Human Monoclonal Antibodies to Human Nucleolin

BACKGROUND OF THE INVENTION

The present invention relates generally to the fields of cell biology and immunology. More particularly, it concerns methods and compositions relating to the production and use of human monoclonal antibodies to human nucleolin (NCL).

Antibodies are a class of agents known as “biologicals.” The source of antibodies can be a polyclonal supply, such as human or horse serum, or derived from a monoclonal source (single cell clone). With the technologic capability to control and select for specific antigen binding, monoclonal antibodies have yielded dramatic therapeutic benefits. However, the difficulty of generating specific antibodies for certain targets has limited the successes, and the potential for therapeutic and diagnostic agents remains largely untapped.

One impediment to the development of monoclonal antibodies for human therapy is the need to “humanize” such antibodies, which are generally made in mice, rats and rabbits. If human patients are administered such antibodies without humanized constant regions, they can suffer from “serum sickness,” meaning that an endogenous immune response is mounted by the recipient against the non-human antibody sequences. Humanizing monoclonal antibodies produced in research animals can avoid this problem. However, the cost in time and expense for humanization of antibodies can be considerable.

Nucleolin is expressed on the cell surface of human chronic lymphocytic leukemia (CLL) cells, acute myeloid leukemia (AML) cells, some forms of breast carcinoma, as well as other tumors. As such, nucleolin constitutes a promising tumor antigen for targeting of therapeutics, including antibodies.

SUMMARY OF THE INVENTION

In one aspect of the invention a method is provided of producing an immortalized human B-cell that secrets an antibody that binds to human nucleolin comprising: obtaining a population of IgM-positive human B-cells; contacting said population with: Epstein-Barr virus (EBV) to immortalize said human B-cells, and a cytokine/growth factor/signaling agent cocktail to induce IgM-to-IgG immunoglobulin isotype class-switching; and culturing cells under conditions supporting said immortalization and immunoglobulin isotype class-switching. In one embodiment the method further comprises, selecting an immortalized human B-cell that expresses an antibody to human nucleolin. In another embodiment, selecting comprises an immunoassay performed on immortalized B-cell culture medium supernatants. In another embodiment, a cytokine cocktail comprises an agent that delivers a costimulatory signal to a human B-cell. In another embodiment, a cytokine cocktail comprises anti-IgM F(ab′)₂interleukin (IL)-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, INFα, BAFF, soluble CD40L. In another embodiment, a population of IgM-positive human B-cells is obtained from peripheral blood, a tonsils bone marrow, a spleen, a lymph node, umbilical cord blood, a liver, an apheresis procedures or a buffy coat. In another embodiment, the method further comprises isolating a nucleic acid encoding an entire heavy and/or light chain from the immortalized human B-cell of step (d). In another embodiment, the method further comprises isolating a nucleic acid encoding a heavy and/or light chain antigen-binding region from the immortalized human B-cell of step (d). In another embodiment, the method further comprises cloning said nucleic acid into a nucleic acid encoding a framework region of a heavy and/or light chain. In another embodiment, contacting said population further comprises an EBV concentration step, a centrifugation step during infection, or both. In another embodiment, the method further comprises freezing said population of human B-cells following step (c). In another embodiment, contacting said population with a cytokine/growth factor/signaling agent cocktail is performed at about 0-96 hours following step (b)(ii). In another embodiment, contacting said population with a cytokine/growth factor/signaling agent cocktail is performed at about 16-20 hours following step (b)(ii). In another embodiment, about 50%-99% of said population are immortalized by EBV infection. In another embodiment, about 95%-99% of said population are immortalized by EBV infection. In another embodiment, selecting an immortalized human B-cell that expresses an antibody to human nucleolin occurs 1-4 weeks following infection. In another embodiment, selecting an immortalized human B-cell that expresses an antibody to human nucleolin occurs 2-3 weeks following infection. In another embodiment, selecting an immortalized human B-cell that expresses an antibody to human nucleolin occurs after thawing stored frozen immortalized B-cells, and/or after thawing stored frozen culture medium supernatants from said immortalized B-cells. In another embodiment, the B-cell is antigen naïve. In another embodiment, the B-cell is antigen experienced.

In another aspect the invention provides an immortalized human B-cell that expresses an IgG antibody that binds to human nucleolin. In one embodiment, the immortalized human B-cell is designated as T-5D1, V-3H11 (3G5), T-2D3, T-7G7 (1H9), T-2H3, T-9F9, T-8G4 or T-P1C6.

In another aspect the invention provides an immortalized human B-cell that expresses an IgG antibody or fragment thereof that binds to a protein of SEQ ID No. 2. In one embodiment, the IgG antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In another embodiment, the IgG antibody is an IgG1 antibody. In another embodiment, the IgG antibody comprises a kappa light chain. In another embodiment, the IgG antibody comprises a lambda light chain. In another embodiment, the B-cell is EBV immortalized. In another embodiment, the antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the antibody or fragment thereof kills at least 10% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 20% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 30% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 40% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 50% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 60% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 70% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 80% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 90% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 100% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 10% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 20% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 30% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 40% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 50% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 60% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 70% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 80% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 90% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 100% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof binds to an RNA binding domain of human nucleolin. In another embodiment, the antibody or fragment thereof inactivates an RNA binding domain of human nucleolin. In another embodiment, the isolated antibody or fragment thereof induces complement-dependent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof induces complement-independent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof induces apoptosis in a cancer cell upon contact. In another embodiment, the isolated antibody or fragment thereof inhibits or kills an AML cancer cell, a CLL cancer cell or a breast cancer cell. In another embodiment, the isolated monoclonal antibody or fragment thereof reduces BCL-2 levels in a cancer cell.

In another aspect the invention provides an immortalized human B-cell that expresses an IgG antibody or fragment thereof that binds to a protein encoded by SEQ ID No. 1. In one embodiment, the IgG antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In another embodiment, the IgG antibody is an IgG1 antibody. In another embodiment, the IgG antibody comprises a kappa light chain. In another embodiment, the IgG antibody comprises a lambda light chain. In another embodiment, the B-cell is EBV immortalized. In another embodiment, the antibody or fragment thereof is a monoclonal antibody or fragment thereof. The isolated antibody or fragment thereof of claim 33, wherein said antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the antibody or fragment thereof kills at least 10% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 20% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 30% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 40% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 50% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 60% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 70% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 80% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 90% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 100% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 10% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 20% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 30% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 40% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 50% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 60% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 70% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 80% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 90% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 100% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof binds to an RNA binding domain of human nucleolin. In another embodiment, the antibody or fragment thereof inactivates an RNA binding domain of human nucleolin. In another embodiment, the isolated antibody or fragment thereof induces complement-dependent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof induces complement-independent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof induces apoptosis in a cancer cell upon contact. In another embodiment, the isolated antibody or fragment thereof inhibits or kills an AML cancer cell, a CLL cancer cell or a breast cancer cell. In another embodiment, the isolated monoclonal antibody or fragment thereof reduces BCL-2 levels in a cancer cell.

In another aspect the invention provides an immortalized human B-cell that expresses an IgG antibody or fragment thereof that binds to a protein comprising SEQ ID No. 4. In one embodiment, the IgG antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In another embodiment, the IgG antibody is an IgG1 antibody. In another embodiment, the IgG antibody comprises a kappa light chain. In another embodiment, the IgG antibody comprises a lambda light chain. In another embodiment, the B-cell is EBV immortalized. In another embodiment, the isolated antibody or fragment thereof of claim 33, wherein said antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the antibody or fragment thereof kills at least 10% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 20% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 30% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 40% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 50% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 60% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 70% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 80% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 90% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 100% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 10% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 20% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 30% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 40% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 50% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 60% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 70% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 80% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 90% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 100% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof binds to an RNA binding domain of human nucleolin. In another embodiment, the antibody or fragment thereof inactivates an RNA binding domain of human nucleolin. In another embodiment, the isolated antibody or fragment thereof induces complement-dependent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof induces complement-independent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof induces apoptosis in a cancer cell upon contact. In another embodiment, the isolated antibody or fragment thereof inhibits or kills an AML cancer cell, a CLL cancer cell or a breast cancer cell. In another embodiment, the isolated monoclonal antibody or fragment thereof reduces BCL-2 levels in a cancer cell.

In another aspect the invention provides an immortalized human B-cell that expresses an IgG antibody or fragment thereof that binds to a protein encoded by SEQ ID No. 3. In one embodiment, the IgG antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In another embodiment, the IgG antibody is an IgG 1 antibody. In another embodiment, the IgG antibody comprises a kappa light chain. In another embodiment, the IgG antibody comprises a lambda light chain. In another embodiment, the B-cell is EBV immortalized. In another embodiment, the antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the antibody or fragment thereof kills at least 20% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 20% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof binds to an RNA binding domain of human nucleolin. In another embodiment, the antibody or fragment thereof inactivates an RNA binding domain of human nucleolin. In another embodiment, the isolated antibody or fragment thereof induces complement-dependent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof induces complement-independent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof induces apoptosis in a cancer cell upon contact. In another embodiment, the isolated antibody or fragment thereof inhibits or kills an AML cancer cell, a CLL cancer cell or a breast cancer cell. In another embodiment, the isolated monoclonal antibody or fragment thereof reduces BCL-2 levels in a cancer cell.

In another aspect the invention provides an isolated human monoclonal antibody or fragment thereof that specifically binds to a protein of SEQ ID No. 4. In one embodiment, the antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the antibody or fragment thereof kills at least 10% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 20% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 30% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 40% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 50% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 60% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 70% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 80% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 90% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 100% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 10% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 20% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 30% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 40% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 50% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 60% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 70% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 80% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 90% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 100% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof binds to an RNA binding domain of human nucleolin. In another embodiment, the antibody or fragment thereof inactivates an RNA binding domain of human nucleolin. In another embodiment, the isolated antibody or fragment thereof induces complement-dependent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof induces complement-independent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof induces apoptosis in a cancer cell upon contact. In another embodiment, the isolated antibody or fragment thereof inhibits or kills an AML cancer cell, a CLL cancer cell or a breast cancer cell. In another embodiment, the isolated monoclonal antibody or fragment thereof reduces BCL-2 levels in a cancer cell.

In another aspect the invention provides an isolated antibody or fragment thereof that specifically binds to a human nucleolin protein, wherein said antibody or fragment thereof kills at least 20% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In one embodiment, the amino acid sequence of said human nucleolin comprises SEQ ID No. 2. In another embodiment, the antibody or fragment thereof binds to an amino acid sequence consisting of amino acid residues 1 to 283 of SEQ ID No. 2. In another embodiment, the antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the antibody or fragment thereof binds to an RNA binding domain of human nucleolin. In another embodiment, the antibody or fragment thereof inactivates an RNA binding domain of human nucleolin. In another embodiment, the isolated antibody or fragment thereof is linked to a diagnostic or therapeutic agent. In another embodiment, the isolated antibody or fragment thereof exhibits complement-dependent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof exhibits complement-independent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof induces apoptosis in a cancer cell upon contact. In another embodiment, the antibody or fragment thereof inhibits or kills an AML cancer cell, a CLL cancer cell or a breast cancer cell. In another embodiment, the isolated antibody or fragment thereof reduces BCL-2 levels in a cancer cell. In another embodiment, the antibody or fragment thereof is linked to a diagnostic agent. In another embodiment, the diagnostic agent is a radionuclide, a fluorophore, a chemilluminescent compound, a fluorescent compound, or an enzyme. In another embodiment, the antibody or fragment thereof is linked to a therapeutic agent. In another embodiment, the therapeutic agent is a radionuclide, a toxin or a chemotherapeutic moiety.

In another aspect the invention provides an isolated antibody or fragment thereof that specifically binds to a human nucleolin protein, wherein said antibody or fragment thereof kills at least 10-100% of a population of MV4-11 cells (such as 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the amino acid sequence of said human nucleolin comprises SEQ ID No. 2. In another embodiment, the antibody or fragment thereof binds to an amino acid sequence consisting of amino acid residues 1 to 283 of SEQ ID No. 2. In another embodiment, the antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the antibody or fragment thereof binds to an RNA binding domain of human nucleolin. In another embodiment, the antibody or fragment thereof inactivates an RNA binding domain of human nucleolin. In another embodiment, the isolated antibody or fragment thereof is linked to a diagnostic or therapeutic agent. In another embodiment, the isolated antibody or fragment thereof exhibits complement-dependent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof exhibits complement-independent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof induces apoptosis in a cancer cell upon contact. In another embodiment, the antibody or fragment thereof inhibits or kills an AML cancer cell, a CLL cancer cell or a breast cancer cell. In another embodiment, the isolated antibody or fragment thereof reduces BCL-2 levels in a cancer cell. In another embodiment, the said antibody or fragment thereof is linked to a diagnostic agent. In another embodiment, the diagnostic agent is a radionuclide, a fluorophore, a chemilluminescent compound, a fluorescent compound, or an enzyme. In another embodiment, the antibody or fragment thereof is linked to a therapeutic agent. In another embodiment, the therapeutic agent is a radionuclide, a toxin or a chemotherapeutic moiety.

In another aspect the invention provides an anti-nucleolin composition comprising one or more isolated antibodies or fragments thereof that specifically binds to a human nucleolin protein, wherein said one or more antibodies kills at least 10-100% of a population of MCF-7 cells (such as 10, 20, 30, 40, 40, 50, 60, 70, 80, 90, or 100%), when incubated with said MCF-7 cells and human AB serum for 48-96 (such as 48, 72 or 96 hours) hours. In one embodiment, the one or more isolated antibodies or fragments thereof is a monoclonal antibody or fragment thereof. In another embodiment, the one or more isolated antibodies or fragments thereof is substantially non-immunogenic to a human. In another embodiment, the one or more isolated antibodies or fragments thereof is a human antibody or fragment thereof. In another embodiment, the one or more isolated antibodies or fragments thereof binds to an RNA binding domain of human nucleolin. In another embodiment, the one or more isolated antibodies or fragments thereof inactivates an RNA binding domain of human nucleolin. In another embodiment, the amino acid sequence of said human nucleolin comprises SEQ ID No. 2. In another embodiment, the one or more isolated antibodies or fragments thereof binds to SEQ ID No. 4. In another embodiment, the anti-nucleolin composition further comprises a radionuclide, a fluorophore, a chemilluminescent compound, a fluorescent compound, an enzyme, a toxin or a chemotherapeutic agent. In another embodiment, the radionuclide, a fluorophore, a chemilluminescent compound, a fluorescent compound, an enzyme, a toxin or a chemotherapeutic agent is conjugated to said one or more isolated antibodies or fragments thereof. In another embodiment, the anti-nucleolin composition comprises two or more isolated antibodies or fragments thereof that specifically binds to said human nucleolin protein, wherein said one or more antibodies kills at least 20% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the anti-nucleolin composition comprises three or more isolated antibodies or fragments thereof that specifically binds to said human nucleolin protein, wherein said one or more antibodies kills at least 20% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours.

In another aspect the invention provides a method of inhibiting or killing a cell expressing nucleolin on its surface comprising contacting said cell with an antibody or fragment thereof that binds to human nucleolin, wherein said antibody or fragment thereof kills at least 20% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In one embodiment, the antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the antibody or fragment thereof binds to SEQ ID No. 2. In another embodiment, the antibody or fragment thereof binds to an amino acid sequence encoded by SEQ ID No. 1. In another embodiment, the antibody or fragment thereof binds to SEQ ID No. 4. In another embodiment, the antibody or fragment thereof binds to an amino acid sequence encoded by SEQ ID No. 3. In another embodiment, the cell is a cancer cell. In another embodiment, the cancer cell is a lung cancer cell, a breast cancer cell, a prostate cancer cell, a colon cancer cell, a pancreatic cancer cell, a renal cell carcinoma cell, an ovarian cancer cell, a leukemia cell, a melanoma cell, a glioblastoma cell, a neuroblastoma cell, a sarcoma cell or a gastric cancer cell. In another embodiment, the cell is an immune cell. In another embodiment, the immune cell is a lymphocyte, dendritic cell, a peripheral blood monocyte, a macrophage or a glial cell. In another embodiment, the immune cell is an activated immune cell. In another embodiment, the immune cell is an activated B cell. In another embodiment, the immune cell is a memory B cell. In another embodiment, the immune cell is an activated T cell. In another embodiment, the immune cell is an activated CD4+ T cell. In another embodiment, the immune cell is an activated CD8+ T cell. In another embodiment, the cell a vascular smooth muscle cell or an endothelial cell. In another embodiment, the antibody or fragment thereof is linked to a therapeutic agent. In another embodiment, the therapeutic agent is a radionuclide, a toxin or a chemotherapeutic agent. In another embodiment, the inhibiting or killing comprises inducing apoptosis in said cell. In another embodiment, the cell is located in a human subject, and said contacting comprising administering said antibody or fragment thereof to said subject. In another embodiment, the method of further comprises contacting said cell with at least one additional inhibitory agent or treatment. In another embodiment, the additional treatment comprises one or more of surgery, radiotherapy, chemotherapy, toxin therapy, immunotherapy, hormone therapy, anti-angiogenic therapy or gene therapy or other biological therapies. In another embodiment, the additional inhibitory agent comprises one or more of radionuclides, chemotherapetic agents, toxins immunotherapeutics, hormones, nucleic acids or polypeptides. In another embodiment, the toxin is diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolaca americana protein, pokeweed antiviral protein, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, calicheamicins or tricothecenes toxin. In another embodiment, the chemotherapeutic agent is an alkylating agent, anthracycline, cytoskeletal disruptor, epothilone, inhibitor of topoisomerase I, inhibitor of topoisomerase II, nucleoside or nucleotide analog, precursor analogs, peptide antibiotic, platinum-based agents retinoids, vinca alkaloids or derivatives thereof. In another embodiment, the wherein said chemotherapeutic agent is 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, or vinorelbine.

In another aspect the invention provides a method of detecting a cell expressing nucleolin on its surface comprising contacting said cell with a human antibody or fragment thereof that binds immunologically to said nucleolin. In one embodiment, the cell is a cancer cell, an immune cell, or a vascular smooth muscle cell that expresses nucleolin on its surface, an endothelial cell that expresses nucleolin on its surface, or a virus infected cell. In another embodiment, the cell is a precancerous cell that expresses nucleolin on its surface. In another embodiment, the cancer cell selected from the group consisting of is a lung cancer cell, a breast cancer cell, a prostate cancer cell, a colon cancer cell, a pancreatic cancer cell, a renal cell carcinoma cell, an ovarian cancer cell, a leukemia cell, a melanoma cell, a glioblastoma cell, a neuroblastoma cell, a sarcoma cell and a gastric cancer cell. In another embodiment, the immune cell is a lymphocyte, dendritic cell, a peripheral blood monocyte, a macrophage and a glial cell. In another embodiment, the cell is an immune cell. In another embodiment, the immune cell is an activated immune cell. In another embodiment, the immune cell is an activated B cell. In another embodiment, the immune cell is a memory B cell. In another embodiment, the immune cell is an activated T cell. In another embodiment, the immune cell is an activated CD4+ T cell. In another embodiment, the immune cell is an activated CD8+ T cell. In another embodiment, the cell is a vascular smooth muscle cell or an endothelial cell. In another embodiment, the antibody or fragment thereof is linked to a diagnostic agent. In another embodiment, the diagnostic agent is a radionuclide, a fluorophore, a chemilluminescent compound, a fluorescent compound, a quantum dot, a nanoparticles or an enzyme. In another embodiment, the cell is located in a human subject and contacting comprises administering said antibody or fragment thereof to said subject. In another embodiment, the cell is located in an isolated, tissue sample or cell suspension.

