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WO2018039107A1 - Binding molecules specific for notch4 and uses thereof - Google Patents

Binding molecules specific for notch4 and uses thereof 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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French (fr)
Inventor
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

The disclosure provides Notch4-binding molecules and antigen binding fragments thereof, for example, monoclonal antibodies capable of inhibiting Notch4 activity, and methods of using the Notch binding molecules, for example, in treating or preventing recurrence of cancer, and in inhibiting or killing cancer stem cells (CSCs).

Description

BINDING MOLECULES SPECIFIC FOR NOTCH4 AND USES THEREOF
BACKGROUND
[0001] Cancer stem cells (CSCs), as their name implies, 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. Several lines of evidence now indicate that CSCs are likely responsible for drug resistance, metastasis, and relapse of cancer, particularly in instances with minimal residual disease. In fact, 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. Sci. USA 106: 13820-13825). In a similar study, patients with 5q deletion MDS were also found to have residual populations of malignant stem cells in their bone marrow, despite having entered clinical and cytogenetic remission (Tehranchi R et al. (2010) Persistent malignant stem cells in del(5q) myelodysplasia in remission. N. Engl. J. Med. 363: 1025-1037). In many clinical indications, it is now believed that despite initial therapeutic response, the retention of a distinct subset of resistant cancer cells can lead to relapse and potentially metastasis.
[0002] 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.
[0003] 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)
Targeting Notch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update. Nat. Rev. Clin. One. 12: 445-464). In particular, the Notch pathway has been implicated in the maintenance of a CSC phenotype in many different malignancies. Four Notch receptors (Notchl-4) and five Notch ligands (DLL1, DLL3, DLL4, JAG1, and JAG2) comprise this family. Upon engagement of a Notch receptor by a ligand, the Notch receptor is
proteolytically cleaved by ADAM proteinases and gamma secretase, leading to the release of the intracellular domain, which translocates to the nucleus and activates down-stream signaling. 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. Oncogene 29:2672-2680) and lung cancer (Hassan KA et al. (2013) 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.
[0004] The availability of additional 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.
SUMMARY OF THE INVENTION
[0005] Some of the main aspects of the present invention are summarized below. Additional aspects are described in the Detailed Description of the Invention, Examples, Drawings, and Claims sections of this disclosure. The description in each section of this disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention. [0006] The disclosure provides 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.
[0007] In one aspect, is provided a Notch4 binding molecule or antigen binding portion
thereof comprising one or more of 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 LCDR1 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.
[0008] 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: 65; SEQ ID NO: 67; SEQ ID NO: 69; SEQ ID NO: 71; SEQ ID NO: 73; SEQ ID NO: 75; SEQ ID NO: 77; SEQ ID NO: 79; SEQ ID NO: 81; SEQ ID NO: 83; SEQ ID NO: 85; SEQ ID NO: 87; SEQ ID NO: 89; and SEQ ID NO: 91; and/or a light chain variable domain (VL) having an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO: 30; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 36; SEQ ID NO: 38; SEQ ID NO: 40; SEQ ID NO: 42; SEQ ID NO: 44; SEQ ID NO: 46; SEQ ID NO: 48; SEQ ID NO: 50; SEQ ID NO: 52; SEQ ID NO: 54; SEQ ID NO: 56; SEQ ID NO: 58; SEQ ID NO: 60; SEQ ID NO: 62; SEQ ID NO: 64; SEQ ID NO: 66; SEQ ID NO: 68; SEQ ID NO: 70; SEQ ID NO: 72; SEQ ID NO: 74; SEQ ID NO: 76; SEQ ID NO: 78; SEQ ID NO: 80; SEQ ID NO: 82; SEQ ID NO: 84; SEQ ID NO: 86; SEQ ID NO: 88; SEQ ID NO: 90; and SEQ ID NO: 92. In some aspects, the binding molecule or antigen binding portion thereof specifically binds to human Notch4, and is cross-reactive with cynomologous monkey, and with mouse.
[0009] In some aspects, 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; SEQ ID NO: 69; SEQ ID NO: 71; SEQ ID NO: 73; SEQ ID NO: 75; SEQ ID NO: 77; SEQ ID NO: 79; SEQ ID NO: 81; SEQ ID NO: 83; SEQ ID NO: 85; SEQ ID NO: 87; SEQ ID NO: 89; and SEQ ID NO: 91; and a light chain variable domain (VL) having an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO: 30; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 36; SEQ ID NO: 38; SEQ ID NO: 40; SEQ ID NO: 42; SEQ ID NO: 44; SEQ ID NO: 46; SEQ ID NO: 48; SEQ ID NO: 50; SEQ ID NO: 52; SEQ ID NO: 54; SEQ ID NO: 56; SEQ ID NO: 58; SEQ ID NO: 60; SEQ ID NO: 62; SEQ ID NO: 64; SEQ ID NO: 66; SEQ ID NO: 68; SEQ ID NO: 70; SEQ ID NO: 72; SEQ ID NO: 74; SEQ ID NO: 76; SEQ ID NO: 78; SEQ ID NO: 80; SEQ ID NO: 82; SEQ ID NO: 84; SEQ ID NO: 86; SEQ ID NO: 88; SEQ ID NO: 90; and SEQ ID NO: 92.
[0010] In some aspects, 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.
[0011] In some aspects, 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.
[0012] In some aspects, the Notch4-binding molecule 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.
[0013] 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. In some aspects, the binding molecule is an IgGl triple mutant. In some aspects, the binding molecule is a YTE mutant. The Notch4-binding molecule or antigen binding fragment thereof can comprise an immunoglobulin light chain constant region.
[0014] In an aspect, the Notch4-binding molecule or antigen binding fragment thereof
specifically binds human Notch4 with an affinity characterized by a dissociation constant (KD) of about 0.2 nM, as measured by an Octet assay.
[0015] In some aspects, the Notch4-binding molecule or antigen binding fragment thereof does not specifically bind to Notch 1, Notch2, or Notch3.
[0016] In some aspects, 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.
[0017] An aspect is a composition comprising a carrier and a Notch-4 binding molecule or antigen binding fragment thereof as described herewith. In some aspects, the composition comprising a carrier and a Notch-4 binding molecule or antigen binding fragment thereof as described herewith is a therapeutic composition. In some aspects, the composition
comprising a carrier and a Notch-4 binding molecule or antigen binding fragment thereof as described herewith is a diagnostic reagent.
[0018] It is provided a method for inhibiting or killing CSCs, the method comprising
administering to the CSCs 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. Similarly, it is provided the use of a No tch4 -binding molecule or antigen binding fragment thereof as described herewith for the manufacture of a medicament for treating or preventing recurrence of cancer. In some aspects, 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.
[0019] In some instances, the methods provided herewith comprise administering a second active agent. In certain aspects, the second active agent is a chemo therapeutic agent.
Examples of chemotherapeutic agents include carboplatin, cisplatin, doxil, and abraxane.
[0020] 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.
[0021] Also within the scope of the disclosure are: an isolated nucleic acid molecule
comprising a nucleotide sequence encoding a Notch4-binding molecule or antigen binding fragment thereof as described herewith, optionally operably linked to a regulatory sequence; a vector comprising the nucleic acid molecule; and a host cell transformed with the nucleic acid molecule or the vector. In some embodiments, the host cell is a mammalian host cell.
[0022] Further provided is a method of making a binding molecule or antigen binding
fragment thereof that specifically binds Notch4, 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.
[0023] Also included is a composition comprising a nucleic acid encoding the binding
molecule or antigen binding fragment thereof that specifically binds Notch4, 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.
[0024] In an additional aspect, is provided a kit 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] 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.
[0026] 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,
NOCH0075, NOCH0090, and NOCH0133.
[0027] 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).
[0028] 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.
[0029] FIG. 5 shows the relative binding of anti-Notch4 antibodies NOCH0090 and GLA to a cell line overexpressing human Notch4.
[0030] FIG. 6A shows a plot of the % maximum inhibition for each of the five GLA-P
antibodies (GLA-P 1; GLA-P2; GLA-P3; GLA-P4; GLA-P5) in the luciferase reporter assay.
FIG. 6B shows the % inhibition of CSC sphere formation of T47D cells caused by GLA-P2,
GLA-P3, and GLA-P4 antibodies.
[0031] FIG. 7A shows the activity of GLA-B 1, GLA-B2, GLA-B3, GLA-B4, GLA-B5,
GLA-B6 antibodies in the luciferase reporter assay. FIG. 7B shows the ability of GLA antibody and GLA-B l, GLA-B3, and GLA-B4 antibodies to inhibit CSC sphere formation of
T47D cells. [0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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).
[0038] FIG. 14A-14D contain a series of graphs illustrating tumor relapse (tumors >500 mm3) 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). [0039] FIG. 15A and 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.
[0040] 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:
binding to N4/N1-LNR3; 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.
[0041] 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.
[0042] 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.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The present invention provides molecules that bind to Notch4. In some embodiments, 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. Related
polynucleotides, compositions comprising the anti-Notch4-binding molecules, and methods of making the anti-Notch4-binding molecules are also provided. Methods of using the novel anti-Notch4 antibodies, such as diagnostic methods and methods of treating cancer and/or inhibiting cancer stem cells are further provided. [0044] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Ausubel et al. eds. (2015) Current Protocols in Molecular Biology (John Wiley and Sons); Greenfield, ed. (2013) Antibodies: A Laboratory Manual (2nd ed., Cold Spring Harbor Press); Green and
Sambrook, eds. (2012), Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press); Krebs et ah, eds. (2012) Lewin's Genes XI (11th ed., Jones & Bartlett Learning); Freshney (2010) Culture Of Animal Cells (6th ed., Wiley).
[0045] In order that the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related. For example, The Dictionary of Cell and Molecular Biology (5th ed. J.M. Lackie ed., 2013), the Oxford Dictionary of Biochemistry and Molecular Biology (2d ed. R. Cammack et al. eds., 2008), and The Concise Dictionary of Biomedicine and Molecular Biology (2d ed. P-S. Juo, 2002) can provide one of skill with general definitions of some terms used herein.
I. Definitions
[0046] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents, unless the context clearly dictates otherwise. The terms "a" (or "an") as well as the terms "one or more" and "at least one" can be used
interchangeably.
[0047] Furthermore, "and/or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).
[0048] Units, prefixes, and symbols are denoted in their Systeme International d'Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation, and nucleic acid sequences are written left to right in 5' to 3' orientation. The headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be completed by reference to the specification as a whole.
Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.
[0049] Wherever embodiments are described with the language "comprising," otherwise analogous embodiments described in terms of "consisting of and/or "consisting essentially of are included.
[0050] 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.
[0051] "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).
[0052] The term "antibody" refers to an immunoglobulin molecule that recognizes and
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. The terms "antibody" or "immunoglobulin" are used interchangeably herein.
[0053] 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.
[0054] 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.
[0055] 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. 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.
[0056] There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (Kabat et ah, (1991) Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD); and (2) an approach based on crystallographic studies of antigen- antibody complexes (Al-lazikani et ah, (1997) J. Mol. Biol. 273:927-948). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.
[0057] The amino acid position numbering as in Kabat, 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
corresponding to a shortening of, or insertion into, a FW or CDR of the variable domain. For example, 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. [0058] 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. 196:901-917 (1987)). 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.
TABLE 1
Figure imgf000014_0001
1 Kabat Numbering
2Chothia Numbering
[0059] IMGT (ImMunoGeneTics) also provides a numbering system for the immunoglobulin variable regions, including the CDRs. 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. According to the IMGT numbering schema 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, and VL-CDR3 is at positions 89 to 97.
