunction, heart failure, congestive heart failure (CHF), and combinations thereof.
  • cardiotoxicity refers to cardiac structural problems, cardiac dysfunction, and heart failure.
  • the screening for, monitoring, preventing, and/or controlling cardiovascular side effects and/or toxicities is screening for, monitoring, preventing, and/or controlling cardiotoxicity.
  • cardiotoxicity is asymptomatic and/or early signs of cardiotoxicity are not evident with, for example, Doppler echocardiograms and/or with left ventricular ejection fraction (LVEF) monitoring.
  • LVEF left ventricular ejection fraction
  • Natriuretic peptides are produced by the heart and vasculature and include 4 identified types.
  • A-type natriuretic peptide (ANP) is secreted largely by the atrial myocardium
  • B-type natriuretic peptide (BNP) is produced mainly by the ventricular myocardium
  • C-type natriuretic peptide (CNP) is produced by endothelial cells that line the blood vessels
  • D-type natriuretic peptide has been isolated in plasma and atrial myocardium.
  • proBNP is a 108 amino acid peptide that is cleaved by the proteolytic enzyme furin into a 32 amino acid C-terminal peptide (BNP) and a 76 amino acid N-terminal peptide (NT-proBNP)
  • proANP is a 126 amino acid peptide that is cleaved by the serine protease enzyme corin into a 28 amino acid C-terminal peptide (ANP) and a 98 amino acid N-terminal peptide (NT-proANP).
  • the present invention provides methods for using natriuretic peptide levels to monitor cardiotoxicity in subjects being treated with a DLL4 antagonist.
  • the methods use natriuretic peptide levels to monitor and/or detect acute cardiotoxicity.
  • monitoring the level of a natriuretic peptide gives an early indication of cardiotoxicity and/or congestive heart failure (CHF).
  • the methods detect cardiotoxicity prior to any evidence of cardiac dysfunction as evaluated by Doppler echocardiograms and/or with LVEF monitoring.
  • the cardiovascular side effects and/or toxicities that are detected, identified, monitored, reduced, prevented, attenuated, and/or screened for are cardiovascular side effects and/or toxicities caused by, associated with, and/or related to administration of a DLL4 antagonist or treatment with a DLL4 antagonist. In certain embodiments, the cardiovascular side effects and/or toxicities are related to the DLL4 antagonist.
  • the cardiovascular side effects and/or toxicities that is detected, identified, monitored, reduced, prevented, attenuated, and/or screened for in a method described herein does not include hypertension. In some embodiments the cardiovascular side effect and/or toxicity does not include a vascular condition. In certain embodiments, the cardiovascular side effect and/or toxicity that is detected, identified, monitored, reduced, prevented, attenuated, and/or screened for in a method described herein is a cardiotoxicity such as a cardiac structural problem, cardiac dysfunction, or heart failure. In certain embodiments, the cardiotoxicity is left ventricular dysfunction. In certain embodiments, the cardiotoxicity is congestive heart failure. In some embodiments, the cardiotoxicity is in an early or reversible stage of development.
  • a cardiotoxicity such as a cardiac structural problem, cardiac dysfunction, or heart failure.
  • the cardiotoxicity is left ventricular dysfunction.
  • the cardiotoxicity is congestive heart failure. In some embodiments, the cardiotoxicity is in an early or reversible stage of development.
  • the invention provides methods for selecting a subject for treatment with a DLL4 antagonist, comprising: determining the level of a biomarker in a sample, and selecting the subject for treatment with the DLL4 antagonist if the level of the biomarker is below a predetermined level.
  • the methods for selecting a subject for treatment with a DLL4 antagonist comprise: obtaining a biological sample from the subject, determining the level of a biomarker in the sample, and selecting the subject for treatment with the DLL4 antagonist if the level of the biomarker is below a predetermined level.
  • the biomarker is a natriuretic peptide.
  • the natriuretic peptide is an atrial natriuretic peptide (ANP)-type peptides, a brain natriuretic peptide (BNP)-type peptides, or variants thereof.
  • ANP-type peptides include pre-proANP, proANP, NT-proANP, and ANP.
  • BNP-type peptides include pre-proBNP, proBNP, NT-proBNP, and BNP.
  • the natriuretic peptide is BNP.
  • the natriuretic peptide is NT-proBNP.
  • the natriuretic peptide is ANP.
  • the method of selecting a subject for treatment with a DLL4 antagonist comprises: obtaining a biological sample from the subject, determining the level of a natriuretic peptide in the sample, and selecting the subject for treatment with the DLL4 antagonist if the level of the natriuretic peptide is below a predetermined level.
  • the biological sample is blood, serum, or plasma.
  • the natriuretic peptide is B-type natriuretic peptide (BNP).
  • the methods of selecting a subject for treatment with a DLL4 antagonist comprising: obtaining a biological sample from the subject, determining the level of BNP in the sample, and selecting the subject for treatment with the DLL4 antagonist if the level of BNP is below a predetermined level.
  • the invention also provides methods of identifying a subject as eligible for treatment with a DLL4 antagonist, comprising: determining the level of a biomarker in a sample, and identifying the subject as eligible for treatment with the DLL4 antagonist if the level of the biomarker is below a predetermined level.
  • the methods of identifying a subject as eligible for treatment with a DLL4 antagonist comprise: obtaining a biological sample from the subject, determining the level of a biomarker in the sample, and identifying the subject as eligible for treatment with the DLL4 antagonist if the level of the biomarker is below a predetermined level.
  • the biomarker is a natriuretic peptide.
  • the natriuretic peptide is an atrial natriuretic peptide (ANP)-type peptides, a brain natriuretic peptide (BNP)-type peptides, or variants thereof.
  • ANP-type peptides include pre-proANP, proANP, NT-proANP, and ANP.
  • BNP-type peptides include pre-proBNP, proBNP, NT-proBNP, and BNP.
  • the natriuretic peptide is BNP.
  • the natriuretic peptide is NT-proBNP.
  • the methods of identifying a subject as eligible for treatment with a DLL4 antagonist comprise: obtaining a biological sample from the subject, determining the level of BNP in the sample, and identifying the subject as eligible for treatment with the DLL4 antagonist if the level of BNP is below a predetermined level.
  • the invention also provides methods of monitoring a subject receiving treatment with a DLL4 antagonist for the development of cardiovascular side effects and/or toxicity, comprising: determining the level of a biomarker in a sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein an increase in the level of the biomarker indicates development of cardiovascular side effects and/or toxicity.
  • the methods of monitoring a subject receiving treatment with a DLL4 antagonist for the development of cardiovascular side effects and/or toxicity comprise: obtaining a biological sample from the subject receiving treatment, determining the level of a biomarker in the sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein an increase in the level of the biomarker indicates development of cardiovascular side effects and/or toxicity.