In another aspect the invention provides a method of treating or preventing cancer in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin agent and a pharmaceutically acceptable carrier; wherein said anti-nucleolin agent comprises an anti-nucleolin antibody or fragment thereof and said antibody or fragment thereof kills at least 10-100% (such as 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 (such as 48, 72, or 96) hours. In one embodiment, the mammal is a human. In another embodiment, the treating cancer in a mammal comprises treating tumor hpoxia. In another embodiment, the treating cancer in a mammal comprises inhibiting tumor angiogenesis. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin monoclonal antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the toxin is diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolaca americana protein, pokeweed antiviral protein, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, calicheamicins or tricothecenes toxin. In another embodiment, the chemotherapeutic agent is an alkylating agent, anthracycline, cytoskeletal disruptor, epothilone, inhibitor of topoisomerase I, inhibitor of topoisomerase II, nucleoside or nucleotide analog, precursor analogs, peptide antibiotic, platinum-based agents retinoids, vinca alkaloids or derivatives thereof. In another embodiment, the chemotherapeutic agent is 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, or vinorelbine.

In another aspect the invention provides a method of treating or preventing cancer in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin antibody or fragment thereof, a toxin or chemotherapeutic agent and a pharmaceutically acceptable carrier, wherein said antibody or fragment thereof kills at least 10-100% (such as 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 (such as 48, 72, or 96) hours. In one embodiment, the mammal is a human. In another embodiment, the treating cancer in a mammal comprises treating tumor hpoxia. In another embodiment, the treating cancer in a mammal comprises inhibiting tumor angiogenesis. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin monoclonal antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the toxin is diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolaca americana protein, pokeweed antiviral protein, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, calicheamicins or tricothecenes toxin. In another embodiment, the chemotherapeutic agent is an alkylating agent, anthracycline, cytoskeletal disruptor, epothilone, inhibitor of topoisomerase I, inhibitor of topoisomerase II, nucleoside or nucleotide analog, precursor analogs, peptide antibiotic, platinum-based agents retinoids, vinca alkaloids or derivatives thereof. In another embodiment, the chemotherapeutic agent is 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, or vinorelbine

In another aspect the invention provides a method of treating or preventing cancer in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin antibody or fragment thereof and a pharmaceutically acceptable carrier, and further treating said mammal with radiation therapy, wherein said antibody or fragment thereof kills at least 10-100% (such as 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 (such as 48, 72, or 96) hours. In one embodiment, the mammal is a human. In another embodiment, the treating cancer in a mammal comprises treating tumor hpoxia. In another embodiment, the treating cancer in a mammal comprises inhibiting tumor angiogenesis. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin monoclonal antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the toxin is diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolaca americana protein, pokeweed antiviral protein, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, calicheamicins or tricothecenes toxin. In another embodiment, the chemotherapeutic agent is an alkylating agent, anthracycline, cytoskeletal disruptor, epothilone, inhibitor of topoisomerase I, inhibitor of topoisomerase H, nucleoside or nucleotide analog, precursor analogs, peptide antibiotic, platinum-based agents retinoids, vinca alkaloids or derivatives thereof. In another embodiment, the chemotherapeutic agent is 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, or vinorelbine.

In another aspect the invention provides a method of treating or preventing cancer in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin agent and a pharmaceutically acceptable carrier; wherein said anti-nucleolin agent comprises a anti-nucleolin antibody or fragment thereof that specifically binds to a protein of SEQ ID No. 4. In one embodiment, the mammal is a human. In another embodiment, the treating cancer in a mammal comprises treating tumor hypoxia. In another embodiment, the treating cancer in a mammal comprises inhibiting tumor angiogenesis. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin monoclonal antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the toxin is diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolaca americana protein, pokeweed antiviral protein, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, calicheamicins or tricothecenes toxin. In another embodiment, the chemotherapeutic agent is an alkylating agent, anthracycline, cytoskeletal disruptor, epothilone, inhibitor of topoisomerase I, inhibitor of topoisomerase II, nucleoside or nucleotide analog, precursor analogs, peptide antibiotic, platinum-based agents retinoids, vinca alkaloids or derivatives thereof. In another embodiment, the chemotherapeutic agent is 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, or vinorelbine.

A method of treating or preventing cancer in a mammal comprising administering to said mammal a therapeutically effective amount of a anti-nucleolin antibody or fragment thereof, a toxin or chemotherapeutic agent and a pharmaceutically acceptable carrier, wherein said antibody or fragment thereof specifically binds to a protein of SEQ ID No. 4. In one embodiment, the mammal is a human. In another embodiment, the treating cancer in a mammal comprises treating tumor hpoxia. In another embodiment, the treating cancer in a mammal comprises inhibiting tumor angiogenesis. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin monoclonal antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the toxin is diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolaca americana protein, pokeweed antiviral protein, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, calicheamicins or tricothecenes toxin. In another embodiment, the chemotherapeutic agent is an alkylating agent, anthracycline, cytoskeletal disruptor, epothilone, inhibitor of topoisomerase 1, inhibitor of topoisomerase II, nucleoside or nucleotide analog, precursor analogs, peptide antibiotic, platinum-based agents retinoids, vinca alkaloids or derivatives thereof. In another embodiment, the chemotherapeutic agent is 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, or vinorelbine.

In another aspect the invention provides a method of treating or preventing cancer in a mammal comprising administering to said mammal a therapeutically effective amount of a anti-nucleolin antibody or fragment thereof and a pharmaceutically acceptable carrier, and further treating said mammal with radiation therapy, wherein said antibody or fragment thereof specifically binds to a protein of SEQ ID No. 4. In one embodiment, the mammal is a human. In another embodiment, the treating cancer in a mammal comprises treating tumor hpoxia. In another embodiment, the treating cancer in a mammal comprises inhibiting tumor angiogenesis. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin monoclonal antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the toxin is diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolaca americana protein, pokeweed antiviral protein, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, calicheamicins or tricothecenes toxin. In another embodiment, the chemotherapeutic agent is an alkylating agent, anthracycline, cytoskeletal disruptor, epothilone, inhibitor of topoisomerase I, inhibitor of topoisomerase II, nucleoside or nucleotide analog, precursor analogs, peptide antibiotic, platinum-based agents retinoids, vinca alkaloids or derivatives thereof. In another embodiment, the chemotherapeutic agent is 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, or vinorelbine.

In another aspect the invention provides a method of treating an autoimmune disease in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin agent and a pharmaceutically acceptable carrier; wherein said anti-nucleolin agent comprises an anti-nucleolin antibody or fragment thereof and said antibody or fragment thereof kills at least 10-100% (such as 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%) of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 (such as 48, 72, or 96) hours. In one embodiment, the autoimmune disease is 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 mellitus (e.g., type 1), 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), myelodysplastic syndrome, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosis (SLE), Sweet's syndrome, Still's disease, lupus erythematosus, takayasu arteritis, temporal arteristis/giant cell arteritis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis. Examples of inflammatory disorders include, but are not limited to, asthma, encephilitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentitated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, graft versus host disease, urticaria, or Vogt-Koyanagi-Hareda syndrome. In another embodiment, the mammal is a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin antibody or fragment thereof is a monoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating an autoimmune disease in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin agent and a pharmaceutically acceptable carrier; wherein said anti-nucleolin agent comprises a human anti-nucleolin antibody or fragment thereof that specifically binds to a protein of SEQ ID No. 2. In one embodiment, the autoimmune disease is 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 mellitus (e.g., type 1), 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), myelodysplastic syndrome, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosis (SLE), Sweet's syndrome, Still's disease, lupus erythematosus, takayasu arteritis, temporal arteristis/giant cell arteritis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis. Examples of inflammatory disorders include, but are not limited to, asthma, encephilitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentitated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, graft versus host disease, urticaria, or Vogt-Koyanagi-Hareda syndrome. In another embodiment, the mammal is a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin antibody or fragment thereof is a monoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating an autoimmune disease in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin agent and a pharmaceutically acceptable carrier; wherein said anti-nucleolin agent comprises an anti-nucleolin antibody or fragment thereof that specifically binds to a protein of SEQ ID No. 4. In one embodiment, the autoimmune disease is 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 mellitus (e.g., type 1), 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), myelodysplastic syndrome, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosis (SLE), Sweet's syndrome, Still's disease, lupus erythematosus, takayasu arteritis, temporal arteristis/giant cell arteritis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis. Examples of inflammatory disorders include, but are not limited to, asthma, encephilitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentitated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, graft versus host disease, urticaria, or Vogt-Koyanagi-Hareda syndrome. In another embodiment, the mammal is a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin antibody or fragment thereof is a monoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating an airway disease in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin agent and a pharmaceutically acceptable carrier; wherein said anti-nucleolin agent comprises an anti-nucleolin antibody or fragment thereof and said antibody or fragment thereof kills at least 10-100% (10, 20, 30, 40, 50, 60, 70, 90, 100%) of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 (48, 72, or 96) hours. In another embodiment, the airway disease is asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, or inflammatory pneumonitis. In another embodiment, the mammal is a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin antibody or fragment thereof is a monoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating an airway disease in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin agent and a pharmaceutically acceptable carrier; wherein said anti-nucleolin agent comprises a human anti-nucleolin antibody or fragment thereof that specifically binds to a protein of SEQ ID No. 2. In another embodiment, the airway disease is asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, or inflammatory pneumonitis. In another embodiment, the mammal is a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin antibody or fragment thereof is a monoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating an airway disease in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin agent and a pharmaceutically acceptable carrier; wherein said anti-nucleolin agent comprises an anti-nucleolin antibody or fragment thereof that specifically binds to a protein of SEQ ID No. 4. In another embodiment, the airway disease is asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, or inflammatory pneumonitis. In another embodiment, the mammal is a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin antibody or fragment thereof is a monoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating a virally infected cell in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin agent and a pharmaceutically acceptable carrier; wherein said anti-nucleolin agent comprises an anti-nucleolin antibody or fragment thereof and said antibody or fragment thereof kills at least 20% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the virally infected cell is infected with an HIV virus. In another embodiment, the mammal is a human. In another embodiment, the anti-nucleolin monoclonal antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin antibody or fragment thereof is a monoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating a virally infected cell in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin agent and a pharmaceutically acceptable carrier; wherein said anti-nucleolin agent comprises a human anti-nucleolin antibody or fragment thereof that specifically binds to a protein of SEQ ID No. 2. In another embodiment, the virally infected cell is infected with an HIV virus. In another embodiment, the mammal is a human. In another embodiment, the anti-nucleolin monoclonal antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin antibody or fragment thereof is a monoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating a virally infected cell in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin agent and a pharmaceutically acceptable carrier; wherein said anti-nucleolin agent comprises an anti-nucleolin antibody or fragment thereof that specifically binds to a protein of SEQ ID No. 4. In another embodiment, the virally infected cell is infected with an HIV virus. In another embodiment, the mammal is a human. In another embodiment, the anti-nucleolin monoclonal antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin antibody or fragment thereof is a monoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating or preventing a non-cancerous condition or disease in a mammal characterized by increased surface expression of nucleolin, comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin agent and a pharmaceutically acceptable carrier; wherein said anti-nucleolin agent comprises an anti-nucleolin antibody or fragment thereof and said antibody or fragment thereof kills at least 10-100% (such as 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%) of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 (48, 72, or 96) hours. In one embodiment, the condition or disease in a mammal characterized by increased surface expression of nucleolin is macular degeneration, diabetic retinopathy, or inflammatory disease. In another embodiment, the mammal is a human. In another embodiment, the anti-nucleolin monoclonal antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin antibody or fragment thereof is a monoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating or preventing a non-cancerous condition or disease in a mammal characterized by increased surface expression of nucleolin, comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin agent and a pharmaceutically acceptable carrier; wherein said anti-nucleolin agent comprises a human anti-nucleolin antibody or fragment thereof that specifically binds to a protein of SEQ ID No. 2. In one embodiment, the condition or disease in a mammal characterized by increased surface expression of nucleolin is macular degeneration, diabetic retinopathy, or inflammatory disease. In another embodiment, the mammal is a human. In another embodiment, the anti-nucleolin monoclonal antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin antibody or fragment thereof is a monoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating or preventing a non-cancerous condition or disease in a mammal characterized by increased surface expression of nucleolin, comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin agent and a pharmaceutically acceptable carrier; wherein said anti-nucleolin agent comprises an anti-nucleolin antibody or fragment thereof that specifically binds to a protein of SEQ ID No. 4. In one embodiment, the condition or disease in a mammal characterized by increased surface expression of nucleolin is macular degeneration, diabetic retinopathy, or inflammatory disease. In another embodiment, the mammal is a human. In another embodiment, the anti-nucleolin monoclonal antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin antibody or fragment thereof is a monoclonal antibody or fragment thereof.

In another aspect the invention provides an anti-nucleolin agent that kills at least 50% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the said anti-nucleolin agent is substantially non-immunogenic to a human.

In another aspect the invention provides an anti-nucleolin agent that kills more MCF-7 cells than MCF10A cells when incubated with separate populations of MCF-7 cells and MCF10A cells and heat inactivated serum for 72 or 96 hours. In another embodiment, the said anti-nucleolin agent is substantially non-immunogenic to a human.

In another aspect the invention provides an anti-nucleolin agent that kills more MCF-7 cells than MCF10A cells when incubated with separate populations of MCF-7 cells and MCF10A cells and human AB serum for 96 hours. In another embodiment, the said anti-nucleolin agent is substantially non-immunogenic to a human.

In another aspect the invention provides an anti-nucleolin agent that specifically binds to a protein of SEQ ID No. 4 and inihibits or kills one or more cancer cells that express nucleolin on their cell surface. In another embodiment, the said anti-nucleolin agent is substantially non-immunogenic to a human.

In another aspect the invention provides a method of determining a likelihood that a subject will develop cancer by detecting increased cell surface nucleolin expression in one or more precancerous cells.

In another aspect the invention provides a antibody of any of the proceeding claims, wherein said antibody fragment is a Fab, Fab′, F(ab′).sub.2, or Fv fragment; diabodie; linear antibody; single-chain antibody; or a multispecific antibodt formed from an antibody fragment.

In another aspect the invention provides a method of any of the proceeding claims comprising the use of an antibody fragment, wherein said antibody fragment is a Fab, Fab′, F(ab′).sub.2, or Fv fragment; diabodie; linear antibody; single-chain antibody; or a multispecific antibodt formed from an antibody fragment.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1. Over-expression of nucleolin and Bcl-2 proteins in CLL cells vs. normal B cells. Peripheral-blood lymphocytes were isolated from CLL patients and healthy volunteers by density gradient centrifugation and the B cells were purified by positive selection with magnetic-activated cell separation (MACS) CD19 immunomagnetic beads. Nucleolin and Bcl-2 protein levels were measured in S10 extracts of the cells by Western blotting. The results were normalized to the values obtained from known amounts of nucleolin and Bcl-2 protein external standards. The labels N and C along the X-axis refer to normal B cells and CLL cells, respectively, from individual subjects. N=CD19+ B cells from healthy volunteers; C=CD19+ CLL cells.

FIGS. 2A-B. Subcellular localization of nucleolin in CLL vs normal B cells and MCF-7 vs MCF-10A cells. The intracellular localization of nucleolin was determined by indirect immunofluorescence using mouse anti-human nucleolin mAb and a secondary FITC-conjugated anti-mouse IgG. Nuclei were counterstained with propidium iodide. FIG. 2A shows a comparison between CLL and normal B cells. FIG. 2B shows a comparison between MCF-7 and MCF-10A cells.

FIG. 3. Subcellular localization of nucleolin in MCF-7 and MCF-10A cells. The intracellular localization of nucleolin was determined by indirect immunofluorescence using a monoclonal antibody against human nucleolin and a secondary FITC-conjugated anti-mouse IgG. Nuclei were counterstained with propidium iodide. Cell morphology is shown as DIC images. Results are representatives of three separate experiments.

FIG. 4. Bone marrow biopsy from a patient with AML-M1 stained with anti-nucleolin antibody (fuchsin stain) shows intense nuclear staining. Interspersed non-leukemic marrow elements are either negative (dark blue counterstain) or show only a nuclear blush. Magnification was 40×.

FIGS. 5A-B. Nucleolin overexpression in spleen sections from human AML-engrafted mice and from human breast cancer cells. (FIG. 5A) Two well-defined nodular human leukemic infiltrates present in the mouse spleen. The leukemic infiltrates are positive for nucleolin (fuchsin immunostain). Surrounding mouse spleen lymphocytes are negative for nucleolin immunostaining (blue counterstain). Magnification was 40×. (FIG. 5B) A tissue array with 50 cases of paraffin embedded formalin fixed human breast tumor and adjacent normal tissue (Biomax BR1006) was stained with anti-nucleolin Mab MS3 (Santa Cruz 1:100) using Vectastain mouse kit, DAB substrate, and hematoxylin counterstain. Representative cases were imaged on an Olympus CKX41 at 100× magnification.

FIG. 6. Relative expression levels of bcl-2 hnRNA and bcl-2 mRNA in CLL and normal B cells. The levels of bcl2 hnRNA and bcl2 mRNA in CLL cells from 4CLL patients and normal B cells from 4 healthy volunteers were determined by RT-PCR. Results are expressed as the means of 4 determinations per group SEM. *P<0.001 compared with normal B cells.

FIG. 7. Decay of bcl-2 mRNA in extracts of CLL and normal B cells. 5′-capped and polyadenylated [³²P]bcl-2-CR RNA and [³²]bcl-2-CR-ARE RNA were incubated with S100 extracts prepared from either CLL cells from 4 patients or normal B cells from 3 human volunteers. At the indicated times aliquots of the reaction mixtures were removed and analyzed by PAGE and phosphorimaging. The results are expressed as the mean percentage of full-length RNA remaining±SEM as a function of time. Symbols: -∘- CLL cell extract+[³²P]bcl-2- CR RNA; --, CLL cell extract+[³²P]bcl-2-CR-ARE RNA; -□-, normal B cell extract+[³²P]bcl-2-CR RNA; -≡-, normal B cell extract+[³²P]bcl-2-CR-ARE RNA; -▴-, normal cell extract+[³²P]bcl-2-CR-ARE RNA+280 nM purified recombinant nucleolin.