[0060] As used throughout this specification, the 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.
[0061] As used herein, the term "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.
[0062] A "monoclonal antibody" (mAb) 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. Furthermore, "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.
[0063] The term "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. Typically, 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. In some instances, the Fv framework region (FW) 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.
[0064] Humanized antibodies can be further modified by the substitution of additional
residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability. In general, 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.
[0065] The term "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.
[0066] The term "chimeric antibodies" refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, 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.
[0067] The term "antigen-binding fragment" refers to a portion of an intact antibody
comprising the complementarity determining variable regions of the antibody. Fragments of a full-length antibody can be an antigen-binding fragment of an antibody. Examples of 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.
[0068] A "blocking" antibody or an "antagonist" antibody is one that inhibits or reduces
biological activity of the antigen it binds, such as Notch4. In certain aspects, 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%. [0069] The term "germlining" means that amino acids at specific positions in an antibody are mutated back to those in the germ line.
[0070] 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.
[0071] The terms "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.
[0072] "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). Unless indicated otherwise, as used herein, "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.
[0073] 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
immunosorbent assay (ELISA), radioimmunoassay (RIA), or kinetics {e.g., KINEXA® or BIACORE™ 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). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD or Kd, Kon, Koff) are made with standardized solutions of antibody and antigen, and a standardized buffer, as known in the art.
[0074] "Potency" of a binding molecule or antibody is normally expressed as an IC50 value, in nM or pM, unless otherwise stated. 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.
[0075] The fold improvement in potency for the antibodies or polypeptides described
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.
[0076] The terms "inhibit," "block," and "suppress" are used interchangeably with respect to the effect of one molecule on another, and refer to any statistically significant decrease in biological activity, including full blocking of the activity. For example, "inhibition" can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in biological activity. Accordingly, when 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
recombinantly produced by introducing a nucleic acid encoding Notch4 into a host cell. In one embodiment, 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. [0077] The term "inhibit," as used herein with respect to inhibition of CSCs by a binding molecule of the invention, refers to a statistically significant reduction in downstream gene expression or expression of sternness genes, in CSC frequency in a tumor, in the rate of tumor growth or in delaying of regrowth of the tumor following treatment, or in the amount and/or rate of CSC sphere formation in an in vitro assay, compared with untreated CSCs. Preferably, 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.
[0078] By "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.
[0079] The term "pharmaceutical 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. Such composition can be sterile and can comprise a
pharmaceutically acceptable carrier, such as physiological saline. 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.
[0080] An "effective amount" of a binding molecule as disclosed herein is an amount
sufficient to carry out a specifically stated purpose. An "effective amount" can be determined empirically and in a routine manner, in relation to the stated purpose.
[0081] Binding molecules of the invention can be naked or conjugated to other molecules such as toxins, labels, etc. The term "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. [0082] 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. In certain embodiments, 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.
[0083] "Prevent" or "prevention" refer to prophylactic or preventative measures that prevent and/or slow the development or recurrence of a targeted pathologic condition or disorder. Thus, 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. In certain embodiments, 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. In some embodiments, 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.
[0084] 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. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. In certain embodiments, the polypeptides can occur as single chains or associated chains.
[0085] 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). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. In certain embodiments, 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.
[0086] A "polynucleotide," as used herein can include one or more "nucleic acids," or
"nucleic acid molecules," and 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.
[0087] The term "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.
Examples of 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.
[0088] 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. In some embodiments, a polypeptide, antibody, binding molecule, polynucleotide, vector, or cell that is isolated is substantially pure. When used herein, the term "substantially pure" refers to purity of greater than 75%, preferably greater than 80% or 90%, and most preferably greater than 95%. [0089] The terms "identical" or percent "identity" in the context of two or more 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.
[0090] One such non-limiting example of a 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. In certain embodiments, 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). In certain alternative embodiments, 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). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller. For example, 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
embodiments, the default parameters of the alignment software are used.
[0091] In certain embodiments, 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.
II. Notch4-Binding Molecules
[0092] Provided herewith are 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), BIACORE™ (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. In certain embodiments, binding molecule that binds to Notch4 has a dissociation constant (KD) of <1 μΜ, <100 nM, <10 nM, <1 nM, <0.1 nM, <10 pM, <1 pM, or <0.1 pM. In particular embodiments, the binding molecule binds to the negative regulatory region of Notch4 (Notch4-NRR).
[0093] 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 Notch4-binding molecules set forth herein. These can contain, for example, conservative amino acid substitutions.
[0094] In certain aspects, this disclosure provides a Notch4-binding molecule that can
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. The term "epitope" refers to a target protein determinant capable of binding to a binding molecule described herewith. 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. Such 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.
[0095] Accordingly, in one embodiment, provided herewith are 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. The ability of 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. In one embodiment, 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. The term "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.
[0096] In some embodiments, 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. In some embodiments, 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' )35 a tetrabody, a triabody, a diabody, a single- domain antibody, DVD-Ig, Fcab, mAb2, a (scFv)2, or a scFv-Fc.
[0097] A Notch4-binding molecule provided herein can include, in addition to a VH and a VL, a heavy chain constant region or fragment thereof. In certain aspects 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.
[0098] In certain embodiments, binding molecules described herewith are produced to
comprise 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). Accordingly, in certain embodiments 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. In particular embodiments, the Notch4- binding molecule has no ADCC activity and/or no CDC activity. In certain aspects, the Notch4-binding molecule does not bind to an Fc receptor and/or complement factors. In certain aspects, 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. In some embodiments, the binding molecule is of the IgGl subtype, and optionally comprises the TM format (L234F/L235E/P331S), as disclosed supra in the Definitions section.
[0099] In certain aspects, 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. For example, 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. In certain aspects 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), Serine (S), Isoleucine (I), Proline (P), or Glutamine (Q), or a substitution of the amino acid at Kabat position 434 with Tryptophan (W), Methionine (M), Serine (S), Histidine (H), Phenylalanine (F), or Tyrosine. More specifically, 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). In some embodiments, the binding molecule is of the IgGl subtype, and optionally comprises the triple mutant YTE, as disclosed supra in the Definitions section.
[00100] A Notch4-binding molecule provided herein can include a light chain constant region or fragment thereof. In certain aspects 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.
[00101] Notch4-binding molecules provided herein can have beneficial properties. For example, 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.
[00102] In certain aspects, the binding molecules provided herein can bind to Notch4 with a binding affinity characterized by a dissociation constant (KD) of about 100 pM to about 0.5 nM as measured by a Biacore™ assay or on a Kinetic Exclusion Assay (KinExA) 3000 platform or on an Octet® instrument. [00103] In certain aspects, 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 KD 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 Biacore™ or KinExA® or Octet®. In a particular aspect, the anti-Notch4 antibody GLA-S4 can bind to human Notch4-NRR with a KD of about 0.2 nM, as measured by an Octet® assay.
[00104] In another embodiment, a Notch4-binding molecule described herewith binds to
Notch4 or an antigenic fragment thereof with a K0ff of less than lxlO-3 s-1, or less than 2xl0~3 s-1. In other embodiments, a Notch4-binding molecule binds to Notch4 or an antigenic fragment thereof with a K0ff 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 5xl0~9 s-1, or less than 10~10 s-1 as measured, e.g., by Biacore™ or KinExA® or Octet®. In a particular aspect, the anti-Notch4 antibody GLA-S4 can bind to human Notch4-NRR with a K0ff of about 1.05 x 10"4 s"1, as measured by an Octet® assay.
[00105] In another embodiment, a Notch4-binding molecule described herewith binds to
Notch4 or an antigenic fragment thereof with an association rate constant or Kon rate of at least 105 M"1 s"1, at least 5xl05 M"1 s"1, at least 106 M"1 s"1, at least 5xl06 M"1 s"1, at least 107 M"1 s"1, at least 5xl07 M"1 s"1, or at least 108 M"1 s"1, or at least 109 M"1 s"1 as measured, e.g., by Biacore™ or KinExA® or Octet®. In a particular aspect, the anti-Notch4 antibody GLA-S4 can bind to human Notch4-NRR with a Kon of about 39.6 x 104 M"1 s"1, as measured by an Octet® assay.
[00106] In some embodiments, 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. In a particular aspect, 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. [00107] 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.
[00108] The disclosure further provides a Notch4-binding molecule that is conjugated to a heterologous agent. In certain aspects, 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. Heteroconjugate anti-Notch4 antibodies are discussed in more detail elsewhere herein.
[00109] The term "binding molecule" includes antibodies and antigen-binding fragments thereof. In certain embodiments, 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. In certain embodiments, phage display technology can be used to identify and/or produce a Notch4-binding polypeptide. In certain embodiments, 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.
[00110] In certain aspects, 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. In certain aspects, the disclosure provides 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.
III. Preparation of Notch4-Binding Molecules
[00111] 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. 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.
[00112] Notch4-binding molecules can also be made using recombinant DNA methods
known in the art. In some instances, 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. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, binding molecules are generated by the host cells. Also, recombinant 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.
[00113] 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. In some embodiments, 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. In some embodiments, 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.
[00114] In certain embodiments, the Notch4-binding molecule is a human antibody or
antigen-binding fragment thereof. 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.
[00115] 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.
[00116] 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.
[00117] In some embodiments, 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. Such imported sequences can be used to reduce immunogenicity or reduce, enhance, or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic, as known in the art. In general, the 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.
[00118] Antibodies can also optionally be humanized, resurfaced, engineered, or human
antibodies engineered with retention of high affinity for Notch4 and other favorable biological properties. To achieve this goal, 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. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate binding molecule sequence, i.e., the analysis of residues that influence the ability of the candidate binding molecule to bind its target, such as Notch4. In this way, FW residues can be selected and combined from the consensus and import sequences so that the desired binding molecule characteristic, such as increased affinity for the target, is achieved.
[00119] Humanization, resurfacing, or engineering of anti-Notch4 antibodies or antigen- binding fragments thereof can be performed using any known method.
[00120] 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.
[00121] In certain embodiments the Notch4-binding molecule is anti-Notch4 antibody
fragment. Various techniques are known for the production of antibody fragments.
Traditionally, these fragments are derived via proteolytic digestion of intact antibodies. In certain embodiments, 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.
[00122] Techniques can be adapted for the production of single-chain antibodies specific to Notch4. In addition, 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.
[00123] In some aspects, 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.
[00124] In certain aspects, 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.
[00125] 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. It is contemplated that 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. For example, 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.
[00126] 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.
IV. Polynucleotides Encoding Notch4-Binding Molecules, Preparation and Expression Thereof
[00127] This disclosure provides polynucleotides comprising nucleic acid sequences that encode a Notch4-binding molecule, e.g., a polypeptide that specifically binds Notch4. For example, it is provided 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.
[00128] In certain embodiments, 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
polynucleotide which aids, for example, in expression and optionally, secretion, of a polypeptide from a host cell (e.g., a promoter or other regulatory sequence, a leader sequence that functions as a secretory sequence for controlling transport of a polypeptide from the cell). The 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.
[00129] The disclosure provides an isolated polynucleotide comprising a nucleic acid
encoding a 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. [00130] In certain embodiments 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. For example, 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.
[00131] Polynucleotide variants are also provided. Polynucleotide variants can contain
alterations in the coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, 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).
[00132] Included herewith are vectors comprising the polynucleotides described above.
