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WO2018039107A1 - Molécules de liaison spécifiques pour notch4 et leurs utilisations - Google Patents

Molécules de liaison spécifiques pour notch4 et leurs utilisations Download PDF

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Publication number
WO2018039107A1
WO2018039107A1 PCT/US2017/047745 US2017047745W WO2018039107A1 WO 2018039107 A1 WO2018039107 A1 WO 2018039107A1 US 2017047745 W US2017047745 W US 2017047745W WO 2018039107 A1 WO2018039107 A1 WO 2018039107A1
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Prior art keywords
seq
notch4
gla
binding molecule
antibody
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Elaine M. HURT
Peter Pavlik
Ping Tsui
David Jenkins
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MedImmune LLC
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MedImmune LLC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4402—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 2, e.g. pheniramine, bisacodyl
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • Cancer stem cells are a subset of cancer cells with the stem cell-like characteristics of pluripotency and unlimited self-renewal. As such, it is believed that this subpopulation of cells is responsible for tumor formation and adaptation to its environment.
  • CSCs are likely responsible for drug resistance, metastasis, and relapse of cancer, particularly in instances with minimal residual disease.
  • recent clinical evidence showed that the fraction of breast cancer cells that survived following standard-of-care therapy was enriched in cells bearing a CSC signature (Creighton CJ et al. (2009) Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Proc. Natl. Acad.
  • CSCs Since their initial identification, CSCs have been discovered and validated in many tumor types. Following the original identification of a CSC in a model of acute myeloid leukemia (AML) (Lapidot T et al. (1994) A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367:645-648), CSCs were discovered and validated in a number of hematological and solid tumor malignancies. Therefore, drugs that target CSCs are emerging as a critical component of any successful therapy against cancer.
  • AML acute myeloid leukemia
  • CSCs are driven by the same major self-renewal pathways as embryonic stem cells, including the wnt, Notch, and hedgehog signaling pathways Takebe N et al. ((2015)
  • Notch pathway has been implicated in the maintenance of a CSC phenotype in many different malignancies.
  • Notchl-4 the Notch receptors
  • DLL1, DLL3, DLL4, JAG1, and JAG2 the Notch receptor is
  • Notch4 specifically has been implicated in CSC maintenance in breast (D'Angelo RC et al. (2015) Notch reporter activity in breast cancer cell lines identifies a subset of cells with stem cell activity. Mol. Cancer Ther. 14:779-787; Harrison H et al. (2010) Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor. Cancer Res. 70:709-18), ovarian (Gao MQ et al. (2010) CD24+ cells from hierarchically organized ovarian cancer are enriched in cancer stem cells.
  • Notch pathway activity identifies cells with cancer stem cell-like properties and correlates with worse survival in lung adenocarcinoma. Clin. Cancer Res. 19: 1972-1980). Therefore, agents that target this Notch receptor in particular should inhibit CSCs, and may lead to better patient prognosis, while sparing some of the toxicity seen with pan-Notch inhibitors, such as gamma secretase inhibitors.
  • compositions and methods for the treatment of cancer including methods for reducing tumorigenicity of cancer, and inhibiting or killing cancer cells, such as CSCs, would provide more therapeutic options and the potential for better clinical outcomes, such as disease remission and/or improvement of patient quality of life.
  • This disclosure provides compositions that specifically bind to Notch4, and methods for the use of such compositions, such as for treating cancer, for reducing tumorigenicity of cancer, and for inhibiting or killing CSCs.
  • Notch4-binding molecules for example, monoclonal antibodies capable of inhibiting Notch4 activity, and methods of using the Notch4-binding molecules, for example, in treating or preventing recurrence of cancer, and in inhibiting or killing CSCs.
  • Notch4 binding molecule or antigen binding portion is provided.
  • An aspect provides a Notch4 binding molecule or antigen binding portion thereof that specifically binds to human Notch4, wherein the binding molecule or portion thereof comprises a heavy chain variable domain (VH) having an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 21; SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO:27; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 33; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 39; SEQ ID NO: 41; SEQ ID NO: 43; SEQ ID NO: 45; SEQ ID NO: 47; SEQ ID NO: 49; SEQ ID NO: 51; SEQ ID NO: 53; SEQ ID NO: 55; SEQ ID NO: 57; SEQ ID NO: 59; SEQ ID NO: 61; SEQ ID NO: 63; SEQ ID NO: 13;
  • the binding molecule or antigen binding portion thereof specifically binds to the same epitope of human Notch4 as an antibody comprising a heavy chain variable domain (VH) having an amino acid sequence selected from SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 21; SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO:27; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 33; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 39; SEQ ID NO: 41; SEQ ID NO: 43; SEQ ID NO: 45; SEQ ID NO: 47; SEQ ID NO: 49; SEQ ID NO: 51; SEQ ID NO: 53; SEQ ID NO: 55; SEQ ID NO: 57; SEQ ID NO: 59; SEQ ID NO: 61; SEQ ID NO: 63; SEQ ID NO: 65; SEQ ID NO: 67
  • VH heavy chain variable
  • the binding molecule or antigen binding fragment thereof competes or cross-competes with a binding molecule or antigen binding portion thereof which comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 93; an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 94; an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 95; an LCDRl having the amino acid sequence set forth in SEQ ID NO: 96; an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 97; and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 98.
  • the Notch4-binding molecule is selected from a murine antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a bi-specific antibody, a multi-specific antibody, and an antigen-binding fragment thereof.
  • the Notch4-binding molecule is selected from an Fv, an Fab, an
  • scFV single chain Fv
  • dsFv disulfide-linked
  • the Notch4-binding molecule or antigen binding fragment thereof can comprise an immunoglobulin (Ig) heavy chain constant region, for instance, a human IgG constant region.
  • the binding molecule is an IgGl triple mutant.
  • the binding molecule is a YTE mutant.
  • the Notch4-binding molecule or antigen binding fragment thereof can comprise an immunoglobulin light chain constant region.
  • K D dissociation constant
  • the Notch4-binding molecule or antigen binding fragment thereof does not specifically bind to Notch 1, Notch2, or Notch3.
  • the Notch4-binding molecule or antigen binding fragment thereof can be conjugated to an agent selected from the group consisting of an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, a polyethylene glycol (PEG), a toxin, and a combination of two or more of any said agents.
  • an agent selected from the group consisting of an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, a polyethylene glycol (PEG), a toxin, and a combination of two or more of any said agents.
  • An aspect is a composition comprising a carrier and a Notch-4 binding molecule or antigen binding fragment thereof as described herewith.
  • the composition comprising a carrier and a Notch-4 binding molecule or antigen binding fragment thereof as described herewith is a therapeutic composition.
  • the composition comprising a carrier and a Notch-4 binding molecule or antigen binding fragment thereof as described herewith is a therapeutic composition.
  • a Notch4-binding molecule or antigen binding fragment thereof as described herewith. It is provided a method of treating or preventing recurrence of cancer in a subject, the method comprising administering to a subject in need of treatment or prevention an effective amount of a Notch4-binding molecule or antigen binding fragment thereof as described herewith, or a composition comprising the Notch4-binding molecule or antigen binding fragment thereof. Another aspect provides a Notch4-binding molecule or antigen binding fragment thereof as described herewith, or a composition comprising the Notch4-binding molecule or antigen binding fragment thereof for treating or preventing recurrence of cancer.
  • the cancer is selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, kidney cancer, thyroid cancer, cancer of the salivary gland, pancreatic cancer, hepatocellular cancer, colorectal cancer, melanoma, and lung cancer.
  • the methods provided herewith comprise administering a second active agent.
  • the second active agent is a chemo therapeutic agent.
  • chemotherapeutic agents include carboplatin, cisplatin, doxil, and abraxane.
  • Another aspect is a method for detecting Notch4 in a sample, the method comprising contacting the sample with a Notch4-binding molecule or antigen binding fragment thereof, and detecting binding of the binding molecule or antigen binding fragment thereof to Notch4, thereby detecting Notch4 in the sample.
  • nucleic acid molecule Also within the scope of the disclosure are: an isolated nucleic acid molecule
  • the host cell is a mammalian host cell.
  • the method comprising culturing a host cell transformed with a nucleic acid encoding the binding molecule or antigen binding fragment thereof that specifically binds Notch4 under suitable conditions for producing the binding molecule or antigen binding fragment thereof.
  • the method can further comprise isolating the binding molecule or antigen binding fragment thereof.
  • composition comprising a nucleic acid encoding the binding
  • the vector encoding the binding molecule or antigen binding fragment thereof that specifically binds Notch4, or the host cell expressing the binding molecule or antigen binding fragment thereof that specifically binds Notch4.
  • kits comprising a Notch4-binding molecule or antigen binding fragment thereof as described herewith, or a nucleic acid molecule comprising a nucleotide sequence encoding the binding molecule or antigen binding fragment thereof that specifically binds Notch4.
  • FIG. 1 shows the percent luciferase activity remaining of Ad293 Notch4-luciferase reporter cells stimulated by plate -bound DLL4 protein after treatment with anti-Notch4 antibodies NOCH0004, NOCH0012, NOCH0075, NOCH0090, and NOCH0133.
  • FIG. 2 shows the percent inhibition of CSC sphere formation in the breast cancer cell lines HCC1937 and T47D treated with anti-Notch4 antibodies NOCH0004, NOCH0012,
  • FIG. 3 A and FIG. 3B show the relative Notch4 signaling in MDA-MB-231 tumors treated with 30 mg/kg of the indicated anti-Notch4 antibody, as determined by down-stream gene expression of Hey2 (FIG. 3A) and Hesl (FIG. 3B).
  • FIG. 4 A and FIG. 4B show the relative gene expression, in MDA-MB-231 tumors treated with 30 mg/kg of the indicated anti-Notch4 antibody, of "sternness" genes EZH2
  • FIG. 4A and BMI1 (FIG. 4B) as an indication of the inhibition of CSCs.
