Antigenic GM-CSF peptides and antibodies to GM-CSF

A method for generating hybridoma cells producing monoclonal antibodies that specifically bind to GM-CSF comprising:

• a. combining peripheral blood mononuclear cells with GM-CSF or an antigenic fragment thereof ex vivo to generate cells that produce antibodies to GM-CSF;
• b. fusing the antibody-producing cells with myeloma cells to generate hybridoma cells;
• c. determining specific binding of antibodies produced from said hybridoma cells to GM-CSF;
• and
• d. selecting hybridoma cells that produce antibodies that specifically bind to GM-CSF,
• thereby generating hybridoma cells that produce antibodies that specifically bind to GM-CSF.

A method for generating hybridoma cells producing monoclonal antibodies that specifically bind to a target antigen comprising combining peripheral blood mononuclear cells with a cell extract comprising an antigen of interest or an antigen presenting cell expressing an antigen of interest ex vivo to generate antibody-producing cells that produce antibodies to the antigen of interest, fusing said antibody-producing cells with myeloma cells to form parental hybridoma cells, wherein the hybridoma cells express a dominant negative allele of a mismatch repair gene;

• incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells;
• performing a screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells or for antibodies produced in higher titer than produced by the parental hybridoma cells;
• and selecting hypermutated hybridoma cells that produce antibodies with greater affinity for antigen or with higher titer than the parental hybridoma cells;
• thereby generating hybridoma cells that produce antibodies that specifically bind to the antigen of interest.

A method for generating hybridoma cells producing monoclonal antibodies that specifically bind to a target antigen comprising combining peripheral blood mononuclear cells with a cell extract comprising an antigen of interest or an antigen presenting cell expressing an antigen of interest ex vivo to generate antibody-producing cells that produce antibodies to the antigen of interest, fusing said antibody-producing cells with myeloma cells to form parental hybridoma cells;

• incubating the parental hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair, thereby forming hypermutated hybridoma cells;
• performing a screen for hypermutated hybridoma cells that produce a higher titer of antibodies than the parental hybridoma cells or for hypermutated hybridoma cells that produce antibodies that bind the antigen with greater affinity than antibodies produced by the parental hybridoma cells;
• and selecting hypermutated hybridoma cells that produce a higher titer of antibody than said parental hybridoma cells or hypermutated hybridoma cells that produce antibodies with greater affinity for said antigen than antibodies produced by said parental hybridoma cells;
• thereby generating hybridoma cells that produce antibodies that specifically bind to the antigen of interest.

A method for generating a mammalian expression cell producing monoclonal antibodies that specifically bind to a target antigen comprising combining peripheral blood mononuclear cells with a cell extract comprising an antigen of interest or an antigen presenting cell expressing an antigen of interest ex vivo to generate cells that antibody-producing produce antibodies to the antigen of interest, fusing said antibody-producing cells with myeloma cells to form hybridoma cells;

• performing a screen for binding of antibodies produced from the hybridoma cells to antigen;
• cloning immunoglobulin genes from the hybridoma cells that produce antibodies to antigen into a mammalian expression cell;
• incubating the mammalian expression cell in the presence of at least one chemical inhibitor of mismatch repair;
• and performing a screen for mammalian expression cells that secrete antibodies with higher affinity for antigen as compared to antibodies produced from the hybridoma cells;
• thereby generating mammalian expression cells that produce antibodies that specifically bind to the antigen of interest.

A method for generating a mammalian expression cell producing monoclonal antibodies that specifically bind to a target antigen comprising combining peripheral blood mononuclear cells with a cell extract comprising an antigen of interest or an antigen presenting cell expressing an antigen of interest ex vivo to generate antibody-producing cells that produce antibodies to the antigen of interest, fusing said antibody-producing cells with myeloma cells to form hybridoma cells;

• incubating the hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair to form hypermutated hybridoma cells;
• performing a screen for hypermutated hybridoma cells that produce antibodies that bind antigen;
• selecting hypermutated hybridoma cells that produce antibodies with greater affinity for antigen than antibodies produced by the parental hybridoma cells;
• and cloning immunoglobulin genes from the hypermutated hybridoma cells into a mammalian expression cell;
• thereby generating mammalian expression cells that produce antibodies that specifically bind to the antigen of interest.