Suitable vectors are described elsewhere herein, and are known to those of ordinary skill in the art. In some embodiments, 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.
Accordingly, the disclosure provides one or more vectors comprising the polynucleotides described above.
[00133] In certain aspects, 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.
[00134] 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. Such 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. [00135] In some embodiments 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
complementary assembly.
[00136] Once assembled (by synthesis, site-directed mutagenesis, or another method), 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.
[00137] In certain embodiments, 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
transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. 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. For example, DNA for a signal peptide (secretory leader) 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. Alternatively, where a recombinant protein is expressed without a leader or transport sequence, 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.
[00138] The choice of expression control sequence and expression vector will depend upon the choice of host. A wide variety of expression host/vector combinations can be employed. 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.
[00139] 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.
[00140] Various mammalian or insect cell culture systems can be advantageously employed to express recombinant Notch4-binding molecules. Expression of recombinant proteins in mammalian cells can be performed because such proteins are generally correctly folded, appropriately modified, and completely functional. Examples of 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. Baculovirus systems for production of heterologous proteins in insect cells are well known in the art.
[00141] Notch4-binding molecules produced by a transformed host can be purified according to any suitable method. Such 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.
[00142] For example, 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
concentration step, the concentrate can be applied to a suitable purification matrix.
Alternatively, 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. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a Notch4-binding molecule. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein. [00143] 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.
V. Treatment Methods Using Notch4-Binding Molecules
[00144] Methods are provided for the use of No tch4 -binding molecules to treat patients
having a disease or disorder associated with or characterized by 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.
[00145] In one embodiment, treatment or prevention includes the application or
administration of a Notch4-binding molecule or a composition comprising Notch4-binding molecule to a subject or patient, or application or administration of the Notch4-binding molecule to an isolated tissue or cell line from a subject or patient, where the subject or patient has a disease, a symptom of a disease, or a predisposition toward a disease. The composition is preferably a pharmaceutical composition.
[00146] Notch4-binding molecules provided herein are useful for the treatment of and/or prevention of recurrence of cancer. Examples of 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.
[00147] 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.
[00148] Methods of preparing and administering No tch4 -binding molecules to a subject in need thereof are well-known to or can be readily determined by those skilled in the art. The route of administration of the Notch4-binding molecule can be, for example, oral, parenteral, by inhalation, or topical. The term "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.
[00149] Usually, 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. 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. In other methods, 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. In one embodiment, the administration is directly to the airway, e.g., by inhalation or intranasal administration.
[00150] As discussed herein, Notch4-binding molecules can be administered in a
therapeutically effective amount for the in vivo treatment of Notch4-mediated diseases such as cancer. In this regard, it will be appreciated that the disclosed binding molecules can be formulated so as to facilitate administration and promote stability of the active agent.
Pharmaceutical compositions can comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like. For the purposes of the instant application, 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.
[00151] 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. This disclosure further provides for the use of 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.
[00152] 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.
VI. Assays and Diagnostics
[00153] 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. By "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. In some aspects, 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. As will be appreciated in the art, once the "standard" Notch4 level is known, it can be used repeatedly as a standard for comparison.
[00154] By "biological sample" is intended any biological sample obtained from an
individual, cell line, tissue culture, or other source of cells potentially expressing Notch4. Methods for obtaining tissue biopsies and body fluids from mammals are known in the art.
[00155] The Notch4-binding molecules described herewith can be used to assay Notch4
protein levels in a biological sample using classical immunohistological methods known to those of skill in the art. 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.
[00156] Detection of Notch4 can be facilitated by coupling the binding molecule to a
detectable substance or label. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. An example of a luminescent material is luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive material include 125I, 131I, 35S, or 3H.
[00157] In situ detection can be accomplished by removing a histological specimen, for
example 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. Through the use of such a procedure, it is possible to determine not only the presence of Notch4, but also its distribution in the examined tissue. Those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
[00158] Methods and reagents suitable for determination of binding characteristics of an isolated Notch4-binding molecule are known in the art and/or are commercially available. Equipment and software designed for such kinetic analyses are commercially available {e.g., Biacore™ BIAevaluation® software, GE Healthcare; KINEXA® Software, Sapidyne Instruments).
VII. Kits comprising Notch4-binding Molecules
[00159] This disclosure further provides kits that comprise a Notch4-binding molecule, which can be used to perform the methods described herein. In certain embodiments, a kit comprises at least one purified Notch4-binding molecule in one or more containers. In some embodiments, 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. One skilled in the art will readily recognize that the disclosed Notch4-binding molecules can be readily incorporated into any of the established kit formats that are well known in the art.
EMBODIMENTS
[00160] 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
complementarity determining region 1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 96; an immunoglobulin variable light chain complementarity determining region 2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 97; and an immunoglobulin variable light chain complementarity determining region 3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 98.
[00161] 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.
[00162] 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 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 106; and an immunoglobulin variable heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 107; and/or (ii) an immunoglobulin variable light chain complementarity determining region 1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 108; an immunoglobulin variable light chain complementarity determining region 2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 109; and an immunoglobulin variable light chain complementarity determining region 3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 1 lO.Embodiment 4 - 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 sequences 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: 65; SEQ ID NO: 67; SEQ ID NO: 69; SEQ ID NO: 71; SEQ ID NO: 73; SEQ ID NO: 75; SEQ ID NO: 77; SEQ ID NO: 79; SEQ ID NO: 81; SEQ ID NO: 83; SEQ ID NO: 85; SEQ ID NO: 87; SEQ ID NO: 89; and SEQ ID NO: 91; and a light chain variable domain (VL) having an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO: 30; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 36; SEQ ID NO: 38; SEQ ID NO: 40; SEQ ID NO: 42; SEQ ID NO: 44; SEQ ID NO: 46; SEQ ID NO: 48; SEQ ID NO: 50; SEQ ID NO: 52; SEQ ID NO: 54; SEQ ID NO: 56; SEQ ID NO: 58; SEQ ID NO: 60; SEQ ID NO: 62; SEQ ID NO: 64; SEQ ID NO: 66; SEQ ID NO: 68; SEQ ID NO: 70; SEQ ID NO: 72; SEQ ID NO: 74; SEQ ID NO: 76; SEQ ID NO: 78; SEQ ID NO: 80; SEQ ID NO: 82; SEQ ID NO: 84; SEQ ID NO: 86; SEQ ID NO: 88; SEQ ID NO: 90; and SEQ ID NO: 92.
63] 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 NO: 67; SEQ ID NO: 69; SEQ ID NO: 71; SEQ ID NO: 73; SEQ ID NO: 75; SEQ ID NO: 77; SEQ ID NO: 79; SEQ ID NO: 81; SEQ ID NO: 83; SEQ ID NO: 85; SEQ ID NO: 87; SEQ ID NO: 89; and SEQ ID NO: 91; and a light chain variable domain (VL) having an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO: 30; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 36; SEQ ID NO: 38; SEQ ID NO: 40; SEQ ID NO: 42; SEQ ID NO: 44; SEQ ID NO: 46; SEQ ID NO: 48; SEQ ID NO: 50; SEQ ID NO: 52; SEQ ID NO: 54; SEQ ID NO: 56; SEQ ID NO: 58; SEQ ID NO: 60; SEQ ID NO: 62; SEQ ID NO: 64; SEQ ID NO: 66; SEQ ID NO: 68; SEQ ID NO: 70; SEQ ID NO: 72; SEQ ID NO: 74; SEQ ID NO: 76; SEQ ID NO: 78; SEQ ID NO: 80; SEQ ID NO: 82; SEQ ID NO: 84; SEQ ID NO: 86; SEQ ID NO: 88; SEQ ID NO: 90; and SEQ ID NO: 92.
[00164] Embodiment 6 - A Notch4 binding molecule or antigen binding portion thereof that competes or cross-competes with the binding molecule of any preceding embodiment.
[00165] 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.
[00166] 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.
[00167] Embodiment 9 - The binding molecule or antigen binding portion thereof of any one preceding embodiment, comprising an immunoglobulin (Ig) heavy chain constant region.
[00168] Embodiment 10 - The binding molecule or antigen binding portion thereof of
embodiment 9, wherein the constant region is a human IgG constant region.
[00169] Embodiment 11 - The binding molecule or antigen binding portion thereof of
embodiment 9, wherein the constant region is an IgGl triple mutant constant region.
[00170] Embodiment 12 - The binding molecule or antigen binding portion thereof of
embodiment 10 or embodiment 11, wherein the constant region comprises a YTE mutation. [00171] Embodiment 13 - The binding molecule or antigen binding portion thereof of any preceding embodiment, comprising an immunoglobulin light chain constant region.
[00172] Embodiment 14 - The binding molecule or antigen binding portion thereof of any preceding embodiment, which specifically binds human Notch4 with an affinity
characterized by a dissociation constant (KD) of about 0.2 nM, as measured by an Octet assay.
[00173] Embodiment 15 - The binding molecule or antigen binding fragment thereof of any preceding embodiment, which does not specifically bind to Notch 1.
[00174] 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.
[00175] Embodiment 17 - A composition comprising the binding molecule or antigen binding fragment thereof of any preceding embodiment and a carrier.
[00176] Embodiment 18 - The composition of embodiment 17, where the composition is a diagnostic reagent.
[00177] 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.
[00178] 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.
[00179] Embodiment 21 - A method of preventing recurrence of cancer in a subject, the
method comprising administering to a subject in need thereof an effective amount of the binding molecule or antigen binding fragment thereof of any one of embodiments 1 to 16 or the composition of embodiment 17.
[00180] 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.
[00181] Embodiment 23 - The method of any one of embodiments 19 to 22, wherein the method comprises administering a second active agent.
[00182] Embodiment 24 - The method of embodiment 17, wherein the second active agent is a chemotherapeutic agent.
[00183] Embodiment 25 - A method for detecting Notch4 in a sample, the method
comprising: contacting the sample with the binding molecule or antigen binding fragment thereof of any one of embodiments 1 to 16, and detecting binding of the binding molecule or fragment thereof to Notch4, thereby detecting Notch4 in the sample.
[00184] Embodiment 26 - An isolated nucleic acid molecule comprising a nucleotide
sequence encoding the binding molecule or antigen binding fragment thereof of any one of embodiments 1 to 16.
[00185] Embodiment 27 - The nucleic acid molecule of embodiment 26 operably linked to a regulatory sequence.
[00186] Embodiment 28 - A vector comprising the nucleic acid molecule of embodiment 26 or embodiment 27.
[00187] Embodiment 29 - A host cell transformed with a nucleic acid molecule of
embodiment 26, or embodiment 27, or the vector of embodiment 28.
[00188] Embodiment 30 - The host cell of embodiment 29, which is a mammalian host cell.
[00189] 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.
[00190] Embodiment 32 - A method of making a binding molecule or antigen binding
fragment thereof that specifically binds Notch4, the method comprising culturing the host cell of embodiment 29 or embodiment 30 under suitable conditions for producing the binding molecule.
[00191] Embodiment 33 - The method of embodiment 32, further comprising isolating the binding molecule or antigen binding fragment thereof. [00192] 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.
[00193] All of the references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers' instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art.
EXAMPLES
[00194] 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.
Example 1. Production of Notch4-Negative Regulatory Region (NRR) Recombinant
Antigens
[00195] All antigens-human, cynomolgus, and mouse-were constructed and expressed in a fusion form (Notch4: last three EGF-like repeats-No tch4-NRR-6x His-Furin cleavage site- mouseIgG2aFc) in Ad293 cells overexpressing Furin. After cell lysis, recombinant antigens were purified using a HisTrap column (GE Healthcare, Little Chalfont, UK). The amino acid sequences of the human Notch4-NRR; cyno Notch4-NRR; mouse Notch4-NRR; and mIgG2aFc are shown below.
