ANTI-NUCLEOLIN AGENTS

CROSS-REFERENCE

[0001] This application claims the priority to U.S. Provisional Application No. 62/323,294, filed on April 15, 2016, and U.S. Provisional Application No.62/414,357, filed on October 28, 2016, both of which are incorporated herein by reference in their entirely.

INCORPORATION BY REFERENCE

[0002] All publications, patents, and patent applications disclosed herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term disclosed herein and a term in an incorporated reference, the term herein controls.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirely. Said ASCII copy, created on April 14, 2017, is named 39723-709_602_SL.txt and is 36,530 bytes in size.

BRIEF SUMMARY

[0004] In some cases, 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. In some instances, the comparable subjects are not resistant to the anti-nucleolin agent. In some instances, 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. [0005] In some cases, 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. In some instances, the comparable subjects are not resistant to the anti-nucleolin agent. In some instances, 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.

[0006] In some cases, 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. In some instances, 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. In some instances, 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.

[0007] In some cases, 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. 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.

[0008] In some cases, 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.

[0009] In some cases, 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.

[00010] In some instances, an immunoassay disclosed herein comprises western blot, flow cytometry, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), or any combination thereof. In some instances, an immunoassay for detecting cell surface nucleolin is flow cytometry or IHC. In some instances, the cell is permeated or permeable for the

immunoassay, e.g., flow cytometry. In some instances, a subject disclosed herein is a human subject. In some instances, 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. In some instances, 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.

[00011] In some instances, 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

combination thereof. In some instances, 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. In some instances, 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. In some instances, the antibody or fragment thereof comprises a light chain CDR1 that binds to E453, R457, or a combination thereof. In some instances, the antibody or fragment thereof comprises a light chain CDR2 that binds to D455. In some instances, the antibody or fragment thereof comprises a light chain CDR3 that binds to K348. In some instances, 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.

[00012] In some instances, an anti-nucleolin agent disclosed herein is an isolated antibody or fragment thereof. In some instances, the anti-nucleolin agent is a polyclonal antibody or fragment thereof. In some instances, the anti-nucleolin agent is a monoclonal antibody or fragment thereof. In some instances, the anti-nucleolin agent is a human, humanized, or chimeric antibody or fragment thereof. In some instances, the anti-nucleolin agent is substantially nonimmunogenic to a human. In some instances, the anti-nucleolin agent is an IgGl, IgG2, IgG3, or IgG4 antibody or fragment thereof. In some instances, 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.

[00013] In some instances, an anti-nucleolin agent disclosed herein is nontotoxic to normal cells or normal tissues. In some instances, the anti-nucleolin agent is cytotoxic to a cancer cell. In some instances, the anti-nucleolin agent is cytotoxic to a cancer cell in presence of human serum. In some instances, 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. In some instances, the anti-nucleolin agent kills at least: 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%; or about 100% of the population of cancer cells. In some instances, the incubation is in presence of human serum. In some instances, the period of time is about 48-96 hours. In some instances, 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. In some instances, 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.

[00014] In some instances, a method disclosed herein comprises administering an anti-nucleolin agent to a subject, and the administering is an injection. In some instances, the administering is intravenous or subcutaneous injection. In some instances, the administering occurs 1-3 times per week. In some instances, the method reduces a size of tumor in the subject by at least: 15%, 25%, 50%, 75%, or 95%. In some instances, the tumor is a solid tumor. In some instances, 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. In some instances, 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.

[00015] In some instances, an anti-nucleolin agent disclosed herein is in a form of an

immunocongjugate and is linked to a therapeutic agent. In some instances, the immunoconjugate is a fusion protein, and wherein the therapeutic agent is a polypeptide. In some instances, the anti-nucleolin agent is a bispecific antibody. In some instances, the anti-nucleolin agent is a probody. In some instances, the anti-nucleolin agent comprises an antigen-binding region that is activated by a tumor cell. In some instances, the antigen-binding region comprises a peptide linked to the N-terminus of a light chain through a protease cleavable linker. In some instances, the anti-nucleolin agent is linked covalently, noncovalently, or recombinantly to the therapeutic agent. In some instances, the therapeutic agent is a cytotoxic agent. In some instances, the cytotoxic agent is doxorubicin, calicheamicin, auristatin, maytansinoid, brentuximab vedotin, tubulysins, duocarmycins, camptothecin, SN-38, pyrrolobenzodiazepine, methotrexate, a- amanitin, ansamitocin, or any combination thereof. In some instances, the therapeutic agent is an immune stimulating agent. In some instances, 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. BRIEF DESCRIPTION OF THE DRAWINGS

[00016] FIG. 1 is a line graph showing IC50 measurements of ANTIBODY I to MCF-7 parental cells and MCF-7 resistant cells (MCF-7R).

[00017] 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 μg/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.

[00018] 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.

[00019] 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.

[00020] FIG. 5 is an image of molecular model of the binding of ANTIBODY I to human nucleolin RNA-binding domains 1 and 2.

DETAILED DESCRIPTION

[00021] The present disclosure provides anti-nucleolin agents and methods of use thereof. 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). In some instances, 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.

[00022] In some of many aspects, the present disclosure includes specific agents that

immunologically recognize, bind to, and/or inactivate nucleolin. In some cases, a nucleolin disclosed herein can be a cell-surface nucleolin or cytoplasmic nucleolin. In some cases, a nucleolin disclosed herein can mean a nucleolin fragment. In some cases, a nucleolin disclosed herein is a human nucleolin.

[00023] In some cases, an anti-nucleolin agent (e.g., antibody) disclosed herein selectively targets cell surface nucleolin (sNu) and cytoplasmic nucleolin (cNu) nucleolin found in most solid tumors and hematological malignancies but not normal cells. In some instances, 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. In some instances, the agent penetrates tumors and exploits the shuttling function of sNu to gain intracellular access. Once inside tumor cells, 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.

[00024] The term "about" means a referenced numeric indication plus or minus 15% of that referenced numeric indication.

[00025] In some cases, 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.

[00026] In some cases, 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.

[00027] In some cases, 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.

[00028] In some cases, 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.

[00029] In some cases, 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.

[00030] In some cases, 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.

[00031] In some cases, an expression level disclosed herein is a protein, DNA, or RNA expression level. In some cases, a threshold value disclosed herein is an average expression level of a cytoplasmic nucleolin of standard subjects in need thereof.

[00032] In some cases, 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. In some instances, 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.

[00033] In some cases, 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.

[00034] In some cases, the present disclosure provides a kit 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.

[00035] In some cases, 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. In some instances, the epitope has/spans about: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, or 80 amino acids. 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 combination thereof. In some instances, the anti-nucleolin agent binds to E453, R457, or a combination thereof. In some instances, 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. In some instances, 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. In some cases, the anti-nucleolin agent is an antibody or fragment thereof, e.g., an IgG antibody or fragment thereof.

[00036] In some instances, 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. In some instances, 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. In some instances, the at least 60% sequence identity is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, or 100% sequence identity. In some instances, 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. In some instances, an amino acid sequence of SEQ ID NO:4 is encoded by nucleotide sequence SEQ ID NO:3. In some instances, an amino acid sequence of SEQ ID NO: 8 is encoded by nucleotide sequence SEQ ID NO:7. In some instances, 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.

[00037] In some cases, an anti-nucleolin agent disclosed herein has a Kd of about: 10 μΜ, 1 μΜ, 0.1 μΜ, 0.05 μΜ, 10 ηΜ, 5 ηΜ, 2.5 ηΜ, 1 ηΜ, 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.

[00038] In some cases, 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.

[00039] In some cases, 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.

[00040] In some cases, an anti-nucleolin agent disclosed herein suppresses expression of cytoplasmic nucleolin in a cell. In some instances, the expression of cytoplasmic nucleolin is suppressed by 1-10 times than a corresponding cell that is not treated with the anti-nucleolin agent. In some instances, the expression of cytoplasmic nucleolin is suppressed by about 6 times.

[00041] In some cases, an anti-nucleolin agent disclosed herein is substantially nonimmunogenic to a human. In some cases, the anti-nucleolin agent is nontotoxic. In some instances, the anti- nucleolin agent is nontotoxic to normal cells or normal tissues. In some cases, the anti-nucleolin agent is cytotoxic. In some instances, the anti-nucleolin agent is cytotoxic to a tumor or cancer cell, e.g., in presence of human serum. In some instances, the anti-nucleolin agent exhibts or induces complement-dependent cytotoxicity to a tumor or cancer cell. In some instances, 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. In some instances, 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.

[00042] In some cases, 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. In some instances, 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.

[00043] In some cases, 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. [00044] In some cases, 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. In some instances, 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.

[00045] 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. In some instances, the anti-nucleolin agent inhibits expression of Bcl-2 protein in the cancer cell.

[00046] In some cases, 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. In some cases, in a method disclosed herein, the anti-nucleolin agent is an isolated antibody or fragment thereof that binds to human nucleolin.

I. Nucleolin

A. General

[00047] 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). It has been proposed that 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.

[00048] 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] A considerable body of evidence supports a role for nucleolin in mRNA stabilization. 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,

2007) , and MCF-7 breast cancer cells (Soundararajan et al., 2008). 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).

[00050] 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). This suggests that the presence of nucleolin on the cell surface is not the result of adsorption of secreted nucleolin by macromolecules on the cell surface of tumor cells. However, 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 Anderer, 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. It is thought that 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. For example, plasma membrane nucleolin has been reported to function as a receptor for intimin-γ 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,

2008) , and the anti-angiogenic pseudopeptide HB-19 (Destouches et al, 2008). Nucleolin is an important protein in the nucleolus involved in ribosome biogenesis and maturation in

exponentially growing eukaryotic cells. In this regard, 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.

