WO2017181049A1 - Agents anti-nucléoline - Google Patents

Agents anti-nucléoline Download PDF

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Publication number
WO2017181049A1
WO2017181049A1 PCT/US2017/027685 US2017027685W WO2017181049A1 WO 2017181049 A1 WO2017181049 A1 WO 2017181049A1 US 2017027685 W US2017027685 W US 2017027685W WO 2017181049 A1 WO2017181049 A1 WO 2017181049A1
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WIPO (PCT)
Prior art keywords
nucleolin
cancer
antibody
agent
instances
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PCT/US2017/027685
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English (en)
Inventor
Daniel Fernandes
Laura SCHWARTZ
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Charlestonpharma, Llc
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Publication of WO2017181049A1 publication Critical patent/WO2017181049A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C07K2317/90—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

  • the present disclosure provides a method for treating cancer in a subject in need thereof with an anti-nucleolin agent, comprising: a) measuring by an immunoassay a level of cytoplasmic nucleolin protein expression in a cancer cell from the subject, b) determining the level of cytoplasmic nucleolin protein expression to be less than about 1-10 times (e.g., less than 5 times) of a threshold value, wherein the threshold value is an average level of cytoplasmic nucleolin protein expression in cancer cells from two or more comparable subjects having the same cancer, and c) administering the anti-nucleolin agent to the subject in need thereof.
  • the comparable subjects are not resistant to the anti-nucleolin agent.
  • the level of cytoplasmic nucleolin protein expression is less than about 1, 2, 3, or 4 times, or less than about 10, 9, 8, 7, 6, or 5 times, of a threshold value. In some instances, the comparable subjects are resistant to the anti-nucleolin agent, and wherein the level of cytoplasmic nucleolin protein expression is lower than the threshold value. In some instances, the level of cytoplasmic nucleolin protein expression is less than 95%, 75%, 50%, 25%, or 10%, or less than about 90%, 80%, 70%, 60%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%, of a threshold value.
  • the present disclosure provides a method for predicting efficacy of treating cancer in a subject in need thereof before a treatment, comprising: a) measuring by an immunoassay a level of cytoplasmic nucleolin protein expression in a cancer cell from the subject, and b) determining the level of cytoplasmic nucleolin protein expression to be less than about 1-10 times (e.g., less than 5 times) of a threshold value, wherein the threshold value is an average level of cytoplasmic nucleolin protein expression in cancer cells from two or more comparable subjects having the same cancer.
  • the comparable subjects are not resistant to the anti-nucleolin agent.
  • the level of cytoplasmic nucleolin protein expression is less than about 4, 3, or 2 times, or less than about 10, 9, 8, 7, 6, or 5 times, of a threshold value. In some instances, the comparable subjects are resistant to the anti-nucleolin agent, and wherein the level of cytoplasmic nucleolin protein expression is lower than the threshold value. In some instances, the level of cytoplasmic nucleolin protein expression is less than 95%, 75%, 50%, 25%, or 10%, or less than about 90%, 80%, 70%, 60%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%, of the threshold value.
  • the present disclosure provides a method for assessing probability of resistance developed over time to treatment of cancer with an anti-nucleolin agent in a subject, comprising: a) measuring by an immunoassay a level of cytoplasmic nucleolin protein expression in a cancer cell from the subject, and b) determining the level of cytoplasmic nucleolin protein expression to be higher than a threshold value, wherein the threshold value is an average level of cytoplasmic nucleolin protein expression in cancer cells from two or more comparable subjects having the same cancer.
  • the comparable subjects are not resistant to the anti-nucleolin agent, and wherein the level of cytoplasmic nucleolin protein expression is > about (1-10) times (e.g., ⁇ 5 times) of the threshold value. In some instances, the level of cytoplasmic nucleolin protein expression is equal or greater than 5, 6, 7, 8, 9, or 10 times, or greater than 1, 2, 3, or 4 times, of the threshold value. In some instances, the comparable subjects are resistant to the anti-nucleolin agent.
  • the level of cytoplasmic nucleolin protein expression is higher than 105%, 110%, 125%, 150%, 175%, or 200%, or higher than about 900%, 800%, 700%, 600%, 500%, 450%, 400%, 350%, 300%, 250%, 200%, 190%, 180%, 170%, 160%, 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of the threshold value.
  • the present disclosure provides a method for treating cancer in a subject in need thereof with an anti-nucleolin agent, comprising: a) measuring by an immunoassay a level of cytoplasmic nucleolin protein expression and a level of surface nucleolin protein expression in a cancer cell from the subject, b) determining a ratio of the level of cytoplasmic nucleolin protein expression to the level of surface nucleolin protein expression to be lower than a threshold value, wherein the threshold value is about 3:1 or lower, about (4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1) or lower, and c) administering the anti-nucleolin agent to the subject in need thereof.
  • the threshold value is about 2:1 or lower, or about 1:1 or lower. In some instances, the threshold value is about (1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1:7, 1:8, 1 :9, or 1 : 10) or lower.
  • the present disclosure provides a method for predicting efficacy of treating cancer in a subject in need thereof before a treatment, comprising: a) measuring by an immunoassay a level of cytoplasmic nucleolin protein expression and a level of surface nucleolin protein expression in a cancer cell from the subject, and b) determining a ratio of the level of cytoplasmic nucleolin protein expression to the level of surface nucleolin protein expression to be lower than a threshold value, wherein the threshold value is about 3:1 or lower, about (4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1) or lower. In some instances, the threshold value is about 2:1 or lower, or about 1 : 1 or lower. In some instances, the threshold value is about (1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10) or lower.
  • the present disclosure provides a method for assessing probability of resistance developed over time to treatment of cancer with an anti-nucleolin agent in a subject, comprising: a) measuring by an immunoassay a level of cytoplasmic nucleolin protein expression and a level of surface nucleolin protein expression in a cancer cell from the subject, and b) determining a ratio of the level of cytoplasmic nucleolin protein expression to the level of surface nucleolin protein expression to be higher than a threshold value, wherein the threshold value is about 3:1 or lower, about (4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1) or lower. In some instances, the threshold value is about 2:1 or lower, or about 1:1 or lower. In some instances, the threshold value is about (1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1:7, 1:8, 1 :9, or 1 : 10) or lower.
  • an immunoassay disclosed herein comprises western blot, flow cytometry, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), or any combination thereof.
  • an immunoassay for detecting cell surface nucleolin is flow cytometry or IHC.
  • the cell is permeated or permeable for the
  • a subject disclosed herein is a human subject.
  • a cancer disclosed herein comprises one, two, three, four, five, or more types selected from the group consisting of lung cancer, skin cancer, breast cancer, liver cancer, colon cancer, lung cancer, kidney cancer, prostate cancer, leukemia, brain cancer, and pancreas cancer.
  • a cancer cell disclosed herein comprises one, two, three, four, five, or more types selected from the group consisting of lung cancer, skin cancer, breast cancer, liver cancer, colon cancer, lung cancer, kidney cancer, prostate cancer, leukemia, brain cancer, and pancreas cancer cells.
  • an anti-nucleolin agent binds to amino acid sequence SEQ ID NO:2. In some instances, an anti-nucleolin agent binds to an epitope within residues G300 to E466 of SEQ ID NO:l. In some instances, the epitope compries one or more amino acids selected from the group consisting of E453, R457, D455, K348, K427, G426, K403, Y402, and any
  • the anti-nucleolin agent is an antibody or fragment thereof that comprises a light chain that binds to E453, R457, D455, K348, or a combination thereof.
  • the anti-nucleolin agent is an isolated antibody or fragment thereof that comprises a heavy chain that binds to K427, G426, K403, Y402, or a combination thereof.
  • the antibody or fragment thereof comprises a light chain CDR1 that binds to E453, R457, or a combination thereof.
  • the antibody or fragment thereof comprises a light chain CDR2 that binds to D455.
  • the antibody or fragment thereof comprises a light chain CDR3 that binds to K348.
  • the antibody or fragment thereof comprises a heavy chain CDR1 that binds to K427. In some instances, the antibody or fragment thereof comprises a heavy chain CDR2 that binds to K427, G426, or a combination thereof. In some instances, the antibody or fragment thereof comprises a heavy chain CDR3 that binds to K403, Y402, or a combination thereof.
  • an anti-nucleolin agent disclosed herein is an isolated antibody or fragment thereof.
  • the anti-nucleolin agent is a polyclonal antibody or fragment thereof.
  • the anti-nucleolin agent is a monoclonal antibody or fragment thereof.
  • the anti-nucleolin agent is a human, humanized, or chimeric antibody or fragment thereof.
  • the anti-nucleolin agent is substantially nonimmunogenic to a human.
  • the anti-nucleolin agent is an IgGl, IgG2, IgG3, or IgG4 antibody or fragment thereof.
  • the anti-nucleolin agent is a fragment that is a Fab fragment, a Fab' fragment, a F(ab') 2 fragment, a Fv fragment, a diabody, a linear antibody, a single-chain antibody, or a multispecific antibody formed from an antibody fragment. In some instances, the anti-nucleolin agent is a fragment that comprises an antigen binding region.
  • an anti-nucleolin agent disclosed herein is nontotoxic to normal cells or normal tissues.
  • the anti-nucleolin agent is cytotoxic to a cancer cell.
  • the anti-nucleolin agent is cytotoxic to a cancer cell in presence of human serum.
  • the anti-nucleolin agent kills at least 10% of a population of cancer cells, when incubated with the cancer cells for a period of time.
  • the anti-nucleolin agent kills at least: 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%; or about 100% of the population of cancer cells.
  • the incubation is in presence of human serum.
  • the period of time is about 48-96 hours.
  • the cancer cell comprises one, two, or more types selected from the group consisting of A549, A375, MCF-7, Hep3B, HCT-116, NCI-H358, 786-0, DU-145, MDA-MB-231, MV4-11, U251, CG-EMT, MIA- PaCa2, and PANC-1 cells.
  • the anti-nucleolin agent does not penetrate into the nucleus of a cancer cell. In some instances, the anti-nucleolin agent localizes in the cytoplasm of a cancer cell.
  • a method disclosed herein comprises administering an anti-nucleolin agent to a subject, and the administering is an injection.
  • the administering is intravenous or subcutaneous injection.
  • the administering occurs 1-3 times per week.
  • the method reduces a size of tumor in the subject by at least: 15%, 25%, 50%, 75%, or 95%.
  • the tumor is a solid tumor.
  • the anti-nucleolin agent is administered at a dose of 0.15 mg to 5 mg per kg of body weight of the subject, e.g., from 0.5 mg to 2 mg per kg of body weight of the subject.
  • the anti-nucleolin agent is administered at a dose of about: 0.1-1, 0.1-2, 0.1-3, 0.1-4, 0.1-5, 0.1-6, 0.1-7, 0.1-8, 0.1-9, 0.1-10, 0.5-1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-25, 5- 10, 5-15, 5-20, or 5-25 per kg of body weight of the subject.
  • an anti-nucleolin agent disclosed herein is in a form of an
  • the immunocongjugate and is linked to a therapeutic agent.
  • the immunoconjugate is a fusion protein, and wherein the therapeutic agent is a polypeptide.
  • the anti-nucleolin agent is a bispecific antibody.
  • the anti-nucleolin agent is a probody.
  • the anti-nucleolin agent comprises an antigen-binding region that is activated by a tumor cell.
  • the antigen-binding region comprises a peptide linked to the N-terminus of a light chain through a protease cleavable linker.
  • the anti-nucleolin agent is linked covalently, noncovalently, or recombinantly to the therapeutic agent.
  • the therapeutic agent is a cytotoxic agent.
  • the cytotoxic agent is doxorubicin, calicheamicin, auristatin, maytansinoid, brentuximab vedotin, tubulysins, duocarmycins, camptothecin, SN-38, pyrrolobenzodiazepine, methotrexate, a- amanitin, ansamitocin, or any combination thereof.
  • the therapeutic agent is an immune stimulating agent.
  • the therapeutic agent is interleukin-2 (IL-2), an immunostimulatory nucleic acid molecule, granulocyte macrophage colony-stimulating factor, resiquimod, gardiquimod, phycocyanobilin, romiplostim, eltrombopag, or any combination thereof.
  • IL-2 interleukin-2
  • an immunostimulatory nucleic acid molecule granulocyte macrophage colony-stimulating factor
  • resiquimod granulocyte macrophage colony-stimulating factor
  • resiquimod granulocyte macrophage colony-stimulating factor
  • resiquimod granulocyte macrophage colony-stimulating factor
  • resiquimod granulocyte macrophage colony-stimulating factor
  • resiquimod granulocyte macrophage colony-stimulating factor
  • resiquimod granulocyte macrophage colony-stimulating factor
  • FIG. 1 is a line graph showing IC50 measurements of ANTIBODY I to MCF-7 parental cells and MCF-7 resistant cells (MCF-7R).
  • FIG. 2 is a set of images showing MCF-7 cells resistant to ANTIBODY I (MCF-7R cells) have increased expression of cytoplasmic nucleolin.
  • MCF-7 cells were exposed to increasing concentrations of ANTIBODY I from 0.5 to 4.0 ⁇ g/ml.
  • Western blot analysis revealed a 5.7-fold increase in SlOO cytoplasmic nucleolin in MCF-7R cells compared to parental MCF-7 sensitive cells. This is consistent with increased cytoplasmic nucleolin being available for oncogene mRNA stabilization.
  • FIG. 3 is an image of western blotting of ANTIBODY I in SlOO cytosolic and nuclear fractions of MCF-7 cells. It demonstrates that ANTIBODY I localizes in the cytoplasm of MCF- 7 cancer cells and does not penetrate into the nucleus of a cancer cell. This may explain why ANTIBODY I is not toxic to normal cells.
  • FIG. 4 is a set of images showing effects of ANTIBODY I on Bcl-2 protein levels in MCF-7 cells. This data confirm that ANTIBODY I leads to downregulation of Bcl-2 protein.
  • FIG. 5 is an image of molecular model of the binding of ANTIBODY I to human nucleolin RNA-binding domains 1 and 2.
  • Anti-nucleolin agents disclosed herein can include organic chemical compounds (e.g., MW less than 500 or 1000 Daltons), peptides or proteins (e.g., antibodies including fragments thereof), nucleic acids (e.g., aptamers).