  • FIG. 5 shows the relative binding of anti-Notch4 antibodies NOCH0090 and GLA to a cell line overexpressing human Notch4.
  • FIG. 6A shows a plot of the % maximum inhibition for each of the five GLA-P
  • GLA-P 1 GLA-P2; GLA-P3; GLA-P4; GLA-P5
  • GLA-P5 antibodies
  • FIG. 6B shows the % inhibition of CSC sphere formation of T47D cells caused by GLA-P2,
  • GLA-P3, and GLA-P4 antibodies are examples of GLA-P3, and GLA-P4 antibodies.
  • FIG. 7A shows the activity of GLA-B 1, GLA-B2, GLA-B3, GLA-B4, GLA-B5,
  • FIG. 7B shows the ability of GLA antibody and GLA-B l, GLA-B3, and GLA-B4 antibodies to inhibit CSC sphere formation of
  • FIG.8A shows the activity of GLA-S 1, GLA-S2, GLA-S3, GLA-S4, and GLA-S5 antibodies in the luciferase reporter assay.
  • FIG. 8B shows the ability of GLA-S 1, GLA-S2, GLA-S 3, and GLA-S 4 antibodies to inhibit CSC sphere formation of T47D cells.
  • FIG. 9A shows anti-CSC activity of the IgGl control antibody dosed at 60 mg/kg, and of anti-Notch4 antibodies GLA-S3 and GLA-S4 dosed at 3 mg/kg, 10 mg/kg, 30 mg/kg, or 60 mg/kg in the MEDI-OVAl model of ovarian cancer.
  • FIG. 9B shows anti-CSC activity of the IgGl control antibody dosed at 60 mg/kg, and the anti-Notch4 antibodies GLA-S3 and GLA-S4 dosed at 3 mg/kg, 10 mg/kg, 30 mg/kg, or 60 mg/kg in the PA-1 model of ovarian cancer.
  • FIG. 10 shows CSC frequency in the OVCAR4 xenograft model as determined by secondary limiting dilution assays, following three doses of IgGl control antibody, or the GLA-S 4 antibody dosed at 3 mg/kg, 10 mg/kg, or 30 mg/kg.
  • FIG. 11A shows the activity of GLA-S4, GLA-S 4F3, GLA-S4F13, GLA-S4F15, GLA-S4F18 and GLA-S4F19 antibodies in the luciferase reporter assay.
  • FIG. 11B shows the ability of antibodies GLA-S4, GLA-S4F13, GLA-S4F15, GLA-S4F18, and GLA-S4F19 to inhibit CSC sphere formation in the OVCAR4 model.
  • FIG. 12A shows tumor regrowth as determined by flow cytometry in the PA-1 ovarian xenograft tumor model, untreated, after treatment with carboplatin, or after treatment with carboplatin and GLA-S4F18 antibody.
  • the black triangles below the X-axis represent the administration of carboplatin and/or antibody.
  • FIG. 12B shows CSC frequency as determined by flow cytometry in the PA-1 ovarian xenograft tumor model, untreated, after treatment with carboplatin, or after treatment with carboplatin and GLA-S4F18 antibody.
  • FIG. 13A and FIG. 13B are graphs depicting upregulation of Notch4 expression by flow cytometry on the surface of SW-780 treated with either cisplatin (FIG. 13A) or doxil (FIG. 13B).
  • FIG. 14A-14D contain a series of graphs illustrating tumor relapse (tumors >500 mm 3 ) of two ovarian patient derived xenograft models, OVA-001 (FIG. 14 A) and OVA-002 (FIG. 14B) treated with either cisplatin plus a control antibody or cisplatin plus GLA- S4F18; and the ex vivo CSC sphere formation of these models (FIG. 14C for OVA-001, and FIG. 14D for OVA-002, respectively).
  • FIG. 15B contain graphs depicting the reduction in the percentage of CSCs as determined by secondary tumor formation in a limiting dilution study of OVCAR-4 xenograft (FIG. 15A) that was treated with GLA-S4F18 and of a patient derived xenograft MEDI-OVA1 (FIG. 15B) treated with GLA-S4F18 either alone or in combination with abraxane.
  • FIG. 16A-16H show flow cytometry results of the binding of GLA-S4 antibody or a commercially available Notch4 antibody (N4) to Notch4/Notchl NRR sub-domain swap constructs to map the GLA-S4 binding epitope.
  • the black open trace is the isotype control and the shaded grey trace is the indicated Notch4 antibody.
  • FIG. 16A binding to N4 NRR;
  • FIG. 16B binding to N4/N1-LNR1;
  • FIG. 16C binding to N4/N1-LNR2;
  • FIG. 16D shows flow cytometry results of the binding of GLA-S4 antibody or a commercially available Notch4 antibody (N4) to Notch4/Notchl NRR sub-domain swap constructs to map the GLA-S4 binding epitope.
  • the black open trace is the isotype control and the shaded grey trace is the indicated Notch4 antibody.
  • FIG. 16A binding to N4 NRR
  • FIG. 16B binding to N4/N1
  • FIG. 16E binding to N4/N1-HD-N
  • FIG. 16F binding to N4/N1 - linker
  • FIG. 16G binding to N4/N1-HD-C
  • FIG. 16H binding to Nl NRR.
  • FIG. 17A and FIG. 17B show an alignment of the amino acid sequences of the heavy chain regions of the germlined leads, block mutants, parsimonious mutants, combination mutants, and optimized GLA-S4 clones.
  • FIG. 17A shows amino acids 1 to 66 of the heavy chain which include HCDR1 and HCDR2;
  • FIG. 17B shows amino acids 67 to 123 of the heavy chain, which includes HDCR3.
  • FIG. 18A and FIG. 18B show an alignment of the amino acid sequences of the light chain regions of the germlined leads, block mutants, parsimonious mutants, combination mutants, and optimized GLA-S4 clones.
  • FIG. 18A shows amino acids 1 to 58 of the light chain which include LCDR1 and LCDR2;
  • FIG. 18B shows amino acids 59 to 110 of the light chain, which includes LCDR3.
  • the present invention provides molecules that bind to Notch4.
  • such molecules are antibodies or antigen-binding fragments thereof, which specifically bind to Notch4 and do not specifically bind to Notch 1, Notch2, or Notch3.
  • antibodies or antigen-binding fragments thereof which specifically bind to Notch4 and do not specifically bind to Notch 1, Notch2, or Notch3.
  • Amino acids are referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter codes.
  • Notch4 refers to the neurogenic locus notch homolog 4 protein.
  • the full-length amino acid and nucleotide sequences for human, cynomolgus monkey (Macaca fasciculari), and mouse Notch4, among other species, are known in the art.
  • Notch4 has an extracellular (EC) domain and a transmembrane (TM) domain.
  • the Notch4 negative regulatory region (NRR) comprises three LIN12/Notch repeats (LNR region) and a heterodimerization (HD) domain. Following cleavage by furin, association of the EC and TM domains is maintained by non-covalent interactions between the N-terminal and C-terminal regions of the HD domain (HD-N and HD-C, respectively).
  • antibody refers to an immunoglobulin molecule that recognizes
  • antibody specifically binds to 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.
  • 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.
  • 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.
  • antibody or “immunoglobulin” are used interchangeably herein.
  • a typical antibody comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2, and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL).
  • the light chain constant region is comprised of one domain, CI.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies can mediate the binding of the
  • immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (Clq) of the classical complement system.
  • various cells of the immune system e.g. effector cells
  • the first component (Clq) of the classical complement system e.g. Clq
  • Antibodies can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, or subclasses (isotypes) thereof (e.g. IgGl, IgG2, IgG3, IgG4, IgAl 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. There are two classes of mammalian light chains, lambda and kappa.
  • the heavy chain and light chain regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with regions that are more conserved, termed framework (FW) regions.
  • CDRs complementarity-determining regions
  • FW framework regions
  • the CDRs in each chain are held together in close proximity by the FW regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies.
  • Each VH and VL is composed of three CDRs and four FWs, arranged from amino-terminus to carboxy- terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4.
  • amino acid position numbering refers to the numbering system used for heavy chain variable domains or light chain variable domains (approximately residues 1- 107 of the light chain and residues 1-113 of the heavy chain). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids
  • a heavy chain variable domain can include a single amino acid insert (residue 52a, according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc., according to Kabat) after heavy chain FW residue 82.
  • residue 52a residue 52 of H2
  • residues 82a, 82b, and 82c, etc., according to Kabat residues 82a, 82b, and 82c, etc., according to Kabat
  • the Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol.
  • the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).
  • the AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular' s AbM antibody modeling software. See Table 1, below.
  • IMGT immunoglobulin variable regions
  • CDR the numbering system for the immunoglobulin variable regions. See, e.g., Lefranc, M.P. et ah, Dev. Comp. Immunol. 27: 55-77 (2003).
  • the IMGT numbering system was based on an alignment of more than 5,000 sequences, structural data, and characterization of hypervariable loops and allows for easy comparison of the variable and CDR regions for all species.
  • VH-CDR1 is at positions 26 to 35
  • VH-CDR2 is at positions 51 to 57
  • VH-CDR3 is at positions 93 to 102
  • VL-CDR1 is at positions 27 to 32
  • VL-CDR2 is at positions 50 to 52
  • VL-CDR3 is at positions 89 to 97.
  • VH CDRs amino acid sequences described correspond to the classical Kabat numbering locations, namely Kabat VH-CDR1 is at positions 31-35, VH-CDR2 is a positions 50-65, and VH-CDR3 is at positions 95-102.
  • VL- CDR1, VL-CDR2 and VL-CDR3 also correspond to classical Kabat numbering locations, namely positions 24-34, 50-56 and 89-97, respectively.
  • antibody encompasses polyclonal antibodies; monoclonal antibodies; multispecific antibodies, such as bispecific antibodies generated from at least two intact antibodies; humanized antibodies; human antibodies; chimeric antibodies; fusion proteins comprising an antigen-determination portion of an antibody; and any other modified immunoglobulin molecule comprising an antigen recognition site, so long as the antibodies exhibit the desired biological activity.