B. Nucleolin in Cancer

[00051] Several lines of evidence suggest that 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). It is of interest that 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).

Table 1 - Cancer Cell Lines That Over-express Nucleolin and/or are Killed Subsequent to Nucleolin Inhibition

Melanoma LOX-IMVI, SK-MEL-2, A375, SK-MEL-28, MDA-MB-435

Glioblastoma SF-268, U87-MG

Neuroblastoma IMR 32, Lan 5

Sarcoma HT-1080

Gastric cancer KATOIII, HGC27

[00052] Anti-nucleolin antibodies can also exploit the shuttling function of plasma membrane nucleolin and become internalized after binding to cell surface nucleolin. Of 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).

C. Antibodies or Fragments thereof

[00053] In some instances, any of the methods disclosed herein can be practiced with an anti - nucleolin antibody or fragment thereof. In some instances, an anti -nucleolin antibody or fragment thereof is used to detect a cell expressing nucleolin on its surface. In some instances, an anti-nucleolin antibody or fragment thereof is used to inhibit or kill a cell expressing nucleolin on its surface. In some instances, 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.

[00054] In some instances 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. In some instances, 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. In some instances, anti - nucleolin antibody or fragment thereof is used to contact a cell expressing nucleolin on its surface. In some instances the cell is pre-cancerous cell, a cancer cell or an immune cell.

[00055] In some instances 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. In some instances, 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.

[00057] "Antibody fragments" comprise a portion of an intact antibody, for example comprising the antigen binding region thereof. Examples of 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.

[00058] 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.

[00059] "Fv" is a minimum antibody fragment which contains a complete antigen-binding site. In some instances, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable region in tight, non-covalent association. In a single-chain Fv (scFv) species, 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. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even 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.

[00060] 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. [00061] "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. Generally, 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. For a review of scFv, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer- Verlag, New York, 1994), pp. 269-315.

[00062] The term "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). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. 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).

[00063] 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. In certain instances, 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. For example, 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. It should be understood that 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. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations can be advantageous in that they are typically uncontaminated by other immunoglobulins. [00064] 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. For example, 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);

Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, PNAS USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies for producing human or humanlike antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., PNAS USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al,

Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

[00065] The modifier "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.

[00066] 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.

[00067] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, 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. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, 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. In general, 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. For further details, see Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1 :105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

[00068] 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. 77 (1985); Boerner et al., J. Immunol., 147(l):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). 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.

II. Cloning and Expression of Ig Light and Heavy Chains

[00069] Various methods can be employed for the cloning and expression of immunoglobulin light and heavy chain sequences. Weltschof et al. (1995), incorporated herein by reference, describes in detail the methods here. The variable regions, or variable + constant regions, can be cloned.

[00070] In some instances, 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.

[00071] Other techniques, such as those described by Takekoshi et al. (2001), are also useful. In that reference, total cellular RNA was isolated from pelleted cells using a commercial kit (RNeasy mini kit, Qiagen). Using random 9-mers, nucleotides and reverse transcriptase (Takara, 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). A

"touchdown" PCR protocol was employed, 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 (κ and λ) and Fc heavy chain genes (y and μ) were introduced into competent E. coli JM109 cells (Toyobo, Osaka). After transformation, the E. coli cells were plated onto Luria-Bertani (LB)/ampicillin (50 μg/ml) plates. Isolated bacterial colonies were incubated at 30°C in 2 ml of Super Broth (SB) with ampicillin (50 μg/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,

Boehringer Mannheim), and shaken for 5 min at room temperature. Cell lysates were centrifuged at 15,000G for 10 min, and the resultant supernatant containing the Fab antibody portion was collected. [00072] In some instances, a heavy chain and a light chain can be in the same cloning construct. In some instances, 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. In some instances, 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. For example, a cell can be genetically modified with a vector containing gene sequences for both a heavy chain and light chain.

[00073] The foregoing is purely exemplary and other methods can be employed.

III. Antibody Production

[00074] Once cloned, 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. 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. First, 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.

Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane

(tetramethylpentadecane) prior to injection. 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. Second, 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.

[00075] Antibodies (e.g., human and/or monoclonal) 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. [00076] In some instances, an anti-nucleolin antibody (e.g., human and/or monoclonal) is produced from an immortalized B cell (e.g., human B cell). In some instances, 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.

[00077] In some instances, the cDNA of an isolated anti-nucleolin antibody (e.g., human and/or monoclonal) 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. In some instances, an isolated anti-nucleolin antibody (e.g., human and/or monoclonal) is produced by a B cell (e.g., human B cell). In some instances, a cell is transfected by one or more

polynucleotide sequences isolated from a B cell (e.g., human 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.

[00078] 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. However, 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.

[00079] It will be understood that variations on the above procedure are within the scope of the present disclosure. In some instances, 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. In some instances, 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. [00080] In some instances, 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. It has long been recognized that Chinese Hamster Ovary cells (CHO cells) make excellent expression vehicles for recombinant or non-endogenous DNA. See U.S. Patent 4,816,567. There has been developed a series of 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. Examples of additional 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.

[00081] Antibodies may be produced using recombinant methods and compositions (See, e.g., U.S. Pat. No. 4,816,567). In some instances, 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. In a further instance, 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.

[00082] In some instances, a host cell comprising such nucleic acid is provided. 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.

Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. In one such instance, 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. In some instances, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NSO, Sp20 cell). In some instances, a method of making a nucleolin antibody is provided, wherein the method 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.

[00083] 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.

[00084] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, 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)). After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[00085] In addition to prokaryotes, 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). Examples of vertebrate cells 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).

IV. Modifications of Antibodies

[00086] In some instances, 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

combination of 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.

[00087] In some instances, 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

immunogenicity, or improved ADCC or CDC.

[00088] 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.

[00089] In some instances, 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. For example, conservative substitutions that do not substantially reduce binding affinity may be made in CDRs. Such alterations may be outside of CDR "hotspots" or SDRs. In some instances, of the variant VH and VL sequences, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.

[00090] Alterations (e.g., substitutions) 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. Alternatively, or

additionally, 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.

[00091] 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. Examples of 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. Examples of intrasequence insertion variants of the antibody molecules include an insertion of 3 amino acids in the light chain. Examples of terminal deletions include an antibody with a deletion of 7 or less amino acids at an end of the light chain.

[00092] In some instances, 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.

Liposomes with enhanced circulation time are disclosed in U.S. Patent 5,013,556.

[00093] 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).

[00094] In some instances, 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

chemotherapeutic agent, see WO 81/01145) to an active drug. See, for example, WO 88/07378 and U.S. Patent 4,975,278. 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.

[00095] 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 β -galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; β-lactamase useful for converting drugs derivatized with β-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes," 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.

[00096] 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. Alternatively, 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).

[00097] In some instances, an anti-nucleolin antibody comprises an antibody fragment, rather than an intact antibody. In this case, 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.

[00098] 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.

[00099] In some instances, 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.

[000100] In some instances, an anti-nucleolin antibody (such as a human antibody) is modified by fusing, or conjugating it to another, heterologous polypeptide or amino acid sequence. In some instances, 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. In some instances, an isolated anti- nucleolin antibody {e.g., human and/or monoclonal) is modified to comprise targeted

immunoconjugate moieties which enable the effective generation of innate and adaptive immune responses against tumors or pathogens. In some instances, an isolated anti-nucleolin antibody (e.g., human and/or monoclonal) produced by a B cell (e.g., human B cell) is modified to comprise 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. Such immunoconjugated antibodies can be used for targeted immunotherapy or immunoprophylaxis of neoplastic diseases, infectious diseases, and other disorders. For example, pattern recognition receptors (PR s), such as Toll like Receptors, recognize pathogen- associated molecular patterns (PAMPs) expressed by diverse infectious microorganisms

(bacteria, fungi, protozoa, viruses) and molecules released by damaged host tissues (damage associated molecular patterns/alarmins). The addition of a PAMP conjugated to an isolated anti- nucleolin antibody (e.g., human and/or monoclonal) 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. Examples of PAMPS that can be conjugated to an anti-nucleolin antibody include but are limited to known viral and pathogenic epitopes, such as polyinosine-polycytidylic acid, lipopolysaccharide (LPS), lipid A, flagellin, GU-rich short single-stranded RNA, unmethylated CpG-oligodeoxynucleotides.

[000101] In some instances, 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. Examples include poly-histidine (poly- his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag and its antibody 12CA5 (Field et al., 1988); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al, 1985); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., 1990). Other 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).

[000102] In some instances, an anti-nucleolin antibody (e.g., human and/or monoclonal) or fragment is linked to a nanoparticle. In some instances, an isolated anti-nucleolin antibody (e.g., human and/or monoclonal) is linked to a nanoparticle. In some instances, an anti-nucleolin antibody (e.g., human and/or monoclonal) produced by a B cell (e.g., human B cell) is linked to a nanoparticle. Cell surface nucleolin has been reported to serve as receptor for DNA

nanoparticles composed of PEGylated polylysine and DNA (Chen et al., 2008). In some instances, the antibody-nanoparticle conjugate can penetrate a cell expressing nucleolin on its surface more rapidly and extensively than the unconjugated antibody. In some instances, the cell is a cancer cell, tumor cell, virally infected cell, lymphocyte, or activated lymphocyte.