  • the cardiovascular side effect and/or toxicity is cardiotoxicity.
  • the biomarker is a natriuretic peptide.
  • the natriuretic peptide is an atrial natriuretic peptide (ANP)-type peptides, a brain natriuretic peptide (BNP)-type peptides, or variants thereof.
  • ANP-type peptides include pre-proANP, proANP, NT-proANP, and ANP.
  • BNP-type peptides include pre-proBNP, proBNP, NT-proBNP, and BNP.
  • the natriuretic peptide is BNP.
  • the natriuretic peptide is NT-proBNP.
  • the natriuretic peptide is ANP.
  • a method of monitoring a subject receiving treatment with a DLL4 antagonist for the development of cardiotoxicity comprises: obtaining a biological sample from the subject receiving treatment, determining the level of BNP in the sample, and comparing the level of BNP in the sample to a predetermined level of BNP, wherein an increase in the level of BNP indicates development of cardiotoxicity.
  • the invention also provides methods of detecting the development of cardiovascular side effects and/or toxicity in a subject receiving treatment with a DLL4 antagonist, comprising: determining the level of a biomarker in a sample, and comparing the level of a biomarker in the sample to a predetermined level of the biomarker, wherein an increase in the level of the biomarker indicates development of cardiovascular side effects and/or toxicity.
  • the methods of detecting the development of cardiovascular side effects and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprise: obtaining a biological sample from the subject receiving treatment, determining the level of a biomarker in the sample, and comparing the level of a biomarker in the sample to a predetermined level of the biomarker, wherein an increase in the level of the biomarker indicates development of cardiovascular side effects and/or toxicity.
  • the cardiovascular side effect and/or toxicity is cardiotoxicity.
  • the biomarker is a natriuretic peptide.
  • the natriuretic peptide is an atrial natriuretic peptide (ANP)-type peptides, a brain natriuretic peptide (BNP)-type peptides, or variants thereof.
  • ANP-type peptides include pre-proANP, proANP, NT-proANP, and ANP.
  • BNP-type peptides include pre-proBNP, proBNP, NT-proBNP, and BNP.
  • the natriuretic peptide is BNP.
  • the natriuretic peptide is NT-proBNP.
  • the natriuretic peptide is ANP.
  • the methods of detecting the development of cardiotoxicity in a subject receiving treatment with a DLL4 antagonist comprise: obtaining a biological sample from the subject receiving treatment, determining the level of BNP in the sample, and comparing the level of BNP in the sample to a predetermined level of BNP, wherein an increase in the level of BNP indicates development of cardiotoxicity.
  • the invention also provides methods for identifying cardiovascular side effects and/or toxicity in a subject receiving treatment with a DLL4 antagonist, comprising: determining the level of a biomarker in a sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein if the level of the biomarker in the sample is higher than the predetermined level of the biomarker then a cardiovascular side effect and/or toxicity is indicated.
  • the methods for identifying cardiovascular side effects and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprise: obtaining a biological sample from the subject receiving treatment, determining the level of a biomarker in the sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein if the level of the biomarker in the sample is higher than the predetermined level of the biomarker then a cardiovascular side effect and/or toxicity is indicated.
  • the cardiovascular side effect and/or toxicity is cardiotoxicity.
  • the biomarker is a natriuretic peptide.
  • the natriuretic peptide is an atrial natriuretic peptide (ANP)-type peptides, a brain natriuretic peptide (BNP)-type peptides, or variants thereof.
  • ANP-type peptides include pre-proANP, proANP, NT-proANP, and ANP.
  • BNP-type peptides include pre-proBNP, proBNP, NT-proBNP, and BNP.
  • the natriuretic peptide is BNP.
  • the natriuretic peptide is NT-proBNP.
  • the natriuretic peptide is ANP.
  • a method for identifying cardiotoxicity in a subject receiving treatment with a DLL4 antagonist comprises: obtaining a biological sample from the subject receiving treatment, determining the level of BNP in the sample, and comparing the level of BNP in the sample to a predetermined level of BNP, wherein if the level of BNP in the sample is higher than the predetermined level of BNP then cardiotoxicity is indicated.
  • the invention also provides methods for monitoring cardiovascular side effects and/or toxicity in a subject receiving treatment with a DLL4 antagonist, comprising: determining the level of a biomarker in a sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein if the level of the biomarker in the sample is higher than the predetermined level of the biomarker then cardiovascular side effects and/or toxicity is indicated.
  • the methods for monitoring cardiovascular side effects and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprise: obtaining a biological sample from the subject receiving treatment, determining the level of a biomarker in the sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein if the level of the biomarker in the sample is higher than the predetermined level of the biomarker then cardiovascular side effects and/or toxicity is indicated.
  • the cardiovascular side effect and/or toxicity is cardiotoxicity.
  • the biomarker is a natriuretic peptide.
  • the natriuretic peptide is an atrial natriuretic peptide (ANP)-type peptides, a brain natriuretic peptide (BNP)-type peptides, or variants thereof.
  • ANP-type peptides include pre-proANP, proANP, NT-proANP, and ANP.
  • BNP-type peptides include pre-proBNP, proBNP, NT-proBNP, and BNP.
  • the natriuretic peptide is BNP.
  • the natriuretic peptide is NT-proBNP.
  • the natriuretic peptide is ANP.
  • a method for monitoring cardiotoxicity in a subject receiving treatment with a DLL4 antagonist comprises: obtaining a biological sample from the subject receiving treatment, determining the level of BNP in the sample, and comparing the level of BNP in the sample to a predetermined level of BNP, wherein if the level of BNP in the sample is higher than the predetermined level of BNP then cardiotoxicity is indicated.
  • the invention also provides methods of reducing cardiovascular side effects and/or toxicity in a subject receiving treatment with a DLL4 antagonist, comprising: determining the level of a biomarker in a sample from the subject, comparing the level of the biomarker in the sample to a predetermined level of the biomarker, and administering to the subject a therapeutically effective amount of a cardioprotective medication such as an ACE inhibitor and/or a (1-blocker if the level of the biomarker in the sample is higher than the predetermined level of the biomarker.
  • a cardioprotective medication such as an ACE inhibitor and/or a (1-blocker if the level of the biomarker in the sample is higher than the predetermined level of the biomarker.
  • the methods of reducing cardiovascular side effects and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprise: obtaining a biological sample from the subject receiving treatment, determining the level of a biomarker in the sample, comparing the level of the biomarker in the sample to a predetermined level of the biomarker, and administering to the subject a therapeutically effective amount of a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker if the level of the biomarker in the sample is higher than the predetermined level of the biomarker.