  • a “monoclonal antibody” refers to a homogeneous antibody population that is involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants.
  • the term “monoclonal” can apply to both intact and full-length monoclonal antibodies, as well as to antibody fragments (such as Fab, Fab' , F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site.
  • “monoclonal antibody” refers to such antibodies made in any number of ways including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals.
  • humanized antibody refers to an antibody derived from a non-human (e.g., murine) immunoglobulin, which has been engineered to contain minimal non-human (e.g., murine) sequences.
  • humanized antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and capability.
  • CDR complementary determining region
  • FW 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 capability.
  • Humanized antibodies can be further modified by the substitution of additional amino acids
  • humanized antibodies will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non- human immunoglobulin whereas all or substantially all of the FW regions are those of a human immunoglobulin consensus sequence.
  • Humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are well known in the art.
  • human antibody means 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 technique known in the art.
  • the definition of a human antibody includes intact or full-length antibodies comprising at least one human heavy and/or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.
  • chimeric antibodies refers to antibodies 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 capability while the constant regions are homologous to the sequences in antibodies derived from another (usually human) to avoid eliciting an immune response in that species.
  • antigen-binding fragment refers to a portion of an intact antibody
  • Fragments of a full-length antibody can be an antigen-binding fragment of an antibody.
  • antibody fragments include, but are not limited to Fab, Fab' , F(ab')2, and Fv fragments, linear antibodies, single chain antibodies (e.g., ScFvs), and multispecific antibodies formed from antibody fragments.
  • a "blocking" antibody or an “antagonist” antibody is one that inhibits or reduces
  • blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. Desirably, the biological activity is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100%.
  • the term "germlining" means that amino acids at specific positions in an antibody are mutated back to those in the germ line.
  • the "IgGl triple mutant" or "IgGl-TM" antibody format is a human IgGl isotype containing three single amino acid substitutions, L234F/L235E/P331S, within the lower hinge and CH2 domain (Oganesyan et ah, Acta Crystallogr. D Biol. Crystallogr. 64:700-704, 2008).
  • the TM causes a profound decrease in binding to human FcyRI, FcyRII, FcyRIII, and Clq, resulting in a human isotype with very low effector function.
  • YTE or "YTE mutant” or “YTE mutation” refer to a mutation in IgGl Fc that results in an increase in the binding to human FcRn and improves the serum half-life of the antibody having the mutation.
  • a YTE mutant comprises a combination of three mutations, M252Y/S254T/T256E (Kabat EU numbering), introduced into the heavy chain of an IgGl . See U.S. Patent No. 7,658,921, which is incorporated by reference herein.
  • the YTE mutant has been shown to increase the serum half-life of antibodies approximately four- times as compared to wild-type versions of the same antibody.
  • Binding affinity generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule ⁇ e.g., an antibody) and its binding partner ⁇ e.g., an antigen).
  • binding affinity refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair ⁇ e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.
  • the affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method known in the art, e.g., flow cytometry, enzyme-linked
  • ELISA immunosorbent assay
  • RIA radioimmunoassay
  • kinetics ⁇ e.g., KINEXA® or BIACORETM or OCTET® analysis.
  • Direct binding assays as well as competitive binding assay formats can be readily employed.
  • the measured affinity of a particular antibody- antigen interaction can vary if measured under different conditions ⁇ e.g., salt concentration, pH, temperature).
  • affinity and other antigen-binding parameters e.g., KD or Kd, K on , Koff
  • KD or Kd, K on , Koff are made with standardized solutions of antibody and antigen, and a standardized buffer, as known in the art.
  • IC50 is the median inhibitory concentration of an antibody molecule. In functional assays, IC50 is the concentration that reduces a biological response by 50% of its maximum. In ligand-binding studies, IC50 is the concentration that reduces receptor binding by 50% of maximal specific binding level. IC50 can be calculated by any number of means known in the art.
  • herewith as compared to a reference antibody can be at least about 2-fold, at least about 4- fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 110-fold, at least about 120-fold, at least about 130-fold, at least about 140-fold, at least about 150-fold, at least about 160-fold, at least about 170-fold, or at least about 180- fold or more.
  • inhibitor can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in biological activity.
  • the terms “inhibition” or “suppression” are applied to describe, e.g., an effect on the Notch signal transduction pathway, the terms refer to the ability of a Notch4-binding molecule to statistically significantly decrease Notch4-mediated cell activation, proliferation, or signal transduction relative to an untreated (control) cell.
  • the cell that expresses Notch4 can be a naturally occurring cell or cell line, or can be
  • the Notch4-binding molecule can inhibit Notch4-mediated cell activation, proliferation, or signal transduction in a Notch4-expressing cell by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% or about 100%, as determined, for example, by flow cytometry, Western blotting, ELISA, or other assays known to those of skill in the art.
  • the binding molecules of the invention inhibit CSCs by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, compared with "control" CSCs, i.e., CSCs that are not contacted with a binding molecule of the invention.
  • subject or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
  • Mammalian subjects include humans, domestic animals, farm animals, sports animals, and zoo animals including, e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and so on.
  • composition refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective and which contains no additional components that are unacceptably toxic to a subject to which the composition would be administered.
  • composition can be sterile and can comprise a
  • Suitable pharmaceutical compositions can comprise one or more of a buffer ⁇ e.g. acetate, phosphate or citrate buffer), a surfactant ⁇ e.g. polysorbate), a stabilizing agent ⁇ e.g. human albumin), a preservative ⁇ e.g. benzyl alcohol), an absorption promoter to enhance bioavailability and/or other conventional solubilizing or dispersing agents.
  • a buffer e.g. acetate, phosphate or citrate buffer
  • a surfactant ⁇ e.g. polysorbate
  • a stabilizing agent e.g. human albumin
  • a preservative ⁇ e.g. benzyl alcohol
  • an absorption promoter to enhance bioavailability and/or other conventional solubilizing or dispersing agents.
  • An "effective amount" of a binding molecule as disclosed herein is an amount
  • An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.
  • Binding molecules of the invention can be naked or conjugated to other molecules such as toxins, labels, etc.
  • label when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to a binding molecule, so as to generate a "labeled" binding molecule.
  • the label can be detectable by itself ⁇ e.g., radioisotope labels or fluorescent labels) or, as in the case of, e.g., an enzymatic label, can catalyze chemical alteration of a substrate compound or composition that is detectable.
  • Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder.
  • a subject is successfully "treated” for a disease or disorder according to the methods provided herein if the patient shows, e.g., total, partial, or transient alleviation or elimination of symptoms associated with the disease or disorder.
  • Prevent refers to prophylactic or preventative measures that prevent and/or slow the development or recurrence of a targeted pathologic condition or disorder.
  • those in need of prevention include those prone to have or susceptible to the disorder, including those who have had the disorder and are susceptible to recurrence.
  • a disease or disorder is successfully prevented according to the methods provided herein if the patient develops, transiently or permanently, e.g., fewer or less severe symptoms or pathology associated with the disease or disorder, or a later onset of symptoms or pathology associated with the disease or disorder, than a patient who has not been subject to the methods of the invention.
  • recurrence of cancer is prevented for at least about 3, 6, 9, 12, 18, or 24 months after the start of treatment with a Notch4-binding molecule of the invention.
  • polypeptide polypeptide
  • peptide protein
  • the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer can be linear or branched, it can comprise modified amino acids and non-amino acids can interrupt it.
  • 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, etc.
  • the polypeptides can occur as single chains or associated chains.
  • 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., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, 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 amino acid sequences of the binding molecules of the invention do not abrogate the binding of the binding molecule to the antigen(s), i.e., Notch4, to which the binding molecule binds.
  • Methods of identifying conservative nucleotide and amino acid substitutions which do not eliminate antigen-binding are well-known in the art.
  • a "polynucleotide,” as used herein can include one or more “nucleic acids,” or
  • nucleic acid molecules refers to a polymer of nucleotides of any length, and includes DNA and RNA.
  • the polynucleotides 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.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
  • vector means a construct, which is capable of delivering and, in some embodiments 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, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
  • an "isolated" polypeptide, antibody, binding molecule, polynucleotide, vector, or cell is in a form not found in nature.
  • Isolated polypeptides, antibodies, binding molecules, polynucleotides, vectors, or cells 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, binding molecule, polynucleotide, vector, or cell that is isolated is substantially pure.
  • substantially pure refers to purity of greater than 75%, preferably greater than 80% or 90%, and most preferably greater than 95%.
  • 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 can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences.
  • sequence alignment algorithm is the algorithm incorporated into the NBLAST and XBLAST programs. Gapped BLAST; BLAST-2; WU- BLAST-2; ALIGN; ALIGN-2; or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6).
  • the GAP program in the GCG software package which incorporates the algorithm of Needleman and Wunsch, can be used to determine the percent identity between two amino acid sequences (e.g., using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5).
  • the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller.
  • the percent identity can be determined using the ALIGN program (version 2.0) and using a PAM120 with residue table, a gap length penalty of 12 and a gap penalty of 4.
  • One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain
  • the default parameters of the alignment software are used.
  • the percentage identity "X" of a first amino acid sequence to a second amino acid sequence is calculated as 100 x (Y/Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.
  • Notch4-binding molecules e.g., anti-Notch4 antibodies and antigen-binding fragments thereof, which specifically bind Notch4.
  • the term "Notch4- binding molecule” or “binding molecule that binds to Notch4" or "anti-Notch4" refers to a binding molecule that is capable of binding Notch4 with sufficient affinity such that the binding molecule is useful as a therapeutic agent or diagnostic reagent in targeting Notch4.
  • a binding molecule that "specifically binds to Notch4" binds to an unrelated, non-Notch4 protein to an extent of less than about 10% of the binding of the binding molecule to Notch4, as measured, e.g., by a radioimmunoassay (RIA), BIACORETM (using recombinant Notch4 as the analyte and binding molecule as the ligand, or vice versa), KINEXA®, OCTET®, or other binding assays known in the art.