V. Therapeutic Use

A. Anti-nucleolin agents

[000103] In some instances, an anti-nucleolin agent (e.g., human and/or monoclonal antibody) is provided that can be used to inhibit or kill a cancer cell. In some instances, the cancer cell expresses nucleolin on its surface or in it cytoplasm. In some instances, 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.

[000104] In some instances, an anti-nucleolin agent (e.g., human and/or monoclonal antibody) is administered to a human subject with one or more forms of cancer. In some instances, the anti- nucleolin agent is administered to a human subject where the cancer is resistant to other cancer treatments. For example, cancers can be resistant to radiation therapy, chemotherapy, or biological therapy. In some instances, 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.

[000105] In some instances, an anti-nucleolin agent (e.g., human and/or monoclonal antibody) is used to inhibit or kill a cell as part of, or in combination with, an adjuvant therapy. 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.

[000106] In some instances, an anti-nucleolin agent (e.g., human and/or monoclonal antibody) 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. For example, specific non-limiting examples of non-malignant cell proliferative disorders that can treat or inhibited with the anti-nucleolin agent include but are not limited to warts, benign prostatic hyperplasia, skin tags, and non- malignant tumors. For example, an isolated anti-nucleolin agent (e.g., human and/or monoclonal antibody) 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). In some instances, an isolated anti-nucleolin agent (e.g., human and/or monoclonal antibody) is used to inhibit or kill in a subject a cell comprising an angiogenic tumor. 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.

[000107] In some instances, an anti-nucleolin agent (e.g., human and/or monoclonal antibody) is used to inhibit or kill in a subject a tumor cell under conditions of tumor hypoxia. 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.

[000108] In some instances, an anti-nucleolin agent (e.g., human and/or monoclonal antibody) is used to inhibit or kill in subject a lymphocyte cell expressing nucleolin on its surface. In some instances, the lymphocyte cell comprises a B cell, T cell, or natural killer cell. In some instances, the lymphocyte cell comprises a CD4-positive or CD8-positive cells.

[000109] In some instances, an anti-nucleolin agent (e.g., human and/or monoclonal antibody) is used to inhibit or kill in a subject an activated lymphocyte or memory cell expressing nucleolin on its surface. In a further instance, the activated lymphocyte comprises an activated B cell, T cell, or natural killer cell. In some instances, an anti-nucleolin agent (e.g., human and/or monoclonal antibody) is used to inhibit or kill a cell in a subject having an autoimmune disorder. In some instances, 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, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, Henoch-Schonlein purpura, idiopathic pulmonary fibrosis, idiopathic/autoimmune thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erthematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus-related disorders (e.g., pemphigus vulgaris), pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus (SLE), Sweet's syndrome, Still's disease, lupus erythematosus, takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis. Examples of inflammatory disorders include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated

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.

[000110] In some instances, an anti-nucleolin agent (e.g., human and/or monoclonal antibody) is used to inhibit or kill a cell in a subject infected by 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);

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);

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); Rous sarcoma virus (RSV), avian leukemia virus (ALV), and avian myeloblastosis virus (AMV)) and 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 lentiviruses, T-cell leukemia viruses and the foamy viruses, lentiviruses including HIV-1, HIV-2, SIV, Visna virus, feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV), simian T-cell leukemia virus (STLV), and bovine leukemia virus (BLV), the foamy viruses including human foamy virus (HFV), simian foamy virus (SFV) and bovine foamy virus (BFV), Poxyiridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g., the etiological agents of

Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1 = internally transmitted; class 2 = parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses), Mycobacterium (Mycobacterium tuberculosis, M. bovis, M. avium-intracellulare, M. leprae), Pneumococcus, Streptococcus, Staphylcococcus, Diphtheria, Listeria, Erysipelothrix, Anthrax, Tetanus, Clostridium, Mixed Anaerobes, Neisseria, Salmonella, Shigella, Hemophilus,

Escherichia coli, Klebsiella, Enterobacter, Serratia, Pseudomonas, Bordatella, Francisella tularensis, Yersinia, Vibrio cholerae, Bartonella, Legionella, Spirochaetes (Treponema,

Leptospira, Borrelia), Fungi, Actinomyces, Rickettsia, Mycoplasma, Chlamydia, Protozoa

(including Entamoeba, Plasmodium, Leishmania, Trypanosoma, Toxoplasma, Pneumocystis, Babasia, Giardia, Cryptosporidium, Trichomonas), Helminths (Trichinella, Wucheraria, Onchocerca, Schistosoma, Nematodes, Cestodes, Trematodes), and viral pneumonias. Additional examples of antigens which can be targets for compositions of the present disclosure are known, such as those disclosed in U.S. Patent Publication No. 2007/0066554. In a further aspect, 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.

B. Conjugates

[000111] In some instances, the present disclosure provides for an anti-nucleolin agent (e.g., human and/or monoclonal antibody) linked to at least one therapeutic agent to form an antibody conjugate. In some instances, 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. In some instances, 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.

[000112] In some instances, an isolated anti-nucleolin agent (e.g., human and/or monoclonal antibody) is conjugated to an enzymatically active toxin or fragment thereof. Examples of enzymatically active toxins and fragments thereof 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 tricothecenes.

[000113] In some instances, an isolated anti-nucleolin agent (e.g., human and/or monoclonal antibody) is conjugated to a radionuclide. Examples of suitable radionuclides include, but are not limited to, 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 iodine, ¹²⁵ iodine, ¹³¹ iodine, ⁱⁿ indium, ¹³¹ In, ¹⁹² iridium, ⁵⁵ iron, ⁵⁹ iron, ⁸⁵ krypton, ²¹⁰ lead, ¹⁷⁷ lutecium, ⁵⁴ manganese, ¹⁹⁷ mercury, ²⁰³ mercury, "molybdenum,

1 ⁴⁷ neodynium, ²³⁷ neptunium, ⁶³ nickel, ⁹⁵ niobium, ^185+191 osmium, ¹⁰³ palladium, ³² phosphorus, ¹⁸⁴ platinum, ¹⁴³ praseodymium, ¹⁴⁷ promethium, ²³³ protactinium, ²²⁶ radium, rhenium ¹⁸⁶ ,

1 ⁸⁸ rhenium, ⁸⁶ rubidium, ¹³⁰ ruthenium, ¹⁰⁶ ruthenium, ⁴⁴ scandium, ⁴⁶ scandium, ⁴⁵ selenium, ⁷⁵ selenium, ^110m silver, ^U1 silver, ²² sodium, ⁸⁵ strontium, ⁸⁹ strontium, ⁹⁰ strontium, ³⁵ sulphur, ¹⁸² tantalum, ^99m technicium, ^125m tellurium, ¹³² tellurium, ¹⁶⁰ terbium, ²⁰⁴ thallium, ²²⁸ thorium, ²³² thorium, ¹⁷⁰ thullium, ¹¹³ tin, ^titanium, ¹⁸⁵ tungsten, ⁴⁸ vanadlum, ⁴⁹ vanadium, ⁸⁸ yttrium, ⁹⁰ yttrium, ⁹¹ yttrium, ¹⁶⁹ ytterbium, ⁶⁵ zinc, and/or ⁹⁵ zirconium.

[000114] 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). For example, 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.

[000115] In some instances, an isolated anti-nucleolin agent (e.g., human and/or monoclonal antibody) is conjugated to a cytokine. The term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are 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 -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin;

vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-growth factor; transforming growth factors (TGFs) such as TGF-a and TGF- β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-a, -β, and -γ; 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). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[000116] In some instances, an isolated anti-nucleolin agent (e.g., human and/or monoclonal antibody) is conjugated to a chemotherapeutic agent. A variety of chemical compounds, also described as "chemotherapeutic agents," function to induce DNA damage. 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. Specific 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, valrubicin, vinblastine , vincristine, vindesine, vinorelbine. The present disclosure also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide.

[000117] In some instances, an isolated anti-nucleolin agent (e.g., human and/or monoclonal antibody) is conjugated to an anti-viral agent. Example of 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.

C. Pharmaceutical Compositions and Administration

[000118] It is envisioned that, for administration to a subject in need thereof, an anti-nucleolin agent will be suspended in a formulation suitable for administration to a host. In some instances, the agent is a monoclonal antibody. In some instances, the monoclonal antibody is an anti- nucleolin antibody. In some instances, 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. The phrases "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.

[000119] As used herein, "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.

[000120] In some instances, an anti-nucleolin agent (e.g., human and/or monoclonal antibody) of the present disclosure can be administered in any 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. In some instances, of the present disclosure, the

pharmaceutically acceptable carrier is pharmaceutically inert. [000121] 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. In addition to the active ingredients, 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

Pharmaceutical Sciences" (Maack Publishing Co, Easton Pa.).

[000122] Pharmaceutical 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.

[000123] Pharmaceutical preparations 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. If desired, 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.

[000124] 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).

[000125] Pharmaceutical preparations 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. In soft capsules, 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. [000126] Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds. For injection, the pharmaceutical 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. Additionally, 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. Optionally, 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.

[000127] The pharmaceutical 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,

isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms. In some instances, 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.

[000128] 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. In many cases, it is suitable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectible compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. [000129] Pharmaceutical 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. Thus, 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 (e.g., a therapeutically effective amount) 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.

[000130] For any compound, 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.

[000131] In an animal, 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.

[000132] With respect to cancer, 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). [000133] With respect to modulation of the immune system, "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. For diseases involving an overactive immune system, "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.