  • the anti-nucleolin agents are microparticles or nanoparticles, e.g., nucleic acid (DNA or RNA) nanoparticles.
  • These anti-nucleolin agents exhibit cytotoxicity towards cells expressing nucleolin, such as cells involved in cancer, autoimmune disorders, and viral disorders. Therefore, the anti-nucleolin agents have therapeutic potential for certain forms of cancer, hyperproliferative and neovascular disorders and autoimmune diseases and can also be used as diagnostic agents.
  • the present disclosure includes specific agents that
  • nucleolin disclosed herein can be a cell-surface nucleolin or cytoplasmic nucleolin.
  • a nucleolin disclosed herein can mean a nucleolin fragment.
  • a nucleolin disclosed herein is a human nucleolin.
  • an anti-nucleolin agent e.g., antibody
  • sNu cell surface nucleolin
  • cNu cytoplasmic nucleolin
  • the agent exploits the temperature-dependent shuttling function of nucleolin to gain access to the cytoplasm of tumor cells and inhibits the stabilization of certain oncogene mRNAs by nucleolin. This induces apoptosis in tumor cells.
  • the agent penetrates tumors and exploits the shuttling function of sNu to gain intracellular access.
  • the agent inhibits the ability of cNu to stabilize oncogenic mRNAs that have the AU-rich nucleolin binding element in their 3'-UTRs (e.g., bcl-2, bcl-XL, IL-2 etc.). This induces apoptosis in most tumor cells and hematological malignancies but not in normal cells which lack sNu and cNu.
  • the anti-nucleolin agent may not be toxic to normal cells observed in vitro, ex vivo, and in vivo, since normal cells lack sNu and cNu.
  • the present disclosure provides a method for treating a subject in need thereof with an anti-nucleolin agent, comprising: a) measuring an expression level of a cytoplasmic nucleolin in a cell from the subject before treatment, b) comparing the expression level from step a) to a threshold value, and c) administering the anti-nucleolin agent to the subject to initiate the treatment if the expression level is below the threshold value.
  • the present disclosure provides a method for treating a subject in need thereof with an anti-nucleolin agent, comprising: a) measuring an expression level of a cytoplasmic nucleolin in a cell from the subject before treatment, and b) comparing the expression level from step a) to a threshold value, wherein the comparing is performed prior to administration of the anti-nucleolin agent to the subject.
  • the present disclosure provides a method for diagnosing a subject in need thereof before a treatment, comprising: a) measuring an expression level of a cytoplasmic nucleolin in a cell from the subject before a treatment to the subject, and b) comparing the expression level from step a) to a threshold value, wherein the comparing is performed prior to the treatment to the subject.
  • the present disclosure provides a method for diagnosing responsiveness of a subject in need thereof to an anti-nucleolin agent, comprising: a) measuring an expression level of a cytoplasmic nucleolin in a cell from the subject before a treatment, and b) comparing the expression level from step a) to a threshold value, wherein the comparing is performed prior to administration of the anti-nucleolin agent to the subject.
  • the present disclosure provides a method for selecting a subject suitable for a treatment with an anti-nucleolin agent, comprising: a) measuring an expression level of a cytoplasmic nucleolin in a cell from the subject, and b) selecting the subject for the treatment with the anti-nucleolin agent if the expression level is lower than a threshold value.
  • the present disclosure provides a method for identifying a subject unsuitable for a treatment with an anti-nucleolin agent, comprising: a) measuring an expression level of cytoplasmic nucleolin in a cell from the subject, and b) identifying the subject as unsuitable for the treatment with the anti-nucleolin agent if the expression level is higher than a threshold value.
  • an expression level disclosed herein is a protein, DNA, or RNA expression level.
  • a threshold value disclosed herein is an average expression level of a cytoplasmic nucleolin of standard subjects in need thereof.
  • a method disclosed herein further comprises measuring an expression level of a surface nucleolin in a cell from the subject and comparing a ratio of the expression level of surface nucleolin over the expression level of cytoplasmic nucleolin to a threshold ratio.
  • the threshold ratio is an average ratio of an expression level of a surface nucleolin over an expression level of a cytoplasmic nucleolin, of standard subjects in need thereof.
  • the present disclosure provides a kit for diagnosing responsiveness to a treatment, comprising: a) a reagent for measuring an expression level of cytoplasmic nucleolin, surface nucleolin, or a combination thereof, in a cell; and b) a comparator module.
  • kits for predicting responsiveness to treatment with an anti-nucleolin agent comprising: a) a reagent for measuring an expression level of cytoplasmic nucleolin, surface nucleolin, or a combination thereof, in a cell; b) a comparator module, and c) the anti-nucleolin agent.
  • an anti-nucleolin agent disclosed herein binds to SEQ ID NO:2 or an epitope within SEQ ID NO:2, i.e., residues #300 to #466 of SEQ ID NO:l.
  • the epitope has/spans about: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, or 80 amino acids.
  • the epitope compries one or more amino acids selected from the group consisting of E453, R457, D455, K348, K427, G426, K403, Y402, and any combination thereof.
  • the anti-nucleolin agent binds to E453, R457, or a combination thereof.
  • the anti-nucleolin agent binds to D455. In some instances, the anti-nucleolin agent binds to K348. In some instances, the anti-nucleolin agent binds to K427. In some instances, the anti-nucleolin agent binds to K427, G426, or a combination thereof. In some instances, the anti-nucleolin agent binds to K403, Y402, or a combination thereof. In some instances, the anti-nucleolin agent is an antibody or fragment thereof that comprises a complementarity determining region (CDR) in a light chain, wherein the CDR binds to E453, R457, D455, K348, or a combination thereof.
  • CDR complementarity determining region
  • the anti-nucleolin agent is an antibody or fragment thereof that comprises a CDR in a heavy chain, wherein the CDR binds to K427, G426, K403, Y402, or a combination thereof.
  • the anti-nucleolin agent is an antibody or fragment thereof, e.g., an IgG antibody or fragment thereof.
  • the isolated antibody or fragment thereof comprises an amino acid sequence (of heavy chain, light chain, variable region, and/or constant region) having at least 60% sequence identity to a corresponding amino acid sequence disclosed herein, for example SEQ ID NOs:4-10.
  • the isolated antibody or fragment thereof comprises: a heavy chain or light chain CDR (complementarity determining region) having at least 60% sequence identity to a corresponding amino acid sequence disclosed herein, for example in Table 6.
  • the at least 60% sequence identity is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, or 100% sequence identity.
  • the isolated antibody or fragment thereof comprises: a heavy chain that has an amino acid sequence of SEQ ID NO:4; and/or a light chain that has an amino acid sequence of SEQ ID NO: 8.
  • an amino acid sequence of SEQ ID NO:4 is encoded by nucleotide sequence SEQ ID NO:3.
  • an amino acid sequence of SEQ ID NO: 8 is encoded by nucleotide sequence SEQ ID NO:7.
  • the isolated antibody or fragment thereof comprises: a heavy chain variable region that has an amino acid sequence of SEQ ID NO: 5; a heavy chain constant region that has an amino acid sequence of SEQ ID NO:6; a light chain variable region that has an amino acid sequence of SEQ ID NO:9; a light chain constant region that has an amino acid sequence of SEQ ID NO: 10, or any combination thereof.
  • an anti-nucleolin agent disclosed herein has a Kd of about: 10 ⁇ , 1 ⁇ , 0.1 ⁇ , 0.05 ⁇ , 10 ⁇ , 5 ⁇ , 2.5 ⁇ , 1 ⁇ , or less, to human nucleolin or a fragment thereof, e.g., SEQ ID NO:2, or an epitope therein. In some instances, the Kd is about 2.5 nM or less.
  • a cell disclosed herein is a human cell or from a human. In some instances, the cell is a cancer cell. In some cases, a method or kit discloses herein uses cells from a blood sample or a tissue sample.
  • an expression level of an anti-nucleolin agent is at least 1 to 10 times higher than a threshold value. In some instances, the expression level is at least 6 times higher than the threshold value.
  • an anti-nucleolin agent disclosed herein suppresses expression of cytoplasmic nucleolin in a cell.
  • the expression of cytoplasmic nucleolin is suppressed by 1-10 times than a corresponding cell that is not treated with the anti-nucleolin agent.
  • the expression of cytoplasmic nucleolin is suppressed by about 6 times.
  • an anti-nucleolin agent disclosed herein is substantially nonimmunogenic to a human.
  • the anti-nucleolin agent is nontotoxic.
  • the anti- nucleolin agent is nontotoxic to normal cells or normal tissues.
  • the anti-nucleolin agent is cytotoxic.
  • the anti-nucleolin agent is cytotoxic to a tumor or cancer cell, e.g., in presence of human serum.
  • the anti-nucleolin agent exhibts or induces complement-dependent cytotoxicity to a tumor or cancer cell.
  • the anti-nucleolin agent exhibts or induces complement-independent cytotoxicity to a tumor or cancer cell. In some instances, the anti-nucleolin agent kills at least 10% of a population of tumor or cancer cells, when incubated with the tumor or cancer cells for a period of time. In some instances, the anti-nucleolin agent kills at least: 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the population of tumor or cancer cells. In some instances, the anti-nucleolin agent kills about 100% of the population of tumor or cancer cells. In some instances, the period of time is about 48-96 hours. In some instances, the period of time is about 96 hours.
  • the tumor or cancer cell is a breast cancer cell. In some instances, the tumor or cancer cell is MCF-7. In some instances, the tumor or cancer cell is an acute myeloid leukemia (AML) cell. In some instances, the tumor or cancer cell is pancreatic ductal adenocarcinoma, or HCT-116, NCI-H358, DU-145, MDA-MB-231, MV4-11, MIA-PaCa2, or PANC-1 cells. In some instances, the tumor or cancer cell is a prostate cancer cell. In some instances, the tumor or cancer cell is a hormone- refractory prostate cancer cell. In some instances, the tumor or cancer cell is a CG-EMT cell.
  • AML acute myeloid leukemia
  • the present disclosure provides a method of treating cancer, or killing cancer cells, with a method or kit disclosed herein. In some cases, the present disclosure provides a method or kit for use in treating cancer or killing cancer cells. In some cases, the present disclosure provides a use of a method or kit in the manufacture of a medicament. In some instances, the medicament is for treatment of cancer. In some instances, the medicament is for killing cancer cells.
  • the cancer or cancer cells include pancreatic ductal adenocarcinoma, breast cancer (cells), prostate cancer (cells), hormone-refractory prostate cancer (cells), or CG-EMT, MCF-7, AML, HCT-116, NCI-H358, DU-145, MDA-MB-231, MV4-11, MIA-PaCa2, or PANC-1 cells.
  • a cancer treated by an anti-nucleolin agent comprises one, two, three, four, five, or more types selected from the group consisting of human lung cancer, skin cancer, breast cancer, liver cancer, colon cancer, lung cancer, kidney cancer, prostate cancer, leukemia, brain cancer, and pancreas cancer.
  • a cancer cell killed by an anti-nucleolin agent comprises one, two, three, four, five, or more types selected from the group consisting of human lung cancer, skin cancer, breast cancer, liver cancer, colon cancer, lung cancer, kidney cancer, prostate cancer, leukemia, brain cancer, and pancreas cancer cells.
  • the cancer cell comprises one, two, three, four, five, or more types selected from the group consisting of A549, A375, MCF-7, Hep3B, HCT-116, NCI-H358, 786-0, DU-145, MDA-MB-231, MV4-11, U251, CG-EMT, MIA- PaCa2, and PANC-1 cells.
  • the present disclosure also provides for a method of modulating Bcl-2 protein level in a cancer cell, comprises administering to the cancer cell an anti-nucleolin agent disclosed herein.
  • the anti-nucleolin agent inhibits expression of Bcl-2 protein in the cancer cell.
  • an anti-nucleolin agent disclosed herein localizes in the cytoplasm of a cancer cell, and/or does not penetrate into the nucleus of a cancer cell.
  • the anti-nucleolin agent is an isolated antibody or fragment thereof that binds to human nucleolin.
  • Nucleolin is a multi-functional protein that binds to DNA, RNA and the external surface of the plasma membrane.
  • the ability of nucleolin to perform numerous and diverse functions within the cell is related to the multiple structural domains within the protein. Its negatively charged N-terminal domain regulates rDNA transcription by inducing nucleolar chromatin decondensation (Srivastava et al, 1989), while the central globular domain contains four RNA binding domains (RBDs) (Serin et al, 1997).
  • nucleolin via binding of its RBD and its RGG-rich C-terminal domains to pre-ribosomal RNA, functions as an assembly factor by bringing together the correctly folded rRNA and other components necessary for rRNA maturation and ribosome assembly (Ginisty et al, 2001). Nucleolin may also be involved in exporting ribosomes to the cytoplasm while shuttling between the cytoplasm and nucleus (Srivastava and Pollard, 1999). The nucleolin gene coding and protein sequences can be accessed at accession number NM_005381, XM 002342275, NP_005372 and XP_002342316. Nucleolin is also known as C23, FLJ45706, FLJ59041, and NCL.
  • Human NCL gene consists of 14 exons with 13 introns and spans approximately llkb.
  • the nucleolin protein contains several functional domains that mediate its functions.
  • the N- terminal part contains multiple phosphorylation sites and is rich in acidic amino acids.
  • the central part of nucleolin includes four RNA binding domains (RBD) and the C-terminal part contains glycine and arginine rich domain (termed RGG or GAR domain). (Farin et al, 2009) [00049]
  • RGG or GAR domain glycine and arginine rich domain
  • Nucleolin binds to the 3'-untranslated region (3'-UTR) of amyloid precursor protein mRNA and stabilizes this mRNA (Westmark and Malter, 2001). It is also required for the stabilization of IL- 2 mRNA that occurs during T cell activation (Chen et al, 2000). More recent studies have demonstrated that nucleolin binds to an A-U rich element (ARE) in the 3'-UTR of bcl-2 mRNA in HL-60 cells (Sengupta et al, 2004), chronic lymphocytic leukemia (CLL) cells (Otake et al,
  • ARE A-U rich element
  • binding of nucleolin to the bcl-2 ARE stabilizes bcl-2 mRNA by protecting it from ribonuclease degradation, while shRNA knockdown of nucleolin in MCF-7 cells leads to bcl-2 mRNA instability and decreased levels of bcl-2 protein (Soundararajan et al., 2008).
