Methods of screening, selecting, and identifying cytotoxic recombinant polypeptides based on an interim diminution of ribotoxicity

A method for identifying an amino acid sequence for use in constructing one or more chimeric cytotoxic protein,

• wherein the one or more chimeric cytotoxic protein comprises:
• i) a ribotoxic region comprising a polypeptide and capable of inactivating a ribosome, and
• ii) a binding region comprising a polypeptide and capable of binding at least one target biomolecule;
• wherein the method comprises the steps of:
• a) providing a plurality of chimeric proteins, each protein comprising:
• i) a modified ribotoxic region having at least one amino acid substitution, deletion, insertion, or addition as compared to a corresponding unmodified ribotoxin region, so as to reduce or eliminate ribotoxicity of the modified ribotoxic region thereby minimizing unwanted selection bias caused by the presence of ribotoxicity, and
• ii) a binding region comprising a polypeptide and capable of binding at least one target biomolecule;
• b) screening the plurality of chimeric proteins to identify a protein with at least one assay-screenable characteristic selected from:
• target biomolecule binding affinity, target biomolecule binding selectivity, target cell binding affinity, target cell binding selectivity, target cell internalization, and improved expression;
• and
• c) identifying the amino acid sequences of the polypeptide regions of the protein identified in step b) in order to construct one or more chimeric cytotoxic protein derived from the identified protein or comprising a binding region in the identified protein associated with a more ribotoxic form of a modified ribotoxic region present in the identified protein.

A method for identifying an amino acid sequence for use in constructing one or more chimeric cytotoxic protein,

• wherein the one or more chimeric cytotoxic protein comprises:
• i) a ribotoxic region comprising a polypeptide and capable of inactivating a ribosome, and
• ii) a binding region comprising a polypeptide and capable of binding at least one target biomolecule, and,
• wherein the method comprises the steps of:
• a) providing a plurality of chimeric cytotoxic proteins, each protein comprising:
• i) a ribotoxic region comprising a polypeptide and capable of inactivating a ribosome, and
• ii) a binding region comprising a polypeptide and capable of binding at least one target biomolecule;
• b) screening the plurality of chimeric cytotoxic proteins in the presence of an inhibitor of the ribotoxic region to identify a protein with at least one assay-screenable characteristic, wherein the at least one characteristic is selected from:
• target biomolecule binding affinity, target biomolecule binding selectivity, target cell binding affinity, target cell binding selectivity, target cell internalization, and improved expression;
• and
• c) identifying the amino acid sequence(s) of the chimeric cytotoxic protein identified in step b).