[000134] 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). 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) may be for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

Administration of a dosage form comprising an anti-nucleolin agent (e.g., human and/or monoclonal antibody) 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 (e.g., human and/or monoclonal antibody) 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 (e.g., human and/or monoclonal antibody) 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;

5,206,344; and 5,225,212, herein incorporated by reference). In some instances, the dosage of a composition comprising anti-nucleolin agent (e.g., human and/or monoclonal antibody) 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. [000135] 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. 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).

[000136] In some liquid compositions, 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. In some compositions, at least one tonicity agent (e.g., D-mannitol) and is present at a concentration of about 1% w/v to about 10% w/v, about 2% w/v to about 6% w/v, or about 4% w/v. In some compositions, at least one buffering agent (e.g., histidine, succinate) 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. In some compositions, an antioxidant (e.g., methionine) is present at a concentration of about 0.1 mM to about 25 mM, about 5 mM to about 15 mM, or about 10 mM. In some compositions, a stabilizer (e.g., polysorbate 80) is present at a concentration of about 0.001% w/v to about 0.01% w/v, about 0.005% w/v to about 0.01% w/v, or about 0.005% w/v. 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.

[000137] In some instances, 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. In various instances, 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 lower limits are intended to be included.

[000138] In some instances, 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. to about 10° C, for example from about 2° C. to about 8° C. In various instances, 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.

[000139] 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. Usually, 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. In some exemplary instances, 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. In some exemplary instances, 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. In some methods, 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.

[000140] 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 μg/ml and in some methods 25- 300 μg/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.

[000141] The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, 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." In this use, 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. Some patients continue to receive treatment for the rest of their lives.

[000142] In some therapeutic applications, 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.

[000143] It is especially advantageous to provide 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. In an exemplary instance, 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. Some pharmaceutical unit dosage forms may require reconstitution with a suitable diluent prior to addition to an intravenous drip bag, for example lyophilized forms. In exemplary instances, the pharmaceutical unit dosage form is a container containing a composition described herein. For example, the container may be a 10 mL glass, type I, tubing vial. Generally, the container should maintain the sterility and stability of the composition. For example, the vial may be closed with a serum stopper. Furthermore, in various instances, 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). In some instances, 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. In some instances, the composition often is supplied as a liquid in unit dosage form.

[000144] In some aspects, 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. Accordingly, the container and the kit may be designed to provide enough composition for multiple uses. In various instances, the kit may further include diluent. The diluent may include excipients, separate or combined. For example, 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.

[000145] Pharmaceutical 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. 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, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrins); chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose or sorbitol); salt-forming counter-ions (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants (e.g., polyethylene glycol (PEG)). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents (e.g., soluble neutral-active hyaluronidase glycoproteins (sHASEGP)). In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases (e.g., chondroitinases). In addition, the compositions may contain an additional agent for pH adjustment (for example, HC1) and a diluent (for example, water). In some instances, different forms of histidine can be used for pH adjustment. In part, 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.

[000146] In various aspects, the composition includes a buffering agent (buffer). The buffer serves to maintain a physiologically suitable pH. In addition, the buffer can serve to enhance isotonicity and chemical stability of the composition. Generally, the composition should have a physiologically suitable pH. In various instances, 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. In some instances, 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. For example, 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)

aminomethane), diethanolamine, citrate, other organic acids and mixtures thereof. In some instances, the buffer is histidine (for example, L-histidine). In some instances, the buffer is succinate. In some instances, 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. In one exemplary instance, the buffer is L-histidine (base) (for example C6H9N ₃ 0 ₂ , FW: 155.15). In some instances, the buffer is L-histidine monochloride monohydrate (for example

C6H9N ₃ 0 ₂ .HC1.H ₂ 0, FW: 209.63). In some instances, the buffer is a mixture of L-histidine (base) and L-histidine monochloride monohydrate. In some instances, 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. In various instances, 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.

[000147] In various aspects, the composition includes a tonicity agent. In part, the tonicity agent contributes to maintaining the isotonicity of the composition, and to maintaining protein levels. In part, the tonicity agent contributes to preserving the level, ratio, or proportion of the therapeutically active polypeptide present in the composition. As used herein, 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. Often, the tonicity agent serves as a bulking agent as well. As such, 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. In some instances, the tonicity agent is mannitol (for example, D-mannitol, for example, C ₆ H ₁₄ 0 ₆ , FW: 182.17). In some instances, 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.

[000148] In various aspects, 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

(eicosapentaenoic acid) or DHA (docosahexaenoic acid) with any natural or synthetic antioxidant with which they can be chemically linked. These include 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) and amino acid antioxidants (for example, methionine, cysteine, carnosine). In some instances, the anti-oxidant is ascorbic acid. The anti-oxidant is methionine, or an analog thereof, for example,

selenomethionine, hydroxy methyl butanoic acid, ethionine, or trifluoromethionine. In some instances, the anti-oxidant (for example, a methionine such as L-methionine, for example

CH ₃ SCH ₂ CH ₂ CH(NH ₂ )C0 ₂ H, FW=149.21) 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, from about 0.1 mM to about 20 mM, or from about 5 mM to about 15 mM. In various instances, 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. For example, the anti-oxidant is present at about 10 mM. In some instances, 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.

[000149] In various aspects, 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. In certain instances, 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. In some instances, 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. For example, the stabilizer concentration is about 0.005% w/v. In some instances, 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. In some instances, 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.

D. Nucleolin-Expressing Cancers and Non-Malignant Cells

[000150] In some instances, an isolated anti-nucleolin agent (e.g., human and/or monoclonal antibody) is used in treating a variety of cells, including both cancerous and non-cancerous cells. In some instances, the isolated anti-nucleolin antibody (e.g., human and/or monoclonal) is a monoclonal antibody. In some instances, the isolated anti-nucleolin antibody (e.g., human and/or monoclonal) is a polyclonal antibody. The term "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

Myeloproliferative Disorders; Colon Cancer; Colorectal Cancer; Cutaneous T-Cell Lymphoma; B-Cell Lymphoma Endometrial Cancer; Ependymoma; Esophageal Cancer; Esophageal Cancer; Ewing's Family of Tumors; Extracranial Germ Cell Tumor; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma; Glioma, Childhood Brain Stem; Glioma, Childhood Cerebral Astrocytoma; Glioma, Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer;

Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin's Lymphoma; Hodgkin's Lymphoma During Pregnancy; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma; Kidney (Renal Cell) Cancer; Kidney Cancer;

Laryngeal Cancer; Leukemia, Acute Lymphoblastic; Leukemia, Acute Lymphoblastic; Leukemia, Acute Myeloid; Leukemia, Acute Myeloid; Leukemia, Chronic Lymphocytic;

Leukemia; Chronic Myelogenous; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer, Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphoma, AIDS-Related; Lymphoma, Burkitt's; Lymphoma, Cutaneous T-Cell, see Mycosis Fungoides and Sezary Syndrome; Lymphoma, Hodgkin's; Lymphoma, Hodgkin's During

Pregnancy; Lymphoma, Non-Hodgkin's; Lymphoma, Non-Hodgkin's During Pregnancy;

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

Syndrome; Multiple Myeloma/Plasma Cell Neoplasm' Mycosis Fungoides; Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Diseases; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Adult Acute; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer;

Nasopharyngeal Cancer; Neuroblastoma; Non-Hodgkin's Lymphoma; Non-Hodgkin's

Lymphoma During Pregnancy; Oral Cancer; Oral Cavity Cancer, Lip and; Oropharyngeal Cancer; Osteosarcoma/Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor;

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

Blastoma; Pregnancy and Breast Cancer; Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma; Primary Central Nervous System Lymphoma; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Cell (Kidney) Cancer; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma; Salivary Gland Cancer; Salivary Gland Cancer; Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma, Soft Tissue; Sarcoma, Soft Tissue; Sarcoma, Uterine; Sezary Syndrome; Skin Cancer (non- Melanoma); Skin Cancer; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma; Soft Tissue Sarcoma; Squamous Cell Carcinoma, see Skin Cancer (non-Melanoma); Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer; Supratentorial Primitive Neuroectodermal Tumors; T-Cell Lymphoma, Cutaneous, see Mycosis Fungoides and Sezary Syndrome; Testicular Cancer; Thymoma; Thymoma and Thymic Carcinoma; Thyroid Cancer; Thyroid Cancer; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Cancer, Endometrial; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma; Vulvar Cancer; Waldenstrom's Macroglobulinemia; and Wilms' Tumor.

[000151] 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. In addition, non-cancer cells that express nucleolin include immune cells such as dendritic cells, peripheral blood monocytes,

macrophages, and glial cells, as well as vascular smooth muscle cells and endothelial cells. In some instances, 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

Publication No. 2009/0191244, incorporated herein by reference.

[000152] In some instances, 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. In some examples, 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. In some examples, the methods for treating cancer provided herein decrease the number of cancer cells. In some examples, the methods for treating cancer provided herein reduce the formation of cancer cells. In some examples, the methods for treating cancer provided herein eradicate, remove, or control primary, regional and/or metastatic cancer cells associated with the cancer. In some examples, the methods for treating cancer provided herein decrease the number or size of metastases associated with the cancer.

[000153] In some instances, methods for treating cancer provided herein 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 70- about 90%, about 80- about 90%, about 10- about 100%, about 20% - about 100%, about 30- about 100%, about 40- about 100%, about 50- about 100%, about 60- about 100%, about 70- about 100%, or by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100%, about 80- about 100%, about 90- about 100%, about 95- about 100%, or any range in between, relative to the number of cancer cells in a subject prior to administration of an anti-nucleolin agent as assessed by, for example, CT Scan, MRI, DCE- MRI, or PET Scan.