  • Nucleolin is present on the external surface of various types of tumor cells (Otake et al., 2007; Soundararajan et al., 2008; Chen et al., 2008; Hovanessian et al., 2000; Sinclair and O'Brien, 2002), despite its lack of a transmembrane domain or signal sequence (Srivastava et al., 1989; Lapeyre et al., 1987). Results show that nucleolin is not secreted from either MV4-11 cells or K-562 cells into the tissue culture medium (Soundararajan et al., 2009).
  • nucleolin undergoes extensive posttranslational modification (Srivastava et al., 1989; Lapeyre et al., 1987). It has been isolated as a glyco-phospho-protein from the surface of various types of proliferating cells (Hovanessian et al., 2000; Pfeifle and Heat, 1983). It is also possible that palmitoylation, prenylation, or myristoylation of nucleolin may allow for insertion or anchoring of these hydrophobic regions of the protein into the plasma membrane.
  • nucleolin functions as a shuttling protein between the plasma membrane and nucleus (Hovanessian et al., 2000). In proliferating tumor cells, nucleolin is often associated with endocytotic vesicles that invaginate from the plasma membrane (Hovanessian et al., 2000). Nucleolin also acts as a cell surface receptor for various ligands, since ligands bound to nucleolin within these vesicles become internalized in a temperature-dependent process.
  • plasma membrane nucleolin has been reported to function as a receptor for intimin- ⁇ of E.coli (Sinclair and O'Brien, 2002), the anti-HIV agent midkine (Said et al, 2002), laminin-1 (Kibbey et al, 1995), DNA nanoparticles (Chen et al,
  • Nucleolin is an important protein in the nucleolus involved in ribosome biogenesis and maturation in
  • nucleolin one important function of nucleolin is as a shuttling protein between cytoplasm and nucleus involving RNA processing and other cell biological processes. While in normal cellular physiology, nucleolin is localized predominantly in the nucleolus and cytoplasm, under certain conditions, especially in various disease states it has also been shown to be present in a phosphorylated form on the cell surface. In this regard, nucleolin in the cell membrane serves as a binding protein for a variety of ligands that drive cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis.
  • nucleolin is an excellent tumor antigen for antibody-based immunotherapy.
  • Nucleolin is overexpressed in the plasma membrane and cytoplasm a variety of human tumors including human chronic lymphocytic leukemia (CLL) (Otake et al, 2007), acute myeloid leukemia (AML) (Soundararajan et al, 2008), and breast cancer cells (Soundararajan et al, 2008), but not in normal CD19+ B cells (Otake et al, 2007), CD33+ myeloid cells (Gattoni-Celli et al, 2009), nor in normal mammary epithelial cells (Soundararajan et al, 2008).
  • CLL chronic lymphocytic leukemia
  • AML acute myeloid leukemia
  • breast cancer cells Soundararajan et al, 2008
  • AML blast cells from patients that engraft in NOD/SCID mice show intense nucleolin staining in the plasma membrane and cytoplasm while the normal mouse bone marrow cells and spleen lymphocytes were negative for nucleolin (Gattoni-Celli et al, 2009).
  • the nucleolin targeting aptamer, AS 1411 targets nucleolin.
  • Plasma membrane nucleolin was recently reported to be a receptor for AS 1411 in human MV4-11 leukemia cells (Soundararajan et al, 2009).
  • AS1411 binds to nucleolin that is overexpressed on the external surface of tumor cells and gains intracellular access when nucleolin is shuttled from the plasma membrane to the cytoplasm and nucleus. Data from NCI Tumor Cell Line Screen of AS1411 (> 50% growth inhibition at 6.3 uM). (Bates et al, 2009).
  • Anti-nucleolin antibodies can also exploit the shuttling function of plasma membrane nucleolin and become internalized after binding to cell surface nucleolin.
  • significance in the present application is the finding that the incubation of human tumor vascular endothelial cells, grown in nude mice or matrigel plugs, with a polyclonal anti-nucleolin antibody resulted in down regulation of bcl-2 mRNA levels and induction of apoptosis (Fogal et al, 2009). This suggests that anti-nucleolin antibodies can elicit anti-tumor effects through intracellular mechanisms, and/or to antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • any of the methods disclosed herein can be practiced with an anti - nucleolin antibody or fragment thereof.
  • an anti -nucleolin antibody or fragment thereof is used to detect a cell expressing nucleolin on its surface.
  • an anti-nucleolin antibody or fragment thereof is used to inhibit or kill a cell expressing nucleolin on its surface.
  • an anti -nucleolin antibody or fragment thereof is used to treat or prevent a neoplastic disease ⁇ e.g., cancer), an autoimmune disease, an inflammatory disease or condition, a respiratory disease, a viral infection, or macular degeneration.
  • an anti -nucleolin antibody or fragment thereof is conjugated, linked or fused to a toxin, chemotherapeutic, an immunostimulatory nucleic acid sequence ⁇ e.g., a CpG sequence), a radionuclide or an immunotherapeutic.
  • an anti -nucleolin antibody or fragment thereof is conjugated, linked or fused to a radionuclide, a fluorophore, a chemilluminescent compound, a fluorescent compound, or an enzyme.
  • anti - nucleolin antibody or fragment thereof is used to contact a cell expressing nucleolin on its surface.
  • the cell is pre-cancerous cell, a cancer cell or an immune cell.
  • the anti-nucleolin antibody fragment thereof is a human anti - nucleolin antibody or fragment. In some instances the anti -nucleolin antibody fragment thereof is a non-human anti -nucleolin antibody fragment thereof. In some instances the anti -nucleolin antibody fragment thereof is a chimeric anti -nucleolin antibody fragment thereof. In some instances the anti -nucleolin antibody fragment thereof is a humanized anti -nucleolin antibody fragment thereof. [00056] In some instances, an anti -nucleolin antibody fragment thereof is generated from an anti -nucleolin antibody. In some instances, the anti-nucleolin antibody fragment has the same binding specificity to nucleolin as the parent antibody.
  • the anti-nucleolin antibody fragment has improved binding specificity to nucleolin as the parent antibody. In some instances, the anti-nucleolin antibody fragment has the same binding affinity to nucleolin as the parent antibody. In some instances, the anti-nucleolin antibody fragment has improved affinity to nucleolin as the parent antibody. In some instances, an anti -nucleolin antibody includes an anti- nucleolin antibody fragment.
  • Antibody fragments comprise a portion of an intact antibody, for example comprising the antigen binding region thereof.
  • antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and
  • multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily.
  • Pepsin treatment yields an F(ab') 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • Fv is a minimum antibody fragment which contains a complete antigen-binding site.
  • a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable region in tight, non-covalent association.
  • scFv single-chain Fv
  • one heavy- and one light-chain variable region can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three HV s of each variable region interact to define an antigen-binding site on the surface of the VH-VL dimer.
  • HVRs confer antigen-binding specificity to the antibody.
  • a single variable region or half of an Fv comprising only three HVRs specific for an antigen has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the Fab fragment contains the heavy- and light-chain variable regions and also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • Single-chain Fv or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • diabodies refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable region (VH) connected to a light-chain variable region (VL) in the same polypeptide chain (VH-VL).
  • VH heavy-chain variable region
  • VL light-chain variable region
  • Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., PNAS USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier "monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
  • such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences.
  • the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of B cell or hybridoma clones, phage clones, or recombinant DNA clones.
  • a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence can also be a monoclonal antibody.
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • monoclonal antibody preparations can be advantageous in that they are typically uncontaminated by other immunoglobulins.
  • the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present disclosure may be made by a variety of techniques, including, for example, the hybridoma or B cell method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);
  • the hybridoma or B cell method e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);
  • polyclonal indicates the character of the antibody as being obtained from a source of a nonhomogeneous population of antibodies.
  • a polyclonal antibody comprises more than one antibody, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 antibodies.
  • the monoclonal antibodies herein include human, non-human, humanized and
  • Chimeric antibodies “Chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., PNAS USA 81 :6851-6855 (1984)). Chimeric antibodies include PRIMATIZED.RTM. antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.
  • humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable regions, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a "human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage- display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE.TM. technology). See also, for example, Li et al., PNAS USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
  • variable regions or variable + constant regions, can be cloned.
  • these antibodies can be prepared by a technique described in WO 2011/062997 (incorporated herein by reference in its entirely) that permits one to directly identify, isolate and characterize, for example in terms of amino acid sequence, human anti- nucleolin antibodies from immortalized antibody-producing cells prepared using human immune cells such as tonsil cells.
  • RNA-PCR kit RNA-PCR kit, Ohtsu
  • cDNAs were synthesized and were amplified by the polymerase chain reaction (PCR), with heavy and light chain primers specific for immunoglobulins (Ig).
  • PCR polymerase chain reaction
  • PCR protocol i.e., three cycles each of denaturation at 95°C for 1 min, annealing for 1 min, and elongation at 72°C for 2 min, for a total of 11 cycles.
  • the annealing temperature was varied from 65-55°C in steps of 1°C.
  • the touchdown cycles were followed by 25 cycles using an annealing temperature of 55°C.
  • the resultant PCR product was gel-purified in agarose and extracted using QIAquick spin-columns (Qiagen). The light chain and heavy chain Fc genes were then cloned into the Nhel/Ascl and the SfiVNotl sites of the expression vector pFabl-His2.
  • the ligated pFabl-His2 vectors with the light chain ( ⁇ and ⁇ ) and Fc heavy chain genes (y and ⁇ ) were introduced into competent E. coli JM109 cells (Toyobo, Osaka). After transformation, the E. coli cells were plated onto Luria-Bertani (LB)/ampicillin (50 ⁇ g/ml) plates. Isolated bacterial colonies were incubated at 30°C in 2 ml of Super Broth (SB) with ampicillin (50 ⁇ g/ml) and MgCl 2 (1.5 mM). Isopropyl-P-D-thiogalactopyranoside (IPTG) was used to induce production of the Fab protein. Cells from the bacterial cultures were pelleted, resuspended in 0.3 ml of B-PER (Pierce) with a protease inhibitor cocktail (Complete,
  • a heavy chain and a light chain can be in the same cloning construct.
  • a heavy chain and a light chain are found in different cloning constructs. Constructs containing sequences for heavy chain genes, light chain genes, or any combination thereof may be cloned simultaneously. Simultaneous cloning can comprise a vector containing both heavy and light chain genes or two separate vectors introduced simultaneously, each containing either a heavy chain or light chain.
  • constructs containing sequences for heavy chain genes, light chain genes, or any combination thereof may be cloned sequentially.
  • Sequential cloning may comprise introducing a vector containing a heavy chain gene followed by the introduction of a second vector containing a light chain gene.
  • a cell can be genetically modified with a vector containing gene sequences for both a heavy chain and light chain.
  • the nucleic acids for the light and heavy chains can be inserted into appropriate expression vectors and transferred into host cells (e.g., antibody-producing cells) that support production of antibodies.
  • host cells e.g., antibody-producing cells
  • Particular cell lines for production are 293 cells, CHO cells, COS cells or various forms of myeloma cells, some lacking IgG. These cells can be exploited for antibodies (e.g., human and/or monoclonal) production in two basic ways.
  • myelomas or immortalized cells can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion (e.g., a syngeneic mouse), or into an immunodeficient animal for injection of incompatible cells.
  • a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion (e.g., a syngeneic mouse), or into an immunodeficient animal for injection of incompatible cells.
  • the animals are primed with a hydrocarbon, especially oils such as pristane
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the transfected myeloma.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide antibodies (e.g., human and/or monoclonal) in high concentration.
  • the individual cell lines could be cultured in vitro, where the antibodies (e.g., human and/or monoclonal) are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
  • Antibodies produced by either means can be further purified, if desired, using ultra filtration, centrifugation and various chromatographic methods such as HPLC, affinity chromatography, or ion exchange chromatography. Fragments of the monoclonal antibodies of the present disclosure can be obtained from the monoclonal antibodies so produced by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction.
  • an anti-nucleolin antibody e.g., human and/or monoclonal
  • an immortalized B cell e.g., human B cell.
  • an anti-nucleolin antibody (e.g., human and/or monoclonal) is produced using a method such as one set forth in PCT/US2008/072124 or US patent application 12/671,936, which are herein incorporated by reference in their entirely.
  • the cDNA of an isolated anti-nucleolin antibody may be produced by cloning cDNA or genomic DNA encoding the immunoglobulin light and heavy chains of the anti-nucleolin antibody from a hybridoma cell (by fusing a specific antibody-producing B cell with a myeloma) that produces an antibody homolog.
  • an isolated anti-nucleolin antibody e.g., human and/or monoclonal
  • a B cell e.g., human B cell.
  • a cell is transfected by one or more
  • polynucleotide sequences isolated from a B cell where the polynucleotide sequence encodes for anti-nucleolin antibody (e.g., human and/or monoclonal).
  • the cDNA or genomic DNA encoding the polypeptides can be inserted into expression vectors so that both genes are operatively linked to their own transcriptional and translational expression control sequences.
  • the expression vector and expression control sequences can then be chosen to be compatible with the expression host cell used. In some instances, separate expression vectors are used for the heavy and light antibody chains.
  • Prokaryotic or eukaryotic cells can be used as expression hosts.
  • Expression in eukaryotic host cells may be suitable because such cells are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.
  • any antibody produced that is inactive due to improper folding may be renaturable according to well known methods (Kim and Baldwin, 1982). It is possible that the host cells will produce portions of intact antibodies, such as light chain dimers or heavy chain dimers, which also are antibody homologs according to the present disclosure.
  • a host cell is transformed with DNA encoding either the light chain or the heavy chain (but not both) of an antibody homolog.
  • Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for nucleolin binding.
  • the molecules expressed from such truncated DNA molecules are antibody homologs.
  • bifunctional antibodies are produced in which one heavy and one light chain are homologs of an anti- nucleolin antibody (e.g., human and/or monoclonal) and the other heavy and light chain are specific for an antigen other than nucleolin, or another epitope of nucleolin.