FIG. 8. Producing human mAbs in vitro. Tonsil B cells are isolated and efficiently immortalized with EBV, then induced to differentiate into antibody-producing cells. Immortalized B cell libraries are plated into multiple wells, then cell supernatants containing IgG are screened for binding to recombinant nucleolin. Reactive cells are cloned by limiting dilution, expanded, and the IgG purified from the culture supernatant. Ig genes can be subcloned into producer cell lines for large scale recombinant IgG production. See PCT/US2008/072124.

FIGS. 9A-B. Efficient EBV infection and induction of differentiation in tonsil B cell libraries. (FIG. 9A) Flow cytometric analysis of tonsil B cells 24 h post-infection with GFP-labeled EBV (EBfaV-GFP) compared with control after conventional supernatant infection (top panels) or after spinfection with concentrated virus (bottom panels). GFP fluorescence indicates infection. (FIG. 9B) Four B95-8 EBV-immortalized tonsil B cell libraries were cultured with or without anti-IgM F(ab′)2, recombinant soluble CD40L and Baff. Cell supernatants were assayed for IgM and IgG by ELISA after 5 (top panel) or 15 (bottom panel) days.

FIGS. 10A-B. Isolation of eight immortalized B cell lines producing nucleolin-specific human antibodies. (FIG. 10A) Culture supernatants obtained from six human B cell lines producing anti-nucleolin antibodies were screened by ELISA for binding to recombinant Δ1-283Nuc-(His)₆nucleolin (nucleolin lacking the N-terminal amino acids 1 through 283), using HRP-labeled goat anti-human IgG and colorimetric substrate for detection. Binding was measured by spectrophotometry at 450 nm. Results were compared with that obtained from culture supernatants containing human anti-HA IgG or mouse anti-nucleolin MS3. (FIG. 10B) Eight B cell lines producing anti-nucleolin antibodies were subcloned by limiting dilution cloning. Three weeks later, fewer than 10% of wells on each 96-well plate contained small monoclonal colonies, which were then tested by ELISA for binding to recombinant nucleolin as described above. Nucleolin binding in the wells was assessed by measuring absorbance at 450 nm.

FIG. 11. Isolation of human anti-nucleolin antibodies. Supernatants from immortalized human B cell libraries were screened by ELISA (top) and Western blotting (bottom) for IgG binding to recombinant Δ1-283Nuc-(His)₆nucleolin and/or endogenous nucleolin isolated from MV411 leukemic cells.

FIG. 12. Binding of HuMAbs to cell surface nucleolin and cytotoxicity to MV4-11 leukemia cells. (FIG. 12A) Anti-nucleolin HuMabs or Rituxan^R(5 μg/ml) were incubated with MV4-11 or normal tonsil cells for 30 min on ice, washed, then incubated with APC-labeled anti-human IgG (Miltenyi). Flow cytometry was performed on a FACSCaliber, and median fluorescence intensity (MFI) is shown.

FIG. 13. Complement-mediated killing of MV4-11 cells. Supernatants from antibody-producing B cell lines were incubated with MV411 cells (50% v/v) for 16 h in the presence of human serum (25% v/v). Controls were treated with staurosporine (25 μM) or media (untreated). Cells were then stained with propidium iodide and annexin V-FITC, and analyzed by flow cytometry.

FIGS. 14A-C. Nucleolin specific killing of MCF-7 and MV4-11 AML cells occurs through complement dependent and independent mechanisms. (FIG. 14A) MCF-7 cells were incubated with anti-nucleolin HuMAbs (2 μg/ml) in the presence of human AB serum (+complement) or heat inactivated serum (−complement). Viability was determined by MTS assay at 48-96 h. (FIG. 14B) MCF-7 and MCF10A cells were incubated with anti-nucleolin HuMAbs (2 μg/ml, top panels; or 1 mg/ml, bottom panels) or AS1411 (20 μM), in the presence of human AB serum (+complement) or heat inactivated serum (−complement). Viability was determined by MTS assay at 72 h (top panels) and 96 h (bottom panels). (FIG. 14C) MV4-11 cells were incubated with anti-nucleolin HuMAbs (2 μg/ml) or AS1411 (20 μM), in the presence of human AB serum (+complement) or heat inactivated serum (−complement). Viability was determined by MTS assay at 48-96 h.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention provides, human monoclonal antibodies that bind specifically to human nucleolin, and methods of use thereof. These antibodies exhibit cytotoxicity towards cells expressing human nucleolin in the plasma membrane, such as cells involved in cancer, autoimmune disorders, viral disorders. Therefore, the antibodies have therapeutic potential for certain forms of cancer and autoimmune disorders and can also be used as diagnostic agents.

I. NUCLEOLIN

A. General

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 are listed in Table 1.

TABLE 1

Nucleolin Sequences

SEQ

ID NO:
Identity
Sequence

1
Human
CTTTCGCCTCAGTCTCGAGCTCTCGCTGGCCTTCGGG

nucleolin
TGTACGTGCTCCGGGATCTTCAGCACCCGCGGCCGCC

coding
ATCGCCGTCGCTTGGCTTCTTCTGGACTCATCTGCGC

sequence
CACTTGTCCGCTTCACACTCCGCCGCCATCATGGTGA

Accession
AGCTCGCGAAGGCAGGTAAAAATCAAGGTGACCCCA

number
AGAAAATGGCTCCTCCTCCAAAGGAGGTAGAAGAAG

NM_005381
ATAGTGAAGATGAGGAAATGTCAGAAGATGAAGAA

XM_002342275
GATGATAGCAGTGGAGAAGAGGTCGTCATACCTCAG

AAGAAAGGCAAGAAGGCTGCTGCAACCTCAGCAAA

GAAGGTGGTCGTTTCCCCAACAAAAAAGGTTGCAGT

TGCCACACCAGCCAAGAAAGCAGCTGTCACTCCAGG

CAAAAAGGCAGCAGCAACACCTGCCAAGAAGACAG

TTACACCAGCCAAAGCAGTTACCACACCTGGCAAGA

AGGGAGCCACACCAGGCAAAGCATTGGTAGCAACTC

CTGGTAAGAAGGGTGCTGCCATCCCAGCCAAGGGGG

CAAAGAATGGCAAGAATGCCAAGAAGGAAGACAGT

GATGAAGAGGAGGATGATGACAGTGAGGAGGATGA

GGAGGATGACGAGGACGAGGATGAGGATGAAGATG

AAATTGAACCAGCAGCGATGAAAGCAGCAGCTGCTG

CCCCTGCCTCAGAGGATGAGGACGATGAGGATGACG

AAGATGATGAGGATGACGATGACGATGAGGAAGAT

GACTCTGAAGAAGAAGCTATGGAGACTACACCAGCC

AAAGGAAAGAAAGCTGCAAAAGTTGTTCCTGTGAAA

GCCAAGAACGTGGCTGAGGATGAAGATGAAGAAGA

GGATGATGAGGACGAGGATGACGACGACGACGAAG

ATGATGAAGATGATGATGATGAAGATGATGACTGAGG

AGGAAGAAGAGGAGGAGGAAGAGCCTGTCAAAGAA

GCACCTGGAAAACGAAAGAAGGAAATGGCCAAACA

GAAAGCAGCTCCTGAAGCCAAGAAACAGAAAGTGG

AAGGCACAGAACCGACTACGGCTTTCAATCTCTTTGT

TGGAAACCTAAACTTTAACAAATCTGCTCCTGAATTA

AAAACTGGTATCAGCGATGTTTTTGCTAAAAATGATC

TTGCTGTTGTGGATGTCAGAATTGGTATGACTAGGAA

ATTTGGTTATGTGGATTTTGAATCTGCTGAAGACCTG

GAGAAAGCGTTGGAACTCACTGGTTTGAAAGTCTTT

GGCAATGAAATTAAACTAGAGAAACCAAAAGGAAA

AGACAGTAAGAAAGAGCGAGATGCGAGAACACTTTT

GGCTAAAAATCTCCCTTACAAAGTCACTCAGGATGA

ATTGAAAGAAGTGTTTGAAGATGCTGCGGAGATCAG

ATTAGTCAGCAAGGATGGGAAAAGTAAAGGGATTGC

TTATATTGAATTTAAGACAGAAGCTGATGCAGAGAA

AACCTTTGAAGAAAAGCAGGGAACAGAGATCGATGG

GCGATCTATTTCCCTGTACTATACTGGAGAGAAAGGT

CAAAATCAAGACTATAGAGGTGGAAAGAATAGCACT

TGGAGTGGTGAATCAAAAACTCTGGTTTTAAGCAAC

CTCTCCTACAGTGCAACAGAAGAAACTCTTCAGGAA

GTATTTGAGAAAGCAACTTTTATCAAAGTACCCCAG

AACCAAAATGGCAAATCTAAAGGGTATGCATTTATA

GAGTTTGCTTCATTCGAAGACGCTAAAGAAGCTTTAA

ATTCCTGTAATAAAAGGGAAATTGAGGGCAGAGCAA

TCAGGCTGGAGTTGCAAGGACCCAGGGGATCACCTA

ATGCCAGAAGCCAGCCATCCAAAACTCTGTTTGTCA

AAGGCCTGTCTGAGGATACCACTGAAGAGACATTAA

AGGAGTCATTTGACGGCTCCGTTCGGGCAAGGATAG

TTACTGACCGGGAAACTGGGTCCTCCAAAGGGTTTG

GTTTTGTAGACTTCAACAGTGAGGAGGATGCCAAAG

CTGCCAAGGAGGCCATGGAAGACGGTGAAATTGATG

GAAATAAAGTTACCTTGGACTGGGCCAAACCTAAGG

GTGAAGGTGGCTTCGGGGGTCGTGGTGGAGGCAGAG

GCGGCTTTGGAGGACGAGGTGGTGGTAGAGGAGGCC

GAGGAGGATTTGGTGGCAGAGGCCGGGGAGGCTTTG

GAGGGCGAGGAGGCTTCCGAGGAGGCAGAGGAGGA

GGAGGTGACCACAAGCCACAAGGAAAGAAGACGAA

GTTTGAATAGCTTCTGTCCCTCTGCTTTCCCTTTTCCA

TTTGAAAGAAAGGACTCTGGGGTTTTTACTGTTACCT

GATCAATGACAGAGGCTTCTGAGGACATTCCAAGAC

AGTATACAGTCCTGTGGTCTCCTTGGAAATCCGTCTA

GTTAACATTTCAAGGGCAATACCGTGTTGGTTTTGAC

TGGATATTCATATAAACTTTTTAAAGAGTTGAGTGAT

AGAGCTAACCCTTATCTGTAAGTTTTGAATTTATATT

GTTTCATCCCATGTACAAAACCATTTTTTTCCTACAAA

TAGTTTGGGTTTTGTTGTTGTTTCTTTTTTTTGTTTTGT

TTTTGTTTTTTTTTTTTTTGCGTTCGTGGGGTTGTAAA

AGAAAAGAAAGCAGAATGTITTATCATGGTITTTGCT

TCAGCGGCTTTAGGACAAATTAAAAGTCAACTCTGG

TGCCAGAAAAAAAAAAAAAAAA

2
Human
MVKLAKAGKNQGDPKKMAPPPKEVEEDSEDEEMSED

nucleolin
EEDDSSGEEVVIPQKKGKKAAATSAKKVVVSPTKKVA

full length
VATPAKKAAVTPGKKAAATPAKKTVTPAKAVTTPGKK

amino acid
GATPGKALVATPGKKGAAIPAKGAKNGKNAKKEDSDE

sequence
EEDDDSEEDEEDDEDEDEDEDEIEPAAMKAAAAAPASE

Accession
DEDDEDDEDDEDDDDDEEDDSEEEAMETTPAKGKKA

number
AKVVPVKAKNVAEDEDEEEDDEDEDDDDDEDDEDDD

NP_005372
DEDDEEEEEEEEEEPVKEAPGKRKKEMAKQKAAPEAK

XP_002342316
KQKVEGTEPTTAFNLFVGNLNFNKSAPELKTGISDVFA

KNDLAVVDVRIGMTRKFGYVDFESAEDLEKALELTGL

KVFGNEIKLEKPKGKDSKKERDARTLLAKNLPYKVTQ

DELKEVFEDAAEIRLVSKDGKSKGIAYIEFKTEADAEKT

FEEKQGTEIDGRSISLYYTGEKGQNQDYRGGKNSTWSG

ESKTLVLSNLSYSATEETLQEVFEKATFIKVPQNQNGKS

KGYAFIEFASFEDAKEALNSCNKREIEGRAIRLELQGPR

GSPNARSQPSKTLFVKGLSEDTTEETLKESFDGSVRARI

VTDRETGSSKGFGFVDFNSEEDAKAAKEAMEDGEIDG

NKVTLDWAKPKGEGGFGGRGGGRGGFGGRGGGRGGR

GGFGGRGRGGFGGRGGFRGGRGGGGDHKPQGKKTKF

E

3
Human
aagaaggaaatggccaaacagaaagcagctcctgaagccaa

nucleolin
gaaacagaaagtggaaggcacagaaccgactacggctttc

coding
aatctctttgttggaaacctaaactttaacaaatctgctc

sequence for
ctgaattaaaaactggtatcagcgatgtttttgctaaaaa

the deletion
tgatcttgctgttgtggatgtcagaattggtatgactagg

construct
aaatttggttatgtggattttgaatctgctgaagacctgg

amino acid
agaaagcgttggaactcactggtttgaaagtctttggcaa

sequence
tgaaattaaactagagaaaccaaaaggaaaagacagtaag

Δ1-283Nuc
aaagagcgagatgcgagaacacttttggctaaaaatctcc

(lacking
cttacaaagtcactcaggatgaattgaaagaagtgtttga

residues 1-
agatgctgcggagatcagattagtcagcaaggatgggaaa

283)
agtaaagggattgcttatattgaatttaagacagaagctg

atgcagagaaaacctttgaagaaaagcagggaacagagat

cgatgggcgatctatttccctgtactatactggagagaaa

ggtcaaaatcaagactatagaggtggaaagaatagcactt

ggagtggtgaatcaaaaactctggttttaagcaacctctc

ctacagtgcaacagaagaaactcttcaggaagtatttgag

aaagcaacttttatcaaagtaccccagaaccaaaatggca

aatctaaagggtatgcatttatagagtttgcttcattcga

agacgctaaagaagctttaaattcctgtaataaaagggaa

attgagggcagagcaatcaggctggagttgcaaggaccca

ggggatcacctaatgccagaagccagccatccaaaactct

gtttgtcaaaggcctgtctgaggataccactgaagagaca

ttaaaggagtcatttgacggctccgttcgggcaaggatag

ttactgaccgggaaactgggtcctccaaagggtttggttt

tgtagacttcaacagtgaggaggatgccaaagctgccaag

gaggccatggaagacggtgaaattgatggaaataaagtta

ccttggactgggccaaacctaagggtgaaggtggcttcgg

gggtcgtggtggaggcagaggcggctttggaggacgaggt

ggtggtagaggaggccgaggaggatttggtggcagaggcc

ggggaggctttggagggcgaggaggcttccgaggaggcag

aggaggaggaggtgaccacaagccacaaggaaagaagacg

aagtttgaagtttaaac

4
Human
MAKQICAAPEAKKQKVEGTEPTTAFNLFVGNLNFNKSA

nucleolin
PELKTGISDVFAKNDLAVVDVRIGMTRKFGYVDFESAE

deletion
DLEKALELTGLKVFGNEIKLEKPKGKDSKKERDARTLL

construct
AKNLPYKVTQDELKEVFEDAAEIRLVSKDGKSKGIAYI

amino acid
EFKTEADAEKTFEEKQGTEIDGRSISLYYTGEKGQNQD

sequence
YRGGKNSTWSGESKTLVLSNLSYSATEETLQEVFEKAT

Δ1-283Nuc
FIKVPQNQNGKSKGYAFIEFASFEDAKEALNSCNKREIE

(lacking
GRAIRLELQGPRGSPNARSQPSKTLFVKGLSEDTTEETL

residues 1-
KESFDGSVRARIVTDRETGSSKGFGFVDFNSEEDAKAA

283)
KEAMEDGEIDGNKVTLDWAKPKGEGGFGGRGGGRGG

FGGRGGGRGGRGGFGGRGRGGFGGRGGFRGGRGGGG

DHKPQGKKTKFE

Listed are the genomic coding region and protein sequence of human nucleolin and N-terminal deletion fragment of human nucleolin. Nucleolin is also known as C23, FLJ45706, F1159041, and NCL.

Human NCL gene consists of 14 exons with 13 introns and spans approximately 11 kb. 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)

In one embodiment, a region of nucleolin containing the RNA binding domain is used for generation of an isolated human anti-nucleolin antibody. In one embodiment, a region of nucleolin lacking residues 1-283 (SEQ ID NO:4) is used for generation of an isolated human anti-nucleolin antibody. In one embodiment, the invention provides for an human anti-nucleolin antibody that specifically targets a RNA domain-containing portion of nucleolin. In one embodiment, the invention provides for an isolated human anti-nucleolin monoclonal antibody that specifically targets a RNA domain-containing portion of nucleolin. In one embodiment, the invention provides for an isolated human anti-nucleolin monoclonal antibody produced by a human B cell that specifically targets a RNA domain-containing portion of nucleolin. In one embodiment, the interaction of a human anti-nucleolin antibody with nucleolin disrupts interactions of the nucleolin RNA domain with other molecules. In one embodiment, the interaction of a human anti-nucleolin antibody with nucleolin disrupts interactions of the nucleolin RNA domain with BCL-2.

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).

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 process. 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

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, AS1411, targets human nucleolin. Plasma membrane nucleolin was recently reported to be a receptor for AS1411 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. AS1411 has been shown to exhibit antiproliferative activity in a broad set of cancer cell lines that over-express nucleolin (Table 2).

TABLE 2

Cancer Cell Lines That Over-express Nucleolin and/or

are Killed Subsequent to Nucleolin Inhibition

Cancer Type:
Cell Line:

Lung cancer
A549, NCI-H322M, NCI-H460, EKVX, HOP-92, NCI-

H299, CaLu1, NCI-H1385, NCI-H82, CaLu6

Breast cancer
MCF7, T-47D, BT-549, MDA-N, MDA-MB-231,

ZR7S-1

Prostate cancer
DU145, PC-3, CA-HPV-10

Colon cancer
HCC 2998, HT-29, KM12, HCT-116, SW620, HCT-15,

LS174T

Pancreatic
PANC-1, MIA-PaCa-2, PANC-1

cancer

Renal cell
786-0, CAKI-1, RXF393, TK10, A498, ACHN, SN12C

carcinoma

Ovarian cancer
IGROV, OVCAR-3, OVCAR-4, OVCAR-5

Cervical cancer
HeLa

Leukemia &
CCRF-CEM, SR, HL60, K-562, RPMI-6226, U937,

Lymphoma
Meg0, MV4-11

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

Data from NCI Tumor Cell Line Screen of AS1411 (≧50% growth inhibition at 6.3 μM). (Bates et al., 2009).

Anti-nucleolin antibodies can also exploit the shuttling function of plasma membrane nucleolin and become internalized after binding to cell surface nucleolin. 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 downregulation 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 cellular cytotoxicity (CDCC).