  • a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker if the level of the biomarker in the sample is higher than the predetermined level of the biomarker.
  • the cardiovascular side effect and/or toxicity is cardiotoxicity.
  • the biomarker is a natriuretic peptide.
  • the natriuretic peptide is an atrial natriuretic peptide (ANP)-type peptides, a brain natriuretic peptide (BNP)-type peptides, or variants thereof.
  • ANP-type peptides include pre-proANP, proANP, NT-proANP, and ANP.
  • BNP-type peptides include pre-proBNP, proBNP, NT-proBNP, and BNP.
  • the natriuretic peptide is BNP.
  • the natriuretic peptide is NT-proBNP.
  • the natriuretic peptide is ANP.
  • a method for reducing cardiotoxicity in a subject receiving treatment with a DLL4 antagonist comprises: obtaining a biological sample from the subject receiving treatment, determining the level of BNP in the sample, and comparing the level of BNP in the sample to a predetermined level of BNP, and administering to the subject a therapeutically effective amount of a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker if the level of BNP in the sample is higher than the predetermined level of BNP.
  • a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker
  • the invention also provides methods of preventing or attenuating the development of cardiovascular side effects and/or toxicity in a subject receiving treatment with a DLL4 antagonist, comprising: determining the level of a biomarker in a sample from the subject, comparing the level of the biomarker in the sample to a predetermined level of the biomarker; administering to the subject a therapeutically effective amount of a cardioprotective medication such as an ACE inhibitor and/or ⁇ -blocker, and administering to the subject the DLL4 antagonist.
  • a cardioprotective medication such as an ACE inhibitor and/or ⁇ -blocker
  • the methods of preventing or attenuating the development of cardiovascular side effects and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprise: obtaining a biological sample from the subject prior to treatment with the DLL4 antagonist, determining the level of a biomarker in the sample, comparing the level of the biomarker in the sample to a predetermined level of the biomarker; administering to the subject a therapeutically effective amount of a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker, and administering to the subject the DLL4 antagonist.
  • the cardiovascular side effect and/or toxicity is cardiotoxicity.
  • the biomarker is a natriuretic peptide.
  • the natriuretic peptide is an atrial natriuretic peptide (ANP)-type peptides, a brain natriuretic peptide (BNP)-type peptides, or variants thereof.
  • ANP-type peptides include pre-proANP, proANP, NT-proANP, and ANP.
  • BNP-type peptides include pre-proBNP, proBNP, NT-proBNP, and BNP.
  • the natriuretic peptide is BNP.
  • the natriuretic peptide is NT-proBNP.
  • the natriuretic peptide is ANP.
  • a method of preventing or attenuating the development of cardiotoxicity in a subject receiving treatment with a DLL4 antagonist comprises: obtaining a biological sample from the subject prior to treatment with the DLL4 antagonist, determining the level of BNP in the sample, comparing the level of BNP in the sample to a predetermined level of BNP; administering to the subject a therapeutically effective amount of a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker if the level of BNP in the sample is higher than the predetermined level of BNP; and administering to the subject the DLL4 antagonist.
  • a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker
  • the predetermined level is about 300 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 200 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 250 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 200 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 150 pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 100 pg/ml or less in a blood, serum, or plasma sample. In the context of predetermined levels of BNP, the term “about” means the referenced amount plus or minus 10% of that referenced amount.
  • the predetermined level of a biomarker is the amount of the biomarker in a sample obtained at an earlier date. In some embodiments, the predetermined level of a biomarker (e.g., natriuretic peptide or BNP) is the amount of the biomarker in a sample obtained prior to treatment. In some embodiments, the predetermined level of a biomarker (e.g., natriuretic peptide or BNP) is a normal reference level. In some embodiments, the predetermined level for BNP is about 100 pg/ml or less in blood, serum, or plasma. In some embodiments, the normal reference level for BNP is about 100 pg/ml or less in blood, serum, or plasma.
  • a biological sample is obtained approximately every week, every 2 weeks, every 3 weeks, or every 4 weeks.
  • the subject is administered a therapeutically effective amount of a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker. In some embodiments, if the BNP level in the biological sample is above 100 pg/ml for two consecutive samples, the subject is administered a therapeutically effective amount of a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker. In some embodiments of the methods described herein, if the BNP level in the biological sample is above a predetermined level for any one sample, the subject is administered a therapeutically effective amount of a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker.
  • the subject is administered a therapeutically effective amount of a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker.
  • a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker and the DLL4 antagonist is withheld.
  • the subject is administered a therapeutically effective amount of a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker and the DLL4 antagonist is withheld.
  • a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker
  • the DLL4 antagonist is resumed.
  • the subjects are evaluated by LVEF monitoring.
  • the LVEF represents the volumetric fraction of blood pumped out of the left ventricle of the heart with each heart beat or cardiac cycle. Generally a normal range for LVEF is 55-70%, and a significantly reduced ejection fraction typically indicates a cardiac dysfunction and/or heart failure.
  • cardiovascular dysfunction and/or cardiotoxicity is indicated if the LVEF is less than about 60%. In some embodiments, cardiovascular dysfunction and/or cardiotoxicity is indicated if the LVEF is less than about 55%. In some embodiments, cardiovascular dysfunction and/or cardiotoxicity is indicated if the LVEF is less than about 50%.
  • cardiovascular dysfunction and/or cardiotoxicity is indicated if there is a 10% or greater decline in LVEF. In some embodiments, cardiovascular dysfunction and/or cardiotoxicity is indicated if there is a 20% or greater decline in LVEF. In some embodiments, cardiovascular dysfunction and/or cardiotoxicity is indicated if there is a 20% or greater decline in LVEF to a value greater than 50%. In some embodiments, cardiovascular dysfunction and/or cardiotoxicity is indicated if there is a 10% or greater decline in LVEF to a value less than 50%.
  • the subjects are evaluated using a Doppler echocardiogram.
  • Doppler echocardiography is a method for detecting the direction and velocity of moving blood within the heart and can be used to detect pulmonary hypertension. Pulmonary hypertension is high blood pressure that occurs in the arteries in the lungs. It is a different measurement altogether from systemic blood pressure and what is generally called “high blood pressure” or “hypertension”.
  • cardiovascular dysfunction and/or cardiotoxicity is indicated if the subject has a peak tricuspid velocity (PTV) greater than 3.4 m/s on Doppler echocardiogram.