  • DNA encoding an isolated anti-nucleolin antibody (e.g., human and/or monoclonal) is transferred to a select mammalian cell line for expression in "production" or commercial amounts.
  • a select mammalian cell line for expression in "production" or commercial amounts.
  • Chinese Hamster Ovary cells (CHO cells) make excellent expression vehicles for recombinant or non-endogenous DNA. See U.S. Patent 4,816,567.
  • DHFR deficient CHO cell strains which permit the amplification of inserted DNA encoding specific proteins or DNA sequences, as set forth in U.S. Patent 5,981,214.
  • mammalian cell lines for expression in "production” or commercial amounts include, but are not limited to 293HEK cells, HeLa cells, COS cells, NIH3T3 cells, Jurkat Cells., NSO cells and HUVEC cells.
  • Other mammalian cell lines suitable for the expression of recombinant proteins have been identified in the literature, and can be equally suitable for use in the present disclosure of this application.
  • Antibodies may be produced using recombinant methods and compositions (See, e.g., U.S. Pat. No. 4,816,567).
  • an isolated nucleic acid encoding a nucleolin antibody described herein is provided.
  • Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody.
  • one or more vectors comprising such nucleic acid are provided.
  • a vector is a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked.
  • a host cell comprising such nucleic acid.
  • Host cells are cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include "transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages.
  • a host cell comprises (e.g., has been transformed with) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody or a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody.
  • the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NSO, Sp20 cell).
  • a method of making a nucleolin antibody comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell or host cell culture medium.
  • nucleolin antibody For recombinant production of a nucleolin antibody, an isolated nucleic acid encoding an antibody, e.g., as described above, is inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures.
  • Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.
  • antibodies may be produced in bacteria, e.g., when glycosylation and Fc effector function are not needed (See, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523; Charlton, Methods in Molecular Biology, Vol. 248, pp. 245-254 (2003)).
  • the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors (See, e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006)).
  • Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms, including invertebrates and vertebrates. Examples of invertebrates include plant and insect cells (See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429).
  • vertebrate cells examples include mammalian cell lines, monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TR1 cells; MRC 5 cells; FS4 cells; Chinese hamster ovary (CHO) cells, including DHFR " CHO cells; and myeloma cell lines such as Y0, NS0 and Sp2/0. (See, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (2003)
  • amino acid sequence variants of the antibodies provided herein are contemplated.
  • a variant typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions.
  • Such variants can be naturally occurring or can be synthetically generated, for example, by modifying one or more of the polypeptide sequences disclosed herein and evaluating one or more biological activities of the polypeptide as described herein and/or using any of a number of techniques well known in the art. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
  • Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any
  • deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding and/or potency.
  • antibody variants having one or more amino acid substitutions are provided.
  • Sites of interest for mutagenesis by substitution include the CDRs and Frameworks (FRs).
  • Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased
  • Hydrophobic amino acids include: Norleucine, Met, Ala, Val, Leu, and He.
  • Neutral hydrophilic amino acids include: Cys, Ser, Thr, Asn, and Gin.
  • Acidic amino acids include: Asp and Glu.
  • Basic amino acids include: His, Lys, and Arg.
  • Amino acids with residues that influence chain orientation include: Gly and Pro.
  • Aromatic amino acids include: Trp, Tyr, and Phe.
  • substitutions, insertions, or deletions may occur within one or more CDRs, wherein the substitutions, insertions, or deletions do not substantially reduce antibody binding to antigen.
  • conservative substitutions that do not substantially reduce binding affinity may be made in CDRs.
  • Such alterations may be outside of CDR "hotspots" or SDRs.
  • each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • Alterations may be made in CDRs, e.g., to improve antibody affinity. Such alterations may be made in CDR encoding codons with a high mutation rate during somatic maturation (See, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and the resulting variant can be tested for binding affinity.
  • Affinity maturation e.g., using error-prone PCR, chain shuffling, randomization of CDRs, or oligonucleotide-directed mutagenesis
  • can be used to improve antibody affinity See, e.g., Hoogenboom et al. in Methods in Molecular Biology 178:1- 37 (2001)).
  • CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling (See, e.g., Cunningham and Wells Science, 244:1081- 1085 (1989)).
  • CDR-H3 and CDR-L3 in particular are often targeted.
  • CDR-H3 and CDR-L3 are often targeted.
  • a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen.
  • Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution.
  • Variants may be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions and deletions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions and deletions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C- terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • intrasequence insertion variants of the antibody molecules include an insertion of 3 amino acids in the light chain.
  • terminal deletions include an antibody with a deletion of 7 or less amino acids at an end of the light chain.
  • an anti-nucleolin antibody may be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al. (1985); Hwang et al. (1980); and U.S. Patents 4,485,045 and 4,544,545.
  • Useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized
  • phosphatidylethanolamine PEG-PE
  • Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments of the antibody of the present disclosure can be conjugated to the liposomes as described in Martin et al. (1982) via a disulfide interchange reaction.
  • a chemotherapeutic agent such as Doxorubicin is optionally contained within the liposome. See Gabizon et al. (1989).
  • an anti-nucleolin antibody is used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug ⁇ e.g., a peptidyl
  • the enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active form exhibiting the desired biological properties.
  • Enzymes that can be useful include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as ⁇ -galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; ⁇ -
  • antibodies with enzymatic activity can be used to convert the prodrugs of the present disclosure into free active drugs (see, e.g., Massey, 1987).
  • Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a desired cell population.
  • the enzymes can be covalently bound to the anti-nucleolin antibodies by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above.
  • fusion proteins comprising at least the antigen binding region of an antibody of the present disclosure linked to at least a functionally active portion of an enzyme of the present disclosure can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al, 1984).
  • an anti-nucleolin antibody comprises an antibody fragment, rather than an intact antibody.
  • the antibody fragment may be modified in order to increase its serum half-life. This may be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment ⁇ e.g., by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle, e.g., by DNA or peptide synthesis). See WO 96/32478 published Oct. 17, 1996.
  • the salvage receptor binding epitope generally constitutes a region wherein any one or more amino acid residues from one or two loops of an Fc domain are transferred to an analogous position of the antibody fragment. In some instances, three or more residues from one or two loops of the Fc domain are transferred. In some instances, the epitope is taken from the CH2 domain of the Fc region ⁇ e.g., of an IgG) and transferred to the CHI, CH3, or V H region, or more than one such region, of the antibody. In some instances, the epitope is taken from the CH2 domain of the Fc region and transferred to the C L region or V L region, or both, of the antibody fragment.
  • an anti-nucleolin antibody is modified by covalent linkages.
  • Covalent linkages may include but are not limited to by chemical synthesis or by enzymatic or chemical cleavage of the antibody.
  • Other types of covalent modifications of the antibody are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues.
  • Exemplary covalent modifications of polypeptides are described in U.S. Patent 5,534,615, specifically incorporated herein by reference.
  • One type of covalent modification of the antibody comprises linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patents 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
  • an anti-nucleolin antibody (such as a human antibody) is modified by fusing, or conjugating it to another, heterologous polypeptide or amino acid sequence.
  • an anti-nucleolin antibody (such as a human antibody) is modified to comprise targeted immunoconjugate moieties which enable the effective generation of innate and adaptive immune responses against tumors or pathogens.
  • an isolated anti- nucleolin antibody ⁇ e.g., human and/or monoclonal) is modified to comprise targeted
  • an isolated anti-nucleolin antibody e.g., human and/or monoclonal
  • a B cell e.g., human B cell
  • targeted immunoconjugate moieties which enable the effective generation of innate and adaptive immune responses against tumors or pathogens.
  • the immunoconjugates can be capable of simultaneously satisfying multiple key requirements for mounting effective antibody- and/or cell-mediated immune responses against the targeted tumor or pathogen, which include but are not limited to : (i) Inducing or augmenting uptake and cross-presentation of tumor- or pathogen antigen(s) or antigenic determinant(s) by antigen presenting cells (APC)/dendritic cells (DC); (ii) promoting the maturation of dendritic cells (DCs) in the target cell milieu; (iii) providing CD4+ T cell help to generate CD8+ T cell memory and antibodies against the tumor or pathogen; (iv) sensitizing the targeted tumor cell to antibody dependent cell cytotoxicity (ADCC) and T-cell mediated death.
  • APC antigen presenting cells
  • DC dendritic cells
  • ADCC antibody dependent cell cytotoxicity
  • Such immunoconjugated antibodies can be used for targeted immunotherapy or immunoprophylaxis of neoplastic diseases, infectious diseases, and other disorders.
  • pattern recognition receptors such as Toll like Receptors
  • PAMPs pathogen- associated molecular patterns
  • a PAMP conjugated to an isolated anti- nucleolin antibody provides a moiety comprising a nucleic acid or protein that is recognized by a PRR, ultimately leading to an immune response which eliminates the target cell with the anti-nucleolin antibody bound to it.
  • known viral and pathogenic epitopes such as polyinosine-polycytidylic acid, lipopolysaccharide (LPS), lipid A, flagellin, GU-rich short single-stranded RNA, unmethylated CpG-oligodeoxynucleotides.
  • an anti-nucleolin antibody (such as a human antibody) is fused or conjugated with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind.
  • the epitope tag can be placed at the amino- or carboxyl-terminus of the anti-nucleolin antibody. The presence of such epitope-tagged forms of an anti-nucleolin antibody can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the anti-nucleolin antibody to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
  • Various tag polypeptides and their respective antibodies are well known in the art.
  • poly-histidine poly- his
  • poly-histidine-glycine poly-his-gly
  • tag polypeptides include the Flag-peptide (Hopp et al., 1988); the KT3 epitope peptide (Martin et al., 1992); an a-tubulin epitope peptide (Skinner et al., 1991); and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., 1990).
  • an anti-nucleolin antibody e.g., human and/or monoclonal or fragment is linked to a nanoparticle.
  • an isolated anti-nucleolin antibody e.g., human and/or monoclonal
  • an anti-nucleolin antibody e.g., human and/or monoclonal
  • a B cell e.g., human B cell
  • Cell surface nucleolin has been reported to serve as receptor for DNA
  • the antibody-nanoparticle conjugate can penetrate a cell expressing nucleolin on its surface more rapidly and extensively than the unconjugated antibody.
  • the cell is a cancer cell, tumor cell, virally infected cell, lymphocyte, or activated lymphocyte.
  • an anti-nucleolin agent e.g., human and/or monoclonal antibody
  • the cancer cell expresses nucleolin on its surface or in it cytoplasm.
  • an anti-nucleolin agent is used to reduce cell viability of a cancer cell in a subject sample by 10-100%, e.g., 20%-90%, or 30%-80%, as compared to cells not exposed to the anti-nucleolin agent.
  • an anti-nucleolin agent e.g., human and/or monoclonal antibody
  • the anti- nucleolin agent is administered to a human subject where the cancer is resistant to other cancer treatments.
  • cancers can be resistant to radiation therapy, chemotherapy, or biological therapy.
  • the immune system of the human subject is more tolerant to the isolated anti-nucleolin agent (e.g., human and/or monoclonal antibody) than a humanized anti-nucleolin agent or a chimeric anti-nucleolin agent.
  • an anti-nucleolin agent e.g., human and/or monoclonal antibody
  • an adjuvant therapy can include chemotherapy, radiation therapy, hormone therapy, targeted therapy, or biological therapy.
  • Adjuvant therapy as used herein refers to treatment given after the primary treatment to lower the risk that the cancer will come back.
  • an anti-nucleolin agent e.g., human and/or monoclonal antibody
  • an anti-nucleolin agent is used to inhibit or kill a cell of a non-malignant cell proliferative disorder wherein nucleolin is expressed on the cell surface or in the cytoplasm.
  • non-malignant cell proliferative disorders include but are not limited to warts, benign prostatic hyperplasia, skin tags, and non- malignant tumors.
  • an isolated anti-nucleolin agent e.g., human and/or monoclonal antibody
  • an isolated anti-nucleolin agent can be used to determine such cell proliferative disorders as benign prostatic hyperplasia or unwanted genital warts by targeting the undesirable cells that characterize such conditions for removal.
  • Expression of nucleolin on the cell surface of endothelial cells in tumors has been shown to be a unique marker of tumor angiogenesis (Christian et al, 2003).
  • an isolated anti-nucleolin agent e.g., human and/or monoclonal antibody
  • An angiogenic tumor as used herein a tumor cell with a proliferation of a network of blood vessels that penetrate into cancerous growths, supplying nutrients and oxygen and removing waste products.
  • an anti-nucleolin agent e.g., human and/or monoclonal antibody
  • Tumor hypoxia occurs in the situation where tumor cells have been deprived of oxygen. Tumor hypoxia can be a result of the high degree of cell proliferation undergone in tumor tissue, causing a higher cell density, and thus taxing the local oxygen supply.
  • an anti-nucleolin agent e.g., human and/or monoclonal antibody
  • a lymphocyte cell expressing nucleolin on its surface.
  • the lymphocyte cell comprises a B cell, T cell, or natural killer cell.
  • the lymphocyte cell comprises a CD4-positive or CD8-positive cells.
  • an anti-nucleolin agent e.g., human and/or monoclonal antibody
  • an activated lymphocyte or memory cell expressing nucleolin on its surface e.g., the activated lymphocyte comprises an activated B cell, T cell, or natural killer cell.
  • an anti-nucleolin agent e.g., human and/or monoclonal antibody
  • the subject has an autoimmune disorder, including but not limited to alopecia greata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, asthma, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg- Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, diabetes, type 1 diabetes mellitus, diabetic retinopathy, eosinophilic fascites, fibromyalgia-fibromyositis, glomerular ery
  • spondyloarthropathy undifferentiated arthropathy, arthritis, inflammatory osteolysis, graft versus host disease, urticaria, Vogt-Koyanagi-Hareda syndrome, chronic inflammatory pneumonitis, and chronic inflammation resulting from chronic viral or bacteria infections.
