Mixtures of binding proteins

A method for producing a single cell expressing at least a single polypeptide chain binding protein (SPCBP) recognizing a first target epitope and a SPCBP recognizing a second target epitope, wherein binding of each SPCBP to its target epitope is mediated by a single protein domain that forms an unpaired binding site, wherein the target epitopes are different from one another, wherein the SPCBPs comprise a scaffold domain selected from the group consisting of domain antibodies (dAbs), camelid antibodies (VHH), anticalins, affibodies, fluorobodies, immunoglobulin heavy chain variable regions, immunoglobulin light chain variable regions, and immunoadhesions, and wherein at least one of the SPCBPs is able to antagonize a function of a target molecule comprising at least one of the target epitopes, the method comprising:

• transfecting cells with a first library of nucleic acid molecules encoding SPCBPs that recognize the first target epitope and a second library of nucleic acid molecules encoding SPCBPs that recognize the second target epitope;
• selecting cells from the transfected cells, which have, integrated into their genomes, a nucleic acid molecule encoding a SPCBP from the first library and a nucleic acid molecule encoding a SPCBP from the second library, thereby producing a first library of cells;
• contacting an aliquot of the first library of cells, or supernatants thereof, with the target epitopes;
• screening for and selecting cells from the contacted first library of cells, or supernatants thereof, expressing SPCBPs able to bind both the target epitopes, thereby producing a second library of cells;
• performing a bioassay on an aliquot of the second library of cells, or supernatants thereof, so as to measure antagonistic activity of the SPCBPs against a molecule comprising at least one of the target epitopes;
• and,
• based upon the results of the bioassay, selecting a cell from the second library that expresses a SPCBP able to antagonize a function of a target molecule comprising at least one of the target epitopes.

A method for producing a single cell expressing at least a single polypeptide chain binding protein (SPCBP) recognizing a first target epitope and a SPCBP recognizing a second target epitope, wherein binding of each SPCBP to its target epitope is mediated by a single protein domain that forms an unpaired binding site, wherein the target epitopes are different from one another, wherein the SPCBPs comprise a scaffold domain selected from the group consisting of domain antibodies (dAbs), camelid antibodies (VHH), anticalins, affibodies, fluorobodies, immunoglobulin heavy chain variable regions, immunoglobulin light chain variable regions, and immunoadhesions, and wherein at least one of the SPCBPs is able to activate a function of a target molecule comprising at least one of the target epitopes, the method comprising:

• transfecting cells with a first library of nucleic acid molecules encoding SPCBPs that recognize the first target epitope and second library of nucleic acid molecules encoding SPCBPs that recognize the second target epitope;
• selecting cells from the transfected cells having integrated into their genomes a nucleic acid molecule encoding a SPCBP from the first library and a nucleic acid molecule encoding a SPCBP from the second library, thereby producing a first library of cells;
• contacting an aliquot of the first library of cells, or supernatants thereof, with the target epitopes;
• screening for and selecting cells from the contacted first library of cells, or supernatants thereof, expressing SPCBPs able to bind both the target epitopes, thereby producing a second library of cells;
• performing a bioassay on an aliquot of the second library of cells, or supernatants thereof, so as to measure antagonistic activity of the SPCBPs against a molecule comprising at least one of the target epitopes;
• and,
• based upon the results of the bioassay, selecting a cell from the second library that expresses a SPCBP able to activate a function of a target molecule comprising at least one of the target epitopes.

A method for producing a single cell expressing at least a first single polypeptide chain binding protein (SPCBP) recognizing a first target epitope and a second SPCBP recognizing a second target epitope, wherein at least one of the SPCBPs is able to antagonize a function of a target molecule comprising at least one of the target epitopes, the method comprising:

• generating a cell comprising a first nucleic acid molecule encoding a first SPCBP that recognizes the first target epitope and a second nucleic acid molecule encoding a second SPCBP that recognizes the second target epitope;
• and
• expressing the first and second SPCBPs;
• wherein the first and second nucleic acid molecules are integrated into the cell's genome;
• wherein the expressed first SPCBP is able to bind the first target epitope and the expressed second SPCBP is able to bind the second target epitope;
• wherein at least one of the first and second SPCBPs is able to antagonize a function of a target molecule comprising at least one of the target epitopes;
• wherein binding of each SPCBP to its target epitope is mediated by a single protein domain that forms an unpaired binding site;
• wherein the target epitopes are different from one another;
• and
• wherein each SPCBP comprises a scaffold domain selected from the group consisting of domain antibodies (dAbs), camelid antibodies (VHH), anticalins, affibodies, fluorobodies, immunoglobulin heavy chain variable regions, immunoglobulin light chain variable regions, and immunoadhesions.

A method for producing a single cell expressing at least a first single polypeptide chain binding protein (SPCBP) recognizing a first target epitope and a second SPCBP recognizing a second target epitope, wherein at least one of the SPCBPs is able to activate a function of a target molecule comprising at least one of the target epitopes, the method comprising:

• generating a cell comprising a first nucleic acid molecule encoding a first SPCBP that recognizes the first target epitope and a second nucleic acid molecule encoding a second SPCBP that recognizes the second target epitope;
• and
• expressing the first and second SPCBPs;
• wherein the first and second nucleic acid molecules are integrated into the cell's genome;
• wherein the expressed first SPCBP is able to bind the first target epitope and the expressed second SPCBP is able to bind the second target epitope;
• wherein at least one of the first and second SPCBPs is able to activate a function of a target molecule comprising at least one of the target epitopes;
• wherein binding of each SPCBP to its target epitope is mediated by a single protein domain that forms an unpaired binding site;
• wherein the target epitopes are different from one another;
• and
• wherein each SPCBP comprises a scaffold domain selected from the group consisting of domain antibodies (dAbs), camelid antibodies (VHH), anticalins, affibodies, fluorobodies, immunoglobulin heavy chain variable regions, immunoglobulin light chain variable regions, and immunoadhesions.