  • PTV peak tricuspid velocity
  • cardiovascular dysfunction and/or cardiotoxicity is indicated if the subject has a peak tricuspid velocity (PTV) greater than 3.4 m/s on Doppler echocardiogram that persists for more than 4 weeks. In some embodiments, cardiovascular dysfunction and/or cardiotoxicity is indicated if the subject has a peak tricuspid velocity (PTV) greater than 3.4 m/s on Doppler echocardiogram that persists for more than 8 weeks.
  • PTV peak tricuspid velocity
  • the invention also provides methods of ameliorating cardiotoxicity in a subject administered a DLL4 antagonist, comprising: administering to the subject a therapeutically effective amount of a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker.
  • a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker.
  • the cardiotoxicity is not systemic hypertension (i.e., high blood pressure).
  • the invention also provides methods of screening a subject for the risk of cardiovascular side effects and/or toxicity from treatment with a DLL4 antagonist, comprising: determining the level of a biomarker in a sample from the subject, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein if the level of the biomarker in the sample is higher than the predetermined level of the biomarker then the subject is at risk for cardiovascular side effects and/or toxicity.
  • the methods of screening a subject for the risk of cardiovascular side effects and/or toxicity from treatment with a DLL4 antagonist comprise: obtaining a biological sample from the subject prior to treatment with the DLL4 antagonist, determining the level of a biomarker in the sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein if the level of the biomarker in the sample is higher than the predetermined level of the biomarker then the subject is at risk for cardiovascular side effects and/or toxicity.
  • the cardiovascular side effect and/or toxicity is cardiotoxicity.
  • the biomarker is a natriuretic peptide.
  • the natriuretic peptide is an atrial natriuretic peptide (ANP)-type peptides, a brain natriuretic peptide (BNP)-type peptides, or variants thereof.
  • ANP-type peptides include pre-proANP, proANP, NT-proANP, and ANP.
  • BNP-type peptides include pre-proBNP, proBNP, NT-proBNP, and BNP.
  • the natriuretic peptide is BNP.
  • the natriuretic peptide is NT-proBNP.
  • the natriuretic peptide is ANP.
  • a method of screening a subject for the risk of cardiotoxicity from treatment with a DLL4 antagonist comprises: obtaining a biological sample from the subject prior to treatment with the DLL4 antagonist, determining the level of BNP in the sample, and comparing the level of BNP in the sample to a predetermined level of BNP, wherein if the level of BNP in the sample is higher than the predetermined level of BNP then the subject is at risk for cardiotoxicity.
  • the predetermined level of BNP is about 100 pg/ml.
  • the subject is administered a therapeutically effective amount of a cardioprotective medication such as an ACE inhibitor and/or a ⁇ -blocker prior to treatment with the DLL4 antagonist.
  • the ACE inhibitor is selected from the group consisting of: captopril, zofenopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, fosinopril, ceronapril, casokinins, lactokinins, teprotide, alacepril, cilazapril, delapril, imidapril, moexipril, rentiapril, spirapril, temocapril, moveltipril, and trandolapril.
  • the ⁇ -blocker is selected from the group consisting of: carvedilol, atenolol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, timolol, acebutolol, bisoprolol, esmolol, labetalol, bucindolol, nebivolol, alprenolol; amosulalol, arotinolol, befunolol, betaxolol, bevantolot, bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, butidrine hydrochloride, butofilolol, carazolol, carteolol, celiprolol, cetamolol
  • the DLL4 antagonist specifically binds human DLL4.
  • the DLL4 antagonist is an antibody that specifically binds the extracellular domain of human DLL4.
  • the DLL4 antagonist specifically binds an epitope within amino acids 27-217 of the extracellular domain of human DLL4 (SEQ ID NO:17).
  • the DLL4 antagonist binds an epitope comprising amino acids 66-73 (QAVVSPGP, SEQ ID NO: 18) of human DLL4.
  • the DLL4 antagonist binds an epitope comprising amino acids 139-146 (LISKIAIQ, SEQ ID NO:19) of human DLL4.
  • the DLL4 antagonist binds an epitope comprising amino acids 66-73 (QAVVSPGP, SEQ ID NO: 18) and amino acids 139-146 (LISKIAIQ, SEQ ID NO: 19) of human DLL4. In some embodiments, the DLL4 antagonist binds human DLL4 with a dissociation constant (K D ) of about 10 nM to about 0.1 nM.
  • K D dissociation constant
  • the DLL4 antagonist is an anti-DLL4 antibody.
  • the DLL4 antagonist is an antibody comprising a heavy chain CDR1 comprising TAYYIH (SEQ ID NO: 1), a heavy chain CDR2 comprising YISSYNGATNYNQKFKG (SEQ ID NO:3), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:5), and a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:6), a light chain CDR2 comprising AASNQGS (SEQ ID NO:7), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:8).
  • the DLL4 antagonist is an antibody comprising a heavy chain variable region comprising the amino acids of SEQ ID NO:10. In certain embodiments, the DLL4 antagonist is an antibody which further comprises a light chain variable region comprising the amino acids of SEQ ID NO: 12. In certain embodiments, the DLL4 antagonist comprises the same heavy and light chain amino acid sequences as an antibody encoded by a plasmid deposited with ATCC having deposit no. PTA-8425 or PTA-8427. In certain embodiments, the DLL4 antagonist comprises the heavy chain CDR amino acid sequences and the light chain CDR amino acid sequences that are contained in the 21M18 antibody produced by the hybridoma deposited with ATCC having deposit no. PTA-8670.
  • the DLL4 antagonist is encoded by the plasmid having ATCC deposit no. PTA-8425 which was deposited with American Type Culture Collection (ATCC), at 10801 University Boulevard, Manassas, Va., 20110, under the conditions of the Budapest Treaty on May 10, 2007.
  • the DLL4 antagonist is encoded by the plasmid having ATCC deposit no. PTA-8427 which was deposited with American Type Culture Collection (ATCC), at 10801 University Boulevard, Manassas, Va., 20110, under the conditions of the Budapest Treaty on May 10, 2007.
  • the DLL4 antagonist is the antibody produced by the hybridoma having ATCC deposit no.
  • the DLL4 antagonist is a humanized version of the antibody produced by the hybridoma having ATCC deposit no. PTA-8670.
  • the DLL4 antagonist competes for specific binding to human DLL4 with an antibody encoded by the plasmid deposited with ATCC having deposit no. PTA-8425 or PTA-8427.
  • the subject has cancer.
  • the cancer is selected from the group consisting of: lung cancer, breast cancer, colon cancer, colorectal cancer, melanoma, pancreatic cancer, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroendocrine cancer, neuroblastoma, glioma, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, and head and neck cancer.
  • the cancer is a hematological cancer, such as a lymphoma or leukemia.
  • the cancer is non-small cell lung cancer (NSCLC).