  • an anti-nucleolin agent e.g., human and/or monoclonal antibody
  • a virus including but not limited to cells infected with Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III); and other isolates, such as HIV-LP);
  • Retroviridae e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III); and other isolates, such as HIV-LP);
  • Picornaviridae e.g., polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g., coronaviruses); Rhabdoviradae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus);
  • Togaviridae e.g., equine encephalitis viruses, rubella viruses
  • Orthomyxoviridae e.g. influenza viruses
  • Bungaviridae e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses
  • Arenaviridae hemorrhagic fever viruses
  • Reoviridae e.g., reoviruses, orbiviurses and rotaviruses
  • Bimaviridae Hepadnaviridae (Hepatitis B virus);
  • Parvovirida Parvoviruses
  • Papovaviridae papilloma viruses, polyoma viruses
  • Adenoviridae most adenoviruses
  • Herpesviridae herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus
  • C-type group B including feline leukemia virus (FeLV), gibbon ape leukemia virus (GALV), spleen necrosis virus (SNV), reticuloendotheliosis virus (RV) and simian sarcoma virus (SSV)
  • D-type retroviruses include Mason-Pfizer monkey virus (MPMV) and simian retrovirus type 1 (SRV-1), the complex retroviruses including the subgroups of
  • Pneumococcus Pneumococcus, Streptococcus, Staphylcococcus, Diphtheria, Listeria, Erysipelothrix, Anthrax, Tetanus, Clostridium, Mixed Anaerobes, Neisseria, Salmonella, Shigella, Hemophilus,
  • Escherichia coli Escherichia coli, Klebsiella, Enterobacter, Serratia, Pseudomonas, Bordatella, Francisella tularensis, Yersinia, Vibrio cholerae, Bartonella, Legionella, Spirochaetes (Treponema,
  • a conjugate can comprise an antigen or cellular component as described herein, but in addition to a targeting moiety and an immunostimulatory nucleic acid molecule.
  • an anti-nucleolin agent e.g., human and/or monoclonal antibody
  • an isolated anti-nucleolin agent is linked, or covalently bound, or complexed to at least one therapeutic agent, such as a molecule or moiety.
  • Therapeutic agents comprise molecules having a desired activity, e.g., cytotoxic activity.
  • a therapeutic agent which can be attached to an antibody includes but is not limited to a toxin (such as a peptide immunotoxin that catalytically inhibit the elongation step of protein synthesis) an anti-tumor agent, a therapeutic enzyme, a radionuclide, an antiviral agent, a chelating agent as described herein, a cytokine, a growth factor, or a oligo- or polynucleotide.
  • Conjugation methodologies are similar to those described above for diagnostic agents.
  • an isolated anti-nucleolin agent e.g., human and/or monoclonal antibody
  • an enzymatically active toxin or fragment thereof examples include, but are not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), pokeweed antiviral protein, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, calicheamicins orthe tricothece
  • an isolated anti-nucleolin agent e.g., human and/or monoclonal antibody
  • a radionuclide e.g., 124 antimony, 125 antimony, 74 arsenic, 211 astatine, 103 barium, 140 barium, 7 beryllium, 206 bismuth, 207 bismuth, 212 Bi, 109 cadmium, 115 cadmium, 45 calcium, 14 carbon, 139 cerium, 141 cerium, 144 cerium, 137 cesium, 51 chromium, 36 chlorine, 56 cobalt, 57 cobalt, 58 cobalt, 60 cobalt, 67 copper, 169 erbium, 152 eurpium, 67 gallium, 153 gadolinium, 195 gold, 199 gold, 175 hafnium, 175 + 181 hafhium, 181 hafhium, 3 hydrogen, 123 io
  • Conjugates can be made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidyl-3-(2-pyri
  • a ricin immunotoxin can be prepared as described in Vitetta et al. (1987).
  • Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO 94/11026.
  • an isolated anti-nucleolin agent e.g., human and/or monoclonal antibody
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
  • lymphokines include lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin;
  • proinsulin relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-a and - ⁇ ; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin;
  • FSH follicle stimulating hormone
  • TSH thyroid stimulating hormone
  • LH luteinizing hormone
  • hepatic growth factor fibroblast growth factor
  • prolactin placental lactogen
  • tumor necrosis factor-a and - ⁇ mullerian-inhibiting substance
  • mouse gonadotropin-associated peptide inhibin
  • activin activin;
  • vascular endothelial growth factor integrin; thrombopoietin (TPO); nerve growth factors such as NGF- ⁇ ; platelet-growth factor; transforming growth factors (TGFs) such as TGF-a and TGF- ⁇ ; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-a, - ⁇ , and - ⁇ ; colony stimulating factors (CSFs) such as macrophage-CSF (M- CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; and other polypeptide factors including LIF and kit ligand (KL).
  • KL kit
  • an isolated anti-nucleolin agent e.g., human and/or monoclonal antibody
  • a chemotherapeutic agent e.g., human and/or monoclonal antibody
  • Categories of chemotherapeutic agents suitable for conjugation include, but are not limited to, alkylating agents, anthracyclines, cytoskeletal disruptors, epothilones, inhibitors of topoisomerase I, inhibitors of topoisomerase II, nucleoside and nucleotide analogs and precursor analogs, peptide antibiotics, platinum-based agents, retinoids, or vinca alkaloids and derivatives.
  • chemotherapeutic agents within these groups include, but are not limited to, actinomycin-D, all- trans retinoic acid azacitidine, adriamycin azathioprine, bleomycin, camptothecin, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, 5-fluorouracil (5FU), gemcitabine, hydroxyurea, hydrogen peroxide, idarubicin, imatinib, mechlorethamine, mercaptopurine, methotrexate, mitomycin C, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, tioguanine, val
  • an isolated anti-nucleolin agent e.g., human and/or monoclonal antibody
  • an anti-viral agent e.g., human and/or monoclonal antibody
  • the anti-viral agents include, but are not limited to, substrates and substrate analogs, inhibitors and other agents that severely impair, debilitate or otherwise destroy virus-infected cells.
  • Substrate analogs include amino acid and nucleoside analogs.
  • Substrates can be conjugated with toxins or other viricidal substances.
  • Inhibitors include integrase inhibitors, protease inhibitors, polymerase inhibitors and
  • transcriptase inhibitors such as reverse transcriptase inhibitors.
  • an anti-nucleolin agent for administration to a subject in need thereof, an anti-nucleolin agent will be suspended in a formulation suitable for administration to a host.
  • the agent is a monoclonal antibody.
  • the monoclonal antibody is an anti- nucleolin antibody.
  • the monoclonal anti-nucleolin antibody is a human monoclonal anti-nucleolin antibody.
  • Aqueous compositions of the present disclosure comprise an effective amount of an antibody dispersed in a pharmaceutically acceptable formulation and/or aqueous medium.
  • pharmaceutically and/or pharmacologically acceptable refer to compositions that do not produce an adverse, allergic and/or other untoward reaction when administered to an animal, and specifically to humans, as appropriate.
  • pharmaceutically acceptable carrier includes any solvents, dispersion media, coatings, antibacterial and/or antifungal agents, isotonic and/or absorption delaying agents and the like.
  • pharmaceutically acceptable carrier includes any solvents, dispersion media, coatings, antibacterial and/or antifungal agents, isotonic and/or absorption delaying agents and the like.
  • the use of such media or agents for pharmaceutical active substances is well known in the art.
  • Supplementary active ingredients can also be incorporated into the
  • compositions for administration to humans, preparations should meet sterility, pyrogenicity, general safety and/or purity standards as required by FDA Office of Biologies standards.
  • an anti-nucleolin agent e.g., human and/or monoclonal antibody
  • a sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. Any of these molecules can be administered to a patient alone, or in combination with other agents, drugs or hormones, in pharmaceutical compositions where it is mixed with suitable excipient(s), adjuvants, and/or pharmaceutically acceptable carriers.
  • the present disclosure the
  • pharmaceutically acceptable carrier is pharmaceutically inert.
  • Administration of pharmaceutical compositions is accomplished orally or parenterally. Methods of parenteral delivery include topical, intra-arterial (e.g., directly to a tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
  • these pharmaceutical compositions can contain suitable pharmaceutically acceptable carriers comprising excipients and other compounds that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of "Remington's
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient.
  • compositions for oral use may be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers include, but are not limited to sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound (i.e., dosage).
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
  • suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
  • Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds.
  • compositions of the present disclosure may be formulated in aqueous solutions, for example in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • compositions of the present disclosure may be manufactured in a manner similar to that known in the art (e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes).
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides,
  • the preparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1%- 2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5, which is combined with buffer prior to use.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • compositions suitable for use in the present disclosure include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • “Therapeutically effective amount” or “pharmacologically effective amount” are well recognized phrases and refer to that amount of an agent effective to produce the intended pharmacological result.
  • a therapeutically effective amount is an amount sufficient to ameliorate the symptoms of the disease being treated.
  • One useful assay in ascertaining an effective amount for a given application is measuring the effect on cell survival.
  • the amount actually administered will be dependent upon the individual to which treatment is to be applied, and can be an optimized amount such that the desired effect is achieved without significant side-effects.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in any appropriate animal model.
  • the animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a “therapeutically effective amount” is the quantity of compound which results in an improved clinical outcome as a result of the treatment compared with a typical clinical outcome in the absence of the treatment.
  • An “improved clinical outcome” refers, for, example, to a longer life expectancy, fewer complications, fewer symptoms, less physical discomfort and/or fewer hospitalizations as a result of the treatment.
  • Improved clinical outcome can be quantified as a certain percent of subjects receiving administration and improving in their disease state over certain period of time. The certain percent of subjects receiving administration and improving in their disease state may be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.
  • the certain percent of subjects receiving administration and improving in their disease state may be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 85%.
  • the certain percent of subjects receiving administration and improving in their disease state may be about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the certain period of time to measure improved clinical outcome may be 1, 2, 3, 4, 5, 6, or 7 days.
  • the certain period of time to measure improved clinical outcome may be 1, 2, 3, or 4 weeks.
  • the certain period of time to measure improved clinical outcome may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years.
  • an “improved clinical outcome” includes a longer life expectancy. It can also include slowing or arresting the rate of growth of a tumor, causing shrinkage in the size of the tumor, a decreased rate of metastasis or an improved quality of life (e.g., a decrease in physical discomfort or an increase in mobility).
  • an improved clinical outcome refers to an increase in the magnitude of the immune response in the individual, if the individual has a disease involving immune suppression.
  • “An improved clinical outcome” for individuals with suppressed immune systems can also refer to a lesser susceptibility to infectious diseases.
  • an improved clinical outcome can refer to a decrease in the magnitude of the immune response. In both cases, an improved clinical outcome can also involve an improvement in the quality of life, as described above.
  • the exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state (e.g., tumor size and location; age, weight and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy).
  • severity of the disease state e.g., tumor size and location; age, weight and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy).
  • Administration may be about every 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, or 24 h. every day, every other day, every week, every other week, every month, every other month, or any variation thereof.
  • Administration of a dosage form comprising an anti-nucleolin agent e.g., human and/or monoclonal antibody
  • Administration of a dosage form comprising an anti-nucleolin agent may be for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.
  • Administration of a dosage form comprising an anti-nucleolin agent may be for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
  • Administration of a dosage form comprising an anti-nucleolin agent may be for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years.
  • Administration of one or more agents can be intermittent; for example, administration can be once every two days, every three days, every five days, once a week, once or twice a month, and the like.
  • Long acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
  • Guidance as to particular dosages and methods of delivery is provided in the literature (see, U.S. Patents 4,657,760;
  • the dosage of a composition comprising anti-nucleolin agent is administered to a patient is about 0.1 mg/kg to 500 mg/kg of the patient's body weight.
  • the amount, forms, and/or amounts of the different forms can be varied at different times of administration.
  • Active ingredients may be entrapped in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) Active ingredients may be entrapped in microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems e.g., liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles (e.g., films or microcapsules).
  • the concentration of the anti-nucleolin agent is about 0.1 mg/ml to about 60 mg/ml, about 40 mg/ml to about 60 mg/ml, about 17 mg/ml to about 23 mg/ml, about 50 mg/ml, about 30 mg/ml, about 17 mg/ml to about 23 mg/ml, about 20 mg/ml, about 17 mg/ml, about 10 mg/ml, about 5 mg/ml, about 2 mg/ml, or about 1 mg/ml.
  • At least one tonicity agent e.g., D-mannitol
  • at least one buffering agent e.g., histidine, succinate
  • at least one buffering agent is present at a concentration of about 0.1 mM to about 25 mM, about 5 mM to about 15 mM, about 5 mM or about 10 mM.
  • an antioxidant e.g., methionine
  • a stabilizer e.g., polysorbate 80
  • a composition disclosed herein can have a pH of about 4 to about 8, about 4.5 to about 7.5, about 5 to about 7, about 5.5 to about 6.5, about 6.0 to about 6.5, about 6.2, about 6.0, or about 5.5.
  • an anti-nucleolin agent disclosed herein is present in a composition from about 0.1 mg/ml to about 100 mg/ml, from about 0.1 mg/ml to about 75 mg/ml, from about 0.1 mg/ml to about 50 mg/ml, from about 0.1 mg/ml to about 40 mg/ml, from about 0.1 mg/ml to about 30 mg/ml, from about 10 mg/ml to about 20 mg/ml, about 12 mg/ml to about 17 mg/ml, about 17 mg/ml to about 23 mg/ml, from about 20 mg/ml to 30 mg/ml, or higher, for example, up to about 100 mg/ml, about 200 mg/ml, about 500 mg/ml, or about 1000 mg/ml or more.
  • the antibody is present at about 1, 2, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 30 mg/ml. Ranges of values using a combination of any of the above recited values as upper and/or
  • compositions disclosed herein are stable to freezing, lyophilization and/or reconstitution. Moreover, exemplary instances are stable over extended periods of time. For example, the compositions are stable for at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 months. In some instances, the compositions are stable for at least about 12 months, for at least about 18 months, for at least about 24 months, or for at least about 30 months. In some instances, the composition may be stored at temperatures from about -80° C. to about 40° C, from about 0° C. to about 25° C, from about 0° C. to about 15° C, or from about 0° C.
  • the composition may be stored at about 0° C, 1° C, 2° C, 3° C, 4° C, 5° C, 6° C, 7° C, 8° C, 9° C. or 10° C. In some instances, the composition is stored at about 5° C. Generally, the composition is stable and retains biological activity at these ranges. Ranges intermediate to the above recited temperatures include from about 2° C. to about 17° C. Ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included.