  • RIA radioimmunoassay
  • BIACORETM using recombinant Notch4 as the analyte and binding molecule as the ligand, or vice versa
  • KINEXA® OCTET®
  • binding molecule that binds to Notch4 has a dissociation constant (KD) of ⁇ 1 ⁇ , ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 10 pM, ⁇ 1 pM, or ⁇ 0.1 pM.
  • the binding molecule binds to the negative regulatory region of Notch4 (Notch4-NRR).
  • Exemplary binding molecules of the present disclosure include humanized, optimized, germlined, and/or other versions of anti-Notch4 antibodies, anti-Notch4 TM antibodies, and serum half-life-optimized anti-Notch4 YTE antibodies (e.g., K44VHa-N56Q, Ka6-N56Q, or K2Ha-N56Q).
  • Exemplary antibodies of the present disclosure include clones GLA, GLA-Pl, GLA-P2, GLA-P3, GLA-P4, GLA-P5, GLA-B 1, GLA-B2, GLA-B3, GLA-B4, GLA-B5, GLA-B6, GLA-S 1, GLA-S2, GLA-S3, GLA-S4, GLA-S5, GLA-S4F1, GLA-S4F2, GLA- S4F3, GLA-S4F4, GLA-S4F5, GLA-S4F6, GLA-S4F8, GLA-S4F9, GLA-S4F10, GLA- S4F11, GLA-S4F12, GLA-S4F13, GLA-S4F14, GLA-S4F15, GLA-S4F16, GLA-S4F18, GLA-S4F19, and GLA-S4F20.
  • the invention also embraces variants and equivalents that are substantially homologous to the Not
  • this disclosure provides a Notch4-binding molecule that can
  • Epitopes specifically bind to the same Notch4 epitope as a binding molecule comprising the heavy chain variable region (VH) and light chain variable region (VL) of any one of clones GLA, GLA-P3, GLA-P4, GLA-S3, GLA-S4, GLA-S4F18, or GLA-S4F19.
  • VH heavy chain variable region
  • VL light chain variable region
  • epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • Conformational and non- conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • binding molecules can be identified based on their ability to cross-compete (e.g., to competitively inhibit the binding of, in a statistically significant manner) with binding molecules, such as GLA, GLA-P3, GLA-P4, GLA-S3, GLA-S4, GLA-S4F18, or GLA-S4F19, in standard Notch4 binding or activity assays.
  • Notch4-binding molecules that compete for binding to Notch4 with another Notch4-binding molecule described herewith, such as one of clones GLA, GLA-P3, GLA-P4, GLA-S3, GLA-S4, GLA-S4F18, or GLA-S4F19.
  • a binding molecule to inhibit the binding of, e.g., GLA, GLA- P3, GLA-P4, GLA-S3, GLA-S4, GLA-S4F18, or GLA-S4F19, demonstrates that the test binding molecule can compete with GLA, GLA-P3, GLA-P4, GLA-S3, GLA-S4, GLA- S4F18, or GLA-S4F19 for binding to Notch4; such a binding molecule can, according to a non-limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on Notch4 as the Notch4-binding molecule with which it competes.
  • an anti-Notch4 antibody or antigen-binding fragment thereof binds to the same epitope on Notch4 as any of clones GLA, GLA-P3, GLA-P4, GLA-S3, GLA-S4, GLA- S4F18, or GLA-S4F19.
  • the term "competes" indicates that a binding molecule competes unidirectionally for binding to Notch4 with any one of clones GLA, GLA-P3, GLA-P4, GLA-S3, GLA-S4, GLA-S4F18, or GLA-S4F19.
  • cross-competes indicates that a binding molecule competes bidirectionally for binding to Notch4 with any one of clones GLA, GLA-P3, GLA-P4, GLA-S3, GLA-S4, GLA-S4F18, or GLA-S4F19.
  • the Notch4-binding molecule is a murine antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a bi-specific antibody, a multispecific antibody, or any combination thereof.
  • Notch4-binding molecules comprise a Fab, a Fab', a F(ab') 2 , a Fd, a Fv, a scFv, a disulfide linked Fv, a V-NAR domain, an IgNar, an intrabody, an IgGACH2, a minibody, a F(ab' )3 5 a tetrabody, a triabody, a diabody, a single- domain antibody, DVD-Ig, Fcab, mAb 2 , a (scFv) 2 , or a scFv-Fc.
  • a Notch4-binding molecule provided herein can include, in addition to a VH and a VL, a heavy chain constant region or fragment thereof.
  • the heavy chain constant region is a human heavy chain constant region, e.g., a human IgG constant region, e.g., a human IgGl constant region.
  • binding molecules described herewith are produced to
  • Fc region comprises an altered Fc region, in which one or more alterations have been made in the Fc region in order to change functional and/or pharmacokinetic properties of the binding molecule. Such alterations may result in altered effector function, reduced immunogenicity, and/or an increased serum half- life.
  • the Fc region interacts with a number of ligands, including Fc receptors, the complement protein Clq, and other molecules, such as proteins A and G. These interactions are essential for a variety of effector functions and downstream signaling events including antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).
  • ADCC antibody dependent cell-mediated cytotoxicity
  • CDC complement dependent cytotoxicity
  • the Notch4-binding molecules described herewith have reduced or ablated affinity for an Fc ligand responsible for facilitating effector function, compared to a Notch4-binding molecule not comprising the modification in the Fc region.
  • the Notch4- binding molecule has no ADCC activity and/or no CDC activity.
  • the Notch4-binding molecule does not bind to an Fc receptor and/or complement factors.
  • the Notch4-binding molecule has no effector function. Selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art.
  • the binding molecule is of the IgGl subtype, and optionally comprises the TM format (L234F/L235E/P331S), as disclosed supra in the Definitions section.
  • a heavy chain constant region or fragment thereof can include one or more amino acid substitutions relative to a wild-type IgG constant domain, wherein the modified IgG has an increased half-life compared to the half-life of an IgG having the wild- type IgG constant domain.
  • the IgG constant domain can contain one or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385- 389, and 428-436, wherein the amino acid position numbering is according to the EU index as set forth in Kabat.
  • the IgG constant domain can contain one or more of a substitution of the amino acid at Kabat position 252 with Tyrosine (Y), Phenylalanine (F), Tryptophan (W), or Threonine (T), a substitution of the amino acid at Kabat position 254 with Threonine (T), a substitution of the amino acid at Kabat position 256 with Serine (S), Arginine (R), Glutamine (Q), Glutamic acid (E), Aspartic acid (D), or Threonine (T), a substitution of the amino acid at Kabat position 257 with Leucine (L), a substitution of the amino acid at Kabat position 309 with Proline (P), a substitution of the amino acid at Kabat position 311 with Serine (S), a substitution of the amino acid at Kabat position 428 with Threonine (T), Leucine (L), Phenylalanine (F), or Serine (S), a substitution of the amino acid at Kabat position 433 with Arginine (R)
  • the IgG constant domain can contain amino acid substitutions relative to a wild- type human IgG constant domain including as substitution of the amino acid at Kabat position 252 with Tyrosine (Y), a substitution of the amino acid at Kabat position 254 with Threonine (T), and a substitution of the amino acid at Kabat position 256 with Glutamic acid (E).
  • the binding molecule is of the IgGl subtype, and optionally comprises the triple mutant YTE, as disclosed supra in the Definitions section.
  • a Notch4-binding molecule provided herein can include a light chain constant region or fragment thereof.
  • the light chain constant region is a kappa constant region or a lambda constant region, e.g., a human kappa constant region or a human lambda constant region.
  • Notch4-binding molecules can have beneficial properties.
  • the binding molecule can inhibit, suppress, or block various Notch4-mediated activities, e.g., CSC activation, CSC proliferation, CSC frequency, and CSC activity, including CSC-mediated tumor initiation, all of which can be measured by assays known in the art.
  • the binding molecules provided herein can bind to Notch4 with a binding affinity characterized by a dissociation constant (K D ) of about 100 pM to about 0.5 nM as measured by a BiacoreTM assay or on a Kinetic Exclusion Assay (KinExA) 3000 platform or on an Octet® instrument.
  • K D dissociation constant
  • an anti-Notch4 antibody or antigen-binding fragment thereof can specifically bind to Notch4, e.g., human Notch4 or cynomolgus monkey Notch4, or an antigenic fragment thereof, with a dissociation constant or K D of less than 10 ⁇ 6 M, or of less than 10 "7 M, or of less than 10 "8 M, or of less than 10 "9 M, or of less than 10 "10 M, or of less than 10 "11 M, of less than 10 "12 M, of less than 10 "13 M, of less than 10 "14 M, or of less than 10 ⁇ 15 M as measured, e.g., by BiacoreTM or KinExA® or Octet®.
  • the anti-Notch4 antibody GLA-S4 can bind to human Notch4-NRR with a K D of about 0.2 nM, as measured by an Octet® assay.
  • a Notch4-binding molecule described herewith binds to
  • a Notch4-binding molecule binds to Notch4 or an antigenic fragment thereof with a K 0 ff of less than 10 ⁇ 3 s -1 , less than 5xl0 ⁇ 3 s -1 , less than 10 ⁇ 4 s -1 , less than 5xl0 ⁇ 4 s -1 , less than 10 ⁇ 5 s -1 , less than 5xl0 ⁇ 5 s -1 , less than 10 ⁇ 6 s -1 , less than 5xl0 "6 s "1 , less than less than 5xl0 "7 s "1 , less than 10 "8 s "1 , less than 5xl0 “8 s "1 , less than 10 ⁇ 9 s -1 , less than 5xl0
  • the anti-Notch4 antibody GLA-S4 can bind to human Notch4-NRR with a K 0 ff of about 1.05 x 10 "4 s "1 , as measured by an Octet® assay.