  • the cancer is ovarian cancer.
  • the cancer is pancreatic cancer.
  • the biological sample is a body fluid.
  • the biological sample is blood, plasma, serum, or urine.
  • the biological sample is a venous whole blood specimen.
  • the biological sample is a venous whole blood specimen using EDTA as an anticoagulant.
  • the biological sample is a plasma specimen.
  • the biological sample is a plasma specimen using EDTA as an anticoagulant. Samples of body fluids may be obtained by any method known in the art.
  • the biological sample is a frozen tissue sample or is fresh tissue sample.
  • natriuretic peptide e.g., NT-proBNP or BNP
  • a fluorescent immunoassay that quantitatively measures BNP levels in whole blood or plasma specimens is used.
  • the sample contains EDTA as an anticoagulant.
  • the test is performed at the bedside.
  • a sample is placed in a test device and the sample moves by capillary action into a reaction chamber containing murine fluorescent antibodies to BNP. The reaction mixture then flows through an elution column. Analyte and fluorescent antibody-BNP conjugates are captured in discrete zones along the column.
  • Bound fluorescent material represents the serum BNP concentration. After about 15 minutes, the test device is placed in an immunofluorescence reader and the BNP concentration is determined. BNP levels less than or equal to 100 pg/ml are considered representative of normal values in patients without congestive heart failure (CHF) by those skilled in the art. BNP levels above 100 pg/ml to 300 ug/ml are suggestive of heart failure, BNP levels above 300 pg/ml indicate mild heart failure, BNP levels above 600 pg/ml indicate moderate heart failure, and BNP levels above 900 pg/ml indicate severe heart failure.
  • CHF congestive heart failure
  • the DLL4 antagonist is administered as an initial dose of about 2.5 mg/kg.
  • antibody OMP-21M18 is diluted with 5% dextrose in water (USP) to a total volume of 250 mL.
  • USP dextrose in water
  • the OMP-21M18 is delivered through a 0.22-micron filter over 30 minutes as an intravenous infusion.
  • subsequent doses are administered in a similar manner.
  • the methods described herein may further comprise administering one or more additional therapeutic agents.
  • An additional therapeutic agent can be administered prior to, concurrently with, and/or subsequently to, administration of the DLL4 antagonist.
  • Pharmaceutical compositions comprising a DLL4 antagonist and an additional therapeutic agent(s) are also provided.
  • the one or more additional therapeutic agents comprise 1, 2, 3, or more additional therapeutic agents.
  • Combination therapy with at least two therapeutic agents often uses agents that work by different mechanisms of action, although this is not required. Combination therapy using agents with different mechanisms of action may result in additive or synergetic effects. Combination therapy may allow for a lower dose of each agent than is used in monotherapy, thereby reducing side effects and/or toxicities. Combination therapy may increase the therapeutic index of one or both of the therapeutic agents. Combination therapy may decrease the likelihood that resistant cancer cells will develop.
  • combination therapy comprises a therapeutic agent that primarily affects (e.g., inhibits or kills) non-tumorigenic cells and a therapeutic agent that primarily affects (e.g., inhibits or kills) tumorigenic CSCs.
  • a DLL4 antagonist and an additional therapeutic agent may be administered in any order or concurrently.
  • the DLL4 antagonist is administered to subjects that have previously undergone treatment with a second therapeutic agent.
  • the DLL4 antagonist and a second therapeutic agent is administered substantially simultaneously or concurrently.
  • a subject may be given a DLL4 antagonist (e.g., an antibody) while undergoing a course of treatment with a second therapeutic agent (e.g., chemotherapy).
  • a DLL4 antagonist is administered within 1 year of the treatment with a second therapeutic agent.
  • a DLL4 antagonist is administered within 10, 8, 6, 4, or 2 months of any treatment with a second therapeutic agent.
  • a DLL4 antagonist is administered within 4, 3, 2, or I weeks of any treatment with a second therapeutic agent. In some embodiments, a DLL4 antagonist is administered within 5, 4, 3, 2, or I days of any treatment with a second therapeutic agent. It will further be appreciated that the two (or more) agents or treatments may be administered to the subject within a matter of hours or minutes (i.e., substantially simultaneously).
  • any therapeutic agent may lead to side effects and/or toxicities.
  • the side effects and/or toxicities are so severe as to preclude administration of the particular agent at a therapeutically effective dose.
  • drug therapy must be discontinued, and other agents may be tried.
  • many agents in the same therapeutic class often display similar side effects and/or toxicities, meaning that the subject either has to stop therapy, or if possible, suffer from the unpleasant side effects associated with the therapeutic agent.
  • Side effects from therapeutic agents may include, but are not limited to, hives, skin rashes, itching, nausea, vomiting, decreased appetite, diarrhea, chills, fever, fatigue, muscle aches and pain, headaches, low blood pressure, high blood pressure, hypokalemia, low blood counts, bleeding, and cardiac problems.
  • the methods described herein include using an intermittent dosing regimen, which may reduce side effects and/or toxicities associated with administration of a DLL4 antagonist.
  • intermittent dosing refers to a dosing regimen using a dosing interval of more than once a week, e.g., dosing once every 2 weeks, once every 3 weeks, once every 4 weeks, etc.
  • a method for treating a subject comprises administering to the subject an effective dose of a DLL4 antagonist (e.g., an anti-DLL4 antibody) according to an intermittent dosing regimen.
  • a DLL4 antagonist e.g., an anti-DLL4 antibody
  • the method comprises administering to the subject an effective dose of a DLL4 antagonist (e.g., an anti-DLL4 antibody) according to an intermittent dosing regimen, and increasing the therapeutic index of the DLL4 antagonist.
  • a DLL4 antagonist e.g., an anti-DLL4 antibody
  • the intermittent dosing regimen comprises administering an initial dose of a DLL4 antagonist to the subject, and administering subsequent doses of the DLL4 antagonist about once every 2 weeks.
  • the intermittent dosing regimen comprises administering an initial dose of a DLL4 antagonist to the subject, and administering subsequent doses of the DLL4 antagonist about once every 3 weeks.
  • the intermittent dosing regimen comprises administering an initial dose of a DLL4 antagonist to the subject, and administering subsequent doses of the DLL4 antagonist about once every 4 weeks.
  • the subsequent doses in an intermittent dosing regimen are about the same amount or less than the initial dose. In other embodiments, the subsequent doses are a greater amount than the initial dose. As is known by those of skill in the art, doses used will vary depending on the clinical goals to be achieved.
  • the initial dose is about 0.25 mg/kg to about 20 mg/kg. In some embodiments, the initial dose is about 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/kg. In certain embodiments, the initial dose is about 0.5 mg/kg. In certain embodiments, the initial dose is about 1 mg/kg. In certain embodiments, the initial dose is about 2.5 mg/kg.