>HumanNotch4-NRR
EIDPCHSQPC FHGGTCEATA GSPLGFICHC PKGFEGPTCS HRAPSCGFHH CHHGGLCLPS PKPGFPPRCA CLSGYGGPDC LTPPAPKGCG PPSPCLYNGS CSETTGLGGP GFRCSCPHSS PGPRCQKPGA KGCEGRSGDG ACDAGCSGPG GNWDGGDCSL GVPDPWKGCP SHSRCWLLFR DGQCHPQCDS EECLFDGYDC ETPPACTPAY DQYCHDHFHN GHCEKGCNTA ECGWDGGDCR PEDGDPEWGP SLALLVVLSP PALDQQLFAL ARVLSLTLRV GLWVRKDRDG RDMVYPYPGA RAEEKLGGTR DPTYQERAAP QTQPLGKETD SLSAGFVVVM GVDLSRCGPD HPASRCPWDP GLLLRFLAAM AAVGALEPLL PGPLLAVHPH AGTAPPANQH HHHHHRRKR (set forth in SEQ ID NO: 1)
>CynoNotch4-NRR
EIDPCHSQPC FHGGTCEATA GSPLGFICHC PKGFEGPTCS HRAPSCGLHH CHHGGLCLPS PKPGFPPRCA CLNGYGGPDC LTPPAPKGCG PPSPCLYNGS CSETTGLGGP GFRCSCPHSS PGPRCQKPGA KGCEGRSGDG ACDAGCSGPG GNWDGGDCSL GVPDPWKGCP SHSRCWLLFR DGQCHPQCDS EECLFDGYDC ETPPACTPAY DQYCHDHFHN GHCEKGCNTA ECGWDGGDCR PEDGDPEWGP SLALLVVLSP PALDQQLFAL ARVLSLTLRL GLWVRKDRDG RDMVYPYPGA RAEEKLGGTR DPSYQERSAP QTQPLGKETD SLSAGFVVVM GVDLSRCGPD HPASRCPWDP GLLLRFLAAM AAVGALEPLL PGPLLAVHPH AGTAPPANQH HHHHHRRKR (set forth in SEQ ID NO: 2)
>MouseNotch4-NRR
EMDLCQSQPC SNGGSCEITT GPPPGFTCHC PKGFEGPTCS HKALSCGIHH CHNGGLCLPS PKPGSPPLCA CLSGFGGPDC LTPPAPPGCG PPSPCLHNGT CTETPGLGNP GFQCTCPPDS PGPRCQRPGA SGCEGRGGDG TCDAGCSGPG GDWDGGDCSL GVPDPWKGCP PHSQCWLLFR DGRCHPQCDS EECLFDGYDC EIPPTCIPAY DQYCRDHFHN GHCEKGCNNA ECGWDGGDCR PEGEDSEGRP SLALLVVLRP PALDQQLLAL ARVLSLTLRV GLWVRKDSEG RNMVFPYPGT RAKEELSGAR DSSSWERQAP PTQPLGKETE SLGAGFVVVM GVDLSRCGPE HPASRCPWDS GLLLRFLAAM AAVGALEPLL PGPLLAAHPQ AGTRPPANQH HHHHHRRKR (set forth in SEQ ID NO: 3)
>mIgG2aFc (present in the expressed proteins, missing in the purified proteins)
GAEPRGPTIK PCPPCKCPAP NLLGGPSVFI FPPKIKDVLM ISLSPIVTCV VVDVSEDDPD VQISWFVNNV EVHTAQTQTH REDYNSTLRV VSALPIQHQD WMSGKEFKCK VNNKDLPAPI ERTISKPKGS VRAPQVYVLP PPEEEMTKKQ VTLTCMVTDF MPEDIYVEWT NNGKTELNYK NTEPVLDSDG SYFMYSKLRV EKKNWVERNS YSCSVVHEGL HNHHTTKSFS RTPGK (set forth in SEQ ID NO: 4)
[00196] Biotinylated reagents were prepared using EZ-Link™ Sulfo-NHS-LC-LC-Biotin (Cat. #21338, Thermo Fisher Scientific, Waltham, MA,) to contain 2-3 biotins per molecule, following the manufacturer's protocol.
Example 2. Isolation of Anti-Notch4 Antibodies
[00197] We panned human scFv (Lloyd C et al. (2009) Modelling the human immune
response: performance of a 1011 human antibody repertoire against a broad panel of therapeutically relevant antigens. PEDS 22: 159-168.; Groves M et al. (2006) Affinity maturation of phage display antibody populations using ribosome display. J. Immunol. Meth. 313: 129-139; Vaughan TJ et al. (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat. Biotechnol. 14:309-314) and Fab (Hoet RM et al. (2005) Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity. Nat. Biotechnol. 23:344-348) phage display antibody libraries against recombinant human and mouse Notch4- NRR antigens prepared above, as described by Lillo AM et al. ((2011) Development of phage-based single chain Fv antibody reagents for detection of Yersinia pestis. PloS one 6:e27756). To evaluate diversity and specificity of individual clones in the panning populations, we used 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.
[00198] 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). [00199] A total of 25,000 monoclonal antibody-containing bacterial culture supernatants were screened for specific binding by homogenous time-resolved fluorescence (HTRF) assay (Cisbio Bioassays, Codolet, France). We identified 850 sequence-unique and human/cyno Notch4-NRR cross-reactive clones. Of these, 120 clones demonstrated strong and specific binding to Notch4-expressing cell lines. We re-formatted 85 clones to IgG, expressed them in 293F mammalian cells, and purified them.
Example 3. Epitope Binning of Anti-Notch4 Antibodies
[00200] We assessed the ability of anti-Notch4 antibodies to compete with each other for binding to human Notch4-NRR on an Octet® instrument (Pall ForteBio LLC, Menlo Park, CA), essentially as described by Abdiche YN et al. (2009) Exploring blocking assays using Octet, ProteOn, and Biacore biosensors. Anal. Biochem. 386: 172-180. Notch4-NRR protein and a first anti-Notch4 antibody were pre-incubated and added to a biotinylated second anti- Notch4 antibody captured on a Streptavidin sensor. If the first anti-Notch4 antibody completely blocked binding of Notch4-NRR to the second anti-Notch4 antibody, 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.
Example 4. Binding of Anti-Notch4 Antibodies to Notch4-NRR and Other Human Anti- Notch Antigens
[00201] The binding affinity and specificity of anti-Notch4 antibodies to recombinant
antigens was determined by Surface Plasmon Resonance (SPR) and ELISA, and binding to Notch4-positive cells by flow cytometry. The binding properties of the anti-Notch4 antibodies are summarized in Table 2 and Table 3, below. All antibodies showed no binding to human Notch 1-NRR, Notch2-NRR, or Notch3-NRR recombinant antigens when tested. Table 2
Binding Properties of Notch4 Antibodies
Figure imgf000052_0001
Table 3
Binding Properties of Notch4 Antibodies
Figure imgf000052_0002
Example 5. In Vitro Activity of Anti-Notch4 Antibodies
[00202] In order to test the activity of the Notch4 antibodies in vitro, we constructed a stable luciferase reporter cell line. We transfected Ad293 cells with an RBP-Jk luciferase reporter and the human Notch4 full-length gene carrying different selectable markers. Following transfection, we assessed single cell clones for activity by stimulation with either plate-bound JAGl or DLL4 recombinant human proteins. We chose a single-cell clone showing both the highest fold luciferase activation and Notch4 expression to use for testing activity of the antibodies. Table 4, below, 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. With the exception of NOCH0075, the antibodies were able to inhibit the luciferase reporter >40%. None of the antibodies characterized showed activation of the reporter.
Table 4
Properties of Notch4 Antibodies
Figure imgf000053_0001
NR = Not Reached
[00203] We also tested the antibodies for their ability to inhibit the establishment and growth of cancer stem cell (CSC) spheres from two different breast cancer cell lines, HCC1937 and T47D. The results are shown in Table 5, below and in FIG. 2. As can be seen in the table and the figure, NOCH0075 failed to inhibit the sphere formation of both cell lines at the tested concentration, but all other anti-Notch4 antibodies tested were able to inhibit the CSC sphere formation of both breast cancer cell lines. The NOCH0090 antibody was the most effective inhibitor, showing a 39% reduction in CSC sphere formation in cell line HCC1937, and 35% reduction in CSC sphere formation in cell line T47D.
Table 5
Activity of Notch4 Antibodies
Figure imgf000054_0001
Example 6. In Vivo Activity of Anti-Notch4 Antibodies
[00204] Additionally, we tested the antibodies in vivo, using the MDA-MB-231 xenograft breast cancer model. Once tumors reached -400 mm3, the animals were dosed three times with 30 mg/kg antibody. We assessed gene expression of both downstream Notch pathway genes, Hey2 and Hesl, and gene expression of sternness genes, EZH2 and BMI1, in the tumors following treatment with IgGl control antibody, or with anti-No tch4 antibodies NOCH0004; NOCH0012; NOCH0075; NOCH0090; or NOCH0133. The results obtained for the Hey2 gene are shown in FIG. 3A, the results obtained for the Hesl gene are shown in FIG. 3B, the results obtained for the EZH2 gene are shown in FIG. 4A, and the results obtained for the BMI1 gene are shown in FIG. 4B. As seen in FIG.3A, FIG. 3B, FIG. 4A, and FIG. 4B, the NOCH0090 antibody showed a trend toward inhibition of both the Notch pathway and the sternness genes.
Example 7. Reversion of Framework Sequences
[00205] 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.
[00206] Following germlining, the binding and activity of the germlined antibody (GLA) was determined and compared to the NOCH0090 antibody. As seen in FIG. 5, GLA binding to the human Notch4-overexpressing cell line was slightly improved compared to NOCH0090.
Example 8. Optimization of the Anti-Notch4 Antibodies
[00207] We optimized the affinity and potency of the GLA antibody by generating
parsimonious libraries (Balint RF et al. (1993) Antibody engineering by parsimonious mutagenesis. Gene 137: 109-118) of CDR variants in IgGl format, and testing the variants for improved binding to Notch4-positive cells. Several mutations with the best improvement in affinity were combined to generate GLA-P antibodies.
[00208] The activity of the GLA-P antibodies was assessed in a luciferase reporter assay.
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. Three of the antibodies, GLA-P2 (ICso=9.9 nM), GLA-P3 (ICso=4.7 nM), and GLA-P4 (ICso=1.9 nM), showed improved activity over the un-optimized GLA molecule (43.6 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.
[00209] We optimized the affinity and potency of the GLA antibody by generating block libraries of CDRs. We isolated No tch4-NRR- specific scFv antibodies from the human scFv phage display library in a series of repeated selection cycles on Notch4-positive cells, essentially as described previously by Lloyd C et al. (cited above). We converted ScFv genes from the selection outputs into IgG and screened for their binding to human, murine, and cynomolgus Notch4-NRR antigen by ELISA or HTRF. IgGl antibodies were expressed in mammalian cells, purified by affinity chromatography, and ranked based on their characteristics in binding and functional assays. Several mutations with the best
improvement were combined to generate GLA-B antibodies. [00210] We assessed the activity of the GLA-B antibodies in a luciferase assay, and the results for six of these antibodies (GLA-B 1; GLA-B2; GLA-B 3; GLA-B4; GLA-B 5 and GLA-B6) are shown in FIG. 7A. 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.