E. Biological Samples

[000154] As used in the present application, "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. In some cases, the biological samples comprise fine needle aspiration samples. In some instances, 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

withdrawing cellular material (fine needle aspiration biopsy). In some instances, 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.

[000155] The biological samples obtained can be used in fresh, frozen, or fixed (e.g.,

formaldehyde fixed-paraffin embedded) form, depending on the nature of the sample, the assay used, and the convenience of the practitioner. Although fresh, frozen and fixed materials are suitable for various RNA and protein assays, generally, fresh tissues can be for ex vivo

measurements of activity.

[000156] Fixed tissue samples can also be employed. 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.

[000157] In some cases, the assay includes a step of cell or tissue culture. For example, 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.

[000158] Detection in a biological sample can be performed by, for example, direct

measurement, immunohistochemistry, immunoblotting, immunofluorescence,

immunoabsorbance, immunoprecipitations, protein array, fluorescence in situ hybridization, FACS analysis, hybridization, in situ hybridization, Northern blots, Southern blots, Western blots, ELISA, radioimmunoassay, gene array/chip, PCR, RT-PCR, flow cytometry, or cytogenetic analysis. 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.

[000159] 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. For example, 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.

[000160] 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

hybridization, laboratory biomarker analysis, immunohistochemistry staining method, flow cytometry, or a combination thereof. The method can further comprise tabulating and/or plotting results of the analytical procedure.

[000161] In some instances, 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. First, 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. To precipitate cell surface proteins, 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 μΐ beads/300 μg 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% β-mercaptoethanol, and 0.001% bromophenol blue) for about 30 minutes at room temperature. Samples can be subjected to gel electrophoresis and

immunoblotting for nucleolin. To normalize for differences in recovery of nucleolin after immunoblotting, Bio-Rad Stain-free technology can be utilized to quantitate housekeeping proteins. Following blotting, 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.

[000162] In some instances, 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. 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.

[000163] In some instances, 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.

F. Computer-Implemented Aspects

[000164] As understood by those of ordinary skill in the art, the methods and information described can be implemented, in all or in part, as computer executable instructions on known computer readable media. For example, the methods described herein can be implemented in hardware. Alternatively, 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. As is known, 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. If implemented in software, 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. Likewise, 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.

[000165] More generally, and as understood by those of ordinary skill in the art, the various steps described above can be implemented as various blocks, operations, tools, modules and techniques which, in turn, can be implemented in hardware, firmware, software, or any combination of hardware, firmware, and/or software. When implemented in hardware, some or all of the blocks, operations, techniques, etc. can be implemented in, for example, a custom integrated circuit (IC), an application specific integrated circuit (ASIC), a field programmable logic array (FPGA), a programmable logic array (PLA), etc.

[000166] 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. In certain instances, 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.

[000167] When implemented in software, 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. Likewise, 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

transportable computer storage mechanism.

[000168] The steps of methods disclosed herein can be operational with numerous other general purpose or special purpose computing system environments or configurations. Examples of well known computing systems, environments, and/or configurations that can be suitable for use with the methods or system herein include, but are not limited to, personal computers, server computers, hand-held or laptop devices, multiprocessor systems, microprocessor-based systems, set top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe computers, distributed computing environments that include any of the above systems or devices, and the like.

[000169] The steps of a method and system disclosed herein can be described in the general context of computer-executable instructions, such as program modules, being executed by a computer. Generally, 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. In both integrated and distributed computing environments, program modules can be located in both local and remote computer storage media including memory storage devices.

[000170] While 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. Thus, 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. When implemented in software, 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. Likewise, 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.

VI. Examples

Example 1. Production of an Anti-Nucleolin Antibody ANTIBODY I from Recombinant Cells

[000171] CH03E7 (Chinese 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. Two, 30 mL cultures of CH03E7 cells in 125 mL shake flasks were transfected with 1 mg of plasmid DNA/L culture using PolyPlus linear Q-PEI at a 1 :4 (w/v) DNA:PEI ratio. Cultures were supplemented with 4.5 mL CD Efficient Feed B (Life Technologies) 24 hours post transfection. Culture parameters were monitored using a ViCell XR for density and viability.

[000172] 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 μηι PES filter system, and stored at 4 °C.

[000173] 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.

[000174] ANTIBODY I bound tightly to human recombinant nucleolin (Kd =2.6 ± 0.7 nM, SEM.) and to plasma membrane nucleolin of human tumor cells. Confocal microscopy of Pane- 1 and DU-145 tumor cells incubated at 37 °C with ANTIBODY I revealed punctate localization of the antibody in the plasma membranes of these cells and internalization of the antibody into the cytoplasm. The localization of the antibody within foci in the plasma membrane suggested that the antibody was bound to nucleolin that was incorporated into lipid rafts within the plasma membrane.

[000175] Plasmid isolation and transient expression in CH03E7 cells at the 6 L culture scale. The goal was to isolate endotoxin-free expression plasmid DNA encoding ANTIBODY I gamma heavy chain. 6 L of CH03E7 cells was transiently transfect with gamma heavy chain and kappa light chain plasmid DNA using linear PEL The culture parameters were monitored, and the conditioned culture supernatant (CCS) was harvested when the viability has dropped to -85-90% (targeting 90%). It was expected to hit viability target 3-4 days post transfection. Protein expression in the CCS was analyzed by reducing and non-reducing SDS-PAGE. Clarified CCS was sterile filtered and stored at 4 °C.

Example 2. Characterization of Resistance of MCF-7 Cells to ANTIBODY I

2.1 Generation of MCF-7 breast cancer cells resistant to ANTIBODY I

[000176] To create resistant cells MCF-7R, MCF-7 cells were exposed to increasing concentrations of ANTIBODY I from 0.5 to 4.0 μg/ml with three week periods between each 0.5 μg/ml dose escalation.

[000177] First, 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 μg/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 μg/ml. Following a one month treatment, the cells were resistant to 0.5 μg/ml of ANTIBODY I so the antibody concentration was increased to 1.0 μg/ml. Again extensive cell kill was observed at 96 h after dose escalation. The remaining viable cells were grown in the presence of 1 μg/ml of antibody until they became resistant to this concentration of antibody after 3 weeks. Then the antibody concentration was increased to 2 μg/ml and then doubled 10 days later to 4 μg/ml. [000178] After a 10 day exposure to 4 μg/ml of ANTIBODY I, 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 μg/ml and greater than 16 μg/ml, respectively. See FIG 1. Thus, MCF-7R cells were at least 64-fold resistant to

ANTIBODY I.

2.2 Comparison of MCF-7 cells and MCF-7R cells

[000179] The levels of nucleolin on the cell surface and in the cytoplasm were determined in MCF-7 and MCF-7R cells.

[000180] 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. Cells were solubilized in RIPA buffer (50 mM Tris-HCl 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 was performed to quantify the concentration of total protein in the cell extracts. To precipitate cell surface proteins, monomeric avidin agarose beads were used. Before use, nonreversible biotin binding sites on the beads were 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 were loaded onto avidin beads, and biotinylated proteins were separated from non-biotinylated proteins by batch affinity

chromatography (250 μΐ beads/300 μg protein). Extracts were incubated with beads for 1 hour at room temperature with constant rotation. The beads were washed four times in 1 ml RIPA buffer, then biotinylated proteins were eluted from the beads by incubation with an equal volume of SDS-polyacrylamide gel electrophoresis sample buffer (62.5 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 0.001% bromophenol blue) for 30 minutes at room temperature. Samples were subjected to gel electrophoresis and immunoblotting for nucleolin. To normalize for differences in recovery of nucleolin between the MCF-7 and MCF-7R groups after immunoblotting, Bio-Rad Stain-free technology was utilized to quantitate housekeeping proteins. Following blotting, the amounts of cell surface nucleolin in the MCF-7 and MCF-7R cells were determined using a ChemiDoc MP Imaging System and the results were normalized to the corresponding amounts of a housekeeping protein in the same sample. 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).

[000181] 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. The resultant S10 supernatants were then centrifuged 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 were transferred to new microcentrifuge tubes and Bradford assay was performed to determine total protein

concentrations in each sample. Samples were subjected to gel electrophoresis and

immunoblotting for nucleolin. To normalize nucleolin expression levels, Bio-Rad Stain-free technology and immunoblotting was utilized as described above for the isolation of cell surface nucleolin. 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. In 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.

Example 3. Western Blotting of ANTIBODY I in S100 Cytosolic and Nuclear Fractions of MCF-7 Cells

[000182] MCF-7 cells were incubated with 2 μ^ιηΐ 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 μg) 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.

Example 4. Western Blot Analysis of BCL-2 Expression Levels in MCF-7 Cells Treated with ANTIBODY I

[000183] This study analyzed the effect of ANTIBODY I on BCL2 protein expression in MCF-7 cells over the course of 8-72 hours. The following materials and experimental procedures were used.

A. Cells:

Cells were thawed from liquid nitrogen and transferred to T25 flask for culturing. Treatment with Control IgG or ANTIBODY I was performed as described in "B" below. B. Treatment Time Course:

1. 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.

2. Day 2: Set up IgG and antibody dilutions at 2 μ^ηιΐ.

6 ml at [2 μ^ηιΐ], add 78.9 μΐ antibody (or [0.152 mg/ml] IgG) to cell growth media in flask.