C. Antibodies or Fragments Thereof.

In one embodiment any of the methods disclosed herein can be practiced with an anti-nucleolin antibody or fragment thereof. In one embodiment an anti-nucleolin antibody or fragment thereof is used to detect a cell expressing nucleolin on its surface. In another embodiment an anti-nucleolin antibody or fragment thereof is used to inhibit or kill a cell expressing nucleolin on its surface. In another embodiment 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.

In one embodiment 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 another embodiment 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 another embodiment anti-nucleolin antibody or fragment thereof is used to contact a cell expressing nucleolin on its surface. In one embodiment the cell is pre-cancerous cell, a cancer cell or an immune cell.

In one embodiment the anti-nucleolin antibody fragment thereof is a human anti-nucleolin antibody or fragment. In one embodiment the anti-nucleolin antibody fragment thereof is a non-human anti-nucleolin antibody fragment thereof. In one embodiment the anti-nucleolin antibody fragment thereof is a chimeric anti-nucleolin antibody fragment thereof. In one embodiment the anti-nucleolin antibody fragment thereof is a humanized anti-nucleolin antibody fragment thereof.

In one embodiment an anti-nucleolin antibody fragment thereof is generated from an anti-nucleolin antibody. In one embodiment the anti-nucleolin antibody fragment has the same binding specificity to human nucleolin as the parent antibody. In another embodiment the anti-nucleolin antibody fragment has improved binding specificity to human nucleolin as the parent antibody. In one embodiment the anti-nucleolin antibody fragment has the same binding affinity to human nucleolin as the parent antibody. In another embodiment the anti-nucleolin antibody fragment has improved affinity to human nucleolin as the parent antibody. In one embodiment an anti-nucleolin antibody or fragment thereof is a human anti-nucleolin antibody fragment.

Antibody fragments” comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′).sub.2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′).sub.2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

“Fv” is a minimum antibody fragment which contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain 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 HVRs of each variable domain 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 domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CH1) 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 CH1 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′).sub.2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. 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.

The term “diabodies” refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (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 tetrabodics are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, 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 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 is also a monoclonal antibody of this invention. 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.

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 invention may be made by a variety of techniques, including, for example, the hybridoma 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 DNA methods (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 human-like 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).

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.

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® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.

“Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. 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 domains, 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).

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(1):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™ 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. PREPARING HUMAN MONOCLONAL ANTIBODIES FROM IgM⁺ B-CELLS

The following are descriptions of the general procedures for obtaining a human monoclonal antibody against nucleolin. These procedures are exemplary and can be modified while retaining the essential aspects of the invention.

A. Obtaining IgM⁺ B-Cell Populations

To prepare B-cells from tonsils, tonsil tissue is mixed with an antibiotic, chopped and minced to approximately 1 mm³pieces, followed by gentle grinding of tonsil pieces and straining through a nylon strainer. The suspension is then centrifuged on a Ficoll cushion. The boundary layer containing mononuclear cells is extracted, washed and re-suspended in DPBS. Further enrichment (>95%) can be achieved by negative selection using antibodies and magnetic beads

Non-human mammalian, chimeric, polyclonal (e.g., sera) and/or monoclonal antibodies (Mabs) and fragments (e.g., proteolytic digestion or fusion protein products thereof) are potential therapeutic agents are often used in an attempt to treat certain diseases. However, such antibodies or fragments can elicit an immune response when administered to humans. Such an immune response can result in an immune complex-mediated clearance of the antibodies or fragments from the circulation, and make repeated administration unsuitable for therapy, thereby reducing the therapeutic benefit to the patient and limiting the readministration of the antibody or fragment. For example, repeated administration of antibodies or fragments comprising non-human portions can lead to serum sickness and/or anaphalaxis. In order to avoid these and other problems, a number of approaches have been taken to reduce the immunogenicity of such antibodies and portions thereof, including chimerization and humanization. These and other approaches, however, still can result in antibodies or fragments having some immunogenicity, low affinity, low avidity, or with problems in cell culture, scale up, production, and/or low yields. Thus, such antibodies or fragments can be less than ideally suited for manufacture or use as therapeutic proteins. Thus, an antibody expressed from human cells can avoid many of these issues and be substantially non-immunogenic to a human. “Immunogenic” as used herein refers to possessing the ability to elicit an immune response.

In one embodiment, a human anti-nucleolin antibody expressed by a human B cell is substantially non-immunogenic a human. In one embodiment, an isolated human anti-nucleolin monoclonal antibody expressed by a human B cell is substantially non-immunogenic a human.

To prepare B-cells from peripheral blood, venous blood is drawn into syringes containing heparin sodium to prevent coagulation, diluted, and centrifuged on a Ficoll cushion. The boundary layer containing mononuclear cells is extracted, washed and re-suspended in DPBS. Further enrichment can be achieved as stated above.

B. EBV Immortalization

For infection by inoculation with EBV supernatant, B-cells are resuspended at 10⁶to 10⁷cells per ml in complete RPMI media, and mixed with an equal volume of filtered EBV supernatant, then incubated for 4 hours at 37° C. and 5% CO₂. The culture volume can be adjusted by the addition of complete RPMI media, such that infected cells were resuspended for cell culture at a desired concentration (generally 10⁵to 10⁶cells per ml). Cells are then dispensed into multi-well plates and transferred to a tissue culture incubator at 37° C. and 5% CO₂.

For spin-fection, B-cells are resuspended at 10⁶to 10⁷cells per ml in complete RPMI media, and mixed with an equal volume of 10-fold ultrafiltration concentrated EBV and placed in a well of a 6-well tissue culture plate. The plate is then centrifuged at 900 g for 1 hr at ambient temperature, at which time infected cells are re-suspended in complete RPMI media at a desired concentration (generally 10⁵to 10⁶cells per ml), dispensed into multi-well plates and transferred to a tissue culture incubator at 37° C. and 5% CO₂.

Optionally, B-cells may be contacted with Toll Like Receptor (TLR) ligands at the time of or subsequent to the infection. The ligands may be added at the following final concentrations: Pam3CSK4 (0.1-1 μg/ml), Zymosan (1-10 μg/ml), poly I:C (1-25 μg/ml), LPS (1-5 μg/ml), Imiquimod (1 μg/ml), and/or CpG (1-10 μg/ml).

Infectivity varies based upon route of infection. Infection of tonsil B cells by inoculation with EBV supernatant results in immortalization of approximately 1-5% of B cells. Addition of TLR ligands approximately doubles infection efficiency. Infection of tonsil B cells by spin-fection with concentrated virus increases infection efficiency to virtually 100% after 24 hours.

C. Culturing to Induce Immunoglobulin Isotype Class Switching and IgG Secretion

To induce B-cell differentiation, immunoglobulin isotype class switching, and/or IgG secretion, cytokines and other signaling agents are added to EBV infected B-cells immediately after infection, 16 to 20 hr after infection, and/or sequentially at weekly intervals (2, 3, 4 or 5 times). Agents may be diluted in media and added to cells at the following final concentrations: recombinant human interleukins (IL) IL-4, 0.2 ng/ml; IL-5, 0.2 ng/ml; IL-6, 0.1 ng/ml; IL-9, 0.2 ng/ml; IL-10, 0.24 ng/ml; IL-13, 1 ng/ml; recombinant human interferon-α2a (IFN-α2a), 2,000 IU/ml; recombinant human BAFF, 1 ng/ml; recombinant human soluble CD40L, 5 ng/ml; goat anti-human, IgM F(ab′)₂, 1.4 μg/ml (amounts are approximate). Particular combinations comprise anti-IgM F(ab′)₂, CD40L+/−BAFF; anti-IgM F(ab′)₂, IL-6+/−BAFF; anti-IgM F(ab′)₂, CD40L, IL-6+/−BAFF; anti-IgM F(ab′)₂, CD40L, IL-6+/−IL4; and anti-IgM F(ab′)₂, CD40L, and IL-9+/−IL-13.

The initiation of immunoglobulin isotype class switching can be detected as early as 5 days following exposure to the cytokine/growth factor/signaling agent cocktail, and the process continues for the following 10 days.

D. Selection of Immortalized B-Cells

Following exposure to the cytokine/growth factor/signaling agent cocktail, culture supernatants are collected about once a week or at days 10-20 post-infection from immortalized tonsil and blood B-cell cultures, pooled, and tested using an ELISA or other screening format, such as dot blot, or flow cytometry, Western blotting, or inhibition/promotion of functional activity. Antigen may be bound directly or through capture antibodies onto the wells of a polystyrene (e.g., 96-well) plate and allowed to bind, e.g., overnight. Plates are then washed, blocked, and contacted with immortalized B cell culture supernatant samples or controls in triplicate or other replicates. Subsequently, the plate is washed extensively, and then e.g., alkaline phosphatase (AP)-coupled goat anti-human IgG or other labeled secondary antibody is added for detection of bound IgG by e.g. AP conversion of colorimetric substrate p-nitrophenyl phosphate disodium salt.

Based upon the discussion above, immunoglobulin isotype class switching and/or IgG secretion starts immediately after and can be detected as early as 5 days following exposure to the cytokine/growth factor/signaling agent cocktail. IgG levels increase in the supernatant over the next 10 days. Thus, from about 7-21 days, about 10-21 days, about 7-10 days or about 10-14 days, or at 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, one will select B-cells that have undergone immunoglobulin isotype class switching and secrete IgG.

III. CLONING AND EXPRESSION OF HUMAN Ig LIGHT AND HEAVY CHAINS

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

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 human 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 NheI/AscI and the SfiI/NotI sites of the expression vector pFab1-His2. The ligated pFab1-His2 vectors with the light chain (κ and λ) and Fc heavy chain genes (γ 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 MgCl₂(1.5 mM). Isopropyl-β-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 15,000 G for 10 min, and the resultant supernatant containing the Fab antibody portion was collected.

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

IV. ANTIBODY PRODUCTION

Once cloned, the nucleic acids for the human 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 contemplated for production are 293 cells, CHO cells, COS cells or various forms of myeloma cells, some lacking IgG. These cells can be exploited for human MAb 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 noncompatible cells. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpcntadecane) 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 human MAbs in high concentration. Second, the individual cell lines could be cultured in vitro, where the human MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.

Human MAbs produced by either means can be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography. Fragments of the monoclonal antibodies of the invention 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.

In one embodiment a human anti-nucleolin antibody is produced from an immortalized human B cell. In one embodiment a human anti-nucleolin antibody is produced using a method such as one set forth in PCT/US2008/072124 or U.S. patent application Ser. No. 12/671,936, which are herein incorporated by reference in their entirety.

In one embodiment, the cDNA of an isolated human anti-nucleolin antibody may be produced by cloning cDNA or genomic DNA encoding the immunoglobulin light and heavy chains of the anti-nucleolin antibody from a hybridoma cell (by fusing a specific antibody-producing B cell with a myeloma) that produces an antibody homolog according to this invention. In one embodiment, an isolated human anti-nucleoline antibody is produced by a human B cell. In one embodiment, a cell is transfected by one or more polynucleotide sequences isolated from a human B cell where the polynucleotide sequence encodes for human anti-nucleolin antibody. 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. Typically, both genes are inserted into the same expression vector.

Prokaryotic or eukaryotic cells can be used as expression hosts. Expression in eukaryotic host cells is preferred 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 invention.

It will be understood that variations on the above procedure are within the scope of the present invention. In one embodiment, a host cell is transformed with DNA encoding either the light chain or the heavy chain (but not both) of an antibody homolog of this invention. 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 according to this invention. In one embodiment, bifunctional antibodies are produced in which one heavy and one light chain are homologs of a human anti-nucleolin antibody and the other heavy and light chain are specific for an antigen other than nucleolin, or another epitope of nucleolin.

In one embodiment, DNA encoding an isolated human anti-nucleolin antibody is transferred to a preferred 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. Pat. No. 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. Pat. No. 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 invention of this application.

A. Amino Acid Sequence Variants of Antibodies

In one embodiment, the anti-nucleolin antibody of the invention comprises a modified amino acid sequence compared to wild type. For example, it can be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the anti-nucleolin antibodies are prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the anti-nucleolin antibody chains, 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 anti-nucleolin antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of the anti-nucleolin antibody, such as changing the number or position of glycosylation sites.

A useful method for identification of certain residues or regions of the anti-nucleolin antibody that are preferred locations for mutagenesis is called “alanine scanning mutagenesis,” as described by Cunningham and Wells Science (1989). Here, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed anti-nucleolin antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an anti-nucleolin antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of the anti-nucleolin antibody molecule include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the anti-nucleolin antibody molecule removed and a different residue inserted in its place. Substantial modifications in the biological properties of the anti-nucleolin antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions may also be done and entail exchanging a member of one of these classes for another class. Any cysteine residue not involved in maintaining the proper conformation of the antagonist also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antagonist to improve its stability (particularly where the anti-nucleolin antibody is an antibody fragment such as an Fv fragment).

A type of substitution variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitution variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., antagonist activity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding.

Alternatively, or in addition, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and nucleolin. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

B. Glycosylation Variants of Antibodies In one embodiment, the anti-nucleolin antibody is modified to have altered glycosylation compared to wild type antibody. Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, 1997; Wright and Morrison, 1997). The oligosaccharide side chains of the immunoglobulins affect the protein's function (Boyd et al., 1996; Wittwe and Howard, 1990), and the intramolecular interaction between portions of the glycoprotein which can affect the conformation and presented three-dimensional surface of the glycoprotein (Jefferis and Lund, 1996). Oligosaccharides may also serve to target a given glycoprotein to certain molecules based upon specific recognition structures. For example, it has been reported that in agalactosylated IgG, the oligosaccharide moiety ‘flips’ out of the inter-CH2 space and terminal N-acetylglucosamine residues become available to bind mannose binding protein (Malhotra et al., 1995). Removal by glycopeptidase of the oligosaccharides from CAMPATH-1H (a recombinant humanized murine monoclonal IgG1 antibody which recognizes the CDw52 antigen of human lymphocytes) produced in Chinese Hamster Ovary (CHO) cells resulted in a complete reduction in complement mediated lysis (CMCL) (Boyd et al., 1996), while selective removal of sialic acid residues using neuraminidase resulted in no loss of DMCL. Glycosylation of antibodies has also been reported to affect antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO cells with tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing formation of bisecting GlcNAc, was reported to have improved ADCC activity (Umana et al. 1999).

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

Glycosylation variants of antibodies are variants in which the glycosylation pattern of an antibody is altered. By altering is meant deleting one or more carbohydrate moieties found in the antibody, adding one or more carbohydrate moieties to the antibody, changing the composition of glycosylation (glycosylation pattern), the extent of glycosylation, etc.

Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites). Similarly, removal of glycosylation sites can be accomplished by amino acid alteration within the native glycosylation sites of the antibody.

The amino acid sequence is usually altered by altering the underlying nucleic acid sequence. Nucleic acid molecules encoding amino acid sequence variants of the anti-nucleolin antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the anti-nucleolin antibody.

The glycosylation (including glycosylation pattern) of antibodies may also be altered without altering the amino acid sequence or the underlying nucleotide sequence. Glycosylation largely depends on the host cell used to express the antibody. Since the cell type used for expression of recombinant glycoproteins, e.g., antibodies, as potential therapeutics is rarely the native cell, significant variations in the glycosylation pattern of the antibodies can be expected (see, e.g., Hse et al., 1997). In addition to the choice of host cells, factors which affect glycosylation during recombinant production of antibodies include growth mode, media formulation, culture density, oxygenation, pH, purification schemes and the like. Various methods have been proposed to alter the glycosylation pattern achieved in a particular host organism including introducing or overexpressing certain enzymes involved in oligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation, or certain types of glycosylation, can be enzymatically removed from the glycoprotein, for example using endoglycosidase H (Endo H). In addition, the recombinant host cell can be genetically engineered, e.g. make defective in processing certain types of polysaccharides. These and similar techniques are well known in the art.

The glycosylation structure of antibodies can be readily analyzed by conventional techniques of carbohydrate analysis, including lectin chromatography, NMR, Mass spectrometry, HPLC, GPC, monosaccharide compositional analysis, sequential enzymatic digestion, and HPAEC-PAD, which uses high pH anion exchange chromatography to separate oligosaccharides based on charge. Methods for releasing oligosaccharides for analytical purposes are also known, and include, without limitation, enzymatic treatment (commonly performed using peptide-N-glycosidase F/endo-β-galactosidase), elimination using harsh alkaline environment to release mainly O-linked structures, and chemical methods using anhydrous hydrazine to release both N- and O-linked oligosaccharides.

C. Other Modifications of Antibodies

In one embodiment, an anti-nucleolin antibody is 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. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.

Particularly 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 invention 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).

In one embodiment, 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. Pat. No. 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.

Enzymes that are useful in the method of this invention 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 invention into free active drugs (see, e.g., Massey, 1987). Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a desired cell population.

The enzymes can be covalently bound to the anti-nucleolin antibodies by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above. Alternatively, fusion proteins comprising at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., 1984).

In one embodiment of the invention, 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.

The salvage receptor binding epitope generally constitutes a region wherein any one or more amino acid residues from one or two loops of a Fc domain are transferred to an analogous position of the antibody fragment. Even more preferably, three or more residues from one or two loops of the Fc domain are transferred. Still more preferred, the epitope is taken from the CH2 domain of the Fe region (e.g., of an IgG) and transferred to the CH1, CH3, or V_Hregion, or more than one such region, of the antibody. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the C_Lregion or V_Lregion, or both, of the antibody fragment.

In one embodiment, 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. Pat. No. 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. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

In another embodiment, 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 one embodiment, an human anti-nucleolin antibody is modified to comprise targeted immunoconjugate moieties which enable the effective generation of innate and adaptive immune responses against tumors or pathogens. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is modified to comprise targeted immunoconjugate moieties which enable the effective generation of innate and adaptive immune responses against tumors or pathogens. In one embodiment, an isolated human anti-nucleolin monoclonal antibody produced by a 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 (PRRs), 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 a isolated human anti-nucleolin antibody provides a moiety comprising a nucleic acid or protein that is recognized by a PRR, ultimately leading to an immune response which eliminates the target cell with the anti-nucleolin antibody bound to it. 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.

In one embodiment 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. A non-limiting summary of example epitope tags that may be fused to an anti-nucleolin antibody of the invention is listed in Table 3. 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 at, 1990). Other tag polypeptides include the Flag-peptide (Hopp et al., 1988); the KT3 epitope peptide (Martin et al., 1992); an α-tubulin epitope peptide (Skinner et al., 1991); and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., 1990).