  • a Notch4-binding molecule described herewith binds to
  • the anti-Notch4 antibody GLA-S4 can bind to human Notch4-NRR with a K on of about 39.6 x 10 4 M “1 s “1 , as measured by an Octet® assay.
  • Notch4-binding molecules described herewith can bind to a Notch4 antigen, e.g., a human Notch4-NRR or a mouse Notch4-NRR at a half maximal effective concentration (EC50) of about 0.05 nM to about 0.3 nM, preferably about 0.09 nM to about 0.26 nM.
  • the anti-Notch4 antibody GLA-S4F18 has an EC50 of about 0.09 nM for human Notch4-NRR, and of about 0.11 nM for mouse Notch4-NRR, as measured by direct ELISA.
  • a VH and/or VL amino acid sequence or portion thereof, including a CDR sequence can be, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% similar to a sequence set forth herein, and/or comprise 1, 2, 3, 4, 5 or more substitutions, e.g., conservative substitutions, relative to a sequence set forth herein, such as a sequence from any of GLA, GLA-P3, GLA-P4, GLASS, GLA-S4, GLA-S4F18, or GLA-S4F19.
  • a Notch4-binding molecule having VH and VL regions with a certain percent identity to a VH region or VL region, or having one or more substitutions, e.g., conservative substitutions, can be obtained by mutagenesis ⁇ e.g., site- directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding VH and/or VL regions described herein, followed by testing of the encoded altered binding molecule for binding to Notch4, and optionally testing for retained function using the functional assays described herein.
  • the disclosure further provides a Notch4-binding molecule that is conjugated to a heterologous agent.
  • the agent can be an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, a polyethylene glycol (PEG), or a combination of two or more of any said agents.
  • PEG polyethylene glycol
  • binding molecule includes antibodies and antigen-binding fragments thereof.
  • the Notch4-binding molecule 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.
  • phage display technology can be used to identify and/or produce a Notch4-binding polypeptide.
  • the polypeptide comprises a protein scaffold of a type selected from the group consisting of protein A, a lipocalin, a fibronectin domain, an ankyrin consensus repeat domain, and thioredoxin.
  • the disclosure provides a composition, e.g., a pharmaceutical
  • composition comprising a Notch4-binding molecule as described herewith, optionally further comprising one or more carriers, diluents, excipients, or other additives.
  • a composition e.g., a pharmaceutical composition, comprising a Notch4-binding molecule as described herewith and a second therapeutic agent such as a chemotherapeutic agent.
  • Monoclonal anti-Notch4 antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein Nature 256:495 (1975). Using the hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized to elicit the production by lymphocytes of antibodies that will specifically bind to an immunizing antigen. Lymphocytes can also be immunized in vitro. Following immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol (PEG), to form hybridoma cells that can then be selected away from unfused lymphocytes and myeloma cells.
  • PEG polyethylene glycol
  • Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen as determined by immunoprecipitation, immunoblotting, or by an in vitro binding assay ⁇ e.g. RIA or ELISA) can then be propagated either in in vitro culture using standard methods known in the art) or in vivo such as ascites tumors in an animal.
  • the monoclonal antibodies can then be purified from the culture medium or ascites fluid.
  • Notch4-binding molecules can also be made using recombinant DNA methods
  • the polynucleotides encoding a monoclonal antibody are isolated from mature B -cells or hybridoma cell, 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 procedures.
  • the isolated polynucleotides encoding the heavy and light chains or antigen-binding fragments thereof are then cloned into suitable expression vectors, which when transfected into host cells such as E.
  • Notch4-binding molecules can be isolated from phage display libraries expressing CDRs of the desired species, as known in the art. Production and expression of nucleic acids comprising nucleotide sequences encoding Notch4-binding molecules are discussed in more detail in the next section.
  • the polynucleotide(s) encoding a binding molecule can further be modified in a number of different manners using recombinant DNA technology to generate alternative binding molecules.
  • the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted (1) for those regions of, for example, a human antibody to generate a chimeric antibody or (2) 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, etc. of a monoclonal antibody.
  • the Notch4-binding molecule is a human antibody or
  • Human antibodies can be directly prepared using various techniques known in the art. Immortalized human B lymphocytes immunized in vitro or isolated from an immunized individual that produce an antibody directed against a target antigen can be generated using methods known in the art.
  • the Notch4-binding molecule can be selected from a phage library, where the phage library expresses human antibodies, as described in the art. Techniques for the generation and use of antibody phage libraries are also described in the art.
  • Affinity maturation strategies and chain shuffling strategies are known in the art and can be employed to generate high affinity human antibodies or antigen-binding fragments thereof.
  • the Notch4-binding molecule can be a humanized antibody or antigen-binding fragment thereof.
  • Methods for engineering, humanizing, or resurfacing non- human or human antibodies can also be used and are well known in the art.
  • a humanized, resurfaced, or similarly engineered antibody can have one or more amino acid residues from a source that is non-human, e.g., mouse, rat, rabbit, non-human primate, or other mammal. These non-human amino acid residues are replaced by residues that are often referred to as "import" residues, which are typically taken from an "import" variable, constant, or other domain of a known human sequence.
  • CDR residues are directly and most substantially involved in influencing Notch4 binding. Accordingly, part or all of the non-human or human CDR sequences are maintained while the non-human sequences of the variable and constant regions can be replaced with human or other amino acids.
  • Antibodies can also optionally be humanized, resurfaced, engineered, or human
  • humanized (or human) or engineered anti-Notch4 antibodies and resurfaced antibodies can be optionally prepared by a process of analyzing the parental sequences and various conceptual humanized and engineered products, using three- dimensional models of the parental, engineered, and humanized sequences.
  • Three- dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences.
  • Anti-Notch4 humanized antibodies and antigen-binding fragments thereof can also be made in transgenic mice containing human immunoglobulin loci that are capable, upon immunization, of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production.
  • the Notch4-binding molecule is anti-Notch4 antibody
  • anti-Notch4 antibody fragments are produced recombinantly. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or other host cells, thus allowing the production of large amounts of these fragments. Such anti-Notch4 antibody fragments can also be isolated from the antibody phage libraries discussed above. Anti-Notch4 antibody fragments can also be linear antibodies. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
  • Techniques can be adapted for the production of single-chain antibodies specific to Notch4.
  • methods can be adapted for the construction of Fab expression libraries to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for Notch4.
  • Antibody fragments can also be produced by techniques in the art including, but not limited to: (a) a F(ab')2 fragment produced by pepsin digestion of an antibody molecule; (b) a Fab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment, (c) a Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent, and (d) Fv fragments.
  • the Notch4-binding molecule can be modified in order to reduce or eliminate effector function. This can be achieved, for example, by the triple mutation (TM) L234F/L235E/P331S in the Fc domain of IgGl. Other mutations that reduce effector function are known in the art.
  • a Notch4-binding molecule can be modified in order to increase its serum half-life. This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the binding molecule by mutation of the appropriate region, or by incorporating the epitope into a peptide tag that is then fused to the binding molecule at either end or in the middle (e.g., by DNA or peptide synthesis), or by YTE mutation.
  • Other methods to increase the serum half-life of an antibody or antigen-binding fragment thereof, e.g., conjugation to a heterologous molecule such as PEG, are known in the art.
  • Heteroconjugate Notch4 antibodies and antigen-binding fragments thereof are also provided herewith.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune cells to unwanted cells.
  • heteroconjugate anti-Notch4 antibodies and antigen- binding fragments thereof can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.
  • a Notch4-binding molecule can be modified to contain additional chemical moieties not normally part of the protein. Such moieties can improve the characteristics of the binding molecule, for example, solubility, biological half-life, or absorption. The moieties can also reduce or eliminate any undesirable side effects of the binding molecule and are known in the art.
  • polynucleotides comprising nucleic acid sequences that encode a Notch4-binding molecule, e.g., a polypeptide that specifically binds Notch4.
  • a polynucleotide comprising a nucleic acid sequence that encodes an anti-Notch4 antibody or encodes an antigen-binding fragment of such an antibody.
  • the polynucleotides can be in the form of RNA or in the form of DNA.
  • DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double- stranded or single- stranded, and, if single stranded, can be the coding strand or non-coding (anti-sense) strand.
  • the polynucleotide can be isolated. In certain embodiments, the polynucleotide can be substantially pure. In certain embodiments, the polynucleotide can be cDNA or are derived from cDNA. In certain embodiments, the polynucleotide can be recombinantly produced. In certain embodiments, the polynucleotide can comprise the coding sequence for a mature polypeptide, fused in the same reading frame to a
  • polypeptide having a leader sequence is a pre-protein and can have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.
  • the polynucleotide can also encode a Notch4-binding pro-protein which is the mature protein plus additional 5' amino acid residues.
  • the disclosure provides an isolated polynucleotide comprising a nucleic acid
  • Notch4-binding molecule comprising an amino acid sequence from a VH and/or VL domain having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence set forth herein, and/or comprising 1, 2, 3, 4, 5 or more amino acid substitutions, e.g., conservative substitutions, relative to an amino acid sequence set forth herein, such as a sequence from any of GLA, GLA-P3, GLA-P4, GLA-S3, GLA-S4, GLA-S4F18, or GLA- S4F19.
  • the polynucleotide that comprises the coding sequence for the Notch4-binding molecule is fused in the same reading frame as a marker sequence that allows, for example, for purification of the encoded polypeptide.
  • the marker sequence can be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence can be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used.
  • a mammalian host e.g., COS-7 cells
  • Polynucleotide variants are also provided. Polynucleotide variants can contain
  • polynucleotide variants contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide.
  • polynucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
  • vectors comprising the polynucleotides described above.
  • a polynucleotide comprising a nucleic acid encoding a VH domain or portion thereof and the polynucleotide comprising a nucleic acid encoding a VL domain or portion thereof can reside in a single vector, or can be on separate vectors.
  • the disclosure provides one or more vectors comprising the polynucleotides described above.