A method for generating hybridoma cells producing monoclonal antibodies that specifically bind to a target antigen comprising combining peripheral blood mononuclear cells with a cell extract comprising an antigen of interest or an antigen presenting cell expressing an antigen of interest ex vivo to generate antibody-producing cells that produce antibodies to the antigen of interest, fusing said antibody-producing cells with myeloma cells to form parental hybridoma cells, wherein the parental hybridoma cells comprise a dominant negative allele of a mismatch repair gene and are deficient in mismatch repair;

• incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells;
• performing a screen for binding of antibodies to antigen for antibodies produced from hypermutated hybridoma cells;
• selecting hypermutated hybridoma cells that produce antibodies with enhanced affinity for the antigen than antibodies produced by the parental hybridoma cells;
• performing a second screen for hypermutated hybridoma cells that produce increased titers of antibodies as compared with the parental hybridoma cells;
• and selecting hypermutated hybridoma cells that produce antibodies in higher titers than produced by said parental hybridoma cells;
• thereby generating hybridoma cells that produce antibodies that specifically bind to the antigen of interest.

A method for generating hybridoma cells producing monoclonal antibodies that specifically bind to a target antigen comprising combining peripheral blood mononuclear cells with a cell extract comprising an antigen of interest or an antigen presenting cell expressing an antigen of interest ex vivo to generate antibody producing cells that produce antibodies to the antigen of interest, fusing said antibody-producing cells with myeloma cells, wherein the myeloma cells are naturally deficient in mismatch repair, thereby forming parental hybridoma cells, wherein the hybridoma cells are deficient in mismatch repair;

• incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells;
• performing a first screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells;
• selecting hypermutated hybridoma cells that produce antibodies with enhanced affinity for the antigen than antibodies produced by the parental hybridoma cells;
• performing a second screen for hypermutated hybridoma cells that produce a higher titer of antibodies than the parental hybridoma cells or for hypermutated hybridoma cells that produce antibodies that bind to the antigen with greater affinity than the antibodies produced by the parental hybridoma cells;
• and selecting hypermutated hybridoma cells that produce a higher titer of antibodies than said parental hybridoma cells or hypermutated hybridoma cells that produce antibodies with greater affinity for said antigen than antibodies produced by said parental cells;
• thereby generating hybridoma cells that produce antibodies that specifically bind to the antigen of interest.

A method for generating a mammalian expression cell producing monoclonal antibodies that specifically bind to a target antigen comprising combining peripheral blood mononuclear cells with a cell extract comprising an antigen of interest or an antigen presenting cell expressing an antigen of interest ex vivo to generate antibody producing cells that produce antibodies to the antigen of interest, wherein said antibody producing cell comprises a dominant negative allele of a mismatch repair gene, fusing said antibody-producing cells with myeloma cells to form parental hybridoma cells, wherein the parental hybridoma cells are deficient in mismatch repair;

• incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells;
• performing a screen for hypermutated hybridoma cells that produce a higher titer of antibodies than the parental hybridoma cells or for hypermutated hybridoma cells that produce antibodies that bind to the antigen with greater affinity than the antibodies produced by the parental hybridoma cells;
• selecting hypermutated hybridoma cells that produce a higher titer of antibodies than said parental hybridoma cells or hypermutated hybridoma cells that produce antibodies with greater affinity for said antigen than antibodies produced by said parental cells;
• thereby generating a mammalian expression cells that produce antibodies that specifically bind to the antigen of interest.

A method for generating a mammalian expression cell producing monoclonal antibodies that specifically bind to a target antigen comprising combining peripheral blood mononuclear cells with a cell extract comprising an antigen of interest or an antigen presenting cell expressing an antigen of interest ex vivo to generate cells that produce antibodies to the antigen of interest, fusing antibody-producing cells with myeloma cells, wherein the myeloma cells are naturally deficient in mismatch repair, thereby forming parental hybridoma cells, wherein the hybridoma cells are deficient in mismatch repair;

• incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells;
• performing a screen for hypermutated hybridoma cells that produce a higher titer of antibodies than the parental hybridoma cells or for hypermutated hybridoma cells that produce antibodies that bind to the antigen with greater affinity than the antibodies produced by the parental hybridoma cells;
• selecting hypermutated hybridoma cells that produce a higher titer of antibodies than said parental hybridoma cells or hypermutated hybridoma cells that produce antibodies with greater affinity for said antigen than antibodies produced by said parental cells;
• and cloning immunoglobulin genes from the hypermutated hybridoma cell into a mammalian expression cell;
• thereby generating a mammalian expression cells that produce antibodies that specifically bind to the antigen of interest.