  • the initial dose is about 5 mg/kg. In certain embodiments, the initial dose is about 7.5 mg/kg. In certain embodiments, the initial dose is about 10 mg/kg. In certain embodiments, the initial dose is about 12.5 mg/kg. In certain embodiments, the initial dose is about 15 mg/kg. In certain embodiments, the initial dose is about 20 mg/kg. In some embodiments, the subsequent doses are about 0.25 mg/kg to about 15 mg/kg. In certain embodiments, the subsequent doses are about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mg/kg. In certain embodiments, the subsequent doses are about 0.5 mg/kg. In certain embodiments, the subsequent doses are about 1 mg/kg.
  • the subsequent doses are about 2.5 mg/kg. In certain embodiments, the subsequent doses are about 5 mg/kg. In some embodiments, the subsequent doses are about 7.5 mg/kg. In some embodiments, the subsequent doses are about 10 mg/kg. In some embodiments, the subsequent doses are about 12.5 mg/kg.
  • the intermittent dosing regimen comprises: (a) administering to the subject an initial dose of a DLL4 antagonist of about 2.5 mg/kg and (b) administering subsequent doses of about 2.5 mg/kg once every 2 weeks. In some embodiments, the intermittent dosing regimen comprises: (a) administering to the subject an initial dose of a DLL4 antagonist of about 5 mg/kg and (b) administering subsequent doses of about 5 mg/kg once every 2 weeks. In some embodiments, the intermittent dosing regimen comprises: (a) administering to the subject an initial dose of a DLL4 antagonist of about 2.5 mg/kg and (b) administering subsequent doses of about 2.5 mg/kg once every 3 weeks.
  • the intermittent dosing regimen comprises: (a) administering to the subject an initial dose of a DLL4 antagonist of about 5 mg/kg and (b) administering subsequent doses of about 5 mg/kg once every 3 weeks. In some embodiments, the intermittent dosing regimen comprises: (a) administering to the subject an initial dose of a DLL4 antagonist of about 2.5 mg/kg and (b) administering subsequent doses of about 2.5 mg/kg once every 4 weeks. In some embodiments, the intermittent dosing regimen comprises: (a) administering to the subject an initial dose of a DLL4 antagonist of about 5 mg/kg and (b) administering subsequent doses of about 5 mg/kg once every 4 weeks.
  • the initial dose and the maintenance doses are different, for example, the initial dose is about 5 mg/kg and the subsequent doses are about 2.5 mg/kg.
  • an intermittent dosing regimen may comprise a loading dose, for example, the initial dose is about 20 mg/kg and the subsequent doses are about 2.5 mg/kg or about 5 mg/kg administered once every 2 weeks, once every 3 weeks, or once every 4 weeks.
  • the choice of delivery method for the initial and subsequent doses is made according to the ability of the subject to tolerate introduction of the DLL4 antagonist into the body.
  • the administration of the DLL4 antagonist may be by intravenous injection or intravenously. In some embodiments, the administration is by intravenous infusion. In any of the aspects and/or embodiments described herein, the administration of the DLL4 antagonist may be by a non-intravenous mute.
  • Therapeutic agents that may be administered in combination with the DLL4 antagonist include chemotherapeutic agents.
  • the method or treatment involves the administration of a DLL4 antagonist of the present invention in combination with a chemotherapeutic agent or cocktail of multiple different chemotherapeutic agents.
  • Treatment with a DLL4 antagonist e.g., an antibody
  • Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously.
  • Preparation and dosing schedules for such chemotherapeutic agents can be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in The Chemotherapy Source Book, 4 th Edition, 2008, M. C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, Pa.
  • Chemotherapeutic agents useful in the instant invention include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN): alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;
  • paclitaxel TAXOL
  • docetaxel TAXOTERE
  • chlorambucil gemcitabine
  • 6-thioguanine mercaptopurine: platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide: mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine (XELODA); and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • DMFO difluoromethylornithine
  • XELODA retinoic acid
  • esperamicins capecitabine
  • XELODA
  • Chemotherapeutic agents also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the additional therapeutic agent is cisplatin.
  • the additional therapeutic agent is carboplatin. In certain embodiments, the additional therapeutic agent is paclitaxel. In certain embodiments, where the chemotherapeutic agent administered in combination with a DLL4 antagonist is carboplatin, the cancer or tumor being treated is lung cancer or a lung tumor.
  • the chemotherapeutic agent is a topoisomerase inhibitor.
  • Topoisomerase inhibitors are chemotherapeutic agents that interfere with the action of a topoisomerase enzyme (e.g., topoisomerase I or II).
  • Topoisomerase inhibitors include, but are not limited to, doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these.
  • the additional therapeutic agent is irinotecan.
  • the chemotherapeutic agent is an anti-metabolite.
  • An anti-metabolite is a chemical with a structure that is similar to a metabolite required for normal biochemical reactions, yet different enough to interfere with one or more normal functions of cells, such as cell division.
  • Anti-metabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these.
  • the additional therapeutic agent is gemcitabine.
  • the additional therapeutic agent is pemetrexed.
  • the cancer or tumor being treated is pancreatic cancer or a pancreatic tumor.
  • the chemotherapeutic agent administered in combination with a DLL4 antagonist is pemetrexed
  • the cancer or tumor being treated is lung cancer or a lung tumor.
  • the DLL4 antagonist is administered in combination with pemetrexed and carboplatin.
  • the chemotherapeutic agent is an antimitotic agent, including, but not limited to, agents that bind tubulin.
  • the agent is a taxane.
  • the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel.
  • the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (ABRAXANE), DHA-paclitaxel, or PG-paclitaxel.
  • the antimitotic agent comprises a vinca alkaloid, such as vincristine, binblastine, vinorelbine, or vindesine, or pharmaceutically acceptable salts, acids, or derivatives thereof.
  • the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor of a mitotic kinase such as Aurora A or Plk1.
  • the chemotherapeutic agent administered in combination with a DLL4 antagonist is an anti-mitotic agent
  • the cancer or tumor being treated is breast cancer or a breast tumor.
  • an additional therapeutic agent comprises an agent such as a small molecule.
  • treatment can involve the combined administration of a DLL4 antagonist (e.g. an antibody) of the present invention with a small molecule that acts as an inhibitor against additional tumor-associated proteins including, but not limited to, EGFR, ErbB2, HER2, and/or VEGF.
  • the additional therapeutic agent is a small molecule that inhibits a cancer stem cell pathway.
  • the additional therapeutic agent is a small molecule inhibitor of the Notch pathway.