[00211] We combined several mutations from GLA-P with mutations from GLA-B to
generate 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.
[00212] We further assessed GLA-S 3 and GLA-S4 for their ability to inhibit CSC sphere formation in vivo. Using FACS, we assessed anti-CSC sphere formation activity in two ovarian models, MEDI-OVAl and PA-1. Twenty-four hours after dosing with a total of three doses, the tumors were excised and assessed by flow cytometry for CSC sphere percentage by measuring ALDEFLUOR™ activity. As shown in FIG. 9A, both antibodies showed a dose-dependent reduction in CSC (ALDEFLUOR™ +) percentage, with similar activity in the MEDI-OVAl model. However, as shown in FIG. 9B, in the PA-1 model there was a greater reduction in CSCs with GLA-S4 as compared to GLA-S3.
[00213] We conducted a re-implantation study using the OVCAR4 ovarian xenograft model to test the ability of GLA-S4 to reduce the CSC fraction. Following treatment with three doses of either IgGl control antibody or GLA-S4, at the indicated dosage level, tumors were removed, dissociated to single cells, and re-implanted into a secondary mouse at a limiting dilution. Animals were monitored for tumors. The CSC frequency was then calculated using L-Calc™ software (StemCell Technologies, Vancouver, Canada) and the results are shown in FIG. 10. The highest dose of GLA-S4 tested produced a 25-fold reduction in CSC frequency compared to the control. These data collectively show that the CSC fraction of multiple different models is effectively decreased by treatment with GLA-S4.
[00214] 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.
Table 6
Binding Properties of Notch4 GLA Antibodies
Figure imgf000057_0001
[00215] The GLA-S4 antibody was further optimized by introducing mutations that removed hydrophobic patches and lowered the isoelectric point to create GLA-S4F antibodies. As shown in FIG. 11A, we tested GLA-S4F antibodies in the luciferase assay, and showed that the antibodies retained similar activity to GLA-S4. 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. 216] Table 7 shows binding properties of some anti-Notch4GLA-S antibodies to human and mouse Notch4-NRR antigen, as measured by direct ELISA.
Table 7
Binding Properties of Anti-Notch4 GLA-S Antibodies
Figure imgf000058_0001
Example 9. In vivo efficacy of GLA-S4F18 in PA-1 ovarian xenograft tumor model
[00217] To further characterize the GLA-S4F18 antibody, we ran an in vivo efficacy study in the PA-1 ovarian xenograft tumor model. Since CSC-targeting agents are likely to be used in combination with standard-of-care (SOC) therapies, we combined GLA-S4F18 with carboplatin, an SOC for ovarian cancer, and monitored for a delay in tumor regrowth following cessation of treatment. The PA-1 cells were subcutaneously implanted in nu/nu animals at 10 million cells per animal. Tumors were allowed to reach -250 mm3 in size before dosing commenced. Carboplatin was administered intraperitoneally at 70 mg/kg on days 24, 28, and 32. GLS-S4F18 was administered intraperitoneally at 30 mg/kg on days 24, 28, 32, 36, and 40. The results are presented in FIG. 12A and FIG. 12B. As seen in FIG. 12A, the combination of GLA-S4F18 and carboplatin resulted in a statistically significant delay in tumor regrowth compared to carboplatin alone (P=0.0212 log rank test).
Additionally, as shown in FIG. 12B, the percentage of CSCs in the combination arm was reduced by approximately 4-fold compared to the control of isotype only.
Example 10. Upregulation of Notch4 on cells treated in vitro with either cisplatin or doxil
[00218] Expression of Notch4 was evaluated on the surface of SW-780 cells following
treatment with either cisplatin or doxil. Briefly SW-780 cells were grown to 80%
confluency, treated with the indicated amounts of either cisplatin or doxil for 72 hours. At 72 hours, cells were harvested using 0.25% trypsin-EDTA, washed in PBS+2% FBS (FACS buffer). Cells were then stained with DAPI (1: 1000, Thermo Fisher Scientific) and Notch4- PE (BioLegend, San Diego CA) for 30 minutes on ice. Cells were then washed and read on an LSRII (BD Biosciences, San Jose, CA) and FCS files were generated. Data was analyzed using Flow Jo with the following gating strategy: compensation was performed using the auto comp matrix in Flow Jo 10 (FLowJo LLC, Ashland, OR) utilizing single stain controls. Cells gated through the FSC-A versus SSC-A plot were then selected for live, single cells through the DAPI versus SSC-W plot. The median fluorescence intensity (MFI) was then determined for the Notch4-PE stain and plotted in GraphPad Prism.
[00219] The results shown in 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
[00220] 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 mm3, 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 mm3 was considered a relapse event. The Kaplan-Meier plot shows the rate at which tumors relapse. OVA-001 was monitored for 150 days (FIG. 14A) and OVA-0002 was monitored for 45 days (FIG. 14B). At the end of the study, tumors were excised and plated for ex vivo CSC sphere formation. Briefly, tumors were minced to 2 mm3 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. Once cells were dissociated they were washed with HBSS, counted and plated in DMEM/F12 containing 20 ng/niL EGF, 10 ng/niL bFGF, 5 μg/mL insulin, 0.4% BSA and 1% knock-out serum replacement (Invitrogen, Waltham MA). Thirty thousand cells per mL were plated in 96-well ultra-low attachment plates (Corning Inc., Corning NY) and incubated at 37°C for 4 days. Plates were then read with CELLTITER-GLO kit (Promega, Madison, WI) following manufacturer's instructions.
[00221] 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
[00222] Subcutaneous xenograft mouse models of ovarian cancer were generated by
implanting 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 mm3 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 mm2. The pieces were then dissociated in DMEM/F12 containing 200 units/ML collagenase IV (Worthington Biochemical
Corporation, Lakewood NJ) with constant shaking at 37°C and trituration every 15 minutes. Once the tumor was disaggregated the cells were spun, washed with DMEM/F12 and put through a 70 micron cell strainer. Cells were then counted and mixed 1 : 1 with MATRIGEL (BD, Biosciences, San Jose CA) and implanted into athymic nude mice at densities of 100, 500, 1000, 5000, 10000, and 50000 cells. Tumor take rates were monitored over time and the CSC frequency was determined by using L-calc software (StemCell Technologies, Vancover BC).
[00223] Results of this example show that GLA-S4F18 is able to reduce CSCs alone (FIG.
15A) and in combination with abraxane (FIG. 15B).
Example 13. Epitope Mapping of Anti-Notch4-NRR Antibody GLA-S4
[00224] We used a full-length, transiently expressed, human 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
AAACCCGGAGCCAAGGGGTGTGAGGGCAGAAGTGGAGATGGGGCCTGCGATGC TGGCTGCAGTGGCCCGGGAGGAAACTGGGATGGAGGGGACTGCTCTCTGGGAGT CCCAGACCCCTGGAAGGGCTGCCCCTCCCACTCTCGGTGCTGGCTTCTCTTCCGG GACGGGCAGTGCCACCCACAGTGTGACTCTGAAGAGTGTCTGTTTGATGGCTAC GACTGTGAGACCCCTCCAGCCTGCACTCCAGCCTATGACCAGTACTGCCATGATC ACTTCCACAACGGGCACTGTGAGAAAGGCTGCAACACTGCAGAGTGTGGCTGGG ATGGAGGTGACTGCAGGCCTGAAGATGGGGACCCAGAGTGGGGGCCCTCCCTGG CCCTGCTGGTGGTACTGAGCCCCCCAGCCCTAGACCAGCAGCTGTTTGCCCTGGC CCGGGTGCTGTCCCTGACTCTGAGGGTAGGACTCTGGGTAAGGAAGGATCGTGA TGGCAGGGACATGGTGTACCCCTATCCTGGGGCCCGGGCTGAAGAAAAGCTAGG AGGAACTCGGGACCCCACCTATCAGGAGAGAGCAGCCCCTCAAACGCAGCCCCT GGGCAAGGAGACCGACTCCCTCAGTGCTGGGTTTGTGGTGGTCATGGGTGTGGA TTTGTCCCGCTGTGGCCCTGACCACCCGGCATCCCGCTGTCCCTGGGACCCTGGG CTTCTACTCCGCTTCCTTGCTGCGATGGCTGCAGTGGGAGCCCTGGAGCCCCTGC TGCCTGGACCACTGCTGGCTGTCCACCCTCATGCAGGGACC (set forth in SEQ ID NO: 5)