TABLE 3

Sequence of Affinity Tags

SEQ ID

NO:
Tag:
Amino Acid Sequence:

5
Poly-Arg
RRRRR (SEQ ID NO: 5)

6
Poly-Lys
KKKK (SEQ ID NO: 6)

7
Poly-His
HHHHHH (SEQ ID NO: 7)

8
FLAG
DYKDDDDK (SEQ ID NO: 8)

9
Strep-tag II
WSHPQFEK (SEQ ID NO: 9)

10
c-myc
EQKLISEEDL (SEQ ID NO: 10)

11
S
KETAAAKFERQHMDS (SEQ ID NO: 11)

12
HAT
KDHLIHNVHKEFHAHAHNK (SEQ ID NO: 12)

13
3x FLAG
DYKDNDGDYKDHDIDYKDDDDK (SEQ ID NO: 13)

14
Calmodulin-
KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID NO: 14)

binding peptide

15
SBP
MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP

(SEQ ID NO: 15)

16
Chitin-binding
TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPS

domain
NVPALWQLQ (SEQ ID NO: 16)

17
Glutathione S-
Protein

transferase

18
Maltose-binding
Protein

protein

In one embodiment, a human anti-nucleolin antibody is linked to a nanoparticle. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is linked to a nanoparticle. In one embodiment, a human anti-nucleolin antibody produced by a 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 one embodiment, the antibody-nanoparticle conjugate can penetrate a cell expressing nucleolin on its surface more rapidly and extensively than the uncongugated antibody. In one embodiment, the cell is a cancer cell, tumor cell, virally infected cell, lymphocyte, or activated lymphocyte.

D. Deposit of Cell Lines

Cell lines that express antibodies that immunospecifically bind one or more nucleolin (NCL) polypeptides (e.g., SEQ ID NO:4 or fragments thereof) were deposited on Nov. 17, 2010, with the American Type Culture Collection (10801 University Boulevard, Manassas, Va. 20110-2209 U.S.A.) as an original deposit and were given the accession numbers ATCC_, ATCC_, ATCC_, ATCC_, ATCC_, ATCC_, ATCC_, and ATCC_. The deposit was made under Budapest Treaty, and all restrictions on the availability to the public of the deposited material will be irrevocably removed upon the granting of the patent, except for the requirements specified in 37 C.F.R. 1.808(b), and the term of the deposit will comply with 37 C.F.R. 1.806.

V. DIAGNOSTICS

In one embodiment, a human anti-nucleolin antibody is used to determine the presence of cancer cells in a human. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used to determine the presence of cancer cells in a human. In one embodiment, an isolated human anti-nucleolin monoclonal antibody produced by a human B cell is used to determine the presence of cancer cells in a human.

In one embodiment, a human anti-nucleolin antibody is used to determine the presence of cancer cells in a human sample. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used to determine the presence of cancer cells in a human sample. In one embodiment, an isolated anti-nucleolin monoclonal antibody expressed by a human B cell is provided and used to determine the presence of cancer cells in a human sample. The term human sample as used refers to biological collections from a subject that include but are not limited to a cell, tissue, plasma, serum, whole blood, sputum, or saliva. In one embodiment, the detection results from a human subject sample are compared to those from a control sample. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used to determine the presence of cancer cells in a subject. In one embodiment, an isolated human anti-nucleolin monoclonal antibody expressed by a human B cell is used to determine the presence of cancer cells in a subject. In one embodiment the determination is made by binding ahuman anti-nucleolin antibody to nucleolin on the surface of a human cancer cell. The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer 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/Ostedsarcoma; 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 Neuroectodennal 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.

In one embodiment, a human anti-nucleolin antibody is used determine the presence of a proliferative disorder. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used determine the presence of a proliferative disorder. In one embodiment, a human anti-nucleolin antibody produced from a human B cell is used determine the presence of a proliferative disorder. The terms “cell proliferative disorder,” “proliferative disorder” and “neoplastic disorder,” as used herein, refer to disorders that are associated with some degree of abnormal cell proliferation.

In one embodiment, a human anti-nucleolin antibody is used determine the presence of a malignant disease wherein nucleolin is expressed on the cell surface or in the cytoplasm. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used determine the presence of a malignant disease wherein nucleolin is expressed on the cell surface or in the cytoplasm. In one embodiment, an isolated anti-nucleolin monoclonal antibody expressed by a human B cell is provided and used to determine the presence of a malignant disease wherein nucleolin is expressed on the cell surface or in the cytoplasm. The term “malignant diseases” as used hereing refers to progressive or metastatic diseases or diseases characterized by small tumor burden such as minimal residual disease. Examples of malignant diseases that a human anti-nucleolin antibody is used determine the presence of include, but are not limited to leukemias (e.g., acute myeloid, acute lymphocytic and chronic myeloid) and cancers (e.g., breast, lung, thyroid or gastrointestinal cancer or a melanoma).

In one embodiment, a human anti-nucleolin antibody is used to determine the presence of a non-malignant cell proliferative disorders wherein nucleolin is expressed on the cell surface or in the cytoplasm. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used to determine the presence of a non-malignant cell proliferative disorders wherein nucleolin is expressed on the cell surface or in the cytoplasm. In one embodiment, an isolated anti-nucleolin monoclonal antibody expressed by a human B cell is provided and used to determine the presence of a non-malignant cell proliferative disorders 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 be used to determine the presence of a non-malignant cell proliferative disorder with an isolated human anti-nucleolin monoclonal antibody include but are not limited to warts, benign prostatic hyperplasia, skin tags, and non-malignant tumors. For example, an isolated human anti-nucleolin 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. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used determine the presence of a angiogenic tumor cell wherein nucleolin is expressed on the cell surface or in the cytoplasm.

In one embodiment, a human anti-nucleolin is to determine the presence of a tumor. “Tumor”, as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

In one embodiment, a human anti-nucleolin antibody is used to determine the presence of a cell expressing human nucleolin on its surface or in its cytoplasm in a subject with an autoimmune disorder. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used to determine the presence of a cell expressing human nucleolin on its surface or in its cytoplasm in a subject with an autoimmune disorder. In one embodiment, an isolated anti-nucleolin monoclonal antibody expressed by a human B cell is provided and used to determine the presence of a cell expressing human nucleolin on its surface or in its cytoplasm in a subject with an autoimmune disorder. In one embodiment, a human anti-nucleolin antibody is used to determine the presence of a cell expressing human nucleolin on its surface or in its cytoplasm in lymphocytes. In one embodiment, a isolated human anti-nucleolin monoclonal antibody is used to determine the presence of a cell expressing human nucleolin on its surface or in its cytoplasm in lymphocytes. In one embodiment, an isolated human anti-nucleolin monoclonal antibody expressed by a human B cell is provided and used to determine the presence of a cell expressing human nucleolin on its surface or in its cytoplasm in lymphocytes. In one embodiment, the lymphocyte comprises a B cell, T cell, or natural killer cell. In one embodiment, the lymphocyte comprises a CD4-positive or CD8-positive cells.

In one embodiment, a human anti-nucleolin antibody is used to determine the presence of a cell expressing human nucleolin on its surface or in its cytoplasm in activated lymphocytes or memory cells. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used to determine the presence of a cell expressing human nucleolin on its surface or in its cytoplasm in activated lymphocytes or memory cells. In one embodiment, an isolated anti-nucleolin monoclonal antibody expressed by a human B cell is provided and used to determine the presence of a cell expressing human nucleolin on its surface or in its cytoplasm in activated lymphocytes or memory cells. In one embodiment, the activated lymphocyte comprises an activated B cell, T cell, or natural killer cell.

The term “autoimmune disease or disorder” refers to a condition in a subject characterized by cellular, tissue and/or organ injury caused by an immunologic reaction of the subject to its own cells, tissues and/or organs. The term “inflammatory disease” is used interchangeably with the term “inflammatory disorder” to refer to a condition in a subject characterized by inflammation, preferably chronic inflammation. Autoimmune disorders may or may not be associated with inflammation. Moreover, inflammation may or may not be caused by an autoimmune disorder. Thus, certain disorders may be characterized as both autoimmune and inflammatory disorders. Exemplary autoimmune diseases or disorders which may be diagnosed with the use of a human anti-nucleolin antibody include, but are 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 I 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), myelodysplastic syndrome, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosis (SLE), Sweet's syndrome, Still's disease, lupus erythematosus, takayasu arteritis, temporal arteristis/giant cell arteritis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis. Examples of inflammatory disorders include, but are not limited to, asthma, encephilitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentitated 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.

In one embodiment, a human anti-nucleolin antibody is used to determine the presence of a cell expressing human nucleolin on its surface or in its cytoplasm in viral infected cells. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used to determine the presence of a cell expressing human nucleolin on its surface or in its cytoplasm in viral infected cells. In one embodiment, an isolated anti-nucleolin monoclonal antibody expressed by a human B cell is provided and used determine the presence of a cell expressing human nucleolin on its surface or in its cytoplasm in viral infected cells. Nucleolin is expressed at the cell surface of virus infected cells (Hovanessian et al., 2006; Bose et al., 2004; Izumi et al., 2001). Examples of virus which can infect cells include but are not limited to: 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); Arena viridae (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 invention are known, such as those disclosed in U.S. Patent Publication No. 2007/0066554. In another embodiment, 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.

In one embodiment, detectable labels are used in a therapeutic and/or diagnostic application using an anti-nucleolin antibody. “Detectable labels” are compounds and/or elements that permit detection of bound antibody. Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated herein by reference). The imaging moieties used can be paramagnetic ions; radioactive isotopes; fluorochromes; NMR-detectable substances; X-ray imaging agents. Other agents include enzymes, haptens, fluorescent labels, phosphorescent molecules, chemilluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin.

In one aspect, the detectable labels comprise paramagnetic ions. Examples of paramagnetic ions include, but are not limited to, chromium (III), manganese (II), iron iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and bismuth (III). In one aspect, the detectable lables comprise radioactive isotopes. Examples of radioactive isotope include, but are not limited to, ¹²⁴antimony, ¹²⁵antimony, ⁷⁴arsenic, ²¹¹astatine, ¹⁰³barium, ¹⁴⁰barium, ⁷beryllium, ²⁰⁶bismuth, ²⁰⁷bismuth, ¹⁰⁹cadmium, ¹¹⁵cadmium, ⁴⁵calcium, ¹⁴carbon, ¹³⁹cerium, ¹⁴¹cerium, ¹⁴⁴cerium, ¹³⁷cesium, ⁵¹chromium, ³⁶chlorine, ⁵⁶cobalt, ⁵⁷cobalt, ⁵⁸cobalt, ⁶⁰cobalt, ⁶⁷copper, ¹⁶⁹erbium, ¹⁵²eurpium, ⁶⁷gallium, ¹⁵³gadolinium, ¹⁹⁵gold, ¹⁹⁹gold, ¹⁷⁵hafnium, ^175+181hafnium, ¹⁸¹hafnium, ³hydrogen, ¹²³iodine, ¹²⁵iodine, ¹³¹iodine, ¹¹¹indium, ¹⁹²iridium, ⁵⁵iron, ⁵⁹iron, ⁸⁵krypton, ²¹⁰lead, ¹⁷⁷lutecium, ⁵⁴manganese, ¹⁹⁷mercury, ²⁰³mercury, ⁹⁹molybdenum, ¹⁴⁷neodynium, ²³⁷neptunium, ⁶³nickel, ⁹⁵niobium, ^185+191osmium, ¹⁰³palladium, ³²phosphorus, ¹⁸⁴platinum, ¹⁴³praseodymium, ¹⁴⁷promethium, ²³³protactinium ²²⁶radium rhenium¹⁸⁶, ¹⁸⁸rhenium, ⁸⁶rubidium, ¹³⁰ruthenium, ¹⁰⁶ruthenium, ⁴⁴scandium, ⁴⁶scandium, ⁴⁵selenium, ⁷⁵selenium, ^110msilver, ¹¹¹silver, ²²sodium, ⁸⁵strontium, ⁸⁹strontium, ⁹⁰strontium, ³⁵sulphur, ¹⁸²tantalum, ^99mtechnicium, ^125mtellurium, ¹³²tellurium, ¹⁶⁰terbium, ²⁰⁴thallium, ²²⁸thorium, ²³²thorium, ¹⁷⁰thullium, ¹¹³tin, ⁴⁴titanium, ¹⁸⁵tungsten, ⁴⁸vanadlum, ⁴⁹vanadium, ⁸⁸yttrium, ⁹⁰yttrium, ⁹¹yttrium, ¹⁶⁹ytterbium, ⁶⁵zinc, and ⁹⁵zirconium.

In one embodiment ¹²⁵Iodine technicium^99mand/or indium¹¹¹are used due to their low energy and suitability for long range detection. Radioactively labeled monoclonal antibodies of the present invention can be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the invention can be labeled with technetium^99mby ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column. Alternatively, direct labeling techniques can be used, e.g., by incubating pertechnate, a reducing agent such as SNCl₂, a buffer solution such as sodium-potassium phthalate solution, and the antibody. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetracetic acid (EDTA).

In one embodiment, a detectable label comprises a fluorescent label. Examples of fluorescent labels include but are not limited to Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.

Another type of antibody conjugates contemplated in the present invention are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include but are not limited to urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. Particular secondary binding ligands are biotin and/or avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.

Yet another known method of site-specific attachment of molecules to antibodies comprises the reaction of antibodies with hapten-based affinity labels. Essentially, hapten-based affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction. However, this can not be advantageous since it results in loss of antigen binding by the antibody conjugate.

Molecules containing azido groups can also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter and Haley, 1983). In particular, 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and can be used as antibody binding agents.

Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody (U.S. Pat. Nos. 4,472,509 and 4,938,948). Monoclonal antibodies can also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. In U.S. Pat. No. 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

In other embodiments, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O'Shannessy et al., 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation.

In one embodiment, the present invention provides a method of transmitting data from a diagnostic result using a human anti-nucleolin antibody. The diagnostic assay may be used to detect cancer or an autoimmune disorder. An example of transmitting data can be disclosing the result of any of the methods and assays described herein across the internet. In one embodiment, the results from diagnostic methods using a human anti-nucleolin antibody with biological samples or a subject is collected and the information is transmitted by digital means, such as by facsimile, electronic mail, telephone, or a global communications network, such as the World Wide Web. For example, data can be transmitted via website posting, such as by subscription or select/secure access thereto and/or via electronic mail and/or via telephone, IR, radio, television or other frequency signal, and/or via electronic signals over cable and/or satellite transmission and/or via transmission of disks, compact discs (CDs), computers, hard drives, or other apparatus containing the information in electronic form, and/or transmission of written forms of the information, e.g., via facsimile transmission and the like. Thus, the invention comprehends a user performing according to the invention and transmitting information therefrom; for instance, to one or more parties who then further utilize some or all of the data or information, e.g., in the manufacture of products, such as therapeutics, assays and diagnostic tests and etc. This invention comprehends disks, CDs, computers, or other apparatus or means for storing or receiving or transmitting data or information containing information from methods and/or use of methods of the invention. Thus, the invention comprehends a method for transmitting information comprising performing a method as discussed herein and transmitting a result thereof.

In one aspect, the invention provides methods of doing business comprising performing or using some or all of the herein methods, and communicating or transmitting or divulging a result of a diagnostic assay using a human anti-nucleolin antibody, advantageously in exchange for compensation, e.g., a fee. Advantageously, the communicating, transmitting or divulging of information is via electronic means, e.g., via internet or email, or by any other transmission means herein discussed. Thus, the invention comprehends methods of doing business. For example, a first party, a “client,” can request information, e.g., via any of the herein mentioned transmission means—either previously prepared information or information specially ordered as to the results of the methods and assays of the invention—of a second party, “vendor”, e.g., requesting information via electronic means such as via internet (for instance request typed into website) or via email. The vendor can transmit that information, e.g., via any of the transmission means herein mentioned, advantageously via electronic means, such as internet (for instance secure or subscription or select access website) or email. The information can come from performing some or all of a herein method or use of a herein method in response to the request, or from performing some or all of a herein method, and generating a library of information from performing some or all of a herein method or use of a herein algorithm. Meeting the request can then be by allowing the client access to the library or selecting data from the library that is responsive to the request.

Accordingly, the invention even further comprehends collections of information, e.g., in electronic form (such as forms of transmission discussed above), from performing or using a herein invention.

For example, a client hospital can find itself in need of determining the presence of a disease, such as cancer. A vendor proficient at the methods of the present invention can be contacted by the hospital to quickly test or screen a sample form a subject using any of the herein described methods or any other method contemplated by the invention. The results of the screen can be transmitted back to the client hospital for a fee.

VI. THERAPEUTIC USE OF MAbs

A. Human Monoclonal Anti-Nucleolin Antibodies

In one embodiment, a human anti-nucleolin antibody is provided that can be used to inhibit or kill a cancer cell. In another embodiment, an isolated human anti-nucleolin monoclonal antibody is provided that can be used to inhibit or kill a cancer cell. In another embodiment, an isolated anti-nucleolin monoclonal antibody expressed by a human B cell is provided that can be used to inhibit or kill a cancer cell. In one embodiment the cancer cell expresses human nucleolin on its surface or in it cytoplasm. Examples of cancer cells that can be inhibited or killed by a human anti-nucleolin antibody 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; Myelodysplastic Syndromes; 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.

In one embodiment, a human anti-nucleolin antibody is used to reduce cell viability of a cancer cell in a subject sample by 30 to 80% as compared to cells not exposed to a human anti-nucleolin antibody. In one embodiment, an isolated human monoclonal anti-nucleolin antibody is used to reduce cell viability of a cancer cell in a subject sample by 30 to 80% as compared cells not exposed to a human anti-nucleolin antibody. In one embodiment, an isolated human monoclonal anti-nucleolin antibody produced from a human B cell is provided and used to reduce cell viability of a cancer cell in a subject sample by 30 to 80% as compared cells not exposed to a human anti-nucleolin antibody.

In one embodiment, a human anti-nucleolin antibody is used to reduce cell viability of a cancer cell in a subject by 30 to 80% as compared cells not exposed to a human anti-nucleolin antibody. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used to reduce cell viability of a cancer cell in a subject by 30 to 80% as compared cells not exposed to a human anti-nucleolin antibody. In one embodiment, an isolated human anti-nucleolin monoclonal antibody produced from a human B cell is provided and used to reduce cell viability of a cancer cell in a subject by 30 to 80% as compared cells not exposed to a human anti-nucleolin antibody.

In one embodiment a human anti-nucleolin antibody is administered to a human subject with one or more forms of cancer. In one embodiment an isolated human anti-nucleolin monoclonal antibody is administered to a human subject with one or more forms of cancer. In one embodiment at least one of the forms of cancer is inhibited or killed by a human anti-nucleolin antibody. In one embodiment an isolated human anti-nucleolin monoclonal antibody is administered to a human subject where the cancer is resistant to other cancer treatments. In one embodiment an isolated human anti-nucleolin monoclonal antibody produced from a human B cell is provided 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 one embodiment the immune system of the human subject is more tolerant to the isolated human anti-nucleolin antibody than to an isolated non human anti-nucleolin antibody. In another embodiment the immune system of the human subject is more tolerant to the isolated human anti-nucleolin antibody than to an isolated humanized anti-nucleolin antibody. In another embodiment the immune system of the human subject is more tolerant to the isolated human anti-nucleolin antibody than to an isolated chimeric anti-nucleolin antibody.