  • the additional therapeutic agent is a small molecule inhibitor of the Wnt pathway.
  • the additional therapeutic agent is a small molecule inhibitor of the BMP pathway.
  • the additional therapeutic agent is a small molecule that inhibits ⁇ -catenin signaling.
  • an additional therapeutic agent comprises a biological molecule, such as an antibody.
  • treatment can involve the combined administration of a DLL4 antagonist (e.g. an antibody) of the present invention with other antibodies against additional tumor-associated proteins including, but not limited to, antibodies that bind EGFR, ErbB2, HER2, and/or VEGF.
  • the additional therapeutic agent is an antibody that is an anti-cancer stem cell marker antibody.
  • the additional therapeutic agent is an antibody that binds a component of the Notch pathway.
  • the additional therapeutic agent is an antibody that binds a component of the Wnt pathway.
  • the additional therapeutic agent is an antibody that inhibits a cancer stem cell pathway.
  • the additional therapeutic agent is an antibody inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an antibody inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an antibody inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an antibody that inhibits ⁇ -catenin signaling. In certain embodiments, the additional therapeutic agent is an antibody that is an angiogenesis inhibitor or modulator (e.g., an anti-VEGF or VEGF receptor antibody). In certain embodiments, the additional therapeutic agent is bevacizumab (AVASTIN), trastuzumab (HERCEPTIN), panitumumab (VECTIBIX), or cetuximab (ERBITUX). Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously.
  • AVASTIN AVASTIN
  • trastuzumab HERCEPTIN
  • panitumumab VECTI
  • treatment with a DLL4 antagonist described herein can include combination treatment with other biologic molecules, such as one or more cytokines (e.g., lymphokines, interleukins, tumor necrosis factors, and/or growth factors) or can be accompanied by surgical removal of tumors, cancer cells, or any other therapy deemed necessary by a treating physician.
  • cytokines e.g., lymphokines, interleukins, tumor necrosis factors, and/or growth factors
  • the treatment involves the administration of a DLL4 antagonist (e.g. an antibody) of the present invention in combination with radiation therapy.
  • a DLL4 antagonist e.g. an antibody
  • Treatment with a DLL4 antagonist can occur prior to, concurrently with, or subsequent to administration of radiation therapy. Dosing schedules for such radiation therapy can be determined by the skilled medical practitioner.
  • Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which describe the use of a DLL4 antagonist for treatment of cancer. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the present disclosure.
  • a method of selecting a subject for treatment with a DLL4 antagonist comprising:
  • a method of identifying a subject as eligible for treatment with a DLL4 antagonist comprising:
  • a method of selecting a subject for treatment with a DLL4 antagonist comprising:
  • a method of identifying a subject as eligible for treatment with a DLL4 antagonist comprising:
  • natriuretic peptide is B-type natriuretic peptide (BNP).
  • a method of monitoring a subject receiving treatment with a DLL4 antagonist for the development of a cardiovascular side effect and/or toxicity comprising:
  • a method of detecting the development of a cardiovascular side effect and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprising:
  • a method for identifying a cardiovascular side effect and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprising:
  • a method for monitoring a cardiovascular side effect and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprising:
  • a method of monitoring a subject receiving treatment with a DLL4 antagonist for the development of a cardiovascular side effect and/or toxicity comprising:
  • a method of detecting the development of a cardiovascular side effect and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprising:
  • a method for identifying a cardiovascular side effect and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprising:
  • a method for monitoring a cardiovascular side effect and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprising:
  • the predetermined level of the natriuretic peptide is the amount of natriuretic peptide in a sample obtained at an earlier date.
  • the predetermined level of the natriuretic peptide is the amount of natriuretic peptide in a sample obtained prior to treatment.
  • any one of embodiments 12-26 wherein if the natriuretic peptide level is above a predetermined level for any one sample, the subject is administered a therapeutically effective amount of an ACE inhibitor and/or a ⁇ -blocker.
  • any one of embodiments 12-26 wherein if the natriuretic peptide level is above a predetermined level for any one sample, the subject is administered a therapeutically effective amount of an ACE inhibitor and/or a ⁇ -blocker and the DLL4 antagonist is withheld.
  • natriuretic peptide is B-type natriuretic peptide (BNP).
  • a method for reducing a cardiovascular side effect and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprising:
  • a method of preventing or attenuating the development of a cardiovascular side effect and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprising:
  • a method of screening a subject for the risk of a cardiovascular side effect and/or toxicity from treatment with a DLL4 antagonist comprising:
  • a method for reducing a cardiovascular side effect and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprising:
  • a method of preventing or attenuating the development of a cardiovascular side effect and/or toxicity in a subject receiving treatment with a DLL4 antagonist comprising:
  • a method of screening a subject for the risk of a cardiovascular side effect and/or toxicity from treatment with a DLL4 antagonist comprising:
  • the DLL4 antagonist is an antibody comprising: a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:1), a heavy chain CDR2 comprising YISSYNGATNYNQKFKG (SEQ ID NO:3), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:5), and a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:6), a light chain CDR2 comprising AASNQGS (SEQ ID NO:7), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:8).
  • the DLL4 antagonist is an antibody comprising: a heavy chain variable region comprising SEQ ID NO:10 and a light chain variable region comprising SEQ ID NO: 12.
  • DLL4 antagonist is an anti-DLL4/anti-VEGF bispecific antibody.
  • ACE inhibitor is selected from the group consisting of: captopril, zofenopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, fosinopril, ceronapril, casokinins, lactokinins, teprotide, alacepril, cilazapril, delapril, imidapril, moexipril, rentiapril, spirapril, temocapril, moveltipril and trandolapril.
  • ⁇ -blocker is selected from the group consisting of: carvedilol, atenolol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, timolol, acebutolol, bisoprolol, esmolol, labetalol, bucindolol, nebivolol, alprenolol; amosulalol, arotinolol, befunolol, betaxolol, bevantolot, bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, butidrine hydrochloride, butofilolol, carazolol, carteolol, carteolol,
  • the cancer is selected from the group consisting of: lung cancer, breast cancer, colon cancer, colorectal cancer, melanoma, pancreatic cancer, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, glioma, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, and head and neck cancer.
  • the study was an open-label Phase 1 dose-escalation study of OMP-21M18 in subjects with advanced solid tumors.
  • the primary objective of the study was to determine the maximum tolerated dose of OMP-21M18.
  • the secondary objectives were to determine the safety, the rate of immunogenicity, the preliminary efficacy, and the pharmacokinetics of OMP-21M18.
  • 15 additional subjects were treated with 10 mg/kg once every other week.