A method for generating a mammalian expression cell producing monoclonal antibodies that specifically bind to a target antigen comprising combining peripheral blood mononuclear cells with a cell extract comprising an antigen of interest or an antigen presenting cell expressing an antigen of interest ex vivo to generate antibody producing cells that produce antibodies to the antigen of interest, fusing antibody-producing cells with myeloma cells, thereby forming parental hybridoma cells, performing a screen for binding of antibodies to antigen for antibodies produced from the hybridoma cells;

• cloning immunoglobulin genes from the hybridoma cell into a mammalian expression cell wherein the expression cell is naturally deficient in mismatch repair, incubating the expression cell to allow for mutagenesis, performing a screen for hypermutated expression cells that produce a higher titer of antibodies than the parental expression cells or for hypermutated expression cells that produce antibodies that bind to the antigen with greater affinity than the antibodies produced by the parental expression cells;
• and selecting hypermutated expression cells that produce a higher titer of antibodies than said parental expression cells or hypermutated expression cells that produce antibodies with greater affinity for the antigen than antibodies produced by said parental cells thereby forming hypermutated expression cells that produce antibodies that specifically bind to the antigen of interest.

A method for generating hybridoma cells producing monoclonal antibodies that specifically bind to a target antigen comprising culturing bone marrow or peripheral blood cells isolated from an animal that has been challenged with an antigen of interest, isolating cells that produce antibodies to the antigen of interest, fusing said antibody-producing cells with myeloma cells to form parental hybridoma cells, wherein the hybridoma cells express a dominant negative allele of a mismatch repair gene;

• incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells;
• performing a screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells or for antibodies produced in higher titer than produced by the parental hybridoma cells;
• and selecting hypermutated hybridoma cells that produce antibodies with greater affinity for antigen or with higher titer than the parental hybridoma cells;
• thereby generating hybridoma cells that produce antibodies that specifically bind to the antigen of interest.

A method for generating hybridoma cells producing monoclonal antibodies that specifically bind to a target antigen comprising culturing bone marrow or peripheral blood cells isolated from an animal that has been challenged with an antigen of interest, isolating cells that produce antibodies to the antigen of interest, fusing said antibody-producing cells with myeloma cells to form parental hybridoma cells;

• incubating the parental hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair, thereby forming hypermutated hybridoma cells;
• performing a screen for hypermutated hybridoma cells that produce a higher titer of antibodies than the parental hybridoma cells or for hypermutated hybridoma cells that produce antibodies that bind the antigen with greater affinity than antibodies produced by the parental hybridoma cells;
• and selecting hypermutated hybridoma cells that produce a higher titer of antibody than said parental hybridoma cells or hypermutated hybridoma cells that produce antibodies with greater affinity for said antigen than antibodies produced by said parental hybridoma cells;
• thereby generating hybridoma cells that produce antibodies that specifically bind to the antigen of interest.

A method for generating mammalian expression cells producing monoclonal antibodies that specifically bind to a target antigen comprising culturing bone marrow or peripheral blood cells isolated from an animal that has been challenged with an antigen of interest, isolating cells that produce antibodies to the antigen of interest, fusing said antibody-producing cells with myeloma cells to form hybridoma cells;

• performing a screen for binding of antibodies produced from the hybridoma cells to antigen;
• cloning immunoglobulin genes from the hybridoma cells that produce antibodies to antigen into a mammalian expression cell;
• incubating the mammalian expression cell in the presence of at least one chemical inhibitor of mismatch repair;
• and performing a screen for mammalian expression cells that secrete antibodies with higher affinity for antigen as compared to antibodies produced from the hybridoma cells;
• thereby generating mammalian expression cells that produce antibodies that specifically bind to the antigen of interest.

A method for generating mammalian expression cells producing monoclonal antibodies that specifically bind to a target antigen comprising culturing bone marrow or peripheral blood cells isolated from an animal that has been challenged with an antigen of interest, isolating cells that produce antibodies to the antigen of interest, fusing said antibody-producing cells with myeloma cells to form hybridoma cells;

• incubating the hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair to form hypermutated hybridoma cells;
• performing a screen for hypermutated hybridoma cells that produce antibodies that bind antigen;
• selecting hypermutated hybridoma cells that produce antibodies with greater affinity for antigen than antibodies produced by the parental hybridoma cells;
• and cloning immunoglobulin genes from the hypermutated hybridoma cells into a mammalian expression cell;
• thereby generating mammalian expression cells that produce antibodies that specifically bind to the antigen of interest.

A method for generating hybridoma cells producing monoclonal antibodies that specifically bind to a target antigen comprising culturing bone marrow or peripheral blood cells isolated from an animal that has been challenged with an antigen of interest, isolating cells that produce antibodies to the antigen of interest, fusing said antibody-producing cells with myeloma cells to form parental hybridoma cells, wherein the parental hybridoma cells comprise a dominant negative allele of a mismatch repair gene and are deficient in mismatch repair;

• incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells;
• performing a screen for binding of antibodies to antigen for antibodies produced from hypermutated hybridoma cells;
• selecting hypermutated hybridoma cells that produce antibodies with enhanced affinity for the antigen than antibodies produced by the parental hybridoma cells;
• performing a second screen for hypermutated hybridoma cells that produce increased titers of antibodies as compared with the parental hybridoma cells;
• and selecting hypermutated hybridoma cells that produce antibodies in higher titers than produced by said parental hybridoma cells;
• thereby generating hybridoma cells that produce antibodies that specifically bind to the antigen of interest.