>Notch4/NotchlLNRl
gaggcgtgcgagctgcccgagtgccaggaggacgcgggcaacaaggtctgcagcctgcagtgcaacaaccacgcgtgcggctg ggacggcggtgactgctccctcaacttcaatGACCCCTGGAAGGGCTGCCCCTCCCACTCTCGGTGC
TGGCTTCTCTTCCGGGACGGGCAGTGCCACCCACAGTGTGACTCTGAAGAGTGTC
TGTTTGATGGCTACGACTGTGAGACCCCTCCAGCCTGCACTCCAGCCTATGACCA
GTACTGCCATGATCACTTCCACAACGGGCACTGTGAGAAAGGCTGCAACACTGC
AGAGTGTGGCTGGGATGGAGGTGACTGCAGGCCTGAAGATGGGGACCCAGAGT
GGGGGCCCTCCCTGGCCCTGCTGGTGGTACTGAGCCCCCCAGCCCTAGACCAGC
AGCTGTTTGCCCTGGCCCGGGTGCTGTCCCTGACTCTGAGGGTAGGACTCTGGGT
AAGGAAGGATCGTGATGGCAGGGACATGGTGTACCCCTATCCTGGGGCCCGGGC
TGAAGAAAAGCTAGGAGGAACTCGGGACCCCACCTATCAGGAGAGAGCAGCCC
CTCAAACGCAGCCCCTGGGCAAGGAGACCGACTCCCTCAGTGCTGGGTTTGTGG
TGGTCATGGGTGTGGATTTGTCCCGCTGTGGCCCTGACCACCCGGCATCCCGCTG
TCCCTGGGACCCTGGGCTTCTACTCCGCTTCCTTGCTGCGATGGCTGCAGTGGGA GCCCTGGAGCCCCTGCTGCCTGGACCACTGCTGGCTGTCCACCCTCATGCAGGGA CC (set forth in SEQ ID NO: 6)
>Notch4/NotchlLNR2
AAACCCGGAGCCAAGGGGTGTGAGGGCAGAAGTGGAGATGGGGCCTGCGATGC
TGGCTGCAGTGGCCCGGGAGGAAACTGGGATGGAGGGGACTGCTCTCTGGGAGT
CCCAgacccctggaagaactgcacgcagtctctgcagtgctggaagtacttcagtgacggccactgtgacagccagtgcaactca gccggctgcctcttcgacggctttgactgccagcgtgcggaaGCCTGCACTCCAGCCTATGACCAGTACT
GCCATGATCACTTCCACAACGGGCACTGTGAGAAAGGCTGCAACACTGCAGAGT
GTGGCTGGGATGGAGGTGACTGCAGGCCTGAAGATGGGGACCCAGAGTGGGGG
CCCTCCCTGGCCCTGCTGGTGGTACTGAGCCCCCCAGCCCTAGACCAGCAGCTGT
TTGCCCTGGCCCGGGTGCTGTCCCTGACTCTGAGGGTAGGACTCTGGGTAAGGAA
GGATCGTGATGGCAGGGACATGGTGTACCCCTATCCTGGGGCCCGGGCTGAAGA
AAAGCTAGGAGGAACTCGGGACCCCACCTATCAGGAGAGAGCAGCCCCTCAAAC
GCAGCCCCTGGGCAAGGAGACCGACTCCCTCAGTGCTGGGTTTGTGGTGGTCAT
GGGTGTGGATTTGTCCCGCTGTGGCCCTGACCACCCGGCATCCCGCTGTCCCTGG
GACCCTGGGCTTCTACTCCGCTTCCTTGCTGCGATGGCTGCAGTGGGAGCCCTGG
AGCCCCTGCTGCCTGGACCACTGCTGGCTGTCCACCCTCATGCAGGGACC (set forth in SEQ ID NO: 7)
>Notch4/NotchlLNR3
AAACCCGGAGCCAAGGGGTGTGAGGGCAGAAGTGGAGATGGGGCCTGCGATGC
TGGCTGCAGTGGCCCGGGAGGAAACTGGGATGGAGGGGACTGCTCTCTGGGAGT
CCCAGACCCCTGGAAGGGCTGCCCCTCCCACTCTCGGTGCTGGCTTCTCTTCCGG
GACGGGCAGTGCCACCCACAGTGTGACTCTGAAGAGTGTCTGTTTGATGGCTAC
GACTGTGAGACCCCTCCAggccagtgcaaccccctgtacgaccagtactgcaaggaccacttcagcgacgggcac tgcgaccagggctgcaacagcgcggagtgcgagtgggacgggctggactgtgcggagGATGGGGACCCAGAGT
GGGGGCCCTCCCTGGCCCTGCTGGTGGTACTGAGCCCCCCAGCCCTAGACCAGC
AGCTGTTTGCCCTGGCCCGGGTGCTGTCCCTGACTCTGAGGGTAGGACTCTGGGT
AAGGAAGGATCGTGATGGCAGGGACATGGTGTACCCCTATCCTGGGGCCCGGGC
TGAAGAAAAGCTAGGAGGAACTCGGGACCCCACCTATCAGGAGAGAGCAGCCC
CTCAAACGCAGCCCCTGGGCAAGGAGACCGACTCCCTCAGTGCTGGGTTTGTGG
TGGTCATGGGTGTGGATTTGTCCCGCTGTGGCCCTGACCACCCGGCATCCCGCTG TCCCTGGGACCCTGGGCTTCTACTCCGCTTCCTTGCTGCGATGGCTGCAGTGGGA GCCCTGGAGCCCCTGCTGCCTGGACCACTGCTGGCTGTCCACCCTCATGCAGGGA CC (set forth in SEQ ID NO: 8)
>Notch4/NotchlHD-N
AAACCCGGAGCCAAGGGGTGTGAGGGCAGAAGTGGAGATGGGGCCTGCGATGC
TGGCTGCAGTGGCCCGGGAGGAAACTGGGATGGAGGGGACTGCTCTCTGGGAGT
CCCAGACCCCTGGAAGGGCTGCCCCTCCCACTCTCGGTGCTGGCTTCTCTTCCGG
GACGGGCAGTGCCACCCACAGTGTGACTCTGAAGAGTGTCTGTTTGATGGCTAC
GACTGTGAGACCCCTCCAGCCTGCACTCCAGCCTATGACCAGTACTGCCATGATC
ACTTCCACAACGGGCACTGTGAGAAAGGCTGCAACACTGCAGAGTGTGGCTGGG
ATGGAGGTGACTGCAGGCCTGAAcatgtacccgagaggctggcggccggcacgctggtggtggtggtgctga tgccgccggagcagctgcgcaacagctccttccacttcctgcgggagctcagccgcgtgctgcacaccaacgtggtcttcaagcgtg acgcacacggccagcagatgatcttcccctactacggcGCCCGGGCTGAAGAAAAGCTAGGAGGAACT
CGGGACCCCACCTATCAGGAGAGAGCAGCCCCTCAAACGCAGCCCCTGGGCAAG
GAGACCGACTCCCTCAGTGCTGGGTTTGTGGTGGTCATGGGTGTGGATTTGTCCC
GCTGTGGCCCTGACCACCCGGCATCCCGCTGTCCCTGGGACCCTGGGCTTCTACT
CCGCTTCCTTGCTGCGATGGCTGCAGTGGGAGCCCTGGAGCCCCTGCTGCCTGGA
CCACTGCTGGCTGTCCACCCTCATGCAGGGACC (set forth in SEQ ID NO: 9).
>Notch4/NotchlHD-linker
AAACCCGGAGCCAAGGGGTGTGAGGGCAGAAGTGGAGATGGGGCCTGCGATGC
TGGCTGCAGTGGCCCGGGAGGAAACTGGGATGGAGGGGACTGCTCTCTGGGAGT
CCCAGACCCCTGGAAGGGCTGCCCCTCCCACTCTCGGTGCTGGCTTCTCTTCCGG
GACGGGCAGTGCCACCCACAGTGTGACTCTGAAGAGTGTCTGTTTGATGGCTAC
GACTGTGAGACCCCTCCAGCCTGCACTCCAGCCTATGACCAGTACTGCCATGATC
ACTTCCACAACGGGCACTGTGAGAAAGGCTGCAACACTGCAGAGTGTGGCTGGG
ATGGAGGTGACTGCAGGCCTGAAGATGGGGACCCAGAGTGGGGGCCCTCCCTGG
CCCTGCTGGTGGTACTGAGCCCCCCAGCCCTAGACCAGCAGCTGTTTGCCCTGGC
CCGGGTGCTGTCCCTGACTCTGAGGGTAGGACTCTGGGTAAGGAAGGATCGTGA
TGGCAGGGACATGGTGTACCCCTATCCTGGGcgcgaggaggagctgcgcaagcaccccatcaagcgt gctgctgaaggctgggcagcacctgacgccctgctgggccaggtgaaggcctcgctgctccctggtggcagcgagggtgggcgg cggcggagggagctggacCTCAGTGCTGGGTTTGTGGTGGTCATGGGTGTGGATTTGTCCC GCTGTGGCCCTGACCACCCGGCATCCCGCTGTCCCTGGGACCCTGGGCTTCTACT CCGCTTCCTTGCTGCGATGGCTGCAGTGGGAGCCCTGGAGCCCCTGCTGCCTGGA CCACTGCTGGCTGTCCACCCTCATGCAGGGACC (set forth in SEQ ID NO: 10)
>Notch4/NotchlHD-C
AAACCCGGAGCCAAGGGGTGTGAGGGCAGAAGTGGAGATGGGGCCTGCGATGC
TGGCTGCAGTGGCCCGGGAGGAAACTGGGATGGAGGGGACTGCTCTCTGGGAGT
CCCAGACCCCTGGAAGGGCTGCCCCTCCCACTCTCGGTGCTGGCTTCTCTTCCGG
GACGGGCAGTGCCACCCACAGTGTGACTCTGAAGAGTGTCTGTTTGATGGCTAC
GACTGTGAGACCCCTCCAGCCTGCACTCCAGCCTATGACCAGTACTGCCATGATC
ACTTCCACAACGGGCACTGTGAGAAAGGCTGCAACACTGCAGAGTGTGGCTGGG
ATGGAGGTGACTGCAGGCCTGAAGATGGGGACCCAGAGTGGGGGCCCTCCCTGG
CCCTGCTGGTGGTACTGAGCCCCCCAGCCCTAGACCAGCAGCTGTTTGCCCTGGC
CCGGGTGCTGTCCCTGACTCTGAGGGTAGGACTCTGGGTAAGGAAGGATCGTGA
TGGCAGGGACATGGTGTACCCCTATCCTGGGGCCCGGGCTGAAGAAAAGCTAGG
AGGAACTCGGGACCCCACCTATCAGGAGAGAGCAGCCCCTCAAACGCAGCCCCT
GGGCAAGGAGACCGACTCCcccatggacgtccgcggctccatcgtctacctggagattgacaaccggcagtgtgt gcaggcctcctcgcagtgcttccagagtgccaccgacgtggccgcattcctgggagcgctcgcctcgctgggcagcctcaacatcc cctacaagatcgaggccgtgcagagtgagaccgtggag (set forth in SEQ ID NO: 11)
>Notchl
gaggcgtgcgagctgcccgagtgccaggaggacgcgggcaacaaggtctgcagcctgcagtgcaacaaccacgcgtgcggctg ggacggcggtgactgctccctcaacttcaatgacccctggaagaactgcacgcagtctctgcagtgctggaagtacttcagtgacggc cactgtgacagccagtgcaactcagccggctgcctcttcgacggctttgactgccagcgtgcggaaggccagtgcaaccccctgtac gaccagtactgcaaggaccacttcagcgacgggcactgcgaccagggctgcaacagcgcggagtgcgagtgggacgggctgga ctgtgcggagcatgtacccgagaggctggcggccggcacgctggtggtggtggtgctgatgccgccggagcagctgcgcaacag ctccttccacttcctgcgggagctcagccgcgtgctgcacaccaacgtggtcttcaagcgtgacgcacacggccagcagatgatcttc ccctactacggccgcgaggaggagctgcgcaagcaccccatcaagcgtgctgctgaaggctgggcagcacctgacgccctgctgg gccaggtgaaggcctcgctgctccctggtggcagcgagggtgggcggcggcggagggagctggaccccatggacgtccgcggc tccatcgtctacctggagattgacaaccggcagtgtgtgcaggcctcctcgcagtgcttccagagtgccaccgacgtggccgcattcc tgggagcgctcgcctcgctgggcagcctcaacatcccctacaagatcgaggccgtgcagagtgagaccgtggag (set forth in
SEQ ID NO: 12) [00225] Constructs were transiently expressed in HEK293 cells. To determine the epitope for GLA-S4, we assayed the transiently transfected clones for binding by flow cytometry. To confirm that all constructs were expressed in the cells, we utilized a commercially available Notch4 antibody (N4; Cat. #349002, BioLegend, San Diego, CA) that binds to the extracellular domain of Notch4 outside of the NRR. As seen in FIG. 16A-FIG. 16H, all constructs were expressed, as assessed by binding of the N4 antibody. Lack of GLA-S4 binding in the constructs containing Notchl LNR3 (FIG. 16D), HD-N (FIG. 16E), and HD- C (FIG. 16G) demonstrates that these domains are critical for GLA-S4 binding. The additional control of a complete swap of the Notch4 NRR for the Notch INRR sequences also showed lack of binding by GLA-S4.
ANTIBODY VH AND VL SEQUENCES
[00226] The 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.
LEAD GENERATION MOLECULES
>NOCH0004 VH
E VQLLES GGGLVQPGGS LRLS C A AS GFTFS W YTMQW VRQ APGKGLE W VS GIGS S G V TRY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC VKGQGPG Y Y Y YG MD V WGQGTTVTVSS (set forth in SEQ ID NO: 13).
>NOCH0004 VL
DIQMTQS PGTLS LS PGERATLS CR AS QS VS RV YLA W YQQKPGQ APRLLIYG AS S RA A GIPDRFS GS GS GTDFTLTINKLEPEDS A V Y YC QQ YGGS PM YTFGQGTKLEIK (set forth in SEQ ID NO: 14).