  • Cohorts of 3 subjects were treated and evaluated for dose-limiting toxicities (DLTs) through Day 28. If 0 of 3 subjects had a DLT, escalation to the next dose cohort occurred. If I of 3 subjects had a DLT, 3 additional subjects were treated and escalation to the next cohort occurred if less than 2 of 6 subjects experienced a DLT. Subjects continued on treatment until Day 56, when an assessment of the tumor respond was performed.
  • DLTs dose-limiting toxicities
  • the maximum tolerated dose was not reached at a dose of 10 mg/kg every other week, however several subjects treated with 10 mg/kg every other week for longer than 100 days showed signs of cardiotoxicity that exceeded the level of acceptable chronic toxicity and enrollment in the study was stopped early.
  • BNP or NT-pro-BNP
  • BNP levels in blood/serum samples may be used to detect cardiac dysfunction and/or heart failure.
  • Increases in BNP levels to greater than 400 pg/ml or NT-proBNP levels to greater than 800 pg/ml considered to be possibly related to OMP-21M18 treatment were observed in six patients who received 10 mg/kg once every other week.
  • the study is a Phase 1b dose-escalation study of OMP-21M18 plus carboplatin and pemetrexed (carbo/PEM) in subjects with unresectable, locally advanced, recurrent, or metastatic non-squamous non-small cell lung cancer (NSCLC). Subjects had not received prior chemotherapy for the cancer.
  • the primary objective of the study was to determine the maximum tolerated dose of OMP-21M18 plus carbo/PEM in subjects with NSCLC.
  • the secondary objectives were to determine the safety, the rate of immunogenicity, the preliminary efficacy, and the pharmacokinetics of OMP-21M18 in combination with carbo/PEM as a first line treatment in subjects with NSCLC.
  • Carboplatin (6 mg/ml ⁇ min ⁇ [creatinine clearance (ml/min)+25]) and pemetrexed (500 mg/m 2 ) were administered once every 21 days for a total of 6 cycles (or for less than 6 full cycles if toxicity necessitates reducing or holding a dose or terminating treatment). Patients with stable disease or a response at the end of the 6 cycles continued to receive OMP-21M18 once every 3 weeks as maintenance therapy.
  • OMP-21M18 was supplied at a concentration of 10 mg/ml in a 25-ml single-use glass vial filled to 20 ml to deliver a total of 200 mg per vial.
  • OMP-21M18 was administered by intravenous (IV) infusion over 30 minutes once every 21 days (on the same day as the scheduled carbo/PEM administration) until disease progression.
  • IV intravenous
  • the first cohort of 6 patients was administered OMP-21M18 at a dosage of 5 mg/kg once every 3 weeks. Treatment in this cohort was paused due to emerging evidence of cardiotoxicity secondary to administration of OMP-21M18 in other ongoing studies (see Example 1). The Phase 1b protocol was amended to include a risk mitigation plan to enhance the therapeutic index of OMP-21M18 and manage tolerability.
  • the second cohort of six patients was administered OMP-21M18 at a dosage of 2.5 mg/kg once every 3 weeks and the third cohort of eight patients was administered OMP-21M18 at a dosage of 5 mg/kg once every 3 weeks.
  • BNP B-type natriuretic peptide
  • Treatment with OMP-21M18 was restarted when BNP levels decreased below 300 pg/ml. If a subject's BNP level was greater than 400 pg/ml in any one sample, treatment with OMP-21M18 was discontinued. In addition, subjects had Doppler echocardiograms every 28 days to assess cardiac function and monitor left ventricular ejection fraction (LVEF) and peak tricuspid velocity (PTV). Baseline BNP and echocardiogram readings were taken just prior to administration of the first dosage.
  • LVEF left ventricular ejection fraction
  • PTV peak tricuspid velocity
  • the study is a Phase 1b dose-escalation study of OMP-21M18 plus gemcitabine in subjects with locally advanced or metastatic pancreatic cancer. Subjects had not received prior chemotherapy for the cancer.
  • the primary objective of the study was to determine the maximum tolerated dose of OMP-21M18 plus gemcitabine in subjects with pancreatic cancer.
  • the secondary objectives were to determine the safety, the rate of immunogenicity, the preliminary efficacy, and the pharmacokinetics of OMP-21M18 in combination with gemcitabine as a first line treatment in subjects with pancreatic.
  • Gemcitabine 1000 mg/m2 was administered once every week for up to 7 weeks (or until toxicity necessitates reducing or holding a dose), followed by a week of rest from treatment. Subsequent cycles consisted of once weekly infusions for 3 consecutive weeks out of every 4 weeks.
  • OMP-21M18 was supplied at a concentration of 10 mg/ml in a 25-ml single-use glass vial filled to 20 ml to deliver a total of 200 mg per vial.
  • OMP-21M18 was administered by intravenous (IV) infusion over 30 minutes once every 14 days or once every 4 weeks until disease progression.
  • the first cohort of eight patients was administered OMP-21M18 at a dosage of 2.5 mg/kg once every 2 weeks. Treatment in this cohort was paused due to emerging evidence of cardiotoxicity secondary to administration of OMP-21M18 in other ongoing studies (see Example 1). The protocol was amended to include a risk mitigation plan to enhance the therapeutic index of OMP-21M18 and manage tolerability.
  • the second cohort of eight patients was administered OMP-21M18 at a dosage of 2.5 mg/kg once every 4 weeks and the third cohort of eight patients will be administered OMP-21M18 at a dosage of 5 mg/kg once every 4 weeks.
  • BNP B-type natriuretic peptide
  • Patients were administered a cardioprotective ACE inhibitor or carvedilol if their BNP levels were greater than or equal to 100 pg/ml in two consecutive samples or greater than or equal to 200 pg/ml in one sample, while still receiving OMP-21M18.
  • Patients were administered an ACE inhibitor or carvedilol if their BNP level was greater than or equal to 300 pg/ml in any one sample and treatment with OMP-21M18 was withheld. Treatment with OMP-21M18 was restarted when BNP levels decreased below 300 pg/ml. If a subject's BNP level was greater than 400 pg/ml in any one sample, treatment with OMP-21M18 was discontinued.
  • subjects had Doppler echocardiograms every 28 days to assess cardiac function and monitor left ventricular ejection fraction (LVEF) and peak tricuspid velocity (PTV). Baseline BNP and echocardiogram readings were taken just prior to administration of the first dosage.
  • LVEF left ventricular ejection fraction
  • PTV peak tricuspid velocity
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US20160334423A1 (en) 2016-11-17
CA2889638A1 (en) 2014-05-08
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US20140220001A1 (en) 2014-08-07
US9599620B2 (en) 2017-03-21
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US20170299598A1 (en) 2017-10-19
JP6371294B2 (ja) 2018-08-08

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