  • Effective doses of the compositions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
  • the patient is a human but non-human mammals including transgenic mammals can also be treated. Treatment dosages need to be titrated to optimize safety and efficacy.
  • Exemplary dosages of an anti-nucleolin agent are from about 0.0001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 5 mg/kg, about 0.15 mg/kg to about 3 mg/kg, 0.5 mg/kg to about 2 mg/kg, for example about 1 mg/kg to about 2 mg/kg, or about 1 mg/kg to about 20 mg/kg of the host body weight.
  • dosages can be about 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0, 1.2, 1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.75, 1.8, 1.9, or 2.0 mg/kg.
  • dosages can be about 5, 10, 15 or 20 mg/kg.
  • Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis.
  • An exemplary treatment entails administration in multiple dosages over a prolonged period, for example, of at least six months. Additional exemplary treatment regimes entail administration once per every two weeks or once a month or once every 3 to 6 months.
  • Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kg weekly.
  • two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated.
  • An anti-nucleolin agent is usually administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibody in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of 1-1000 ⁇ g/ml and in some methods 25- 300 ⁇ g/ml. In some instances, antibody can be administered as a sustained release composition, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half- life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies.
  • compositions containing the present antibodies or a cocktail thereof are administered to a patient not already in the disease state to enhance the patient's resistance. Such an amount is defined to be a "prophylactic effective dose.”
  • prophylactic effective dose the precise amounts again depend upon the patient's state of health and general immunity, but generally range from 0.1 to 25 mg per dose, especially 0.5 to 2.5 mg per dose.
  • a relatively low dosage is administered at relatively infrequent intervals over a long period of time.
  • a relatively high dosage for example, from about 0.5 or 1 to about 200 mg/kg of anti-nucleolin agent per dose (for example 0.5, 1, 1.5, 2, 5, 10, 20, 25, 50, or 100 mg/kg), with dosages of from 5 to 25 mg/kg being more commonly used) at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and for example until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
  • compositions in unit dosage form for ease of administration and uniformity of dosage.
  • Compositions may be presented in capsules, ampules, lyophilized form, or in multi-dose containers.
  • the term "container” refers to something, for example, a holder, receptacle, or vessel, into which an object or liquid can be placed or contained, for example, for storage.
  • the unit dosage form may comprise any composition described herein including suspensions, solutions or emulsions of the active ingredient together with formulating agents such as suspending, stabilizing and/or dispersing agents.
  • the pharmaceutical dosage unit form may be added to an intravenous drip bag (for example a 50 ml, 100 ml, or 250 ml, or 500 ml drip bag) with a suitable diluent, for example, sterile pyrogen-free water or saline solution, before administration to the patient, for example, by intravenous infusion.
  • a suitable diluent for example, sterile pyrogen-free water or saline solution
  • Some pharmaceutical unit dosage forms may require reconstitution with a suitable diluent prior to addition to an intravenous drip bag, for example lyophilized forms.
  • the pharmaceutical unit dosage form is a container containing a composition described herein.
  • the container may be a 10 mL glass, type I, tubing vial.
  • the container should maintain the sterility and stability of the composition.
  • the vial may be closed with a serum stopper.
  • the container should be designed so as to allow for withdrawal of about 100 mg of composition or active ingredient (for example, for single use).
  • the container may be suitable for larger amounts, of composition or active ingredient, for example, from about 10 mg to about 5000 mg, from about 100 mg to about 1000 mg, and from about 100 mg to about 500 mg, about 40 mg to about 250 mg, about 60 mg to about 80 mg, about 80 mg to about 120 mg, about 120 mg to about 160 mg, or ranges or intervals thereof, for example, about 100 mg to about 200 mg. Ranges intermediate to the above recited amounts include from about 25 mg to about 195 mg. Ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included.
  • the composition often is supplied as a liquid in unit dosage form.
  • the present disclosure provides a kit including a pharmaceutical dosage unit form (for example, a container with a composition disclosed herein), and instructions for use.
  • the container and the kit may be designed to provide enough composition for multiple uses.
  • the kit may further include diluent.
  • the diluent may include excipients, separate or combined.
  • the diluent may include a tonicity modifier such as mannitol, a buffering agent such as histidine, a stabilizer such as polysorbate 80, an anti-oxidant such as methionine, and/or combinations thereof.
  • the diluent may contain other excipients, for example, lyoprotectant, as deemed necessary by one skilled in the art.
  • compositions of an anti-nucleolin agent as described herein are prepared by mixing such anti-nucleolin agent having the desired degree of purity with one or more optional pharmaceutically acceptable excipients (See, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized compositions or aqueous solutions.
  • pharmaceutically acceptable excipients See, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)
  • Exemplary pharmaceutical acceptable excipients include buffers (e.g., phosphate, citrate, and other organic acids); antioxidants (e.g., ascorbic acid and methionine); preservatives (e.g., octadecyldimethylbenzyl ammonium chloride); hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens (e.g., methyl or propyl paraben); catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight (less than about 10 residues) polypeptides; proteins, (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, aspara
  • Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents (e.g., soluble neutral-active hyaluronidase glycoproteins (sHASEGP)).
  • a sHASEGP is combined with one or more additional glycosaminoglycanases (e.g., chondroitinases).
  • the compositions may contain an additional agent for pH adjustment (for example, HC1) and a diluent (for example, water).
  • HC1 a diluent
  • different forms of histidine can be used for pH adjustment.
  • the excipients serve to maintain the stability and the biological activity of the antibody (for example, by maintaining the proper conformation of the protein), and/or to maintain pH.
  • the composition includes a buffering agent (buffer).
  • the buffer serves to maintain a physiologically suitable pH.
  • the buffer can serve to enhance isotonicity and chemical stability of the composition.
  • the composition should have a physiologically suitable pH.
  • the composition has a pH of about 5 to about 7, about 5.5 to about 6.5, for example about 6.0 to about 6.5.
  • the composition has a pH of about 6. Ranges intermediate to the above recited pH levels include about pH 5.2 to about pH 6.3, for example pH 6.0 or pH 6.2. Ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included.
  • the pH may be adjusted as necessary by techniques known in the art.
  • HC1 may be added as necessary to adjust the pH to desired levels or different forms of histidine may be used to adjust the pH to desired levels.
  • the buffer may include, but is not limited to, succinate (sodium or phosphate), histidine, phosphate (sodium or potassium), Tris (tris (hydroxymethyl)
  • the buffer is histidine (for example, L-histidine).
  • the buffer is succinate.
  • the composition includes an amino acid such as histidine that is present in an amount sufficient to maintain the composition at a physiologically suitable pH. Histidine is an exemplary amino acid having buffering capabilities in the physiological pH range. Histidine derives its buffering capabilities spanning from its imidazole group.
  • the buffer is L-histidine (base) (for example C6H9N 3 0 2 , FW: 155.15).
  • the buffer is L-histidine monochloride monohydrate (for example
  • the buffer is a mixture of L-histidine (base) and L-histidine monochloride monohydrate.
  • the buffer for example, L- histidine or succinate
  • concentration is present from about 0.1 mM to about 50 mM, from about 0.1 mM to about 40 mM, from about 0.1 mM to about 30 mM, about 0.1 mM to about 25 mM, from about 0.1 mM to about 20 mM, or from about 5 mM to about 15 mM, for example 5 mM or 10 mM.
  • the buffer may be present at about 6 mM, 7 mM, 8 mM, 9 mM, 11 mM, 12 mM, 13 mM, 14 mM, or 15 mM. In some instances, the buffer is present at about 10 mM. Ranges intermediate to the above recited concentrations include about 12 mM to about 17 mM. Ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included. In certain instances, the buffer is present in an amount sufficient to maintain a physiologically suitable pH.
  • the composition includes a tonicity agent.
  • the tonicity agent contributes to maintaining the isotonicity of the composition, and to maintaining protein levels.
  • the tonicity agent contributes to preserving the level, ratio, or proportion of the therapeutically active polypeptide present in the composition.
  • the term "tonicity" refers to the behavior of biologic components in a fluid environment or solution. Isotonic solutions possess the same osmotic pressure as blood plasma, and so can be intravenously infused into a subject without changing the osmotic pressure of the subject's blood plasma. In some instances, tonicity agent is present in an amount sufficient to render the composition suitable for intravenous infusion.
  • the tonicity agent serves as a bulking agent as well.
  • the agent may allow the protein to overcome various stresses such as freezing and shear.
  • the tonicity agent may include, but is not limited to, CaCl 2 , NaCl, MgCl 2 , lactose, sorbitol, sucrose, mannitol, trehalose, raffinose, polyethylene glycol, hydroxyethyl starch, glycine and mixtures thereof.
  • the tonicity agent is mannitol (for example, D-mannitol, for example, C 6 H 14 0 6 , FW: 182.17).
  • the tonicity agent is present at about 2% to about 6% w/v, or about 3% to about 5% w/v. In some instances, the tonicity agent is present at about 3.5% to about 4.5% w/v. In some instances, the tonicity agent is percent at about 20 mg/ml to about 60 mg/ml, at about 30 mg/ml to about 50 mg/ml, or at about 35 mg/ml to about 45 mg/ml. For example, the tonicity agent is present at about 4% w/v or at about 40 mg/ml. In some instances, the tonicity agent is present at about 6% w/v. In some instances, the tonicity agent is present at about 10% w/v.
  • Ranges intermediate to the above recited concentrations include about 3.2% to about 4.3% w/v or about 32 to about 43 mg/ml. Ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included.
  • the tonicity agent should be present in a sufficient amount so as to maintain tonicity of the composition.
  • the composition includes an anti-oxidant so as to, in part, preserve the composition (for example, by preventing oxidation).
  • the anti-oxidant may include, but is not limited to, GLA (gamma-linolenic acid)-lipoic acid, DHA (docosahexaenoic acid)-lipoic acid, GLA-tocopherol, di-GLA-3,3'-thiodipropionic acid and in general any of, for example, GLA, DGLA (dihomo-gamma-linolenic acid), AA (arachidonic acid), SA (salicylic acid), EPA
  • phenolic anti-oxidants for example, eugenol, carnosic acid, caffeic acid, BHT (butylated hydroxyanisol), gallic acid, tocopherols, tocotrienols and flavenoid anti-oxidants (such as myricetin and fisetin)
  • polyenes for example, retinoic acid
  • unsaturated sterols for example, A 5 -avenosterol
  • organosulfur compounds for example, allicin
  • terpenes for example, geraniol, abietic acid
  • amino acid antioxidants for example, methionine, cysteine, carnosine
  • the anti-oxidant is ascorbic acid.
  • the anti-oxidant is methionine, or an analog thereof, for example,
  • the anti-oxidant for example, a methionine such as L-methionine, for example
  • the anti-oxidant may be present at about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, or 15 mM.
  • the anti-oxidant is present at about 10 mM.
  • the anti-oxidant is present at about 15 mM. Ranges intermediate to the above recited concentrations include about 12 mM to about 17 mM. Ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included. In certain instances, the anti-oxidant should be present in a sufficient amount so as to preserve the composition, in part, by preventing oxidation.
  • the composition includes a stabilizer, also known as a surfactant.
  • Stabilizers are specific chemical compounds that interact and stabilize biological molecules and/or general pharmaceutical excipients in a composition.
  • stabilizers may be used in conjunction with lower temperature storage.
  • Stabilizers generally protect the protein from air/solution interface induced stresses and solution/surface induced stresses, which may otherwise result in protein aggregation.
  • the stabilizer may include, but is not limited to, glycerin, polysorbates such as polysorbate 80, dicarboxylic acids, oxalic acid, succinic acid, adipic acid, fumaric acid, phthalic acids, and combinations thereof. In some instances, the stabilizer is polysorbate 80.
  • the stabilizer (for example, polysorbate 80) concentration is about 0.001% w/v to about 0.01% w/v, about 0.001% w/v to about 0.009% w/v, or about 0.003% w/v to about 0.007% w/v.
  • the stabilizer concentration is about 0.005% w/v.
  • the stabilizer is present at about 0.01% w/v. Ranges intermediate to the above recited concentrations include about 0.002% w/v to about 0.006% w/v. Ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included.
  • the composition is substantially free of preservatives. In some instances, preservatives may be added as necessary. For example, cryoprotectants or lyoprotectants may be included, for example, should the composition be lyophilized.
  • an isolated anti-nucleolin agent e.g., human and/or monoclonal antibody
  • a variety of cells including both cancerous and non-cancerous cells.
  • the isolated anti-nucleolin antibody e.g., human and/or monoclonal
  • the isolated anti-nucleolin antibody is a polyclonal antibody.
  • cancer is described previously herein.
  • Examples of types cancer that can be inhibited or treated include, but are not limited to: Acute Lymphoblastic Leukemia; Myeloid Leukemia; Acute Myeloid Leukemia; Chronic Myeloid Leukemia; Adrenocortical Carcinoma Adrenocortical Carcinoma; AIDS-Related Cancers; AIDS- Related Lymphoma; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Basal Cell Carcinoma; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer; Bone Cancer, osteosarcoma/Malignant Fibrous Histiocytoma; Brain Stem Glioma; Brain Tumor; Brain Tumor, Brain Stem Glioma; Brain Tumor, Cerebellar Astrocytoma; Brain Tumor, Cerebral Astrocytoma/Malignant Glioma; Brain Tumor, Ependymoma; Brain Tumor,
  • Medulloblastoma Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors; Brain Tumor, Visual Pathway and Hypothalamic Glioma; Breast Cancer, Female; Breast Cancer, Male; Bronchial Adenomas/Carcinoids; Burkitt's Lymphoma; Carcinoid Tumor; Central Nervous System Lymphoma; Cerebellar Astrocytoma; Cerebral Astrocytoma/Malignant Glioma; Cervical Cancer; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic
  • Leukemia Acute Lymphoblastic
  • Leukemia Acute Lymphoblastic
  • Leukemia Acute Myeloid
  • Leukemia Acute Myeloid
  • Leukemia Chronic Lymphocytic
  • Lymphoma Primary Central Nervous System; Macroglobulinemia, Waldenstrom's; Malignant Fibrous Histiocytoma of Bone/Osteosarcoma; Medulloblastoma; Melanoma; Melanoma, Intraocular (Eye); Merkel Cell Carcinoma; Mesothelioma, Adult Malignant; Mesothelioma; Metastatic Squamous Neck Cancer with Occult Primary; Multiple Endocrine Neoplasia
  • Pancreatic Cancer Pancreatic Cancer; Pancreatic Cancer; Pancreatic Cancer, Islet Cell; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineoblastoma and Supratentorial Primitive Neuroectodermal Tumors; Pituitary Tumor; Plasma Cell Neoplasm/Multiple Myeloma; Pleuropulmonary
  • Cancer cells known to express nucleolin include lung cancers (e.g., non-small cell lung cancers), breast cancers, prostate cancers, colon cancers, pancreatic cancers, renal cell carcinomas, ovarian cancers, leukemias (e.g., AML, CLL), melanomas, glioblastomas, neuroblastomas, sarcomas and gastric cancers.