  • the disclosure provides a composition, e.g., a pharmaceutical composition, comprising a polynucleotide or vector as described above, optionally further comprising one or more carriers, diluents, excipients, or other additives.
  • a composition e.g., a pharmaceutical composition, comprising a polynucleotide or vector as described above, optionally further comprising one or more carriers, diluents, excipients, or other additives.
  • the disclosure further provides a host cell comprising a polynucleotide or vector as described herewith, wherein the host cell can, in some instances, express a binding molecule that specifically binds to Notch4.
  • a host cell can be utilized in a method of making a Notch4-binding molecule, where the method includes (a) culturing the host cell and (b) isolating the binding molecule from the host cell or from the culture medium, if the binding molecule is secreted by the host cell.
  • a nucleotide sequence encoding a Notch4-binding molecule can be constructed by chemical synthesis using an oligonucleotide synthesizer.
  • Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and 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 an isolated polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. Further, a nucleotide oligomer containing a nucleotide sequence coding for the particular isolated 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' or 3' overhangs for
  • 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 protein in a desired host. Proper assembly can be confirmed, e.g., by nucleotide sequencing, restriction mapping, and/or expression of a biologically active polypeptide in a suitable host. In order to obtain high expression levels of a transfected gene in a host, the gene can be operatively linked to or associated with 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 Notch4-binding molecules.
  • Recombinant expression vectors are replicable DNA constructs that have synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of a Notch4-binding molecule, operatively linked to suitable
  • a transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters 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, as described in detail below.
  • Such 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 additionally 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.
  • the protein 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 pCR 1, pBR322, pMB9 and their derivatives, wider host range plasmids, such as M13, and filamentous single- stranded DNA phages.
  • Suitable host cells for expression of a Notch4-binding molecule 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 could also be employed. Additional information regarding methods of protein production, including antibody production, can be found in the art.
  • Suitable mammalian host cell lines include 293 cells (e.g., HEK-293, HEK-293T, AD293), the COS-7 lines of monkey kidney cells, and other cell lines including, for example, L cells, C 127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines, and cell lines available from international depository agencies.
  • 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.
  • 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 in the art.
  • Notch4-binding molecules produced by a transformed host can be purified according to any suitable method.
  • standard 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 hexahistidine, 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 also be physically characterized using such techniques as proteolysis, nuclear magnetic resonance and x-ray crystallography.
  • supernatants from systems that 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
  • the concentrate can be applied to a suitable purification matrix.
  • an anion exchange resin can be 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 can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups.
  • RP-HPLC reversed-phase high performance liquid chromatography
  • a recombinant Notch4-binding molecule produced in bacterial culture can be 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. High performance liquid chromatography (HPLC) can be 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.
  • Notch4 activation or signaling e.g., cancer.
  • the following discussion refers to diagnostic methods and methods of treatment with a Notch4-binding molecule that is capable of specifically binding Notch4 and antagonizing Notch4 activity.
  • treatment or prevention includes the application or
  • the composition is preferably a pharmaceutical composition.
  • Notch4-binding molecules provided herein are useful for the treatment of and/or prevention of recurrence of cancer.
  • cancers that may be treated or the recurrence of which may be prevented using the Notch4-binding molecules described herewith can include breast cancer, ovarian cancer, prostate cancer, kidney cancer, thyroid cancer, cancer of the salivary gland, pancreatic cancer, hepatocellular cancer, colorectal cancer, melanoma, and lung cancer.
  • No tch4 -binding molecules described herewith are also useful for inhibiting or killing CSCs associated with various cancers, including breast cancer, ovarian cancer, prostate cancer, kidney cancer, thyroid cancer, cancer of the salivary gland, colorectal cancer, melanoma, and lung cancer, particularly non-small cell lung carcinoma.
  • Clinical response to administration of a Notch4-binding molecule can be assessed using screening techniques such as magnetic resonance imaging (MRI), x-radiographic imaging, computed tomographic (CT) scan, flow cytometry or fluorescence-activated cell sorter (FACS) analysis, histology, gross pathology, and blood chemistry, including but not limited to changes detectable by ELISA, ELISPOT, RIA, chromatography, and the like. Further, the subject undergoing therapy with the Notch4-binding molecule can experience improvement in the symptoms associated with the disease or disorder.
  • screening techniques such as magnetic resonance imaging (MRI), x-radiographic imaging, computed tomographic (CT) scan, flow cytometry or fluorescence-activated cell sorter (FACS) analysis, histology, gross pathology, and blood chemistry, including but not limited to changes detectable by ELISA, ELISPOT, RIA, chromatography, and the like.
  • the route of administration of the Notch4-binding molecule can be, for example, oral, parenteral, by inhalation, or topical.
  • parenteral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, and vaginal administration.
  • Oral dosage forms include, e.g., capsules, tablets, aqueous suspensions, and solutions.
  • Nasal aerosol or inhalation dosage forms can be prepared, for example, as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other conventional solubilizing or dispersing agents.
  • a suitable pharmaceutical composition can comprise a buffer (e.g. acetate, phosphate or citrate buffer), optionally a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc.
  • a buffer e.g. acetate, phosphate or citrate buffer
  • a surfactant e.g. polysorbate
  • a stabilizer agent e.g. human albumin
  • the form and character of the pharmaceutically acceptable carrier or diluent can be dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.
  • a cocktail comprising one or more species of Notch4-binding molecules, e.g., anti-Notch4 antibodies, or antigen-binding fragments, variants, or derivatives thereof, can also be used.
  • Notch4-binding molecules can be delivered directly to the site of the adverse cellular population, thereby increasing the exposure of the diseased tissue to the therapeutic agent.
  • the administration is directly to the airway, e.g., by inhalation or intranasal administration.
  • Notch4-binding molecules can be administered in a
  • the disclosed binding molecules can be formulated so as to facilitate administration and promote stability of the active agent.
  • compositions can comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like.
  • a "therapeutically effective amount" of a Notch4-binding molecule means an amount sufficient to achieve a benefit, e.g., to ameliorate symptoms of a disease or condition or to detect a substance or a cell.
  • This disclosure also provides for the use of a Notch4-binding molecule as described herein to treat or prevent recurrence of cancer, such as breast cancer, ovarian cancer, prostate cancer, kidney cancer, thyroid cancer, cancer of the salivary gland, pancreatic cancer, hepatocellular cancer, colorectal cancer, melanoma, and lung cancer.
  • a Notch4-binding molecule for inhibiting or killing CSCs associated with various cancers, including breast cancer, ovarian cancer, pancreatic cancer,
  • hepatocellular cancer prostate cancer kidney cancer, thyroid cancer, cancer of the salivary gland, colorectal cancer, melanoma, and lung cancer.
  • This disclosure also provides for the use of a Notch4-binding molecule as described herein in the manufacture of a medicament for treating or preventing recurrence of breast cancer, ovarian cancer, pancreatic cancer, hepatocellular cancer, prostate cancer, kidney cancer, thyroid cancer, cancer of the salivary gland, colorectal cancer, melanoma, and lung cancer.
  • This disclosure additionally provides for the use of a Notch4-binding molecule as described herein in the manufacture of a medicament for inhibiting or killing CSCs associated with various cancers, including breast cancer, ovarian cancer, pancreatic cancer, hepatocellular cancer, prostate cancer, kidney cancer, thyroid cancer, cancer of the salivary gland, colorectal cancer, melanoma, and lung cancer.
  • This disclosure also provides a Notch4- binding molecule as described herein for treating or preventing recurrence of breast cancer, ovarian cancer, pancreatic cancer, hepatocellular cancer, prostate cancer, kidney cancer, thyroid cancer, cancer of the salivary gland, colorectal cancer, melanoma, and lung cancer.
  • This disclosure additionally provides a Notch4-binding molecule as described herein for inhibiting or killing CSCs associated with various cancers, including breast cancer, ovarian cancer, pancreatic cancer, hepatocellular cancer, prostate cancer, kidney cancer, thyroid cancer, cancer of the salivary gland, colorectal cancer, melanoma, and lung cancer.
  • Notch4-binding molecules provided herewith can be used for diagnosis of Notch4- mediated diseases, and/or for diagnostic monitoring as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Such methods typically involve assaying the expression level of Notch4.
  • assaying the expression level of Notch4 is intended to mean qualitatively or quantitatively measuring or estimating the level of Notch4 in a first biological sample either directly (e.g., by determining or estimating absolute protein level) or relatively (e.g., by comparing to the disease associated polypeptide level in a second biological sample).
  • the Notch4 expression level in the first biological sample can be measured or estimated and compared to a standard Notch4 level, the standard being taken from a second biological sample obtained from an individual not having the disorder, or being determined by averaging levels from a population of individuals not having the disorder.
  • a standard Notch4 level the standard being taken from a second biological sample obtained from an individual not having the disorder, or being determined by averaging levels from a population of individuals not having the disorder.
  • an increase in the protein level of the test sample compared to the standard sample is indicative of a disease or disorder treatable by a No tch4 -binding molecule described herewith.
  • the "standard" Notch4 level can be used repeatedly as a standard for comparison.
  • biological sample any biological sample obtained from an
  • tissue biopsies and body fluids from mammals are known in the art.
  • Notch4-binding molecules described herewith can be used to assay Notch4
  • Immunoassays that can be used include but are not limited to competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), ELISPOT, "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and immunoelectron microscopy, to name some examples.
  • Such assays are routine and well known in the art. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • Detection of Notch4 can be facilitated by coupling the binding molecule to a
  • detectable substance or label examples include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.
  • An example of a luminescent material is luminol.
  • bioluminescent materials include luciferase, luciferin, and aequorin.
  • suitable radioactive material include 125 I, 1 31 I, 35 S, or 3 H.
  • In situ detection can be accomplished by removing a histological specimen, for
  • a blood sample from a patient, and applying thereto a labeled Notch4-binding molecule, applied by overlaying the labeled Notch4-binding molecule onto a biological sample.