C. WCL Preparation:

1. Reagents:

RIPA Lysis Buffer: 5 ml

50 mM Tris, pH 8.0 250 μΐ 1M stock

150 mM NaCl 150 μΐ 5M stock

1% Triton X- 100 50 μΐ

0.5% sodium deoxycholate 250 μΐ 10% stock

0.1% sodium dodecyl sulfate 50 μΐ 10% stock

Protease inhibitors see below in section 2

Protease Inhibitors: For 5 ml lysis buffer, add in the indicated volume of stocks as below:

1 mM PMSF: 20 μΐ 0.25 M stock ([43.6 mg/ml]) in EtOH (fresh)

Trypsin inhibitor (10 μ^ηιΐ): 10 μΐ [5 mg/ml] stock in dH ₂ 0 @-20°C

Pepstatin A (1 μ^ιηΐ): 10 μΐ [0.5 mg/ml] stock in EtOH @-20°C

Aprotinin (2 μ^ιοΐ): 2 μΐ [5 mg/ml] stock in dH ₂ 0 @-20°C

2. Protocol:

a. Wash the cells three times with ice-cold lx PBS buffer.

b. Add 250 μΐ RIPA buffer directly to cells on flask and scrape cells in RIPA buffer.

c. Transfer cell suspension to microcentrifuge tube and incubate on ice.

d. RIPA cell suspensions were stored at -70°C until the last time point (72 h). e. WCL Preparation:

i. Removed cell lysates from -70°C freezer and allowed to thaw slowly on ice.

ii. Incubate on ice for 30 minutes.

iii. At 30 minutes, briefly sonicate all samples for 3 pulses of 5 seconds each to shear chromatin.

iv. Centrifuge at high speed for 15 minutes at 4°C to pellet chromatin and DNA.

v. Transfer the supernatant into a new labeled 2.0 ml microfuge tube and keep on ice. f. Protein Quantitation: Coomassie Plus (Bradford) protein assay. Thermo Scientific Pierce (Cat # PI-23238). Standard Microplate Protocol (Working Range = 100-1,500 μg/ml) according to manufacturer's protocol.

Prepare BSA standard solution as below (10 μΐ x 2).

Final BSA Cone. [μ¾/π.1] | Vol. of BSA Stock (μΐ) | Vol. Diluent (μΐ) ii. Prepare sample solution as below. Assay 10 μΐ in triplicate each.

Prepare l/5x, /10x, l/20x, and l/40x sample solution each of WCL and total cell extracts.

l/5x: 20 μΐ lx sample + 80 μΐ H20

1/1 Ox: 50 μΐ l/5x sample + 50 μΐ H20

l/20x: 50 μΐ 1/1 Ox sample + 50 μΐ H20

l/40x: 50 μΐ l/20x sample + 50 μΐ H20 iii. Pipette 10 μΐ of each standard or unknown sample into the appropriate microplate wells as below:

Al, 2: 0 μg BSA (blank)

A3, 4: [31.5 μg/ml] BSA

A5, 6: [62.5 μg/ml] BSA

A7, 8: [125 μg/ml] BSA

A9, 10: [250 μg/ml] BSA

Al l, 12: [500 μg/ml] BSA

Bl, 2: [750 μg/ml] BSA

B3, 4: [1,000 μg/ml] BSA

Cl-3: MCF-7 WCL at l/40x

C4-6: MCF-7 WCL at l/20x

C7-9: MCF-7 WCL at 1/lOx

ClO-12: MCF-7 WCL at l/5x

Dl-3: MCF-7R F3 WCL at l/40x

D4-6: MCF-7R F3 WCL at l/20x

D7-9: MCF-7R F3 WCL at 1/lOx

D10-12: MCF-7R F3 WCL at l/5x iv. Add 200 μΐ 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

temperature (RT) away from light. vi. Measure the absorbance at 620 nm with a plate reader. Subtract the average 620 nm measurement for the Blank replicates from the 620 nm measurements of all other individual standard and unknown sample replicates. vii. Prepare a standard curve by plotting the average Blank-corrected 595 nm measurement for each protein standard vs. its concentration in μg/ml. Use the standard curve to determine the protein concentration of each unknown sample.

D. MCF-7 cells:

-Loaded at 25 μg WCL/each.

Assay Protocol:

1. Set up the gel apparatus with pre-cast PAGE gel. Pour lx running buffer all the way to the top of the gels (-500 ml).

2. Prepared samples in IX sample buffer with β-mercaptoethanol (8 μΐ of 4X sample buffer added to each sample). Samples were heated for 5 minutes in 100°C boiling water, then centrifuged briefly to collect the sample at the bottom of the tube.

3. Load samples into each well of gel as below:

Gel Loading Key:

Used Bio-Rad Criterion TGX 4-20% Stain-Free Protein Gel (18 well)

Lane 1 : MW Marker

Lane 2: 25 μg MCF-7 IgG Oh WCL

Lane 3: 25 μg MCF-7 IgG 8h WCL

Lane 4: 25 μg MCF-7 IgG 24h WCL

Lane 5: 25 μg MCF-7 IgG 48h WCL

Lane 6: 25 μg MCF-7 IgG 72h WCL

Lane 7: 25 μg MCF-7 ANTIBODY I Oh WCL

Lane 8: 25 μg MCF-7 ANTIBODY I 8h WCL

Lane 9: 25 μg MCF-7 ANTIBODY 1 24h WCL

Lane 10: 25 μg MCF-7 ANTIBODY 1 48h WCL

Lane 11: 25 μg MCF-7 ANTIBODY I 72h WCL

Lane 12: MW Marker

4. Run at 180V until the loading dye is near the bottom of the gel.

5. Separated plastic plates of gel, then cut around unused edges of gel.

Measured gel dimensions.

6. Activate stain-free technology by exposing gel to UV light for 2 minutes in BioRad ChemiDoc MP Imager.

7. Cut PVDF membrane to dimensions of gel. Solubilized PVDF membrane in 100% methanol, then equilibrated membrane in IX Transfer Buffer for 10 minutes. 8. Assembled transfer sandwich as follows:

a. Sponge, dipped in IX Transfer Buffer.

b. 2 sheets of Whatman chromatography paper, dipped in IX

Transfer Buffer, then all bubbles between layers of paper were smoothed out using a 10 ml serological pipet.

c. Gel, dipped in IX Transfer Buffer, then all bubbles between

Whatman Chromatography paper and gel were smoothed out as described above.

d. 2 sheets of Whatman chromatography paper, dipped in IX

Transfer Buffer, all bubbles smoothed out.

e. Sponge, dipped in IX Transfer Buffer.

f. Placed sandwich inside transfer cassette and closed.

9. Placed transfer cassette in transfer apparatus and connected leads. Place

frozen cold pack in tank to keep transfer buffer cold. Transferred proteins at 100V for 0.5 hours.

10. Blocked membrane in blocking buffer (5% milk in IX TBS-T) for 1 hour at room temperature with rocking.

11. Incubated each membrane overnight at 4°C with Santa Cruz a-BCL2 antibody in an incubation tray on a rocker at a dilution of 1 : 1000 in 5% milk/lX TBS- T.

12. Rinse the membrane in IX TBS-T 3 times for 15 minutes each on rocker.

13. Incubate membrane for 1 hour at room temperature with appropriate 2° Ab (mouse 2° antibody at 1-M 0,000 dilution).

14. Rinse the membrane in IX TBS-T 3 times for 15 minutes each on rocker.

15. Place ECL (Luminata Forte) on sheet protector in droplets to the approximate dimensions of each membrane piece, then carefully place membrane pieces onto ECL.

16. Cover with sheet protector and visualize immediately using Bio-Rad ChemiDoc MP and ImageLab software.

17. Statistical was done using a one tailed paired t-test.

Results are shown in FIG. 4.

Reagents:

4X SDS Sample Loading Buffer: 200 mM Tris/HCl, pH 6.8; 8% SDS; 0.4%

bromophenol blue; 20% glycerol; and 400 mM β-mercaptoethanol

Gel: Bio-Rad Criterion TGX Precast gel (18 well, 4-20%).

MW marker: Bio-Rad Precision Plus All Blue Standard

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

Santa Cruz Biotechnology GAPDH

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

Examle 5. Effects of ANTIBODY I on Viability of Tumor Cells

Illustrative IC50 Value Determination

[000184] The antiproliferative activity of ANTIBODY I against the human tumor cell lines was investigated with Promega's Cell Titer-Glo® Luminescent Cell Viability assay.

Table 2. Cells and antibodies used in the IC50 study.

[000185] 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 μίΛνβΙΙ. 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 μg/mL). After 72 hours of culture in a humidified incubator at 37°C, in an atmosphere of 5% C0 ₂ and 95% air, the plated cells and Cell Titer-Glo ^® (Promega G7571) reagents were brought to room temperature to equilibrate for 30 minutes. A picture was taken of the control wells to depict confluency at endpoint. 100 μΐ _^ of Cell Titer- Glo ^® reagent was added to each well. The plate was shaken for two minutes and then left to equilibrate for ten minutes. 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.

Table 3. Results of IC50 measurements.