In one embodiment, a human anti-nucleolin antibody is used to inhibit or kill a cell as part of an adjuvant therapy. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used to inhibit or kill a cell as part of an adjuvant therapy. In one embodiment, an isolated anti-nucleolin monoclonal antibody expressed by a human B cell is provided and used as part of 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.

In one embodiment, a human anti-nucleolin antibody is used to inhibit or kill a cell used in combination with an adjuvant therapy. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used to inhibit or kill a cell used in combination with an adjuvant therapy. In one embodiment, an isolated anti-nucleolin monoclonal antibody expressed by a human B cell is provided as part of an adjuvant therapy. Adjuvant therapy may include chemotherapy, radiation therapy, hormone therapy, targeted therapy, or biological therapy.

In one embodiment, a human anti-nucleolin 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. In one embodiment, an isolated human anti-nucleolin 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. In one embodiment, the isolated anti-nucleolin monoclonal antibody is expressed by a human B cell. For example, specific non-limiting examples of non-malignant cell proliferative disorders that can treated or inhibited with an anti-nucleolin antibody include but are not limited to warts, benign prostatic hyperplasia, skin tags, and non-malignant tumors. For example, an isolated human anti-nucleolin 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 one embodiment, a human anti-nucleolin antibody is used to inhibit or kill in a subject a cell comprising anangiogenic tumor. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used to inhibit or kill in a subject a cell comprising an angiogenic tumor. In one embodiment, an isolated anti-nucleolin monoclonal antibody expressed by a human B cell is provided that can 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.

In one embodiment, a human anti-nucleolin antibody is used to inhibit or kill in a subject a tumor cell under conditions of tumor hypoxia. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used to inhibit or kill in a subject a tumor cell under conditions of tumor hypoxia. In one embodiment, an isolated anti-nucleolin monoclonal antibody expressed by a human B cell is provided that can 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.

In one embodiment, a human anti-nucleolin antibody is used to inhibit or kill in subject a lymphocyte cell expressing human nucleolin on its surface. In one embodiment, a isolated human anti-nucleolin monoclonal antibody is used to inhibit or kill in subject a lymphocyte cell expressing human nucleolin on its surface. In one embodiment, an isolated human anti-nucleolin monoclonal antibody expressed by a human B cell is provided that is used to inhibit or kill in subject a lymphocyte cell expressing human nucleolin on its surface. In one embodiment, the lymphocyte cell comprises a B cell, T cell, or natural killer cell. In one embodiment, the lymphocyte cell comprises a CD4-positive or CD8-positive cells.

In one embodiment, a human anti-nucleolin antibody is used to inhibit or kill in a subject an activated lymphocyte or memory cell expressing human nucleolin on its surface. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used to inhibit or kill in a subject an activated lymphocyte or memory cell expressing human nucleolin on its surface. In one embodiment, an isolated anti-nucleolin monoclonal antibody expressed by a human B cell is provided that is used to inhibit or kill in subject an activated lymphocyte cell or memory cell expressing human nucleolin on its surface. In a further embodiment, the activated lymphocyte comprises an activated B cell, T cell, or natural killer cell. In one embodiment, a human anti-nucleolin antibody is used to inhibit or kill a cell in a subject having an autoimmune disorder. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used to inhibit or kill a cell in a subject having an autoimmune disorder. In one embodiment, an isolated anti-nucleolin monoclonal antibody expressed by a human B cell is provided that is used to inhibit or kill a cell in a subject having an autoimmune disorder.

In one embodiment, ahuman anti-nucleolin antibody is used to inhibit or kill a cell in a subject having an autoimmune disorder. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used to inhibit or kill a cell in a subject having an autoimmune disorder. In one embodiment, an isolated anti-nucleolin monoclonal antibody expressed by a human B cell is provided that can inhibit or kill cell in a subject having an autoimmune disorder. CD40 and CD40 ligand are interactions mediate T-dependent B cell response and efficient T cell priming and nucleolin has been shown to interact with CD40 ligand. (Geahlen et al., 1984) In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used to inhibit or kill a cell in a subject having an autoimmune disorder wherein the cell is characterized by activated CD40 receptors. In one embodiment the cell expresses human nucleolin on its surface or in its cytoplasm. In one embodiment the cell is a lymphocyte. In one embodiment the lymphocyte is a B cell or T cell. In one embodiment the lymphocyte is activated. Exemplary autoimmune diseases or disorders which may be diagnosed with the use of a human anti-nucleolin antibody include, but are 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, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosis (SLE), Sweet's syndrome, Still's disease, lupus erythematosus, takayasu arteritis, temporal arteristis/giant cell arteritis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis. Examples of inflammatory disorders include, but are not limited to, asthma, encephilitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentitated 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.

In another embodiment, an human anti-nucleolin antibody is used to inhibit or kill a cell in a subject infected by a virus. In another embodiment, an isolated human anti-nucleolin monoclonal antibody is used to inhibit or kill a cell in a subject infected by a virus. In one embodiment, an isolated anti-nucleolin monoclonal antibody expressed by a human B cell is provided that can inhibit or kill cell in a subject infected by a virus. Examples of virus which can infect cells include but are not limited to: 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 (HEV), 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 invention are known, such as those disclosed in U.S. Patent Publication No. 2007/0066554. In a further aspect of the invention, 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.

In one embodiment, a human anti-nucleolin antibody is used to inhibit or kill a cell in a sample from a subject as a indicator for the presence of a disease. In one embodiment, an isolated human anti-nucleolin monoclonal antibody is used to inhibit or kill a cell in a sample from a subject as a prognostic indicator for a disease. In one embodiment, an isolated anti-nucleolin monoclonal antibody expressed by a human B cell is provided that is used to inhibit or kill a cell in a sample from a subject as a prognostic indicator for a disease. Examples of diseases tested include but are not limited to malignant tumor, non-malignant tumor, cancer, autoimmune disease, inflammatory disease, and infectious disease.

B. Antibody Conjugates

In one embodiment, the present invention provides for an isolated human anti-nucleolin antibody linked to at least one therapeutic agent to form an antibody conjugate. In one embodiment efficacy of an isolated human anti-nucleolin antibody, 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 one embodiment 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.

In one embodiment, an isolated human anti-nucleolin 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 or the tricothecenes.

In another embodiment, an isolated human anti-nucleolin antibody is conjugated to an a radionuclide. Examples of suitable radionuclides include, but are not limited to, ¹²⁴antimony, ¹²⁵antimony, ⁷⁴arsenic, ²¹¹astatine, ¹⁰³barium, ¹⁴⁰barium, ⁷beryllium, ²⁰⁶bismuth, ²⁰⁷bismuth, ¹⁰⁹cadmium, ¹¹⁵cadmium, ⁴⁵calcium, ¹⁴carbon, ¹³⁹cerium, ¹⁴¹cerium, ¹⁴⁴cerium, ¹³⁷cesium, ⁵¹chromium, ³⁶chlorine, ⁵⁶cobalt, ⁵⁷cobalt, ⁵⁸cobalt, ⁶⁰cobalt, ⁶⁷copper, ¹⁶⁹erbium, ¹⁵²eurpium, ⁶⁷gallium, ¹⁵³gadolinium, ¹⁹⁵gold, ¹⁹⁹gold, ¹⁷⁵hafnium, ^175+181hafnium, ¹⁸¹hafnium, ³hydrogen, ¹²³iodine, ¹²⁵iodine, ¹³¹iodine, ¹¹¹indium, ¹⁹²iridium, ⁵⁵iron, ⁵⁹iron, ⁸⁵krypton, ²¹⁰lead, ¹⁷⁷lutecium, ⁵⁴manganese, ¹⁹⁷mercury, ²⁰³mercury, ⁹⁹molybdenum, ¹⁴⁷neodynium, ²³⁷neptunium, ⁶³nickel, ⁹⁵niobium, ^185+191osmium, ¹⁰³palladium, ³²phosphorus, ¹⁸⁴platinum, ¹⁴³praseodymium, ¹⁴⁷promethium, ²³³protactinium ²²⁶radium rhenium¹⁸⁶, ¹⁸⁸rhenium, ⁸⁶rubidium, ¹³⁰ruthenium, ¹⁰⁶ruthenium, ⁴⁴scandium, ⁴⁶scandium, ⁴⁵selenium, ⁷⁵selenium, ^110msilver, ¹¹¹silver, ²²sodium, ⁸⁵strontium, ⁸⁹strontium, ⁹⁰strontium, ³⁵sulphur, ¹⁸²tantalum, ^99mtechnicium, ^125mtellurium, ¹³²tellurium, ¹⁶⁰terbium, ²⁰⁴thallium, ²²⁸thorium, ²³²thorium, ¹⁷⁰thullium, ¹¹³tin, ⁴⁴titanium, ¹⁸⁵tungsten, ⁴⁸vanadlum, ⁴⁹vanadium, ⁸⁸yttrium, ⁹⁰yttrium, ⁹¹yttrium, ¹⁶⁹ytterbium, ⁶⁵zinc, and ⁹⁵zirconium.

Conjugates of the antibody and cytotoxic agent 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 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al. (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO 94/11026.

In one embodiment, an isolated human anti-nucleolin antibody is conjugated to an 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-α 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-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -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-1a, 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.

In another embodiment, an isolated human anti-nucleolin antibody is conjugated to an chemotherapeutic agent. A variety of chemical compounds, also described as “chemotherapeutic agents,” function to induce DNA damage. Categories of chemotherapeutic agents suitable for conjugation with a an isolated human anti-nucleolin antibody 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 invention 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.

In another embodiment, an isolated human anti-nucleolin antibody is conjugated to an anti-viral agent. Example of anti-viral agents that can be used with an isolated human anti-nucleolin antibody 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.

Specific antiviral agents that can be used with an isolated human anti-nucleolin antibody include, but are not limited to, ganciclovir, valganciclovir, oseltamivir (Tamiflu), zanamivir (Relenza), abacavir, aciclovir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, fusion inhibitors (e.g., enfuvirtide), ibacitabine, immunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitor, interferon type III, interferon type II, interferon type I, interferon, lamivudine, lopinavir, loviride, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, nucleoside analogues, peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitor, raltegravir, reverse transcriptase inhibitor, ribavirin, rimantadine, ritonavir, pyrimidine antiviral, saquinavir, stavudine, synergistic enhancer (antiretroviral), tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir (Valtrex), vicriviroc, vidarabine, viramidine, zalcitabine, and zidovudine.

Examples of nucleoside analogs that can be used with an isolated human anti-nucleolin antibody include acyclovir (ACV), ganciclovir (GCV), famciclovir, foscarnet, ribavirin, zalcitabine (ddC), zidovudine (AZT), stavudine (D4T), lamivudine (3TC), didanosine (ddI), cytarabine, dideoxyadenosine, edoxudine, floxuridine, idozuridine, inosine pranobex, 2′-deoxy-5-(methylamino)uridine, trifluridine and vidarabine.

C. Pharmaceutical Compositions and Administration

It is envisioned that, for administration to a subject in need thereof, a antibody will be suspended in a formulation suitable for administration to a host. In one embodiment the antibody is a monoclonal antibody. In one embodiment the monoclonal antibody is an anti-nucleolin antibody. In one embodiment the monoclonal anti-nucleolin antibody is a human monoclonal anti-nucleolin antibody. Aqueous compositions of the present invention 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.

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. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. 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 Biologics standards.

In one embodiment, a human anti-nucleolin antibody of the invention 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 one embodiment of the present invention, the pharmaceutically acceptable carrier is pharmaceutically inert.

Administration of pharmaceutical compositions is accomplished orally or parenterally. Methods of parenteral delivery include topical, intra-arterial (e.g., directly to a tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. 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.).

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. See PCT publication WO 93/23572.

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.

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).

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.

Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds. For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably 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.

For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention 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 other cases, 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, that is combined with buffer prior to use.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

After pharmaceutical compositions comprising a compound of the invention formulated in an acceptable carrier have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of human telomerase proteins and nucleic acids, such labeling would include amount, frequency and method of administration.

Pharmaceutical compositions suitable for use in the present invention 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 will preferably be an optimized amount such that the desired effect is achieved without significant side-effects.

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.

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.

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 a 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).

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.

A therapeutically effective amount refers to that amount of protein, polypeptide, peptide, antibody, oligo- or polynucleotide, agonist or antagonists which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., ED₅₀, the dose therapeutically effective in 50% of the population; and LD₅₀, the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, ED₅₀/LD₅₀Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

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 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 a human anti-nucleolin antibody may be for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. Administration of a dosage form comprising a human anti-nucleolin antibody may be for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. Administration of a dosage form comprising a human anti-nucleolin 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 a human anti-nucleolin 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 (e.g., a human anti-nucleolin antibody and an other agent) 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. Pat. Nos. 4,657,760; 5,206,344; and 5,225,212, herein incorporated by reference). In one embodiment, the dosage of a composition comprising human anti-nucleolin 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. In one embodiment, a human anti-nucleolin antibody is administered to a subject. The subjects can be male or female and may be of any race or ethnicity, including, but not limited to, Caucasian, African-American, African, Asian, Hispanic, Indian, etc. The subjects can be of any age, including newborn, neonate, infant, child, adolescent, adult, and geriatric. Subjects can also include animal subjects, particularly mammalian subjects such as dog, cat, horse, mouse, rat, etc., screened for veterinary medicine or pharmaceutical drug development purposes. Subjects further include, but are not limited, to those who have, have been exposed to, or have been previously diagnosed as afflicted with a proliferative disorder, such as cancer, or an autoimmune disorder, such as a viral disorder such as HIV or AIDS.

D. Nucleolin-Expressing Cancers and Non-Malignant Cells

In one embodiment, an isolated human anti-nucleolin antibody produced in accordance with the present invention is used in treating a variety of cells, including both cancerous and non-cancerous cells. In one embodiment the isolated human anti-nucleolin antibody is a monoclonal antibody. In another embodiment the isolated human anti-nucleolin antibody is a polyclonal antibody. The term “cancer” is described previously herein. Examples of types cancer that can be inhibited or treated with an isolated human anti-nucleolin antibody 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. 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 one embodiment, a antibody of the present invention 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.

i. Acute Myeloid Leukemia

Acute myeloid leukemia (AML), also known as acute myelogenous leukemia, is a cancer of the myeloid line of blood cells, characterized by the rapid growth of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells. AML is the most common acute leukemia affecting adults, and its incidence increases with age. Although AML is a relatively rare disease, accounting for approximately 1.2% of cancer deaths in the United States, its incidence is expected to increase as the population ages.

The symptoms of AML are caused by replacement of normal bone marrow with leukemic cells, which causes a drop in red blood cells, platelets, and normal white blood cells. These symptoms include fatigue, shortness of breath, easy bruising and bleeding, and increased risk of infection. Although several risk factors for AML have been identified, the specific cause of the disease remains unclear. As an acute leukemia, AML progresses rapidly and is typically fatal within weeks or months if left untreated.

AML has several subtypes; treatment and prognosis varies among subtypes. Five-year survival varies from 5-70%, and relapse rate varies from 30-95%, depending on subtype. AML is treated initially with chemotherapy aimed at inducing a remission; patients can go on to receive additional chemotherapy or a hematopoietic stem cell transplant. Recent research into the genetics of AML has developed tests that better predict how long a patient is likely to survive and whether a drug is likely to be effective.

The first symptom leading to a diagnosis of AML is typically non-specific, related to one or more of the cytopenias, e.g., anemia, neutropenia, and/or thrombocytopenia. While an excess of abnormal white blood cells (leukocytosis) with immature blood cells (blasts) in the peripheral blood are a common finding, and leukemic blasts are sometimes seen, AML can also present with isolated decreases in platelets, red blood cells, or even with a low white blood cell count (leukopenia).

While a presumptive diagnosis of AML can be made via examination of the peripheral blood smear when there are circulating leukemic blasts, a definitive diagnosis usually requires an examination of the cells taken from a bone marrow aspiration and biopsy.

Marrow or blood is examined via light microscopy as well as flow cytometry to diagnose the presence of leukemia, to differentiate AML from other types of leukemia (e.g., acute lymphoblastic leukemia), and to classify the subtype of disease (see below). A sample of marrow or blood is typically also tested for various chromosomal aberrations by routine cytogenetics or fluorescent in situ hybridization.

Genetic studies can also be performed to look for specific mutations in genes such as FLT3, nucleophosmin, and bcr/able among others, which can influence the outcome of the disease.

Cytochemical stains on blood and bone marrow smears are helpful in the distinction of AML from ALL and in subclassification of AML. The combination of a myeloperoxidase or Sudan black stain and a non specific esterase stain will provide the desired information in most cases. The myeloperoxidase or Sudan black reactions are most useful in establishing the identity of AML and distinguishing from ALL. The non-specific esterase stain is used to identify a monocytic component in AMLs and to distinguish a poorly differentiated monoblastic leukemia from ALL.

The diagnosis and classification of AML can be challenging, and should be performed by a qualified hematopathologist or hematologist. In straightforward cases, the presence of certain morphologic features (such as Auer rods) or specific flow cytometry results can distinguish AML from other leukemias; however, in the absence of such features, diagnosis can be more difficult.

According to the widely used WHO criteria, the diagnosis of AML is established by demonstrating involvement of more than 20% of the blood and/or bone marrow by leukemic myeloblasts. AML must be carefully differentiated from “pre-leukemic” conditions such as myelodysplastic or myeloproliferative syndromes, which are treated differently.

Because acute promyelocytic leukemia (APL) has the highest curability and requires a unique form of treatment, it is important to quickly establish or exclude the diagnosis of this subtype of leukemia. Fluorescent in situ hybridization performed on blood or bone marrow is often used for this purpose, as it readily identifies the chromosomal translocation (t[15;17]) that characterizes APL.

The malignant cell in AML is the myeloblast. In normal hematopoiesis, the myeloblast is an immature precursor of myeloid white blood cells; a normal myeloblast will gradually mature into a mature white blood cell. However, in AML, a single myeloblast accumulates genetic changes which “freeze” the cell in its immature state and prevent differentiation. Such a mutation alone does not cause leukemia; however, when such a “differentiation arrest” is combined with other mutations which disrupt genes controlling proliferation, the result is the uncontrolled growth of an immature clone of cells, leading to the clinical entity of AML.

Much of the diversity and heterogeneity of AML stems from the fact that leukemic transformation can occur at a number of different steps along the differentiation pathway. Modern classification schemes for AML recognize that the characteristics and behavior of the leukemic cell (and the leukemia) can depend on the stage at which differentiation was halted.

Specific cytogenetic abnormalities can be found in many patients with AML; the types of chromosomal abnormalities often have prognostic significance. The chromosomal translocations encode abnormal fusion proteins, usually transcription factors whose altered properties can cause the “differentiation arrest.” For example, in acute promyelocytic leukemia, the t(15;17) translocation produces a PML-RARα fusion protein which binds to the retinoic acid receptor element in the promoters of several myeloid-specific genes and inhibits myeloid differentiation.