>NOCH0012 VH
Q VQLQES GPGLVKPS QTLS LTCT VS GGS IS S GGN YWS WIRQHPGKGLE WIG YIYNS EI IY YNPS LRNP VS IS IDTS KNQFS LNLNS VT V ADT A V Y YC AR ADS N YE YFEHWGRGTL VTVSS (set forth in SEQ ID NO: 15). >NOCH0012 VL
QSALTQPASVSGSPGQSITISCTGTGSDVGGYNYVSWYQQHPGKAPKLMIYDVSDRP S GVS NRFS GS KS GNT AS LS IS GLQ ADDE AD Y YC S S FS TS PTP VLFGGGTKVT VL (set forth in SEQ ID NO: 16).
>NOCH0075 VH
E 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).
>NOCH0075 VL
NFMLTQPHS VS ES PGKT VTIS CTGS S GS IAS N Y VQW YKQRPGS APTT VIYEDNKRPS G VPDRFS GSIDSSSNSASLTIS GLKTEDE AD Y YCQS YDNNNQG VFGGGTKVT VL (set forth in SEQ ID NO: 18).
>NOCH0090 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW VS TIS GS GD NTYYADSVKGRFTISRDNSKNTLYLHMNSLRAEDTAIYYCAKGAVKGYYYYYYMD VWGQGTMVTVSS (set forth in SEQ ID NO: 19).
>NOCH0090 VL
QS VLTQP AS VSGSPGQSITIS CTGTS S D VGG YN Y VS W YQQHPGKAPKLMIYEGS KRP S GVS NRFS GS KS GNT AS LT VS GLQ AEDE AD Y YCS S YTTRS TR VFGGGTKLT VL (set forth in SEQ ID NO: 20).
>NOCH0133 VH
QVQLQES GPGLVKPS QTLS LTCT VS GGS IS S GN Y YWS WIRQHPGKGLE WIG YIY YS G S T Y YS PS LQS R ATIS LDTS KNQFS LKLS S MT A ADT A V Y YC ART A YS GW YGQ AFD YW GRGTLVTVSS (set forth in SEQ ID NO: 21).
>NOCH0133VL QPVLTQPPSVSVAPGQTARIPCGGDNLGRKNVHWYQQKPGQAPVLVVFDDRDRPSG IPERFS GFNS GDT ATLTIS RVE AGDE AD Y YC Q VS HS GS AH V VFGGGTKLT VL (set forth in SEQ ID NO: 22).
GERMLINED LEAD >GLA VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIS GS G DNT Y Y ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A VKG Y Y Y YY Y MD VWGQGTM VT VS S (set forth in SEQ ID NO: 23).
>GLA VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YN Y VS W YQQHPGKAPKLMIYEGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTTRS TR VFGGGTKLT VL (set forth in SEQ ID NO: 24).
BLOCK MUTANTS >GLA-B1 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIS GS G DNT YY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A VKG Y Y Y YIW WDVWGQGTMVTVSS (set forth in SEQ ID NO: 25).
>GLA-B1 VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YNYVSW YQQHPGKAPKLMIYEGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRLS QRVFGGGTKLT VL (set forth in SEQ ID NO: 26).
>GLA-B2 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIS GS G DNT YY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A VKG Y Y Y YIW WDVWGQGTMVTVSS (set forth in SEQ ID NO: 27). >GLA-B2 VL
QS ALTQP VS VSGSPGQSITIS CTGTS S D VGG YN Y VS W YQQHPGKAPKLMIYEGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S FTRRS TRVFGGGTKLT VL (set forth in SEQ ID NO: 28).
>GLA-B3 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIS GS G DNT Y Y ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A VKG Y Y Y YLYL FPWGQGTMVT VS S (set forth in SEQ ID NO: 29).
>GLA-B3 VL
QS ALTQP VS VSGSPGQSITIS CTGTS S D VGG YNYVSW YQQHPGKAPKLMIYEGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S FTRRS TRVFGGGTKLT VL (set forth in SEQ ID NO: 30).
>GLA-B4 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIS GS G DNT YY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A VKG Y Y Y YLYL YPWGQGTM VTVS S (set forth in SEQ ID NO: 31).
>GLA-B4 VL
QS ALTQP VS VSGSPGQSITIS CTGTS S D VGG YNYVSW YQQHPGKAPKLMIYEGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S FTRRS TRVFGGGTKLT VL (set forth in SEQ ID NO: 32).
>GLA-B5 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIS GS G DNT YY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A VKG Y Y Y YLY M YPWGQGTM VTVS S (set forth in SEQ ID NO: 33).
>GLA-B5 VL QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YN Y VS W YQQHPGKAPKLMIYEGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRRS QRTFGGGTKLT VL (set forth in SEQ ID NO: 34).
>GLA-B6 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIS GS G DNT Y Y ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A VKG Y Y Y YLY M YPWGQGTM VT VS S (set forth in SEQ ID NO: 35).
>GLA-B6 VL
QS ALTQP VS VSGSPGQSITIS CTGTS S D VGG YNYVSW YQQHPGKAPKLMIYEGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S FTRRS TRVFGGGTKLT VL (set forth in SEQ ID NO: 36).
PARSIMONIOUS MUTANTS >GLA-P1 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW VATIP AS G DNT YY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YR Y MDPWGQGTM VT VS S (set forth in SEQ ID NO: 37).
>GLA-P1 VL
QS ALTQP AS VSGSPGQSITIS CTGTS KD VGG YNYVSW YQQHPGKAPKLMIYEVS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRRS TRVFGGGTKLT VL (set forth in SEQ ID NO: 38).
>GLA-P2 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIS AS G DNT YY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YIYM DPWGQGTM VTVS S (set forth in SEQ ID NO: 39).
>GLA-P2 VL QS ALTQP AS VSGSPGQSITIS CTGTS KD VGG YN Y VS W YQQHPGKAPKLMIYLGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRRS TR VFGGGTKLT VL (set forth in SEQ ID NO: 40).
>GLA-P3 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YG YHW VRQ APGKGLEW V ATIS AS GD NT Y Y ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y Y V YM DPWGQGTM VTVS S (set forth in SEQ ID NO: 41).
>GLA-P3 VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YN Y VS W YQQHPGKAPKLMIYE VS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRRS TR VFGGGTKLT VL (set forth in SEQ ID NO: 42).
>GLA-P4 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIS VS G DNTYY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YY Y MDPWGQGTM VT VS S (set forth in SEQ ID NO: 43).
>GLA-P4 VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YNYVSW YQQHPGKAPKLMIYLGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRRS TR VFGGGTKLT VL (set forth in SEQ ID NO: 44).
>GLA-P5 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW VATIP AS G DNTYY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YR Y MDPWGQGTM VTVS S (set forth in SEQ ID NO: 45).
>GLA-P5 VL
QS ALTQP AS VSGSPGQSITIS CTGTS KD VGG YNYVSW YQQHPGKAPKLMIYLGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRRS TR VFGGGTKLT VL (set forth in SEQ ID NO: 46). COMBINATION MUTANTS >GLA-S1 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIP AS G DNT Y Y ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YLYL FPWGQGTMVTVSS (set forth in SEQ ID NO: 47).
>GLA-S1 VL
QS ALTQP AS VS GS PGQS ΓΤΚ CTGTS KD VGG YNYVSWYQQHPGKAPKLMIYEVS KRP S G VS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YC S S FTRRS TR VFGGGTKLT VL (set forth in SEQ ID NO: 48).
>GLA-S2 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW VATIS AS G DNT YY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YIW WDVWGQGTMVTVSS (set forth in SEQ ID NO: 49).
>GLA-S2 VL
QS ALTQP AS VSGSPGQSITIS CTGTS KD VGG YN Y VS W YQQHPGKAPKLMIYLGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRLS QRVFGGGTKLT VL (set forth in SEQ ID NO: 50).
>GLA-S3 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGYHW VRQ APGKGLEW VATIS AS GD NT YY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YLYLF PWGQGTMVTVSS (set forth in SEQ ID NO: 51).
>GLA-S3 VL
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSKTP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD YYC S S FTRRS TR VFGGGTKLT VL (set forth in SEQ ID NO: 52). >GLA-S4 VH
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVATISVSGDNT YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGAYKGYYYYIWWDVWG QGTMVTVSS (set forth in SEQ ID NO: 53).
>GLA-S4 VL
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYLGSKRPSG VS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YC S S YTRLS QR VFGGGTKLT VL (set forth in SEQ ID NO: 54).
>GLA-S5 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIP AS G DNT Y Y ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YLYL FPWGQGTMVT VS S (set forth in SEQ ID NO: 55).
>GLA-S5 VL
QS ALTQP AS VSGSPGQSITIS CTGTS KD VGG YN Y VS W YQQHPGKAPKLMIYLGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S FTRRS TRVFGGGTKLT VL (set forth in SEQ ID NO: 56).
OPTIMIZED GLA-S4 CLONES >GLA-S4F1 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW VATIS VS G DNT YY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YIW WDVWGQGTMVTVSS (set forth in SEQ ID NO: 57).
>GLA-S4F1 VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YNYVSW YQQHPGKAPKLMIYLGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRRS QR VFGGGTKLT VL (set forth in SEQ ID NO: 58).
>GLA-S4F2 VH QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIS VS G DNT Y Y ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YIW WDVWGQGTMVTVSS (set forth in SEQ ID NO: 59).
>GLA-S4F2 VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YN Y VS W YQQHPGKAPKLMIYEGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRLS QRVFGGGTKLT VL (set forth in SEQ ID NO: 60).
>GLA-S4F3 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIS VS G DNT YY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YIW WDVWGQGTMVTVSS (set forth in SEQ ID NO: 61).
>GLA-S4F3 VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YN Y VS W YQQHPGKAPKLMIYE VS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRLS QRVFGGGTKLT VL (set forth in SEQ ID NO: 62).
>GLA-S4F4 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIS AS G DNT YY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YIW WDVWGQGTMVTVSS (set forth in SEQ ID NO: 63).
>GLA-S4F4 VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YN Y VS W YQQHPGKAPKLMIYLGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRLS QRVFGGGTKLT VL (set forth in SEQ ID NO: 64).
>GLA-S4F5 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW VATIP AS G DNT YY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YIW WDVWGQGTMVTVSS (set forth in SEQ ID NO: 65). >GLA-S4F5 VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YN Y VS W YQQHPGKAPKLMIYLGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRLS QRVFGGGTKLT VL (set forth in SEQ ID NO: 66).
>GLA-S4F6 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIS VS G DNT Y Y ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YIW WDVWGQGTMVTVSS (set forth in SEQ ID NO: 67).
>GLA-S4F6 VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YN Y VS W YQQHPGKAPKLMIYEGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRRS QRVFGGGTKLT VL (set forth in SEQ ID NO: 68).
>GLA-S4F8 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIS AS G DNT YY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YIW WDVWGQGTMVTVSS (set forth in SEQ OD NO: 69).
>GLA-S4F8 VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YNYVSW YQQHPGKAPKLMIYLGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRRS QRVFGGGTKLT VL (set forth in SEQ ID NO: 70).
>GLA-S4F9 VH
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVATIPASG
DNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGAYKGYYYYIW WDVWGQGTMVTVSS (set forth in SEQ ID NO: 71).
>GLA-S4F9 VL QSALTQPASVSGSPGQSmSCTGTSSDVGGYNYVSWYQQHPG APKLMIYLGSKRP SGVSNRFSGSKSGNTASLTiSGLQAEDEADYYCSSYTRRSQRVFGGGTKLTVL (set forth in SEQ ID NO: 72).