Example 6. Methods of Predicting CDRs

[000186] Amino acid numbering based on framework and complementary determining regions (CDR) are defined by one of the following:

Rosie Rosetta. Reference: Lyskov S, et al., "Serverification of Molecular Modeling Applications: The Rosetta Online Server That Includes Everyone (ROSIE)". PLoS One. 2013 May 22;8(5):e63906. doi: 10.1371/journal.pone.0063906. Print 2013. The ROSIE app interface to the RosettaAntibody3 program was used to model the 3-D structure of the hypervariable region of ANTIBODY I and identify the six CDRs. The first stage utilizes canonical template selection and assembly based on the Chothia definition described below and the lowest energy structures. They are assembled using a Rosetta protocol, resulting in a crude structure. In the second stage, CDR-H3 is remodeled de novo. Paratope side chains and loop backbones were refined simultaneously based on the Paratome method described below. CDR-H3 contained less than 10 amino acids, which indicates that the root mean square deviation is approximately 1.5 Angstroms and the model is highly accurate.

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.

P.I.G.S. (Prediction of Immunoglobulin Structure) numbering system. Reference:

Marcatili P. et al. PIGS: automatic prediction of antibody structures. Bioinformatics 2008 24:

1953.

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.

Kabat. Reference: Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

A set of rules were followed to identify the CDRs, as published Dr. Andrew C.R.

Martin's Bioinformatics Group at University College London in UK, or below.

CDR-L1 (L24-L34)

Start Approx residue 24

Residue before always a Cys

Residue after always a Trp. Typically Trp-Tyr-Gln, but also, Trp-Leu-Gln, Trp-Phe-Gln,

Trp-Tyr-Leu

Length 10 to 17 residues

CDR-L2 (L50-L56)

Start always 16 residues after the end of LI

Residues before generally Ile-Tyr, but also, Val-Tyr, Ile-Lys, Ile-Phe

Length always 7 residues (except NEW (7FAB) which has a deletion in this region)

CDR-L3 (L89-L97)

Start always 33 residues after end of L2 (except NEW (7FAB) which has the deletion at the end of CDR-L2)

Residue before always Cys

Residues after always Phe-Gly-XXX-Gly

Length 7 to 11 residues

CDR-H1 (H31-H35B) Start Approx residue 31

Residues before always Cys-XXX-XXX-XXX

Residues after always a Trp. Typically Trp-Val, but also, Trp-Ile, Trp-Ala

Length 10 to 12 residues [AbM definition]; Chothia definition excludes the last 4 residues

CDR-H2 (H50-H65)

Start always 15 residues after the end of CDR-H1

Residues before typically Leu-Glu-Trp-Ile-Gly (SEQ ID NO:56), but a number of variations

Residues after Lys/Arg-Leu/HeA^al/Phe/Thr/Ala-Thr/Ser/Ile/Ala

Length Kabat definition 16 to 19 residues; AbM (and recent Chothia) definition ends 7 residues earlier

CDR-H3 (H95-H102)

Start always 33 residues after end of CDR-H2 (always 2 after a Cys)

Residues before always Cys-XXX-XXX (typically Cys-Ala-Arg)

Residues after always Trp-Gly-XXX-Gly

Length 3 to 25 residues

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)).

Table 4. Definition of the FR and CDR according to IMGT

TRP as described in the text. 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.

Example 7. Methods of Computer Modeling the Binding of ANTIBODY I to Nucleolin

[000187] ClusPro 2.0 protein-protein docking software (available at http://cluspro.bu.edu) was utilized to predict the interaction between the Rosetta ROSIE-predicted structure of ANTIBODY I and the solution structure of RNA-binding domains (RBD) 1 and 2 of human nucleolin. The solution structure of human nucleolin RBDs 1 and 2, also known as "2KRR", is publically available through the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB).

[000188] To begin docking in ClusPro 2.0, the .pdb file containing the Rosetta ROSIE-predicted structure of the antibody was assigned as the "Receptor" and the .pdb file containing the 2KRR structure was assigned as the "Ligand". Under "Advanced Options", "Use Antibody Mode" was selected. In Antibody Mode, ClusPro 2.0 uses an asymmetric potential for docking antibody and antigen pairs. This asymmetric potential was arrived at following the discovery that antibody- antigen interactions do not exhibit high degrees of surface complementarity, as in enzyme- substrate interactions. Rather, antibody-antigen interactions exhibit mostly flat, less hydrophobic interfaces. Finally, since antibodies interact with their antigens via their complementarity- determining regions (CDRs), the option to "Automatically Mask non-CDR Regions" was selected.

[000189] To execute docking, 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

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.

[000190] 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.

[000191] 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). Some of the closest distances between residues in antibody CDRs and residues of nucleolin (NCL) are listed in Table 5.

Table 5. Distances of select amino acids in the binding model of nucleolin - ANTIBODY I.

VII. Sequences of Nucleolin and ANTIBODY I

1) Nucleolin SEQ ID NO: 1

1 MVKLAKAGKN QGDPKKMAPP PKEVEEDSED EEMSEDEEDD S SGEEWIPQ KKGKKAAATS

61 AKKVWSPTK KVAVATPAKK AAVTPGKKAA ATPAKKTVTP AKAVTTPGKK GATPGKALVA

12 1 TPGKKGAAIP AKGAKNGKNA KKEDSDEEED DDSEEDEEDD EDEDEDEDE I EPAAMKAAAA

1 81 APASEDEDDE DDEDDEDDDD DEEDDSEEEA METTPAKGKK AAKWPVKAK NVAEDEDEEE

2 4 1 DDEDEDDDDD EDDEDDDDED DEEEEEEEEE EPVKEAPGKR KKEMAKQKAA PEAKKQKVEG

3 01 TEPTTAFNLF VGNLNFNKSA PELKTGI SDV FAKNDLAWD VRIGMTRKFG YVDFESAEDL

3 61 EKALELTGLK VFGNE IKLEK PKGKDSKKER DARTLLAKNL PYKVTQDELK EVFEDAAE IR 42 1 LVSKDGKSKG IAYIEFKTEA DAEKTFEEKQ GTE I DGRS I S LYYTGEKGQN QDYRGGKNST

4 81 WSGE SKTLVL SNLSYSATEE TLQEVFEKAT FIKVPQNQNG KSKGYAFIEF ASFEDAKEAL 54 1 NSCNKRE IEG RAIRLELQGP RGSPNARSQP SKTLFVKGLS EDTTEETLKE SFDGSVRARI 601 VTDRETGS SK GFGFVDFNSE EDAKAAKEAM EDGE IDGNKV TLDWAKPKGE GGFGGRGGGR 6 61 GGFGGRGGGR GGRGGFGGRG RGGFGGRGGF RGGRGGGGDH KPQGKKTKFE

Residues G300 to E466 of nucleolin, amino acid sequence SEQ ID NO: 2

GTEPTTAFNLFVGNLNFNKSAPELKTGI SDVFAKNDLAWDVRIGMTRKFGYVDFE SAEDLEKALELTGL KVFGNE IKLEKPKGKDSKKERDARTLLAKNLPYKVTQDELKEVFEDAAE IRLVSKDGKSKGIAYIEFKTE ADAEKTFEEKQGTE I DGRS I SLYYTGE

2) ANTIBODY I

ANTIBODY I Gamma heavy chain, nucleotide sequence SEQ ID NO:3

ATGAAACAC CTGTGGTTCT TTCTCCTGCT GGTGGCTGCT

CCCAGGTGGG TGCTGAGCCA GGTGCAGCTG CAGGAAAGCG GACCTGGCCT GGTCAAACCC TCCCAGACAC TGAGCCTGAC CTGCACCGTC AGCGGCGGAT CCATCAACTC CGGCGGCTTC TACTGGAGCT GGATCAGACA GCATCCTGGC AAGGGCCTCG AGTGGATCGG CTACATTAGC TATACCGGCA GCACCTACTA CAATCCCTCC CTGAAGAGCA GGGTGAACAT TAGCGCCGAC ACCTCCAAGA ACAGGTTCAG CCTGAAGCTC AGCAGCGTCA CCGCCGCCGA TACCGCCGTG TACTACTGCG CCAGGGACAT GAACGACGGC CTGCAGATCT GGGGACAGGG CACACTGGTC ACCGTGTCCG CTGCCAGCAC CAAGGGACCC AGCGTGTTCC CCCTGGCTCC CTCCTCCAAG AGCACCTCCG GAGGCACCGC CGCCCTGGGC TGCCTGGTGA AGGATTACTT CCCCGAGCCC GTGACCGTGA GCTGGAACAG CGGAGCCCTG ACAAGCGGAG TGCACACATT CCCTGCCGTG CTGCAGAGCA GCGGCCTGTA CTCCCTGAGC TCCGTGGTCA CAGTGCCTAG CTCCTCCCTC GGCACCCAGA CCTACATCTG CAACGTGAAC CATAAGCCCT CCAATACCAA GGTGGACAAG AGGGTCGAGC CCAAATCCTG CGACAAGACA CACACCTGTC CTCCTTGCCC CGCCCCCGAA CTGCTGGGCG GACCCTCCGT CTTCCTCTTC CCTCCTAAGC CCAAGGATAC CCTGATGATC AGCAGGACAC CTGAGGTGAC CTGCGTGGTG GTGGACGTCT CCCACGAGGA CCCCGAGGTG AAGTTCAACT GGTACGTGGA TGGCGTGGAG GTCCACAACG CCAAGACCAA GCCCAGAGAG GAGCAGTACA ACAGCACATA CAGGGTGGTC TCCGTCCTGA CAGTGCTCCA CCAGGACTGG CTGAATGGCA AGGAGTACAA GTGCAAGGTC AGCAACAAAG CCCTGCCCGC CCCTATCGAG AAGACCATCA GCAAGGCTAA GGGCCAGCCC AGGGAGCCCC AGGTCTATAC CCTGCCCCCC AGCAGGGAAG AGATGACCAA GAATCAGGTC TCCCTGACCT GTCTGGTGAA GGGCTTCTAC CCTAGCGACA TCGCCGTGGA GTGGGAGAGC AACGGCCAGC CTGAAAACAA CTACAAGACC ACCCCTCCTG TGCTGGACTC CGACGGATCC TTCTTCCTGT ACTCCAAGCT GACCGTGGAT AAAAGCAGGT GGCAACAGGG CAACGTGTTC TCCTGCTCCG TCATGCACGA AGCTCTGCAC AACCACTACA CCCAGAAGAG CCTGTCCCTG AGCCCTGGCA AG