  • lung cancers e.g., non-small cell lung cancers
  • breast cancers e.g., prostate cancers, colon cancers, pancreatic cancers, renal cell carcinomas, ovarian cancers
  • leukemias e.g., AML, CLL
  • melanomas e.g., glioblastomas
  • neuroblastomas e.g., sarcomas
  • gastric cancers e.g., pneumas, pneumomediastinum, pneumas, pneumomediastinum, pneumomediastinum, pneumomediasti
  • an antibody of the present disclosure is used in a treatment for subjects with hyper-immune and hyper-angiogenic diseases, the latter being described in U.S. Patent
  • the methods for treating a cancer provided herein inhibit, reduce, diminish, arrest, or stabilize a cancer cell associated with cancer. In some instances, the methods for treating cancer provided herein inhibit, reduce, diminish, arrest, or stabilize the symptoms associated with the cancer or two or more symptoms thereof. In some examples, the methods for treating cancer provided herein cause the reduction in the number of cancer cells and/or one or more symptoms associated with the cancer.
  • the methods for treating cancer provided herein maintain the number of cancer cells so that they do not increase, or so that the number of cancer cells increases by less than the increase of a number of cancer cells after administration of a standard therapy as measured by, for example, conventional methods available to one of skill in the art, such as ultrasound, CT Scan, MRI, dynamic contrast-enhanced MRI, or PET Scan.
  • the methods for treating cancer provided herein decrease the number of cancer cells.
  • the methods for treating cancer provided herein reduce the formation of cancer cells.
  • the methods for treating cancer provided herein eradicate, remove, or control primary, regional and/or metastatic cancer cells associated with the cancer.
  • the methods for treating cancer provided herein decrease the number or size of metastases associated with the cancer.
  • methods for treating cancer reduce the number of cancer cells, inhibit or decrease cancer cell metabolism, and/or reduce cancer cell perfusion in a subject by an amount in the range of about 5- about 10%, about 5- about 20%, about 10- about 20%, about 15- about 20%, about 10- about 30%, about 20- about 30%, about 20- about 40%, about 30- about 40%, about 10- about 50%, about 20- about 50%, about 30- about 50%, about 40- about 50%, about 10- about 60%, about 20- about 60%, about 30- about 60%, about 40- about 60%, about 50- about 60%, about 10- about 70%, about 20- about 70%, about 30- about 70%, about 40- about 70%, about 50- about 70%, about 60- about 70%, about 10- about 80%, about 20- about 80%, about 30- about 80%, about 40- about 80%, about 50- about 80%, about 60- about 80%, about 70- about 80%, about 10- about 90%, about 20- about 90%, about 30- about 90%, about 40- about 90%, about 50- about 90%, about 60- about 90%, about 30- about 90%, about 40- about
  • biological sample means any fluid or other material derived from the body of a normal or diseased subject, such as blood, serum, plasma, lymph, urine, saliva, tears, cerebrospinal fluid, milk, amniotic fluid, bile, ascites fluid, pus, and the like. Also included within the meaning of the term “biological sample” is an organ or tissue extract and culture fluid in which any cells or tissue preparation from a subject has been incubated.
  • the biological samples can be any samples from which genetic material can be obtained.
  • Biological samples can also include solid or liquid cancer cell samples or specimens.
  • the cancer cell sample can be a cancer cell tissue sample. In some instances, the cancer cell tissue sample can obtained from surgically excised tissue.
  • Exemplary sources of biological samples include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy.
  • the biological samples comprise fine needle aspiration samples.
  • the biological samples comprise tissue samples, including, for example, excisional biopsy, incisional biopsy, or other biopsy.
  • the biological samples can comprise a mixture of two or more sources; for example, fine needle aspirates and tissue samples. Tissue samples and cellular samples can also be obtained without invasive surgery, for example by punctuating the chest wall or the abdominal wall or from masses of breast, thyroid or other sites with a fine needle and
  • a biological sample is a bone marrow aspirate sample.
  • a biological sample can be obtained by methods known in the art such as the biopsy methods provided herein, swabbing, scraping, phlebotomy, or any other suitable method.
  • the biological samples obtained can be used in fresh, frozen, or fixed (e.g.,
  • fresh, frozen and fixed materials are suitable for various RNA and protein assays, generally, fresh tissues can be for ex vivo
  • Tissue obtained by biopsy is often fixed, usually by formalin, formaldehyde, or gluteraldehyde, for example, or by alcohol immersion.
  • Fixed biological samples are often dehydrated and embedded in paraffin or other solid supports. See the reference Plenat et ah, 2001, Ann. Pathol. 21 :29-47.
  • Non-embedded, fixed tissue, as well as fixed and embedded tissue, can be used in the present methods. Solid supports for embedding fixed tissue can be removed with organic solvents to enable subsequent rehydration of preserved tissue.
  • the assay includes a step of cell or tissue culture.
  • cells from a biopsy can be disaggregated using enzymes (such as collagenase and hyaluronidase) and or physical disruption (e.g., repeated passage through a 25-gauge needle) to dissociate the cells, collected by centrifugation, and resuspended in desired buffer or culture medium for culture, immediate analysis, or further processing.
  • enzymes such as collagenase and hyaluronidase
  • physical disruption e.g., repeated passage through a 25-gauge needle
  • Detection in a biological sample can be performed by, for example, direct
  • the biological sample for pharmacodynamic analysis can be a blood sample or a cancer cell specimen from the subject, for example, a biological sample for pharmacodynamic analysis can be a blood sample or a cancer cell specimen from the human subject.
  • the methods of the disclosure can optionally include collecting one or more biological sample from a subject at one or more specific time point before analyzing the one or more biological sample for levels of an anti-nucleolin agent and/or cytoplasmic or surface nucleolin.
  • the biological samples can be collected any time before, during, or after the administration of the agent to the subject.
  • the biological sample can be collected within 72 h, within 48 h, within 24 h, within 23 h, within 22 h, within 21 h, within 20 h, within 19 h, within 18 h, within 17 h, within 16 h, within 15 h, within 14 h, within 13 h, within 12 h, within 11 h, within 10 h, within 9 h, within 8 h, within 7 h, within 6 h, within 5 h, within 4 h, within 3 h, within 2 h, within 1 h, within 30 min, within 15 min of, or immediately after or before the administration of the agent to the subject.
  • the method of the disclosure can optionally include clinical activity analysis of an agent disclosed herein. Accordingly, the methods can comprise analyzing one or more biological samples collected from the subject at one or more specific time points. Any appropriate analytical procedure can be used for the analysis of the biological samples. For example, imaging techniques like radiographs, ultrasound, CT scan, PET scan, MRI scan, chest x-ray, laparoscopy, complete blood count (CBC) test, bone scanning and fecal occult blood test can be used. Further analytical procedures that can be used include blood chemistry analysis, chromosomal translocation analysis, needle biopsy, tissue biopsy, fluorescence in situ
  • the method can further comprise tabulating and/or plotting results of the analytical procedure.
  • cell surface nucleolin are extracted and measured using one or more of the following steps.
  • Cell surface nucleolin can be extracted by first biotinylating the cells and then pulling down surface proteins on streptavidin agarose beads.
  • cell surface proteins can be biotinylated using EZ-link-sulfo-NHS-biotin.
  • Biotin coupled to highly reactive N- hydroxysuccinimide ester group can be used as a membrane-impermeant probe to covalently tag cell surface proteins.
  • Unbound biotin can be removed from cells by washing once or twice with a buffer, e.g., 0.1 M glycine in D-PBS (PBS supplemented with 0.1 mM CaCl 2 and 0.1 mM MgCl 2 ), or D-PBS without glycine.
  • Cells can be solubilized in RIPA buffer (e.g., 50 mM Tris- HC1 pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate and protease inhibitors) to generate whole cell extracts.
  • the Bradford assay can be performed to quantify the concentration of total protein in the cell extracts.
  • monomeric avidin agarose beads can be used. Before use, nonreversible biotin binding sites on the beads can be blocked with 2 mM D-biotin in PBS for 15 minutes at room temperature, then washed three times in 0.1 M glycine in D-PBS, followed by washing four times in D-PBS without glycine. Equal amounts protein from the cell extracts can be loaded onto avidin beads, and biotinylated proteins can be separated from non-biotinylated proteins by batch affinity chromatography (250 ⁇ beads/300 ⁇ g protein). Extracts can be incubated with beads for 1 hour at room temperature with constant rotation.
  • the beads can be washed four times in 1 ml RIPA buffer, then biotinylated proteins can be eluted from the beads by incubation with an equal volume of SDS-polyacrylamide gel electrophoresis sample buffer (e.g., 62.5 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 5% ⁇ -mercaptoethanol, and 0.001% bromophenol blue) for about 30 minutes at room temperature. Samples can be subjected to gel electrophoresis and
  • Bio-Rad Stain-free technology can be utilized to quantitate housekeeping proteins.
  • the amounts of cell surface nucleolin can be determined using a ChemiDoc MP Imaging System and the results can be normalized to the corresponding amounts of a housekeeping protein in the same sample.
  • cytoplasmic nucleolin are extracted and measured using one or more of the following steps.
  • Cytoplasmic nucleolin can be isolated by first gently lysing the cells for 30 min on ice in hypotonic lysis buffer (e.g., 10 mM HEPES-KOH pH 8.0, 40 mM KC1, 3 mM MgCl 2 , 10% glycerol, 1% NP-40, 1 mM DTT, protease inhibitors), allowing for the separation of nuclei from the rest of the cellular contents.
  • hypotonic lysis buffer e.g. 10 mM HEPES-KOH pH 8.0, 40 mM KC1, 3 mM MgCl 2 , 10% glycerol, 1% NP-40, 1 mM DTT, protease inhibitors
  • the resultant S10 supernatants can be centrifuged, e.g., at 100,000 x g for 1 hour at 4°C, to pellet insoluble cellular material and generate soluble SI 00 fractions.
  • SI 00 supernatants can be transferred to new microcentrifuge tubes and Bradford assay can be performed to determine total protein concentrations in each sample.
  • Samples can be subjected to gel electrophoresis and immunoblotting for nucleolin. To normalize nucleolin expression levels, Bio-Rad Stain-free technology and immunoblotting can be utilized as described above for the isolation of cell surface nucleolin.
  • the cytoplasmic, surface, and/or nuclear nucleolin can be isolated by differential centrifugation and analyzed by non-reducing SDS-PAGE followed by Western blotting.
  • GAPDH and Histone H2B can serve as cytoplasmic and nuclear markers, respectively.
  • the methods and information described can be implemented, in all or in part, as computer executable instructions on known computer readable media.
  • the methods described herein can be implemented in hardware.
  • the method can be implemented in software stored in, for example, one or more memories or other computer readable medium and implemented on one or more processors.
  • the processors can be associated with one or more controllers, calculation units and/or other units of a computer system, or implanted in firmware as desired.
  • the routines can be stored in any computer readable memory such as in RAM, ROM, flash memory, a magnetic disk, a laser disk, or other storage medium, as is also known.
  • this software can be delivered to a computing device via any known delivery method including, for example, over a communication channel such as a telephone line, the Internet, a wireless connection, etc., or via a transportable medium, such as a computer readable disk, flash drive, etc.
  • a communication channel such as a telephone line, the Internet, a wireless connection, etc.
  • a transportable medium such as a computer readable disk, flash drive, etc.
  • Results from such genotyping can be stored in a data storage unit, such as a data carrier, including computer databases, data storage disks, or by other convenient data storage means.
  • a data storage unit such as a data carrier, including computer databases, data storage disks, or by other convenient data storage means.
  • the computer database is an object database, a relational database or a post- relational database. Data can be retrieved from the data storage unit using any convenient data query method.
  • the software can be stored in any known computer readable medium such as on a magnetic disk, an optical disk, or other storage medium, in a RAM or ROM or flash memory of a computer, processor, hard disk drive, optical disk drive, tape drive, etc.
  • the software can be delivered to a user or a computing system via any known delivery method including, for example, on a computer readable disk or other
  • program modules include routines, programs, objects, components, and/or data structures that perform particular tasks or implement particular abstract data types.
  • the methods and apparatus can also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network.
  • program modules can be located in both local and remote computer storage media including memory storage devices.
  • the risk evaluation system and method, and other elements have been described as being implemented in software, they can be implemented in hardware, firmware, etc., and can be implemented by any other processor.
  • the elements described herein can be implemented in a standard multi-purpose CPU or on specifically designed hardware or firmware such as an application-specific integrated circuit (ASIC) or other hard-wired device as desired.
  • ASIC application-specific integrated circuit
  • the software routine can be stored in any computer readable memory such as on a magnetic disk, a laser disk, or other storage medium, in a RAM or ROM of a computer or processor, in any database, etc.
  • this software can be delivered to a user or a screening system via any known or desired delivery method including, for example, on a computer readable disk or other transportable computer storage mechanism or over a
  • communication channel for example, a telephone line, the internet, or wireless communication. Modifications and variations can be made in the techniques and structures described and illustrated herein without departing from the spirit and scope of the present disclosure.
  • CH03E7 Choinese hamster ovary cells with an initial density of 2.1 x 10 6 cells/mL, in a culture medium of F17 supplemented with 0.1% Pluronic F-68, 4 mM GlutaMAX, was transfected with either ANTIBODY I gamma heavy chain/kappa light chain or ANTIBODY I gamma heavy chain/lambda light chain.
  • cDNA encoding was synthetically produced with codon optimization for mammalian cell expression and cloned into expression vector pTT5 at the indicated restriction sites by standard methods.