A method for generating hybridoma cells producing monoclonal antibodies that specifically bind to a target antigen comprising culturing bone marrow or peripheral blood cells isolated from an animal that has been challenged with an antigen of interest, isolating cells that produce antibodies to the antigen of interest, fusing said antibody-producing cells with myeloma cells, wherein the myeloma cells are naturally deficient in mismatch repair, thereby forming parental hybridoma cells, wherein the hybridoma cells are deficient in mismatch repair;

• incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells;
• performing a first screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells;
• selecting hypermutated hybridoma cells that produce antibodies with enhanced affinity for the antigen than antibodies produced by the parental hybridoma cells;
• performing a second screen for hypermutated hybridoma cells that produce a higher titer of antibodies than the parental hybridoma cells or for hypermutated hybridoma cells that produce antibodies that bind to the antigen with greater affinity than the antibodies produced by the parental hybridoma cells;
• and selecting hypermutated hybridoma cells that produce a higher titer of antibodies than said parental hybridoma cells or hypermutated hybridoma cells that produce antibodies with greater affinity for said antigen than antibodies produced by said parental cells;
• thereby generating hybridoma cells that produce antibodies that specifically bind to the antigen of interest.

A method for generating mammalian expression cells producing monoclonal antibodies that specifically bind to a target antigen comprising culturing bone marrow or peripheral blood cells isolated from an animal that has been challenged with an antigen of interest, isolating cells that produce antibodies to the antigen of interest, introducing into said antibody producing cell a dominant negative allele of a mismatch repair gene, fusing said antibody-producing cells with myeloma cells to form parental hybridoma cells, wherein the parental hybridoma cells are deficient in mismatch repair;

• incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells;
• performing a screen for hypermutated hybridoma cells that produce a higher titer of antibodies than the parental hybridoma cells or for hypermutated hybridoma cells that produce antibodies that bind to the antigen with greater affinity than the antibodies produced by the parental hybridoma cells;
• selecting hypermutated hybridoma cells that produce a higher titer of antibodies than said parental hybridoma cells or hypermutated hybridoma cells that produce antibodies with greater affinity for said antigen than antibodies produced by said parental cells;
• thereby generating a mammalian expression cells that produce antibodies that specifically bind to the antigen of interest.

A method for generating mammalian expression cells producing monoclonal antibodies that specifically bind to a target antigen comprising culturing bone marrow or peripheral blood cells isolated from an animal that has been challenged with an antigen of interest, isolating cells that produce antibodies to the antigen of interest, fusing antibody-producing cells with myeloma cells, wherein the myeloma cells are naturally deficient in mismatch repair, thereby forming parental hybridoma cells, wherein the hybridoma cells are deficient in mismatch repair;

• incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells;
• performing a screen for hypermutated hybridoma cells that produce a higher titer of antibodies than the parental hybridoma cells or for hypermutated hybridoma cells that produce antibodies that bind to the antigen with greater affinity than the antibodies produced by the parental hybridoma cells;
• selecting hypermutated hybridoma cells that produce a higher titer of antibodies than said parental hybridoma cells or hypermutated hybridoma cells that produce antibodies with greater affinity for said antigen than antibodies produced by said parental cells;
• and cloning immunoglobulin genes from the hypermutated hybridoma cell into a mammalian expression cell;
• thereby generating a mammalian expression cells that produce antibodies that specifically bind to the antigen of interest.

A method for generating mammalian expression cells producing monoclonal antibodies that specifically bind to a target antigen comprising culturing bone marrow or peripheral blood cells isolated from an animal that has been challenged with an antigen of interest, isolating cells that produce antibodies to the antigen of interest, fusing antibody-producing cells with myeloma cells, thereby forming parental hybridoma cells, performing a screen for binding of antibodies to antigen for antibodies produced from the hybridoma cells;

• cloning immunoglobulin genes from the hybridoma cell into a mammalian expression cell wherein the expression cell is naturally deficient in mismatch repair, incubating the expression cell to allow for mutagenesis, performing a screen for hypermutated expression cells that produce a higher titer of antibodies than the parental expression cells or for hypermutated expression cells that produce antibodies that bind to the antigen with greater affinity than the antibodies produced by the parental expression cells;
• and selecting hypermutated expression cells that produce a higher titer of antibodies than said parental expression cells or hypermutated expression cells that produce antibodies with greater affinity for the antigen than antibodies produced by said parental cells thereby forming hypermutated expression cells that produce antibodies that specifically bind to the antigen of interest.