The clinical signs and symptoms of AML result from the fact that, as the leukemic clone of cells grows, it tends to displace or interfere with the development of normal blood cells in the bone marrow. This leads to neutropenia, anemia, and thrombocytopenia. The symptoms of AML are in turn often due to the low numbers of these normal blood elements. In rare cases, patients can develop a chloroma, or solid tumor of leukemic cells outside the bone marrow, which can cause various symptoms depending on its location.

Treatment of AML consists primarily of chemotherapy, and is divided into two phases: induction and postremission (or consolidation) therapy. The goal of induction therapy is to achieve a complete remission by reducing the amount of leukemic cells to an undetectable level; the goal of consolidation therapy is to eliminate any residual undetectable disease and achieve a cure.

All FAB subtypes except M3 are usually given induction chemotherapy with cytarabine (ara-C) and an anthracycline (such as daunorubicin or idarubicin). This induction chemotherapy regimen is known as “7+3” (or “3+7”), because the cytarabine is given as a continuous IV infusion for seven consecutive days while the anthracycline is given for three consecutive days as an IV push. Up to 70% of patients will achieve a remission with this protocol. Other alternative induction regimens, including high-dose cytarabine alone or investigational agents, can also be used. Because of the toxic effects of therapy, including myelosuppression and an increased risk of infection, induction chemotherapy can not be offered to the very elderly, and the options can include less intense chemotherapy or palliative care.

The M3 subtype of AML, also known as acute promyelocytic leukemia, is almost universally treated with the drug ATRA (all-trans-retinoic acid) in addition to induction chemotherapy. Care must be taken to prevent disseminated intravascular coagulation (DIC), complicating the treatment of APL when the promyelocytes release the contents of their granules into the peripheral circulation. APL is eminently curable with well-documented treatment protocols.

The goal of the induction phase is to reach a complete remission. Complete remission does not mean that the disease has been cured; rather, it signifies that no disease can be detected with available diagnostic methods. Complete remission is obtained in about 50%-75% of newly diagnosed adults, although this can vary based on the prognostic factors described above. The length of remission depends on the prognostic features of the original leukemia. In general, all remissions will fail without additional consolidation therapy.

Even after complete remission is achieved, leukemic cells likely remain in numbers too small to be detected with current diagnostic techniques. If no further post-remission or consolidation therapy is given, almost all patients will eventually relapse. Therefore, more therapy is necessary to eliminate non-detectable disease and prevent relapse—that is, to achieve a cure.

The specific type of postremission therapy is individualized based on a patient's prognostic factors (see above) and general health. For good-prognosis leukemias (i.e., inv(16), t(8;21), and t(15;17)), patients will typically undergo an additional 3-5 courses of intensive chemotherapy, known as consolidation chemotherapy. For patients at high risk of relapse (e.g. those with high-risk cytogenetics, underlying MDS, or therapy-related AML), allogeneic stem cell transplantation is usually recommended if the patient is able to tolerate a transplant and has a suitable donor. The best postremission therapy for intermediate-risk AML (normal cytogenetics or cytogenetic changes not falling into good-risk or high-risk groups) is less clear and depends on the specific situation, including the age and overall health of the patient, the patient's personal values, and whether a suitable stem cell donor is available.

Despite aggressive therapy, however, only 20%-30% of patients enjoy long-term disease-free survival. For patients with relapsed AML, the only proven potentially curative therapy is a stem cell transplant, if one has not already been performed. In 2000, the monoclonal antibody-linked cytotoxic agent gemtuzumab ozogamicin (Mylotarg) was approved in the United States for patients aged more than 60 years with relapsed AML who are not candidates for high-dose chemotherapy.

Patients with relapsed AML who are not candidates for stem cell transplantion, or who have relapsed after a stem cell transplant, can be offered treatment in a clinical trial, as conventional treatment options are limited. Agents under investigation include cytotoxic drugs such as clofarabine as well as targeted therapies such as farnesyl transferase inhibitors, decitabine, and inhibitors of MDR1 (multidrug-resistance protein). Since treatment options for relapsed AML are so limited, another option which can be offered is palliative care.

For relapsed acute promyelocytic leukemia (APL), arsenic trioxide has been tested in trials and approved by the Food and Drug Administration. Like ATRA, arsenic trioxide does not work with other subtypes of AML.

Acute myeloid leukemia is a curable disease; the chance of cure for a specific patient depends on a number of prognostic factors. The single most important prognostic factor in AML is cytogenetics, or the chromosomal structure of the leukemic cell. Certain cytogenetic abnormalities are associated with very good outcomes (for example, the t(15;17) translocation in acute promyelocytic leukemia). About half of AML patients have “normal” cytogenetics; they fall into an intermediate risk group. A number of other cytogenetic abnormalities are known to associate with a poor prognosis and a high risk of relapse after treatment.

AML which arises from a pre-existing myelodysplastic syndrome or myeloproliferative disease (so-called secondary AML) has a worse prognosis, as does treatment-related AML arising after chemotherapy for another previous malignancy. Both of these entities are associated with a high rate of unfavorable cytogenetic abnormalities.

In some studies, age >60 years and elevated lactate dehydrogenase level were also associated with poorer outcomes. As with most forms of cancer, performance status (i.e., the general physical condition and activity level of the patient) plays a major role in prognosis as well.

FLT3 internal tandem duplications (ITDs) have been shown to confer a poorer prognosis in AML. Treating these patients with more aggressive therapy, such as stem-cell transplantation in first remission, has not been shown to enhance long-term survival, so this prognostic feature is of uncertain clinical significance at this point. ITDs of FLT3 can be associated with leukostasis.

Researchers are investigating the clinical significance of c-KIT mutations in AML. These are prevalent, and clinically relevant because of the availability of tyrosine kinase inhibitors, such as imatinib and sunitinib that can block the activity of c-KIT pharmacologically. Other genes being investigated as prognostic factors or therapeutic targets include CEBPA, BAALC, ERG, and NPM1.

Cure rates in clinical trials have ranged from 20-45%; however, it should be noted that clinical trials often include only younger patients and those able to tolerate aggressive therapies. The overall cure rate for all patients with AML (including the elderly and those unable to tolerate aggressive therapy) is likely lower. Cure rates for promyelocytic leukemia can be as high as 98%.

ii. Chronic Lymphocytic Leukemia

B-cell chronic lymphocytic leukemia (B-CLL), also known as chronic lymphoid leukemia (CLL), is the most common type of leukemia. Leukemias are abnormal and malignant neoplastic proliferations of the white blood cells (leukocytes). CLL involves a particular subtype of white blood cells, which is a lymphocyte called a B cell. B cells originate in the bone marrow, develop in the lymph nodes, and normally fight infection. In CLL, the DNA of a B cell is damaged, so that it can't fight infection by producing antibodies. Additionally, they grow out of control and accumulate in the bone marrow and blood, where they crowd out healthy blood cells.

CLL is a disease of adults, but in rare cases it can occur in teenagers and occasionally in children (inherited). Most (>75%) people newly diagnosed with CLL are over the age of 50, and the majority are men. Most people are diagnosed without symptoms as the result of a routine blood test that returns a high white blood cell count, but as it advances CLL results in swollen lymph nodes, spleen, and liver, and eventually anemia and infections. Early CLL is not treated, and late CLL is treated with chemotherapy and monoclonal antibodies. Survival varies from 5 years to more than 25 years. It is now possible to predict survival length more precisely by examining the DNA mutations; patients with slowly-progressing disease can be reassured and can not need any treatment in their lifetimes.

Although not originally appreciated, CLL is now felt to be identical to a disease called small lymphocytic lymphoma (SLL), a type of non-Hodgkin's lymphoma which presents primarily in the lymph nodes. The World Health Organization considers CLL and SLL to be one disease at different stages, not two separate entities.

Staging, determining the extent of the disease, is done with the Rai staging system or the Binet classification and is based primarily on the presence, or not, of a low platelet or red cell count. Early stage disease does not need to be treated.

Recent publications suggest that two or three prognostic groups of CLL exist based on the maturational state of the cell. This distinction is based on the maturity of the lymphocytes as discerned by the immunoglobulin variable-region heavy chain (IgV_H) gene mutation status. High risk patients have an immature cell pattern with few mutations in the DNA in the IgV_Hantibody gene region whereas low risk patients show considerable mutations of the DNA in the antibody gene region indicating mature lymphocytes.

Since assessment of the IgV_Hantibody DNA changes is difficult to perform, the presence of either cluster of differentiation 38 (CD38) or Z-chain-associated protein kinase-70 (ZAP-70) can be surrogate markers of high risk subtype of CLL. Their expression correlates with a more immature cellular state and a more rapid disease course.

In addition to the maturational state, the prognosis of patients with CLL is dependent on the genetic changes within the neoplastic cell population. These genetic changes can be identified by fluorescent probes to chromosomal parts using a technique referred to as fluorescent in situ hybridization (FISH). Four main genetic aberrations are recognized in CLL cells that have a major impact on disease behavior.

Deletions of part of the short arm of chromosome 17 (del 17p) which target the cell cycle regulating protein p53 are particularly deleterious. Patients with this abnormality have significantly short interval before they require therapy and a shorter survival. This abnormality is found in 5-10% of patients with CLL. Deletions of the long arm on chromosome 11 (del 11q) are also unfavorable although not to the degree seen with del 17p. The abnormality targets the ATM gene and occurs infrequently in CLL (5-10%). Trisomy 12, an additional chromosome 12, is a relatively frequent finding occurring in 20-25% of patients and imparts an intermediate prognosis. Deletion of the long arm of chromosome 13 (del 13q) is the most common abnormality in CLL with roughly 50% of patients with cells containing this defect. These patients have the best prognosis and most will live many years, even decades, without the need for therapy. The gene targeted by this deletion is a segment that likely produces small inhibitory RNA molecules that affect expression of important death inhibiting gene products.

Most people are diagnosed without symptoms as the result of a routine blood test that returns a high white blood cell count. Uncommonly, CLL presents as enlargement of the lymph nodes without a high white blood cell count or no evidence of the disease in the blood. This is referred to as small lymphocytic lymphoma. In some individuals the disease comes to light only after the neoplastic cells overwhelm the bone marrow resulting in anemia producing tiredness or weakness.

The disease is easily diagnosed. CLL is usually first suspected by the presence of a lymphocytosis, an increase in one type of the white blood cell, on a complete blood count (CBC) test. This frequently is an incidental finding on a routine physician visit. Most often the lymphocyte count is greater than 4000 cells per mm³(microliter) of blood but can be much higher. The presence of a lymphocytosis in an elderly individual should raise strong suspicion for CLL and a confirmatory diagnostic test, in particular flow cytometry, should be performed unless clinically unnecessary.

The diagnosis of CLL is based on the demonstration of an abnormal population of B lymphocytes in the blood, bone marrow, or tissues that display an unusual but characteristic pattern of molecules on the cell surface. This atypical molecular pattern includes the co-expression of cells surface markers cluster of differentiation 5 (CD5) and cluster of differentiation 23 (CD23). In addition, all the CLL cells within one individual are clonal, that is genetically identical. In practice, this is inferred by the detection of only one of the mutually exclusive antibody light chains, kappa or lambda, on the entire population of the abnormal B cells. Normal B lymphocytes consist of a stew of different antibody producing cells resulting in a mixture of both kappa and lambda expressing cells. The lack of the normal distribution of kappa and lambda producing B cells is one basis for demonstrating clonality, the key element for establishing a diagnosis of any B cell malignancy (B cell Non-Hodgkin's lymphoma).

The combination of the microscopic examination of the peripheral blood and analysis of the lymphocytes by flow cytometry to confirm clonality and marker molecule expression is needed to establish the diagnosis of CLL. Both are easily accomplished on a small amount of blood. A flow cytometer is an instrument that can examine the expression of molecules on individual cells in fluids. This requires the use of specific antibodies to marker molecules with fluorescent tags recognized by the instrument. In CLL, the lymphocytes are genetically clonal, of the B cell lineage (express marker molecules cluster of differentiation 19 (CD19) and CD20), and characteristically express the marker molecules CD5 and CD23. Morphologically, the cells resemble normal lymphocytes under the microscope, although slightly smaller, and are fragile when smeared onto a glass slide giving rise to many broken cells (smudge cells).

CLL treatment focuses on controlling the disease and its symptoms rather than on an outright cure. CLL is treated by chemotherapy, radiation therapy, biological therapy, or bone marrow transplantation. Symptoms are sometimes treated surgically (splenectomy removal of enlarged spleen) or by radiation therapy (“de-bulking” swollen lymph nodes).

Initial CLL treatments vary depending on the exact diagnosis and the progression of the disease, and even with the preference and experience of the health care practitioner. There are dozens of agents used for CLL therapy, and there is considerable research activity studying them individually or in combination with each other.

While generally considered incurable, CLL progresses slowly in most cases. Many people with CLL lead normal and active lives for many years—in some cases for decades. Because of its slow onset, early-stage CLL is generally not treated since it is believed that early CLL intervention does not improve survival time or quality of life. Instead, the condition is monitored over time to detect any change in the disease pattern.

The decision to start CLL treatment is taken when the patient's clinical symptoms or blood counts indicate that the disease has progressed to a point where it can affect the patient's quality of life. Determining when to start treatment and by what means is often difficult; studies have shown there is no survival advantage to treating the disease too early. The National Cancer Institute Working Group has issued guidelines for treatment, with specific markers that should be met before it is initiated.

Although the purine analogue fludarabine was shown to give superior response rates than chlorambucil as primary therapy, there is no evidence that early use of fludarabine improves overall survival, and some clinicians prefer to reserve fludarabine for relapsed disease. Mab therapies include alemtuzumab (directed against CD52) and rituximab (directed against CD20).

Combination chemotherapy options are effective in both newly-diagnosed and relapsed CLL. Recently, randomized trials have shown that combinations of purine analogues (fludarabine) with alkylating agents (cyclophosphamide) produce higher response rates and a longer progression-free survival than single agents:

FC (fludarabine with cyclophosphamide)

FR (fludarabine with rituximab)

FCR (fludarabine, cyclophosphamide, and rituximab)

CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone)

Allogeneic bone marrow (stem cell) transplantation is rarely used as a first-line treatment for CLL due to its risk. There is increasing interest in the use of reduced intensity allogeneic stem cell transplantation, which offers the prospect of cure for selected patients with a suitable donor.

Current research is comparing different forms of bone marrow transplants to determine which patients are the best candidates and which approach is best in different situations. Younger patients that are at high risk for dying from CLL might consider hematopoietic stem cell transplantation (HSCT). Autologous stem cell transplantation, a lower-risk form of treatment using the patient's own blood cells, is not curative. Myeloablative (bone marrow killing) forms of allogeneic stem cell transplantation, a high-risk treatment using blood cells from a healthy donor, can be curative for some patients, but most patients cannot tolerate the treatment. An intermediate level, called reduced-intensity conditioning allogeneic stem cell transplantation, can be better tolerated by older or frail patients.

“Refractory” CLL is a disease that no longer responds favorably to treatment.

In this case more aggressive therapies, including lenalidomide, flavopiridol, and bone marrow (stem cell) transplantation, are considered. The monoclonal antibody, alemtuzumab (directed against CD52), can be used in patients with refractory, bone marrow-based disease.

iii. Breast Cancer

Breast cancer is a cancer that starts in the breast, usually in the inner lining of the milk ducts or lobules. There are different types of breast cancer, with different stages (spread), aggressiveness, and genetic makeup. With best treatment, 10-year disease-free survival varies from 98% to 10%. Treatment is selected from surgery, drugs (chemotherapy), and radiation. In the United States, there were 216,000 cases of invasive breast cancer and 40,000 deaths in 2004. Worldwide, breast cancer is the second most common type of cancer after lung cancer (10.4% of all cancer incidence, both sexes counted) and the fifth most common cause of cancer death. In 2004, breast cancer caused 519,000 deaths worldwide (7% of cancer deaths; almost 1% of all deaths). Breast cancer is about 100 times as frequent among women as among men, but survival rates are equal in both sexes.

The first symptom, or subjective sign, of breast cancer is typically a lump that feels different from the surrounding breast tissue. According to the The Merck Manual, more than 80% of breast cancer cases are discovered when the woman feels a lump. According to the American Cancer Society, the first medical sign, or objective indication of breast cancer as detected by a physician, is discovered by mammogram. Lumps found in lymph nodes located in the armpits can also indicate breast cancer. Indications of breast cancer other than a lump can include changes in breast size or shape, skin dimpling, nipple inversion, or spontaneous single-nipple discharge. Pain (“mastodynia”) is an unreliable tool in determining the presence or absence of breast cancer, but can be indicative of other breast health issues.

When breast cancer cells invade the dermal lymphatics—small lymph vessels in the skin of the breast—its presentation can resemble skin inflammation and thus is known as inflammatory breast cancer (IBC). Symptoms of inflammatory breast cancer include pain, swelling, warmth and redness throughout the breast, as well as an orange-peel texture to the skin referred to as “peau d′ orange.” Another reported symptom complex of breast cancer is Paget's disease of the breast. This syndrome presents as eczematoid skin changes such as redness and mild flaking of the nipple skin. As Paget's advances, symptoms can include tingling, itching, increased sensitivity, burning, and pain. There can also be discharge from the nipple. Approximately half of women diagnosed with Paget's also have a lump in the breast.

Occasionally, breast cancer presents as metastatic disease, that is, cancer that has spread beyond the original organ. Metastatic breast cancer will cause symptoms that depend on the location of metastasis. Common sites of metastasis include bone, liver, lung and brain. Unexplained weight loss can occasionally herald an occult breast cancer, as can symptoms of fevers or chills. Bone or joint pains can sometimes be manifestations of metastatic breast cancer, as can jaundice or neurological symptoms. These symptoms are “non-specific,” meaning they can also be manifestations of many other illnesses.

The primary risk factors that have been identified are sex, age, childbearing, hormones, a high-fat diet, alcohol intake, obesity, and environmental factors such as tobacco use, radiation and shiftwork. No etiology is known for 95% of breast cancer cases, while approximately 5% of new breast cancers are attributable to hereditary syndromes. In particular, carriers of the breast cancer susceptibility genes, BRCA1 and BRCA2, are at a 30-40% increased risk for breast and ovarian cancer, depending on in which portion of the protein the mutation occurs. Experts believe that 95% of inherited breast cancer can be traced to one of these two genes. Hereditary breast cancers can take the form of a site-specific hereditary breast cancer—cancers affecting the breast only—or breast-ovarian and other cancer syndromes. Breast cancer can be inherited both from female and male relatives.