  • Culture supernatants were harvested 10 days post transfection (6 days for the second transfection) via centrifugation for 5 minutes at 1000 xg.
  • the conditioned culture supernatants (CCS) were clarified by centrifugation for 30 minutes at 9100 xg, filter sterilized with a 0.2 ⁇ PES filter system, and stored at 4 °C.
  • Protein expression was analyzed via reducing and non-reducing SDS-PAGE. See FIG. 11. Expression verification SDS-PAGE. Forty microliter samples of conditioned culture supernatant (CCS) were evaluated via reducing (left panels) and non-reducing (right panels) SDS-PAGE on 4-20% Tris-glycine TGX gels (Bio-Rad) and stained with Instant Blue gel stain.
  • CCS conditioned culture supernatant
  • MCF-7R To create resistant cells MCF-7R, MCF-7 cells were exposed to increasing concentrations of ANTIBODY I from 0.5 to 4.0 ⁇ g/ml with three week periods between each 0.5 ⁇ g/ml dose escalation.
  • MCF-7 parental cells were grown in RPMI medium containing 10% human A/B serum overnight. The cells were then incubated with an initial concentration of 0.5 ⁇ g/ml of an anti-nucleolin antibody ANTIBODY I. After 96 h extensive cell kill was observed ( ⁇ 70%). Therefore, the cells were grown in the absence of antibody to allow them to resume growth at a rate similar to MCF-7 parental cells. After a 3 week recovery period, the incubation of the cells with ANTIBODY I was resumed at a concentration of 0.5 ⁇ g/ml. Following a one month treatment, the cells were resistant to 0.5 ⁇ g/ml of ANTIBODY I so the antibody concentration was increased to 1.0 ⁇ g/ml.
  • MCF-7 resistant cells and MCF-7 parental cells were tested for sensitivity to the antibody.
  • the IC 50 values for MCF-7 parental cells and MCF-7 resistant (MCF-7R) cells were determined to be 0.25 ⁇ g/ml and greater than 16 ⁇ g/ml, respectively. See FIG 1. Thus, MCF-7R cells were at least 64-fold resistant to
  • Cell Surface Nucleolin- Cell surface nucleolin was extracted by first biotinylating the cells and then pulling down surface proteins on streptavidin agarose beads. First, cell surface proteins were biotinylated using EZ-link-sulfo-NHS-biotin. Biotin coupled to highly reactive N- hydroxysuccinimide ester group was used as a membrane-impermeant probe to covalently tag cell surface proteins. Unbound biotin was removed from cells by washing twice with 0.1 M glycine in D-PBS (PBS supplemented with 0.1 mM CaCl 2 and 0.1 mM MgCl 2 ), then washing twice in D-PBS without glycine.
  • FIG 2 shows that there was no statistically significant difference between the levels of cell surface nucleolin in MCF-7 versus MCF-7R cells (p ⁇ 0.12, two tailed t-test).
  • Cytoplasmic Nucleolin- Cytoplasmic nucleolin levels were compared between MCF-7 parental cells and ANTIBODY I resistant MCF-7R cells. Cytoplasmic nucleolin was isolated by first gently lysing the cells for 30 min on ice in hypotonic lysis buffer (10 mM HEPES-KOH pH 8.0, 40 mM KC1, 3 mM MgCl 2 , 10% glycerol, 1% NP-40, 1 mM DTT, protease inhibitors), allowing for the separation of nuclei from the rest of the cellular contents.
  • hypotonic lysis buffer (10 mM HEPES-KOH pH 8.0, 40 mM KC1, 3 mM MgCl 2 , 10% glycerol, 1% NP-40, 1 mM DTT, protease inhibitors
  • FIG 2 shows that cytoplasmic nucleolin levels were increased 5.7-fold in the cytoplasm of MCF-7R cells compared to parental MCF-7 cells. This is consistent with both the concept that antibody ANTIBODY I targets nucleolin as well as our proposed mechanism of action of the antibody.
  • MCF-7R cells more nucleolin is available in the cytoplasm to stabilize oncogene mRNAs (e.g., BCL-2, BCL-XL), which promotes tumor cell growth and allows the resistant cells to better avoid apoptosis when exposed to ANTIBODY I.
  • oncogene mRNAs e.g., BCL-2, BCL-XL
  • MCF-7 cells were incubated with 2 ⁇ of ANTIBODY I for 0, 24, or 48 h.
  • the cytoplasmic and nuclear fractions were isolated by differential centrifugation and analyzed by non-reducing SDS-PAGE followed by Western blotting. Equal amounts (25 ⁇ g) of protein were added to each gel lane.
  • GAPDH and Histone H2B served as cytoplasmic and nuclear markers, respectively. Results are shown in FIG 3.
  • Day 1 Seed MCF-7 cells. Allow cells to settle and attach before placing flasks in incubator (-10 mins). Cells are seeded in RPMI + 10% human serum + 1% antibiotics.
  • Pepstatin A (1 ⁇ ): 10 ⁇ [0.5 mg/ml] stock in EtOH @-20°C
  • D10-12 MCF-7R F3 WCL at l/5x iv. Add 200 ⁇ of the Coomassie Plus Reagent to each well and mix with plate shaker for 30 seconds. v. For most consistent results, incubate plate for 10 minutes at room
  • RT temperature away from light.
  • Lane 1 MW Marker
  • Lane 2 25 ⁇ g MCF-7 IgG Oh WCL
  • Lane 3 25 ⁇ g MCF-7 IgG 8h WCL
  • Lane 5 25 ⁇ g MCF-7 IgG 48h WCL
  • Lane 7 25 ⁇ g MCF-7 ANTIBODY I Oh WCL
  • Lane 8 25 ⁇ g MCF-7 ANTIBODY I 8h WCL
  • Lane 9 25 ⁇ g MCF-7 ANTIBODY 1 24h WCL
  • Lane 10 25 ⁇ g MCF-7 ANTIBODY 1 48h WCL
  • Lane 11 25 ⁇ g MCF-7 ANTIBODY I 72h WCL
  • ECL Luminata Forte
  • Bands 1-10 250 kDa, 150 kDa, 100 kDa, 75 kDa, 50 kDa, 37 kDa, 25 kDa, 20 kDa,15 kDa, 10 kDa, respectively
  • ECL Western Blotting Detection Reagent Luminata Forte (EMD Millipore, # WBLUF0100).
  • Tris-Glycine-SDS running buffer 25 mM Tris, 192 mM Glycine, 0.1% SDS Transfer buffer: 25 mM Tris, 192 mM Glycine, 20% (v/v) methanol Antibodies: Santa Cruz Biotechnology BCL-2 (C-2) sc7382
  • IX TBS-T buffer 20 mM Tris, 150 mM NaCl, 0.1% Tween-20
  • Blocking buffer 5% (w/v) milk dissolved in IX TBS-T
  • the human tumor cells were seeded in a clear polystyrene 96-well microculture plate (Corning ® Costar ® 96-well flat bottom plate, Cat.# 3997) in a total volume of 90 ⁇ . After 24 hours of incubation in a humidified incubator at 37 °C with 5% C02 and 95% air, 10iL of 10X, serially diluted test agents in growth medium were added to each well in duplicate (10 pt dose response, highest concentration 8 ⁇ g/mL).
  • the medium/Cell Titer-Glo ® reagent was transferred to a white polystyrene 96-well microculture plate (Corning ® Costar ® 96-well flat bottom plate, Cat.# 3917) before reading luminescence on the BioTek Synergy II microplate reader.
  • the ICJO value for the test agents were estimated using Graph Pad Prism 7.0 by plotting compound concentration (Log uM) versus % C and fitting the four parameter logistic equation to the normalized data by nonlinear regression.
  • CDR complementary determining regions
  • Paratome Reference: Kunik V, et al. (2012). Paratome: An online tool for systematic identification of antigen binding regions in antibodies based on sequence or structure. Nucleic Acids Res. 2012 Jul;40(Web Server issue):W521-4. doi: 10.1093/nar/gks480. Epub 2012 Jun 6.
  • the Paratome web server (http://www.ofranlab.org/paratome/) was used to identify the six Antibody Binding Regions (ABRs) within the hypervariable region of ANTIBODY I.
  • the ABRs are similar to CDRs but also contain amino acid side-chains near the CDRs that also contribute to binding of the antibody to the antigen. From the primary structure of ANTIBODY I, the Paratome web server was able to identify the ABRs of the antibody by comparison to the structural consensus regions within a multiple structure alignment of a non-redundant set of all antibody-antigen complexes.
  • Chothia Definition Reference: Chothia C et al. Conformations of immunoglobulin hypervariable regions. Nature 1989 342: 887).
  • the Chothia definition is a scheme for numbering the amino acid residues of the hypervariable regions of antibodies and the beginning and ending of each of the six complementary regions (CDRs) within the hypervariable regions. The scheme is based on the analysis of the canonical structures of numerous antibodies. Using the Chothia definition, we were able to identify the positions of the amino acids at the beginning and ending of the six CDRs and the remaining amino acids within the CDRs by comparison to the primary structure of ANTIBODY I.
  • Trp-Tyr-Gln Residue after always a Trp.
  • Trp-Tyr-Gln Residue after always a Trp.
  • Trp-Leu-Gln Trp-Phe-Gln
  • Trp-Val Typically Trp-Val, but also, Trp-Ile, Trp-Ala
  • IMGT (ImMunoGeneTics).
  • the rules of IMGT as listed in Table 4 are followed to identify the CDRs. Reference: Lefranc, M.-P., The Immunologist, 7, 132-136 (1999)).
  • IMGT notes: (1) The geimline CDR3-IMGT is specific of the V-REGION of geimline V-GENEs. It comprises 0, 1 or 2 nucleotide(s) before the V-HEPTAMER. (2) The rearranged CDR3-IMGT and the FR4-IMGT are specific of the V-DOMAIN (V-J-REGION or V-D-J- REGION). They are characteristic of rearranged V-J-GENEs and V-D-J-GENEs, and corresponding cDNAs and proteins.
  • ClusPro 2.0 utilizes the protein-protein docking program PIPER, an FFT (fast Fourier transform)-based docking program that uses a structure-based pairwise potential as one component of its energy function; in Antibody Mode, the asymmetric potential is used. While the antibody is held in place on a three-dimensional grid, the ligand is rotated in increments of 1.0 A every 5°, resulting in 70,000 rotations total. For each rotation, the ligand is translated in x,y,z relative to the receptor on a grid. The lowest scoring 1000
  • ClusPro 2.0 structures/translation combinations from PIPER are then exported to ClusPro 2.0, where they are clustered within a 9 Angstrom C-alpha rmsd radius. This means that ClusPro 2.0 finds the ligand position with the most "neighbors" within 9 Angstroms, and it becomes a cluster center, and its neighbors the members of the cluster. These are then removed from the set and ClusPro 2.0 then looks for a second cluster center, and so on. The docking predictions can be ranked by cluster size. Those with the highest number of cluster members scored better than those with fewer. The model having the highest number of cluster members was chosen as the working model.
  • ANTIBODY I binds to cell surface nucleolin and the complex appears to utilize lipid raft mediated endocytosis for cellular entry. In the cytoplasm ANTIBODY I binds to RNA binding domains 1 and 2 of human nucleolin.
  • FIG. 5 is an image of molecular model of the binding of ANTIBODY I to human nucleolin, where the CDRs in contact with nucleolin are labeled.
  • ClusPro 2.0 protein-protein docking software was utilized in antibody mode to predict the interaction between the hypervariable region of ANTIBODY I and the structures of various nucleolin fragments in the Protein Data Bank (PDB).
  • the model as shown was obtained with the binding of ANTIBODY I (its CDR predictions are done with Rosie Rosetta) and the RNA-binding domains 1 and 2 (RBD 1 & 2) of human nucleolin (PDB 2krr).
  • Residues Ml to SI 9 native leader (signal peptide)
  • Residues E236 to P250 human Gl Hinge
  • Residues A251 to G361 human CH2
  • Residues Q362 to K467 human CH3
  • R234, E376, M378 are allotype residues identified by comparing the sequencing of ANTIBODY I to known allotype sequences in other human antibodies.
  • A175 and V213 are allotype residues identified by comparing the sequencing of ANTIBODY I to known allotype sequences in other human antibodies.
  • Residues Ml to C22 are a native leader (signal peptide). Residues D23 to K129 of ANTIBODY I Kappa light chain variable region, amino acid sequence SEQ ID NO:9
  • Residues R130 to C236 of ANTIBODY I Kappa light chain constant region amino acid sequence SEQ ID NO: 10
  • CDR Complementarity Determining Region
  • VH Heavy Chain Variable Region
  • VH Framework H1 CDR HI Framework H2 CDR H2 Framework H3 CDR H3 Framework H4
  • VL Framework L1 CDR L1 Framework L2 CDR L2 Framework L3 CDR L3 Framework L4

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Abstract

L'invention concerne une méthode destinée à traiter les cancers, à prédire l'efficacité du traitement des cancers, à évaluer la probabilité de développer une résistance au traitement du cancer par mesure du taux de nucléoline cytoplasmique ou du rapport de l'expression cytoplasmique à superficielle de la protéine de nucléoline.
PCT/US2017/027685 2016-04-15 2017-04-14 Agents anti-nucléoline WO2017181049A1 (fr)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
US20030194754A1 (en) * 2002-04-08 2003-10-16 Miller Donald M. Method for the diagnosis and prognosis of malignant diseases
US20060258605A1 (en) * 2005-05-12 2006-11-16 Yongzhang Luo Nucleolin-mediated cancer diagnostics and therapy
WO2011062997A2 (fr) * 2009-11-17 2011-05-26 Musc Foundation For Research Development Anticorps monoclonaux humains pour nucléoline humaine

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US20030194754A1 (en) * 2002-04-08 2003-10-16 Miller Donald M. Method for the diagnosis and prognosis of malignant diseases
US20060258605A1 (en) * 2005-05-12 2006-11-16 Yongzhang Luo Nucleolin-mediated cancer diagnostics and therapy
WO2011062997A2 (fr) * 2009-11-17 2011-05-26 Musc Foundation For Research Development Anticorps monoclonaux humains pour nucléoline humaine

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