  • a labeled Notch4-binding molecule applied by overlaying the labeled Notch4-binding molecule onto a biological sample.
  • Kits comprising Notch4-binding Molecules
  • kits that comprise a Notch4-binding molecule, which can be used to perform the methods described herein.
  • a kit comprises at least one purified Notch4-binding molecule in one or more containers.
  • the kit contains all of the components necessary and/or sufficient to perform a detection assay, including all controls, directions for performing assays, and any necessary software for analysis and presentation of results.
  • the disclosed Notch4-binding molecules can be readily incorporated into any of the established kit formats that are well known in the art.
  • Embodiment 1 - A Notch4 binding molecule or antigen binding portion thereof comprising one or more of: an immunoglobulin variable heavy chain complementarity determining region 1 (HCDRl) having the amino acid sequence set forth in SEQ ID NO: 93; an immunoglobulin variable heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 94; an immunoglobulin variable heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 95; an immunoglobulin variable light chain
  • HCDRl immunoglobulin variable heavy chain complementarity determining region 1
  • HCDR2 immunoglobulin variable heavy chain complementarity determining region 2
  • HCDR3 an immunoglobulin variable heavy chain complementarity determining region 3
  • LCDR1 complementarity determining region 1
  • LCDR2 immunoglobulin variable light chain complementarity determining region 2
  • LCDR3 immunoglobulin variable light chain complementarity determining region 3
  • Embodiment 2 - A Notch4 binding molecule or antigen binding portion thereof comprising: (i) an immunoglobulin variable heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 99; an immunoglobulin variable heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 100; and an immunoglobulin variable heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 101; and/or (ii) an immunoglobulin variable light chain complementarity determining region 1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 102; an immunoglobulin variable light chain complementarity determining region 2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 103; and an immunoglobulin variable light chain complementarity determining region 3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 104.
  • HCDR1 immunoglobulin variable heavy
  • Embodiment 3 A Notch4 binding molecule or antigen binding portion thereof comprising (i) an immunoglobulin variable heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 105; an
  • immunoglobulin variable heavy chain complementarity determining region 2 having the amino acid sequence set forth in SEQ ID NO: 106
  • an immunoglobulin variable heavy chain complementarity determining region 3 having the amino acid sequence set forth in SEQ ID NO: 107
  • an immunoglobulin variable light chain complementarity determining region 1 having the amino acid sequence set forth in SEQ ID NO: 108
  • an immunoglobulin variable light chain complementarity determining region 2 having the amino acid sequence set forth in SEQ ID NO: 109
  • an immunoglobulin variable light chain complementarity determining region 3 having the amino acid sequence set forth in SEQ ID NO: 1 lO.Embodiment 4 -
  • Embodiment 5 -A Notch4 binding molecule or antigen binding portion thereof that specifically binds to the same epitope of human Notch4 as an antibody comprising a heavy chain variable domain (VH) having an amino acid sequence selected from SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 21; SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO:27; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 33; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 39; SEQ ID NO: 41; SEQ ID NO: 43; SEQ ID NO: 45; SEQ ID NO: 47; SEQ ID NO: 49; SEQ ID NO: 51; SEQ ID NO: 53; SEQ ID NO: 55; SEQ ID NO: 57; SEQ ID NO: 59; SEQ ID NO: 61; SEQ ID NO: 63; SEQ ID NO: 65; SEQ ID
  • Embodiment 6 A Notch4 binding molecule or antigen binding portion thereof that competes or cross-competes with the binding molecule of any preceding embodiment.
  • Embodiment 7 The Notch4 binding molecule or portion thereof of any preceding embodiment, which is selected from a murine antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a bi-specific antibody, a multi- specific antibody, and an antigen-binding fragment thereof.
  • Embodiment 8 The Notch4 binding molecule or portion thereof of any preceding embodiment, which is selected from an Fv, an Fab, an F(ab')2, an Fab', a dsFv fragment, a single chain Fv (scFV), an sc(Fv)2, a disulfide-linked (dsFv), a diabody, a triabody, a tetrabody, a minibody, or a single chain antibody.
  • scFV single chain Fv
  • dsFv disulfide-linked
  • Embodiment 9 The binding molecule or antigen binding portion thereof of any one preceding embodiment, comprising an immunoglobulin (Ig) heavy chain constant region.
  • Ig immunoglobulin
  • Embodiment 10 The binding molecule or antigen binding portion thereof of
  • Embodiment 11 The binding molecule or antigen binding portion thereof of
  • Embodiment 12 The binding molecule or antigen binding portion thereof of
  • Embodiment 13 The binding molecule or antigen binding portion thereof of any preceding embodiment, comprising an immunoglobulin light chain constant region.
  • Embodiment 14 The binding molecule or antigen binding portion thereof of any preceding embodiment, which specifically binds human Notch4 with an affinity
  • K D dissociation constant
  • Embodiment 15 The binding molecule or antigen binding fragment thereof of any preceding embodiment, which does not specifically bind to Notch 1.
  • Embodiment 16 The binding molecule or antigen binding fragment thereof of any one preceding embodiment, which is conjugated to an agent selected from the group consisting of an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, a polyethylene glycol (PEG), a toxin, and a combination of two or more of any said agents.
  • an agent selected from the group consisting of an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, a polyethylene glycol (PEG), a toxin, and a combination of two or more of any said agents.
  • PEG polyethylene glycol
  • Embodiment 17 - A composition comprising the binding molecule or antigen binding fragment thereof of any preceding embodiment and a carrier.
  • Embodiment 18 The composition of embodiment 17, where the composition is a diagnostic reagent.
  • Embodiment 19 A method for inhibiting or killing cancer stem cells (CSCs), the method comprising administering to the CSCs the binding molecule or antigen binding fragment thereof of any one of embodiments 1 to 16.
  • CSCs cancer stem cells
  • Embodiment 20 - A method of treating cancer in a subject, the method comprising administering to a subject in need of treatment an effective amount of the Notch4 binding molecule or fragment thereof of any one of embodiments 1 to 16 or the composition of embodiment 17.
  • Embodiment 21 - A method of preventing recurrence of cancer in a subject, the
  • Embodiment 22 The method of any one of embodiments 19 to 21, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, kidney cancer, thyroid cancer, cancer of the salivary gland, colorectal cancer, melanoma, and lung cancer.
  • Embodiment 23 The method of any one of embodiments 19 to 22, wherein the method comprises administering a second active agent.
  • Embodiment 24 - The method of embodiment 17, wherein the second active agent is a chemotherapeutic agent.
  • Embodiment 25 A method for detecting Notch4 in a sample, the method
  • Embodiment 26 An isolated nucleic acid molecule comprising a nucleotide
  • Embodiment 27 The nucleic acid molecule of embodiment 26 operably linked to a regulatory sequence.
  • Embodiment 28 - A vector comprising the nucleic acid molecule of embodiment 26 or embodiment 27.
  • Embodiment 29 - A host cell transformed with a nucleic acid molecule of
  • Embodiment 30 The host cell of embodiment 29, which is a mammalian host cell.
  • Embodiment 31 - A composition comprising the nucleic acid molecule of
  • embodiment 26 or embodiment 27 the vector of embodiment 28, or the host cell of embodiment 29 or embodiment 30.
  • Embodiment 32 - A method of making a binding molecule or antigen binding
  • the method comprising culturing the host cell of embodiment 29 or embodiment 30 under suitable conditions for producing the binding molecule.
  • Embodiment 33 The method of embodiment 32, further comprising isolating the binding molecule or antigen binding fragment thereof.
  • Embodiment 34 - A kit comprising the binding molecule or antigen binding fragment thereof of any one of embodiments 1 to 16 or the nucleic acid molecule of embodiment 26 or embodiment 27.
  • Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which describe in detail preparation of certain binding molecules of the present disclosure and methods for using binding molecules of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.
  • Biotinylated reagents were prepared using EZ-LinkTM Sulfo-NHS-LC-LC-Biotin (Cat. #21338, Thermo Fisher Scientific, Waltham, MA,) to contain 2-3 biotins per molecule, following the manufacturer's protocol.
  • capture phage ELISA to screen 94 single colonies from all panning outputs for binding to human Notch4-NRR, mouse Notch4-NRR, and irrelevant control antigens. Positive clones were sequenced.
  • Panning outputs with the highest diversity of antigen- specific and human/mouse cross-reactive clones were sub-cloned into screening vectors.
  • ScFv libraries were sub-cloned into a pSpliceV4 screening vector that allows for scFv-Fc expression in bacterial and mammalian cells, as described by Xiao X et al. ((2015) A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format. PloS one 10:e0140691).
  • the Fab library was sub-cloned into a pXP vector for bacterial expression of soluble Fab fragments (Dyax Corp., Burlington, MA).
  • HTRF time-resolved fluorescence
  • both antibodies were placed in the full overlap epitope bin. If the binding of the second antibody was reduced, antibodies were placed in a partial overlap bin. If both antibodies could bind simultaneously to Notch4-NRR, they were placed in the no overlap epitope bin. Pairwise testing of the anti-Notch4 antibodies demonstrated that they belong to seven distinct epitope bins. We characterized five antibodies, NOCH0004, NOCH0012, NOCH0075, NOCH0090, and NOCH0133, in greater detail. These antibodies are also referred interchangeably herein as N0004, N0012, N0075, N0090, and N133.
  • Table 4 shows the IC50 and the maximum inhibition seen in the luciferase reporter cell line when activated by plate-bound DLL4 protein and tested with antibodies NOCH0004, NOCH0012, NOCH0075, NOCH0090, and NOCH0133.
  • the data is graphed in FIG. 1, which shows the activity of the antibodies in comparison to an isotype-matched control, when stimulated with plate-bound recombinant human DLL4.
  • NOCH0075 the antibodies were able to inhibit the luciferase reporter >40%. None of the antibodies characterized showed activation of the reporter.