Breast cancer subtypes are categorized on an immunohistochemical basis. Subtype definitions are generally as follows:

normal (ER+, PR+, HER2+, cytokeratin 5/6+, and HER1+)

luminal A (ER+ and/or PR+, HER2−)

luminal B (ER+ and/or PR+, HER2+)

triple-negative (ER−, PR−, HER2−)

HER2+/ER−(ER−, PR−, and HER2+)

unclassified (ER−, PR−, HER2−, cytokeratin 5/6−, and HER1−)

In the case of triple-negative breast cancer cells, the cancer's growth is not driven by estrogen or progesterone, or by growth signals coming from the HER2 protein. By the same token, such cancer cells do not respond to hormonal therapy, such as tamoxifen or aromatase inhibitors, or therapies that target HER2 receptors, such as Herceptin®. About 10-20% of breast cancers are found to be triple-negative. It is important to identify these types of cancer to that one can avoid costly and toxic effects of therapies that are unlike to succeed, and to focus on treatments that can be used to treat triple-negative breast cancer. Like other forms of breast cancer, triple-negative breast cancer can be treated with surgery, radiation therapy, and/or chemotherapy.

One particularly promising approach is “neoadjuvant” therapy, where chemo- and/or radiotherapy is provided prior to surgery. Another new drug therapy is the use of poly (ADP-ribose) polymerase, or PARP inhibitors.

While screening techniques discussed above are useful in determining the possibility of cancer, a further testing is necessary to confirm whether a lump detected on screening is cancer, as opposed to a benign alternative such as a simple cyst. In a clinical setting, breast cancer is commonly diagnosed using a “triple test” of clinical breast examination (breast examination by a trained medical practitioner), mammography, and fine needle aspiration cytology. Both mammography and clinical breast exam, also used for screening, can indicate an approximate likelihood that a lump is cancer, and can also identify any other lesions. Fine Needle Aspiration and Cytology (FNAC), which can be done in a GP's office using local anaesthetic if required, involves attempting to extract a small portion of fluid from the lump. Clear fluid makes the lump highly unlikely to be cancerous, but bloody fluid can be sent off for inspection under a microscope for cancerous cells. Together, these three tools can be used to diagnose breast cancer with a good degree of accuracy. Other options for biopsy include core biopsy, where a section of the breast lump is removed, and an excisional biopsy, where the entire lump is removed.

Breast cancer screening is an attempt to find cancer in otherwise healthy individuals. The most common screening method for women is a combination of x-ray mammography and clinical breast exam. In women at higher than normal risk, such as those with a strong family history of cancer, additional tools can include genetic testing or breast Magnetic Resonance Imaging.

Breast self-examination was a form of screening that was heavily advocated in the past, but has since fallen into disfavour since several large studies have shown that it does not have a survival benefit for women and often causes considerably anxiety. This is thought to be because cancers that could be detected tended to be at a relatively advanced stage already, whereas other methods push to identify the cancer at an earlier stage where curative treatment is more often possible.

X-ray mammography uses x-rays to examine the breast for any uncharacteristic masses or lumps. Regular mammograms is recommended in several countries in women over a certain age as a screening tool.

Genetic testing for breast cancer typically involves testing for mutations in the BRCA genes. This is not generally a recommended technique except for those at elevated risk for breast cancer.

The mainstay of breast cancer treatment is surgery when the tumor is localized, with possible adjuvant hormonal therapy (with tamoxifen or an aromatase inhibitor), chemotherapy, and/or radiotherapy. At present, the treatment recommendations after surgery (adjuvant therapy) follow a pattern. Depending on clinical criteria (age, type of cancer, size, metastasis) patients are roughly divided to high risk and low risk cases, with each risk category following different rules for therapy. Treatment possibilities include radiation therapy, chemotherapy, hormone therapy, and immune therapy.

Targeted cancer therapies are treatments that target specific characteristics of cancer cells, such as a protein that allows the cancer cells to grow in a rapid or abnormal way. Targeted therapies are generally less likely than chemotherapy to harm normal, healthy cells. Some targeted therapies are antibodies that work like the antibodies made naturally by our immune systems. These types of targeted therapies are sometimes called immune-targeted therapies.

There are currently 3 targeted therapies doctors use to treat breast cancer. Herceptin® (trastuzumab) works against HER2-positive breast cancers by blocking the ability of the cancer cells to receive chemical signals that tell the cells to grow. Tykerb® (lapatinib) works against HER2-positive breast cancers by blocking certain proteins that can cause uncontrolled cell growth. Avastin® (bevacizumab) works by blocking the growth of new blood vessels that cancer cells depend on to grow and function.

Hormonal (anti-estrogen) therapy works against hormone-receptor-positive breast cancer in two ways: first, by lowering the amount of the hormone estrogen in the body, and second, by blocking the action of estrogen in the body. Most of the estrogen in women's bodies is made by the ovaries. Estrogen makes hormone-receptor-positive breast cancers grow. So reducing the amount of estrogen or blocking its action can help shrink hormone-receptor-positive breast cancers and reduce the risk of hormone-receptor-positive breast cancers coming back (recurring). Hormonal therapy medicines are not effective against hormone-receptor-negative breast cancers. There are several types of hormonal therapy medicines, including aromatase inhibitors, selective estrogen receptor modulators, and estrogen receptor downregulators. In some cases, the ovaries and fallopian tubes can be surgically removed to treat hormone-receptor-positive breast cancer or as a preventive measure for women at very high risk of breast cancer. The ovaries also can be shut down temporarily using medication.

In planning treatment, doctors can also use PCR tests like Oncotype DX or microarray tests that predict breast cancer recurrence risk based on gene expression. In February 2007, the first breast cancer predictor test won formal approval from the Food and Drug Administration. This is a new gene test to help predict whether women with early-stage breast cancer will relapse in 5 or 10 years, this could help influence how aggressively the initial tumor is treated.

Radiation therapy is also used to help destroy cancer cells that can linger after surgery. Radiation can reduce the risk of recurrence by 50-66% when delivered in the correct dose.

E. Combination Therapy

Tumor cell resistance to single agents represents a major problem in clinical oncology. One goal of current cancer research is to find ways to improve the efficacy of existing therapies. One way is by combining therapies in what is known as combination therapy. In one embodiment, the present invention provides a combination therapy wherein an anti-nucleolin antibody and at least one other agent comprise a composition for use in contacting a “target cell.” In one aspect, the combination of anti-nucleolin antibody and at least one other agent are used in a combined amount effective to kill or inhibit proliferation of the cell.

In one embodiment, the method further comprises can contacting the cells with a human anti-nucleolin antibody and one or more additional agent(s) or factor(s). In one embodiment, the method further comprises contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the anti-nucleolin antibody and the other includes the agent.

In another embodiment, the method comprises contact by the anti-nucleolin antibody before or after contact by the other agent by intervals ranging from minutes to weeks. In embodiments where the other agent and anti-nucleolin antibody are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and anti-nucleolin antibody would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one would contact the cell with both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. In some situations, it can be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

In another embodiment, the method comprises more than one administration of either the anti-nucleolin antibody or the other agent. Combinations of the anti-nucleolin antibody or the other agent, include, where the anti-nucleolin antibody is “A” and the other agent is “B,” as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B

A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A

A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

Other combinations are contemplated. Again, to achieve cell killing, both agents are delivered to a cell in a combined amount effective to kill the cell.

Agents or factors suitable for use in a combined therapy are any chemical compound or treatment method that induces DNA damage when applied to a cell. In particular, such agents are those discussed in Section VI(C)(i)-(iii) above. In treating cancer according to the invention, one would contact the tumor cells with an agent in addition to the anti-nucleolin antibody. This can be achieved by irradiating the localized tumor site with radiation or administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a chemotherapeutic compound.

A variety of chemical compounds, also described as “chemotherapeutic agents,” function to induce DNA damage, all of which are intended to be of use in the combined treatment methods disclosed herein. In one embodiment, the “other agents” comprise chemotherapeutic agents. Categories of chemotherapeutic agents of the invention include, but are not limited to, alkylating agents, anthracyclines, cytoskeletal disruptors, epothilones, inhibitors of topoisomerase II, 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 invention 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.

Agents that directly cross-link nucleic acids, specifically DNA, are envisaged to facilitate DNA damage leading to a synergistic, antineoplastic combination with anti-nucleolin antibody. Agents such as cisplatin, and other DNA alkylating agents can be used. Cisplatin has been widely used to treat cancer, with efficacious doses used in clinical applications of 20 mg/m²for 5 days every three weeks for a total of three courses. Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally.

Agents that damage DNA also include compounds that interfere with DNA replication, mitosis and chromosomal segregation. Such chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and the like. Widely used in a clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m²for etoposide intravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of nucleic acid precursors and subunits also lead to DNA damage. As such a number of nucleic acid precursors have been developed. Particularly useful are agents that have undergone extensive testing and are readily available. As such, agents such as 5-fluorouracil (5-FU), are preferentially used by neoplastic tissue, making this agent particularly useful for targeting to neoplastic cells. Although quite toxic, 5-FU, is applicable in a wide range of carriers, including topical, however intravenous administration with doses ranging from 3 to 15 mg/kg/day being commonly used.

Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage DNA, on the precursors of DNA, the replication and repair of DNA, and the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

F. Immunodetection Kits

In still further embodiments, the present invention concerns immunodetection kits for use with the immunodetection methods described above. The immunodetection kits will comprise, in suitable container means, a human anti-nucleolin antibody, and optionally an immunodetection reagent.

In certain embodiments, the antibody can be pre-bound to a solid support, such as a column matrix and/or well of a microtitre plate. The immunodetection reagents of the kit can take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

Further suitable immunodetection reagents for use in the present kits include the two-component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and all such labels can be employed in connection with the present invention.

The kits can further comprise a suitably aliquoted composition of the nucleolin, whether labeled or unlabeled, as can be used to prepare a standard curve for a detection assay. The kits can contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits can be packaged either in aqueous media or in lyophilized form.

The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody can be placed, or preferably, suitably aliquoted. The kits of the present invention will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers can include injection or blow-molded plastic containers into which the desired vials are retained.

VII. EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

In another aspect the invention provides an immortalized human B-cell that expresses an IgG antibody or fragment thereof that binds to a protein encoded by SEQ ID No. 1. In one embodiment, the IgG antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In another embodiment, the IgG antibody is an IgG1 antibody. In another embodiment, the IgG antibody comprises a kappa light chain. In another embodiment, the IgG antibody comprises a lambda light chain. In another embodiment, the B-cell is EBV immortalized. In another embodiment, the antibody or fragment thereof is a monoclonal antibody or fragment thereof. The isolated antibody or fragment thereof of claim 33, wherein said antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the antibody or fragment thereof kills at least 10% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 20% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 30% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 40% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 50% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 60% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 70% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 80% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 90% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 100% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 10% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 20% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 30% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 40% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 50% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB scrum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 60% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 70% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 80% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 90% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof kills at least 100% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the antibody or fragment thereof binds to an RNA binding domain of human nucleolin. In another embodiment, the antibody or fragment thereof inactivates an RNA binding domain of human nucleolin. In another embodiment, the isolated antibody or fragment thereof induces complement-dependent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof induces complement-independent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof induces apoptosis in a cancer cell upon contact. In another embodiment, the isolated antibody or fragment thereof inhibits or kills an AML cancer cell, a CLL cancer cell or a breast cancer cell. In another embodiment, the isolated monoclonal antibody or fragment thereof reduces BCL-2 levels in a cancer cell.

In another aspect the invention provides an isolated antibody or fragment thereof that specifically binds to a human nucleolin protein, wherein said antibody or fragment thereof kills at least 10-100% of a population of MV4-11 cells (such as 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of a population of MV4-11 cells, when incubated with said MCF-7 cells and human AB serum for 48 hours. In another embodiment, the amino acid sequence of said human nucleolin comprises SEQ ID No. 2. In another embodiment, the antibody or fragment thereof binds to an amino acid sequence consisting of amino acid residues 1 to 283 of SEQ ID No. 2. In another embodiment, the antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the antibody or fragment thereof binds to an RNA binding domain of human nucleolin. In another embodiment, the antibody or fragment thereof inactivates an RNA binding domain of human nucleolin. In another embodiment, the isolated antibody or fragment thereof is linked to a diagnostic or therapeutic agent. In another embodiment, the isolated antibody or fragment thereof exhibits complement-dependent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof exhibits complement-independent cytotoxicity to a cancer cell. In another embodiment, the isolated antibody or fragment thereof induces apoptosis in a cancer cell upon contact. In another embodiment, the antibody or fragment thereof inhibits or kills an AML cancer cell, a CLL cancer cell or a breast cancer cell. In another embodiment, the isolated antibody or fragment thereof reduces BCL-2 levels in a cancer cell. In another embodiment, the said antibody or fragment thereof is linked to a diagnostic agent. In another embodiment, the diagnostic agent is a radionuclide, a fluorophore, a chemilluminescent compound, a fluorescent compound, or an enzyme. In another embodiment, the antibody or fragment thereof is linked to a therapeutic agent. In another embodiment, the therapeutic agent is a radionuclide, a toxin or a chemotherapeutic moiety.

In another aspect the invention provides a method of treating or preventing cancer in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin antibody or fragment thereof, a toxin or chemotherapeutic agent and a pharmaceutically acceptable carrier, wherein said antibody or fragment thereof kills at least 10-100% (such as 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 (such as 48, 72, or 96) hours. In one embodiment, the mammal is a human. In another embodiment, the treating cancer in a mammal comprises treating tumor hpoxia. In another embodiment, the treating cancer in a mammal comprises inhibiting tumor angiogenesis. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin monoclonal antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the toxin is diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolaca americana protein, pokeweed antiviral protein, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, calicheamicins or tricothecenes toxin. In another embodiment, the chemotherapeutic agent is an alkylating agent, anthracycline, cytoskeletal disruptor, epothilone, inhibitor of topoisomerase I, inhibitor of topoisomerase II, nucleoside or nucleotide analog, precursor analogs, peptide antibiotic, platinum-based agents retinoids, vinca alkaloids or derivatives thereof. In another embodiment, the chemotherapeutic agent is 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, or vinorelbine

In another aspect the invention provides a method of treating or preventing cancer in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin antibody or fragment thereof and a pharmaceutically acceptable carrier, and further treating said mammal with radiation therapy, wherein said antibody or fragment thereof kills at least 10-100% (such as 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 (such as 48, 72, or 96) hours. In one embodiment, the mammal is a human. In another embodiment, the treating cancer in a mammal comprises treating tumor hpoxia. In another embodiment, the treating cancer in a mammal comprises inhibiting tumor angiogenesis. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin monoclonal antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the toxin is diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolaca americana protein, pokeweed antiviral protein, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, calicheamicins or tricothecenes toxin. In another embodiment, the chemotherapeutic agent is an alkylating agent, anthracycline, cytoskeletal disruptor, epothilone, inhibitor of topoisomerase I, inhibitor of topoisomerase II, nucleoside or nucleotide analog, precursor analogs, peptide antibiotic, platinum-based agents retinoids, vinca alkaloids or derivatives thereof. In another embodiment, the chemotherapeutic agent is 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, or vinorelbine.

In another aspect the invention provides a method of treating or preventing cancer in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin agent and a pharmaceutically acceptable carrier; wherein said anti-nucleolin agent comprises a anti-nucleolin antibody or fragment thereof that specifically binds to a protein of SEQ ID No. 4. In one embodiment, the mammal is a human. In another embodiment, the treating cancer in a mammal comprises treating tumor hypoxia. In another embodiment, the treating cancer in a mammal comprises inhibiting tumor angiogenesis. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin monoclonal antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the toxin is diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolaca americana protein, pokeweed antiviral protein, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, calicheamicins or tricothecenes toxin. In another embodiment, the chemotherapeutic agent is an alkylating agent, anthracycline, cytoskeletal disruptor, epothilone, inhibitor of topoisomerase I, inhibitor of topoisomerase II, nucleoside or nucleotide analog, precursor analogs, peptide antibiotic, platinum-based agents retinoids, vinca alkaloids or derivatives thereof. In another embodiment, the chemotherapeutic agent is 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, or vinorelbine.

A method of treating or preventing cancer in a mammal comprising administering to said mammal a therapeutically effective amount of a anti-nucleolin antibody or fragment thereof, a toxin or chemotherapeutic agent and a pharmaceutically acceptable carrier, wherein said antibody or fragment thereof specifically binds to a protein of SEQ ID No. 4. In one embodiment, the mammal is a human. In another embodiment, the treating cancer in a mammal comprises treating tumor hpoxia. In another embodiment, the treating cancer in a mammal comprises inhibiting tumor angiogenesis. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin monoclonal antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the toxin is diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolaca americana protein, pokeweed antiviral protein, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, calicheamicins or tricothecenes toxin. In another embodiment, the chemotherapeutic agent is an alkylating agent, anthracycline, cytoskeletal disruptor, epothilone, inhibitor of topoisomerase I, inhibitor of topoisomerase II, nucleoside or nucleotide analog, precursor analogs, peptide antibiotic, platinum-based agents retinoids, vinca alkaloids or derivatives thereof. In another embodiment, the chemotherapeutic agent is 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, or vinorelbine.

In another aspect the invention provides a method of treating or preventing cancer in a mammal comprising administering to said mammal a therapeutically effective amount of a anti-nucleolin antibody or fragment thereof and a pharmaceutically acceptable carrier, and further treating said mammal with radiation therapy, wherein said antibody or fragment thereof specifically binds to a protein of SEQ ID No. 4. In one embodiment, the mammal is a human. In another embodiment, the treating cancer in a mammal comprises treating tumor hpoxia. In another embodiment, the treating cancer in a mammal comprises inhibiting tumor angiogenesis. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin monoclonal antibody or fragment thereof is a monoclonal antibody or fragment thereof. In another embodiment, the toxin is diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolaca americana protein, pokeweed antiviral protein, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, calicheamicins or tricothecenes toxin. In another embodiment, the chemotherapeutic agent is an alkylating agent, anthracycline, cytoskeletal disruptor, epothilone, inhibitor of topoisomerase I, inhibitor of topoisomerase II, nucleoside or nucleotide analog, precursor analogs, peptide antibiotic, platinum-based agents retinoids, vinca alkaloids or derivatives thereof. In another embodiment, the chemotherapeutic agent is 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, or vinorelbine.

In another aspect the invention provides a method of treating an autoimmune disease in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-nucleolin agent and a pharmaceutically acceptable carrier; wherein said anti-nucleolin agent comprises an anti-nucleolin antibody or fragment thereof and said antibody or fragment thereof kills at least 10-100% (such as 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%) of a population of MCF-7 cells, when incubated with said MCF-7 cells and human AB serum for 48 (such as 48, 72, or 96) hours. In one embodiment, the autoimmune disease is 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 mellitus (e.g., type 1), eosinophilic fascitis, 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), myelodysplastic syndrome, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosis (SLE), Sweet's syndrome, Still's disease, lupus erythematosus, takayasu arteritis, temporal arteristis/giant cell arteritis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis. Examples of inflammatory disorders include, but are not limited to, asthma, encephilitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentitated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, graft versus host disease, urticaria, or Vogt-Koyanagi-Hareda syndrome. In another embodiment, the mammal is a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is substantially non-immunogenic to a human. In another embodiment, the anti-nucleolin antibody or fragment thereof is a human antibody or fragment thereof. In another embodiment, the anti-nucleolin antibody or fragment thereof is a monoclonal antibody or fragment thereof.