ANTIBODY I Gamma heavy chain, amino acid sequence SEQ ID NO:4

1 MKHLWFFLLL VAAPRWVLSQ VQLQE SGPGL VKPSQTLSLT CTVSGGS INS GGFYWSWIRQ 61 HPGKGLEWIG YI SYTGSTYY NPSLKSRVNI SADTSKNRFS LKLSSVTAAD TAVYYCARDM 12 1 NDGLQIWGQG TLVTVSAAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS 181 GALTSGVHTF PAVLQSSGLY SLSSWTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC

241 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD

301 GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK

361 GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS

421 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK

Residues Ml to SI 9: native leader (signal peptide)

Residues A138 to V235: human CHI

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.

Residues Q20 to A137 of ANTIBODY I Gamma heavy chain variable region, amino acid sequence SEQ ID NO:5

QVQLQESGPGLVKPSQTLSLTCTVSGGSINSGGFYWSWIRQHPGKGLEWIGYISYTGSTY YNPSLKSRVN ISADTSKNRFSLKLSSVTAADTAVYYCARDM DGLQIWGQGTLVTVSA

Residues A138 to K467 of ANTIBODY I Gamma heavy chain constant region, amino acid sequence SEQ ID NO:6

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY KCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

ANTIBODY I Kappa light chain, nucleotide sequence SEQ ID NO:7

ATGGACATG AGGGTGCCTG CCCAGCTGCT CGGACTGCTG CTGCTGTGGC TGCCCGGAGC TAAGTGCGAC ATCCAGATGA CCCAGAGCCC TTCCACACTC TCCGCCAGCG TGGGCGATAG GGTGACCATC ACCTGCAGGG CCAGCCAGTC CATCAGCAGG TGGCTGGCCT GGTACCAGCA GAAGCCCGGC AAGGCCCCCA AGCTGCTGAT CTACAAGGCC AGCACACTCG AGTCCGGCGT GCCCAGCAGA TTCAGCGGAA GCGGCAGCGG CACCGAGTTT ACCCTGACCA TCAGCAGCCT GCAGCCCGAC GACTTCGCCA CCTACTACTG CCAGCAGTAC AACTCCTATA GCAGGGCCTT CGGCCAGGGC ACCAAAGTGG AGATCAAGAG GACCGTGGCC GCCCCTAGCG TCTTCATCTT CCCCCCCTCC GACGAGCAGC TGAAGAGCGG CACAGCCTCC GTGGTGTGCC TGCTGAACAA CTTCTACCCC AGGGAGGCCA AGGTGCAGTG GAAGGTGGAC AACGCCCTGC AGAGCGGCAA CTCCCAGGAG AGCGTGACCG AGCAGGACTC CAAGGACAGC ACCTACAGCC TGAGCAGCAC CCTCACCCTG AGCAAGGCCG ACTACGAGAA GCACAAGGTG TACGCCTGCG AGGTGACACA CCAGGGCCTG AGCAGCCCTG TGACCAAGTC TTTTAACAGG GGCGAATGC

ANTIBODY I Kappa light chain, amino acid sequence SEQ ID NO:8

1 MDMRVPAQLL GLLLLWLPGA KCDIQMTQSP STLSASVGDR VTITCRASQS ISRWLAWYQQ 61 KPGKAPKLLI YKASTLESGV PSRFSGSGSG TEFTLTISSL QPDDFATYYC QQYNSYSRAF 121 GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASWCLLNN FYPREAKVQW KVDNALQSGN 181 SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

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

DIQMTQSPSTLSASVGDRVTI TCRASQS I SRWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTE FTLTI SSLQPDDFATYYCQQYNSYSRAFGQGTKVE IK

Residues R130 to C236 of ANTIBODY I Kappa light chain constant region, amino acid sequence SEQ ID NO: 10

RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLS S TLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC

Table 6. Part 1. Complementarity Determining Region (CDR) of Heavy Chain Variable Region (VH) of ANTIBODY I, SEQ ID NOS. 11 to 31

VH Framework H1 CDR HI Framework H2 CDR H2 Framework H3 CDR H3 Framework H4

Rosie Rosetta QVQLQESGPG GGSINSGGFY WIRQHPGKGLE YISYTGSTYYN RVNISADTSK DMNDGLQI WGQGTLVTVS

LVKPSQTLSLT WS WIG PSLKS NRFSLKLSSV A CTVS (SEQ ID NO:26)

TAADTAVYYC

(SEQ ID NO: 13) (SEQ ID NO:16) (SEQ ID NO:19)

AR (SEQ ID NO:29)

(SEQ ID NO:11)

(SEQ ID

NO:22)

Paratome QVQLQESGPG GSINSGG FYWS WIRQHPGKGLE WIGYISYTGST NPSLKSRVNI RDMNDGLQI WGQGTLVTVS

LVKPSQTLSLT YY SADTSKNRFS A

(SEQ ID NO:14) (SEQ ID NO:17)

CTVSG LKLSSVTAAD (SEQ ID NO:27)

(SEQ ID NO:20)

TAVYYCA (SEQ ID NO:29)

(SEQ ID NO: 12)

(SEQ ID

NO:23)

P.I.G.S. QVQLQESGPG GGSINSGG FYWSWIRQHP YIS YTGSTYYNPS YCARDM NDGLQIWGQG

LVKPSQTLSLT GKGLEWIG LKSRVNISAD TLVTVSA

(SEQ ID NO: 15)

CTVS TSKNRFSLKL (SEQ ID NO:28)

(SEQ ID NO:18)

SSVTAADTAV (SEQ ID NO:30)

(SEQ ID NO:11)

Y

(SEQ ID

NO:24)

Chothia QVQLQESGPG GGSINSGGFY WIRQHPGKGLE YISYTGSTYYN KSRVNISADT DMNDGLQI WGQGTLVTVS Definition LVKPSQTLSLT WS WIG PSL SKNRFSLKLS A

CTVS SVTAADTAVY (SEQ ID NO:26)

(SEQ ID NO: 13) (SEQ ID NO:16) (SEQ ID NO:21 )

YCAR (SEQ ID NO:29)

(SEQ ID NO:11)

(SEQ ID

NO:25)

GSINSGG (SEQ ID NO:31)

Table 6. Part 2. CDR of Light Chain Variable Region (VL) of ANTIBODY I, SEQ ID NOS. 32 to 55

VL Framework L1 CDR L1 Framework L2 CDR L2 Framework L3 CDR L3 Framework L4

Rosie Rosetta DIQMTQSPSTL RASQSISRWLA WYQQKPGKAP KASTLES GVPSRFSGSG QQYNSY SRAFGQGTKV

SASVGDRVTIT KLLIY SGTEFTLTISSL EIK

(SEQ ID NO:35) (SEQ ID NO:41 ) (SEQ ID NO:47)

C QPDDFATYYC

(SEQ ID NO:38) (SEQ ID NO:51)

(SEQ ID NO:32) (SEQ ID NO:44)

Paratome DIQMTQSPSTL QSISRWLA WYQQKPGKAP LLIYKASTLES GVPSRFSGSG QQYNSYSRA FGQGTKVEIK

SASVGDRVTIT K SGTEFTLTISSL

(SEQ ID NO:36) (SEQ ID NO:42) (SEQ ID NO:48) (SEQ ID NO:52) CRAS QPDDFATYYC

(SEQ ID NO:39)

(SEQ ID NO:33) (SEQ ID NO:44)

P.I.G.S. DIQMTQSPSTL SQSISRWL AWYQQKPGKA KAST LESGVPSRFSG YNSYSR AFGQGTKVEIK

SASVGDRVTIT PKLLIY SGSGTEFTLTIS

(SEQ ID NO:37) (SEQ ID NO:43) (SEQ ID NO:49) (SEQ ID NO:53) CRA SLQPDDFATYY

(SEQ ID NO:40)

CQQ

(SEQ ID NO:34)

(SEQ ID NO:45)

Chothia DIQMTQSPSTL RASQSISRWLA WYQQKPGKAP KASTLES GVPSRFSGSG YNSYSRA FGQGTKVEIK Definition SASVGDRVTIT KLLIY SGTEFTLTISSL

(SEQ ID NO:35) (SEQ ID NO:41 ) (SEQ ID NO:50) (SEQ ID NO:52) C QPDDFATYYC

(SEQ ID NO:38)

QQ

(SEQ ID NO:32)

(SEQ ID NO:46)

QSISRWL (SEQ ID NO:54)

YNSY (SEQ ID NO:55)

[000192] All of the methods or embodiments disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. It will be apparent to those of skill in the art that variations can be applied to the methods or embodiments and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention as defined by the appended claims. More specifically, it will be apparent that certain agents which are both chemically and physiologically related can be substituted for agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.