  • FIG. 4A the results obtained for the EZH2 gene are shown in FIG. 4A
  • FIG. 4B the results obtained for the BMI1 gene are shown in FIG. 4B.
  • the NOCH0090 antibody showed a trend toward inhibition of both the Notch pathway and the sternness genes.
  • the NOCH0090 antibody was selected for further optimization based on its ability to inhibit the Notch pathway as well as the sternness genes. Prior to optimization, we attempted to revert as many framework residues of NOCH0090 to the closest human germline sequences without impairing affinity. This was done to minimize the potential immunogenicity of the final antibody drug in humans. All framework residues of the VL and VH domains could be reverted to match the amino acid sequence of human germlines IGLV2-23, IGLJ3, IGHV3-30, and IGHJ3, without loss of binding or potency.
  • GLA germlined antibody
  • FIG. 6A shows a plot of the percent maximum inhibition for each of 5 GLA-P antibodies vs the log of the antibody concentration in nM.
  • These three antibodies were also tested for their ability to inhibit CSC sphere formation of T47D cells and the results are shown in FIG. 6B. As seen in the figure, both GLA-P3 and GLA-P4 were able to inhibit CSC sphere formation to a similar extent, while GLA-P2 inhibited CSC sphere formation to a lower extent.
  • GLA-B 1 showed a decreased IC50 (16.53 nM) compared to the GLA molecule (43.6 nM), while GLA-B3 and GLA-B4 did not show improvements but showed similar IC50s (49.8 nM and 55.79 nM, respectively).
  • GLA-B2, GLA-B 5 and GLA- B6 all had IC50s higher than GLA.
  • the antibodies with either similar IC50s (GLA-B3 and GLA-B4), or those that showed improvements (GLA-B 1) in the luciferase assay as compared with GLA were also tested for their ability to inhibit T47D CSC sphere formation. As seen in FIG. 7B, all three antibodies showed improved activity over GLA.
  • GLA-S antibodies with further improved binding and potency.
  • the GLA-S series mutants were tested in both the luciferase assay and in the T47D CSC sphere formation assay. The results from testing using the luciferase assay are shown in FIG. 8A, and the results from the CSC sphere formation assay are shown in FIG. 8B. These figures show that GLA-S3 and GLA-S4 showed the best activity in both assays.
  • Table 6 shows the binding properties of anti-Notch4 antibodies to human Notch4- NRR antigen, measured by an Octet instrument (Pall ForteBio LLC, Menlo Park, CA) using anti-human IgG-Fc capture biosensors.
  • the GLA-S4 antibody was further optimized by introducing mutations that removed hydrophobic patches and lowered the isoelectric point to create GLA-S4F antibodies.
  • FIG. 11A we tested GLA-S4F antibodies in the luciferase assay, and showed that the antibodies retained similar activity to GLA-S4.
  • FIG. 11B We also tested these clones to determine if they retained the ability to inhibit OVCAR4 spheres, and the results are shown in FIG. 11B. Both GLA-S4F18 and GLA-S4F19 showed similar activity to GLA-S4 in this last assay.
  • Table 7 shows binding properties of some anti-Notch4GLA-S antibodies to human and mouse Notch4-NRR antigen, as measured by direct ELISA.
  • Example 9 In vivo efficacy of GLA-S4F18 in PA-1 ovarian xenograft tumor model
  • the percentage of CSCs in the combination arm was reduced by approximately 4-fold compared to the control of isotype only.
  • SW-780 cells were grown to 80%
  • FIG. 13A and FIG. 13B demonstrate that Notch4 expression is increased on the surface of SW-780 cells following treatment with either cisplatin or doxil.
  • Example 11 Treatment with the combination of GLA-S4F18 with cisplatin delays tumor regrowth (relapse) in ovarian PDX models
  • Ovarian PDX models were established by implanting patient tumor pieces into NSG mice. Following growth in animals, the tumors were expanded into additional mice via trocar implantation. Once the tumors reached approximately 200 mm 3 , the mice were randomized and either left untreated or were treated with 5 mg/kg cisplatin once weekly for 3 doses along with either 10 mg/kg of the control antibody (IgGl isotype control) or GLA- S4F18 twice weekly until the end of study. Following treatment with cisplatin at day 21 the animals were monitored for tumor regrowth. A tumor reaching 500 mm 3 was considered a relapse event. The Kaplan-Meier plot shows the rate at which tumors relapse. OVA-001 was monitored for 150 days (FIG.
  • tumors were excised and plated for ex vivo CSC sphere formation. Briefly, tumors were minced to 2 mm 3 pieces with scalpel blades on ice. The pieces were then dissociated in DMEM/F12 containing 200 units/ML collagenase IV (Worthington Biocehmical Corporation, Lakewood NJ) with constant shaking at 37°C and trituration every 15 minutes.
  • DMEM/F12 200 units/ML collagenase IV (Worthington Biocehmical Corporation, Lakewood NJ) with constant shaking at 37°C and trituration every 15 minutes.
  • Results shown in FIG. 14A-14D demonstrate that the combination of cisplatin with GLA-S4F18 leads to a slower rate of tumor regrowth as compared to cisplatin alone. This slower rate of tumor regrowth is coincident with a decrease in the CSCs as measured by the ex vivo sphere assay.
  • Example 12 Reduction of CSCs present in tumors treated with GLA-S4F18 alone or in combination with abraxane
  • Notch4 expressing OVCAR-4 cells into female athymic nude mice (FIG. 15 A) or by passing tumor pieces into NSG mice using a trocar. Once tumors reached approximately 300 mm 3 they were treated with either 3, 10 or 30 mg/kg twice weekly for 3 doses for OVCAR 4 or with 10 mg/kg of antibodies with or without 30 mg/kg Abraxane twice weekly for a total of five doses. Tumors were then excised and processed to single cells. Briefly, tumors were minced to approximately 200 mm 2 . The pieces were then dissociated in DMEM/F12 containing 200 units/ML collagenase IV (Worthington Biochemical
  • Notch4/Notchl sub-domain swap approach for epitope mapping.
  • GLA-S4 does not bind to human Notch 1.
  • Sub-domains of Notch4-NRR LNR1, LNR2, LNR3, HD-N, HD-linker, and HD-C, were replaced with NotchlNRR corresponding sub-domains.
  • the nucleotide sequences of Notch4/Notchl NRR sub-domain swap constructs are shown below. Nucleotides written in lower case are from human Notch 1, all other nucleotide sequences including sequences preceding and following NRR domain (not shown) were human Notch4. >Notch4 NRR
  • amino acid sequences of the VH and VL of the anti-NOTCH4 antibodies tested follow. Included are sequences for the lead generation molecules, germlined leads, block mutants, parsimonious mutants, combination mutants, and optimized GLA-S4 clones.
  • VQLLES GGGLVQPGGS LRLS C A AS GFTFS S Y AMS W VRQ APGKGLEW VS AIS GS GG S T Y Y ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDRA V Y YC ARDRTRMD VWGRGTT VTVSS (set forth in SEQ ID NO: 17).
  • QVQLVES GGGVVQPGRS LRLSC AAS GFTFS S YGMHW VRQ APGEGLEWVATIPAS GDNT YYADSVEGRFTISRDNSENTLYLQMNSLRAEDTAVYYCAKGAYKGYYYYIWWDVWG QGTMVTVSS (set forth in SEQ ID NO: 87).
  • FIG. 17A and FIG. 17B show an alignment of the amino acid sequences of the heavy chain regions of the germlined leads, block mutants, parsimonious mutants, combination mutants, and optimized GLA-S4 clones.
  • FIG. 18A and FIG. 18B show an alignment of the amino acid sequences of the light chain regions of the same antibodies as in
  • FIG. 17A and FIG. 17B The amino acid sequences of framework 1 (FW1); CDR1; FW2; and CDR2 are shown in FIG. 17A and FIG. 18A.
  • the amino acid sequences of FW3; CDR3 and FW4 are shown in FIG. 17B and FIG. 18B.
  • the CDR sequences are boxed, and the consensus sequences of the CDRs follow:
  • Xi is M or Y.
  • Xi is S or P
  • X2 is G; V; or A;
  • X3 is K or E.
  • Xi is V or Y
  • X 2 is Y; V; R; I; or L;
  • X 3 is Y or W
  • X 4 is M; W; or L;
  • X 5 is D; Y; or F; X 6 is V or P.
  • Xi is S or K
  • Xi is E or L
  • X 2 is G or V
  • Xi is Y or F
  • X 2 is T or R
  • X3 is R or L
  • X 4 is T or Q
  • >GLA-S4 HCDR1 SYGMH (set forth in SEQ ID NO: 99), >GLA-S4 HCDR2: TISVSGDNTYYADSVKG (set forth in SEQ ID NO: 100), >GLA-S4 HCDR3: GAYKGYYYYIWWDV (set forth in SEQ ID NO: 101) >GLA-S4 LCDR1: TGTSSDVGGYNYVS (set forth in SEQ ID NO: 102), >GLA-S4 LCDR2: LGSKRPS (set forth in SEQ ID NO: 103), >GLA-S4 LCDR3: SSYTRLSQRV (set forth in SEQ ID NO: 104) >GLA-S4F18 HCDR1: SYGMH (set forth in SEQ ID NO: 105) >GLA-S4F18 HCDR2: TIPASGDNTYYADSVEG (set forth in SEQ ID NO: 106) >GLA-S4F18 HCDR3: GAYKGYY

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Abstract

L'invention concerne des molécules de liaison à Notch4 et des fragments de liaison à l'antigène de celles-ci, par exemple, des anticorps monoclonaux capables d'inhiber l'activité de Notch4, et des méthodes d'utilisation des molécules de liaison Notch, par exemple, dans le traitement ou la prévention de la récurrence du cancer, et dans l'inhibition ou la destruction de cellules souches cancéreuses (CSC).
PCT/US2017/047745 2016-08-22 2017-08-21 Molécules de liaison spécifiques pour notch4 et leurs utilisations Ceased WO2018039107A1 (fr)

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