>GLA-S4F10 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V AT IS AS GDNT Y Y ADS VKGRFTIS RDNS KNTLYLQMNS LRAEDT A V Y YC AKG A YKG Y Y Y YrWWD VWGQGTM VT VS S (Set forth in SEQ ID NO: 73).
>GLA-S4F10 VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YN Y VS W YQQHPGKAPKLMIYEGS KRP S G VS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRLS QRVFGGGTKLT VL (Set forth in SEQ ID NO: 74).
>GLA-S4F11 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW VATIS AS G DNTYY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YIW WDVWGQGTMVTVSS (set forth in SEQ ID NO: 75).
>GLA-S4F11 VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YN Y VS W YQQHPGKAPKLMIYE VS KRP S G VS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRLS QRVFGGGTKLT VL (set forth in SEQ ID NO: 76).
>GLA-S4F12 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW VATIP AS G DNTYY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YIW WDVWGQGTMVTVSS (set forth in SEQ ID NO: 77).
>GLA-S4F12 VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YNYVSW YQQHPGKAPKLMIYEGS KRP S G VS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRLS QRVFGGGTKLT VL (set forth in SEQ ID NO: 78). >GLA-S4F13 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIP AS G DNT Y Y ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YIW WDVWGQGTMVTVSS (set forth in SEQ ID NO: 79).
>GLA-S4F13 VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YN Y VS W YQQHPGKAPKLMIYE VS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRLS QRVFGGGTKLT VL (set forth in SEQ ID NO: 80).
>GLA-S4F14 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW VATIS AS G DNT YY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YIW WDVWGQGTMVTVSS (set forth in SEQ ID NO: 81).
>GLA-S4F14 VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YN Y VS W YQQHPGKAPKLMIYEGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRRS QRVFGGGTKLT VL (set forth in SEQ ID NO: 82).
>GLA-S4F15 VH
QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW VATIS AS G DNT YY ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YIW WDVWGQGTMVTVSS (set forth in SEQ ID NO: 83).
>GLA-S4F15 VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YNYVSW YQQHPGKAPKLMIYE VS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRRS QRVFGGGTKLT VL (set forth in SEQ ID NO: 84).
>GLA-S4F16 VH QVQLVES GGG V VQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGKGLEW V ATIP AS G DNT Y Y ADS VKGRFTIS RDNS KNTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YIW WDVWGQGTMVTVSS (set forth in SEQ ID NO: 85).
>GLA-S4F16 VL
QS ALTQP AS VSGSPGQSITIS CTGTS S D VGG YN Y VS W YQQHPGKAPKLMIYEGS KRP S GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRRS QRVFGGGTKLT VL (set forth in SEQ ID NO: 86).
>GLA-S4F18 VH
QVQLVES GGGVVQPGRS LRLSC AAS GFTFS S YGMHW VRQ APGEGLEWVATIPAS GDNT YYADSVEGRFTISRDNSENTLYLQMNSLRAEDTAVYYCAKGAYKGYYYYIWWDVWG QGTMVTVSS (set forth in SEQ ID NO: 87).
>GLA-S4F18 VL
QS ALTQP AS VS GS PGQS ΓΤΚ CTGTS S D VGG YN Y VS W YQQHPGE APKLMIYE VS KRPS G V S NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YCS S YTRRS QRVFGGGTKLT VL (set forth in SEQ ID NO: 88).
>GLA-S4F19 VH
QVQLVES GGGVVQPGRS LRLS C A AS GFTFS S YGMHW VRQ APGEGLEWVATIS VS GD NT Y Y ADS VEGRFTIS RDNS ENTLYLQMNS LR AEDT A V Y YC AKG A YKG Y Y Y YIW WD VWGQGTMVTVSS (set forth in SEQ ID NO: 89).
>GLA-S4F19 VL
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGEAPKLMIYLGSKRPS GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD YYC S S YTRLS QRVFGGGTKLT VL (set forth in SEQ ID NO: 90).
>GLA-S4F20 VH
QVQLVES GGG V VQPGGS LTLS C A AS GFTFS S YGMHW VRQ APGEGLEWVATIS VS GD NT YY ADS VKGRFTIS RDNS ENTLYLQMNS LT AEDT A V YYC AKG A YKG YYY YIW WD VWGQGTMVTVSS (set forth in SEQ ID NO: 91). >GLA-S4F20 VL
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGEAPKLMIYLGSKRPS GVS NRFS GS KS GNT AS LTIS GLQ AEDE AD Y YC S S YTRLS QR VFGGGTKLT VL (set forth in SEQ ID NO: 92).
ANTIBODY HCDR AND LCDR SEQUENCES
[00227] The amino acid sequences of the VH and VL some of the anti-NOTCH4 antibodies listed above where aligned, and the CDRs determined following the Kabat numbering system. 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:
>HCDR1 (set forth in SEQ ID NO: 93)
SYGXiH; where
Xi is M or Y.
>HCDR2 (set forth in SEQ ID NO: 94)
TIX1X2SGDNTYYADSVX3G; where
Xi is S or P;
X2 is G; V; or A; and
X3 is K or E.
>HCDR3 (set forth in SEQ ID NO: 95)
GAX 1 KG Y Y Y YX2X3X4X5X6 ; where
Xi is V or Y;
X2 is Y; V; R; I; or L;
X3 is Y or W;
X4 is M; W; or L;
X5 is D; Y; or F; X6 is V or P.
>LCDR1 (set forth in SEQ ID NO: 96)
TGTSXiDVGGYNYVS; where
Xi is S or K;
>LCDR2 (set forth in SEQ ID NO: 97)
X1X2SKRPS; where
Xi is E or L;
X2 is G or V;
>LCDR3 (set forth in SEQ ID NO: 98)
SSX1TX2X3SX4RV; where
Xi is Y or F;
X2 is T or R;
X3 is R or L;
X4 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: GAYKGYYYYIWWDV (set forth in SEQ ID NO: 107) >GLA-S4F18 LCDR1: TGTSSDVGGYNYVS (set forth in SEQ ID NO: 108) >GLA-S4F18 LCDR2: EVSKRPS (set forth in SEQ ID NO: 109) >GLA-S4F18 LCDR3: SSYTRRSQRV (set forth in SEQ ID NO: 110) t-t-t- 28] The foregoing description of the specific embodiments will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance. The present invention is further described by the following claims.

Claims

1. A Notch4 binding molecule or antigen binding portion thereof comprising one or more of: an immunoglobulin variable heavy chain complementarity determining region 1
(HCDR1) 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 complementarity determining region 1
(LCDR1) having the amino acid sequence set forth in SEQ ID NO: 96;
an immunoglobulin variable light chain complementarity determining region 2
(LCDR2) having the amino acid sequence set forth in SEQ ID NO: 97; and an immunoglobulin variable light chain complementarity determining region 3
(LCDR3) having the amino acid sequence set forth in SEQ ID NO: 98.
2. 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 sequences 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: 65; SEQ ID NO: 67; SEQ ID NO: 69; SEQ ID NO: 71; SEQ ID NO: 73; SEQ ID NO: 75; SEQ ID NO: 77; SEQ ID NO: 79; SEQ ID NO: 81; SEQ ID NO: 83; SEQ ID NO: 85; SEQ ID NO: 87; SEQ ID NO: 89; and SEQ ID NO: 91; and/or a light chain variable domain (VL) having an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO: 30; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 36; SEQ ID NO: 38; SEQ ID NO: 40; SEQ ID NO: 42; SEQ ID NO: 44; SEQ ID NO: 46; SEQ ID NO: 48; SEQ ID NO: 50; SEQ ID NO: 52; SEQ ID NO: 54; SEQ ID NO: 56; SEQ ID NO: 58; SEQ ID NO: 60; SEQ ID NO: 62; SEQ ID NO: 64; SEQ ID NO: 66; SEQ ID NO: 68; SEQ ID NO: 70; SEQ ID NO: 72; SEQ ID NO: 74; SEQ ID NO: 76; SEQ ID NO: 78; SEQ ID NO: 80; SEQ ID NO: 82; SEQ ID NO: 84; SEQ ID NO: 86; SEQ ID NO: 88; SEQ ID NO: 90; and SEQ ID NO: 92.
3. 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 NO: 67; SEQ ID NO: 69; SEQ ID NO: 71; SEQ ID NO: 73; SEQ ID NO: 75; SEQ ID NO: 77; SEQ ID NO: 79; SEQ ID NO: 81; SEQ ID NO: 83; SEQ ID NO: 85; SEQ ID NO: 87; SEQ ID NO: 89; and SEQ ID NO: 91; and a light chain variable domain (VL) having an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO: 30; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 36; SEQ ID NO: 38; SEQ ID NO: 40; SEQ ID NO: 42; SEQ ID NO: 44; SEQ ID NO: 46; SEQ ID NO: 48; SEQ ID NO: 50; SEQ ID NO: 52; SEQ ID NO: 54; SEQ ID NO: 56; SEQ ID NO: 58; SEQ ID NO: 60; SEQ ID NO: 62; SEQ ID NO: 64; SEQ ID NO: 66; SEQ ID NO: 68; SEQ ID NO: 70; SEQ ID NO: 72; SEQ ID NO: 74; SEQ ID NO: 76; SEQ ID NO: 78; SEQ ID NO: 80; SEQ ID NO: 82; SEQ ID NO: 84; SEQ ID NO: 86; SEQ ID NO: 88; SEQ ID NO: 90; and SEQ ID NO: 92.
4. A Notch4 binding molecule or antigen binding portion thereof that competes or cross-competes with the binding molecule of any preceding claim.
5. The Notch4 binding molecule or antigen binding portion thereof of any preceding claim, which is selected from a murine antibody, a human antibody, a humanized antibody, a chimeric antibody, monoclonal antibody, a polyclonal antibody, a recombinant antibody, a bi- specific antibody, a multi-specific antibody, and an antigen-binding fragment thereof.
6. The Notch4 binding molecule or antigen binding portion thereof of any preceding claim, 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.
7. The binding molecule or antigen binding portion thereof of any preceding claim, comprising an immunoglobulin (Ig) heavy chain constant region.
8. The binding molecule or antigen binding portion thereof of any preceding claim, which specifically binds human Notch4 with an affinity characterized by a dissociation constant (KD) of about 0.2 nM, as measured by an Octet assay.
9. The binding molecule or antigen binding portion thereof of any preceding claim, which does not specifically bind to Notch 1.
10. A composition comprising the binding molecule or antigen binding fragment thereof of any preceding claim and a carrier.
11. A method for inhibiting or killing cancer stem cells (CSCs), the method comprising administering to the CSCs the binding molecule of any one of claims 1 to 9.
12. 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 claims 1 to 9 or the composition of claim 10.
13. A method of preventing recurrence of cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of the binding molecule or fragment thereof of any one of claims 1 to 9 or the composition of claim 10.
14. The method of any one of claims 11 to 13, 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.
15. The method of any one of claims 11 to 13, wherein the method comprises administering a second active agent.
16. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the binding molecule of any one of claims 1 to 9.
17. A host cell transformed with a nucleic acid molecule of claim 16.
18. A method of making a binding molecule or antigen binding fragment thereof that specifically binds Notch4, the method comprising culturing the host cell of claim 17 under suitable conditions for producing the binding molecule or antigen binding fragment thereof.
19. The method of claim 18, further comprising isolating the binding molecule.
20. A kit comprising the binding molecule or antigen binding fragment thereof of any one of claims 1 to 9.
PCT/US2017/047745 2016-08-22 2017-08-21 Binding molecules specific for notch4 and uses thereof Ceased WO2018039107A1 (en)

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