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

Abstract

The present invention relates to methods of screening libraries of chimeric molecules comprising ribotoxic polypeptides, where screening is based on the interim reduction or elimination of ribotoxicity and the methods can identify cytotoxic molecules, each comprising a binding region and a ribotoxic region which jointly possess a desired assay-selectable characteristic, such as, e.g., binding to a target biomolecule, binding to a target cell, and/or cellular internalization.

The invention is claimed as follows: 
     
         1 . A method for isolating a binding region capable of binding a target biomolecule, the method comprising the steps of:
 a) providing a plurality of chimeric proteins, each protein comprising:
 i) a modified Shiga toxin ribotoxic region comprising at least one amino acid substitution, deletion, insertion, or addition as compared to a corresponding unmodified Shiga toxin ribotoxic region, so as to reduce or eliminate ribotoxicity of the modified Shiga toxin ribotoxic region, 
 wherein the unmodified Shiga toxin ribotoxin region comprises an amino acid sequence having at least 85% sequence identity to a naturally occurring Shiga toxin A subunit sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:4, or a Shiga toxin ribotoxic fragment thereof; and wherein the unmodified Shiga toxin ribotoxic region is capable of inactivating a ribosome, and 
 ii) a different candidate binding region comprising a polypeptide and capable of binding at least one target biomolecule; 
   b) screening the plurality of chimeric proteins to isolate a chimeric 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, or expression level; and   c) identifying the amino acid sequence of a binding region of the chimeric protein isolated in step b), thereby isolating a binding region capable of binding a target biomolecule.   
     
     
         2 . The method of  claim 1 , wherein an additional amino acid sequence of the protein isolated in step (b) is identified in step (c), such as the amino acid sequences of one or more additional polypeptide regions of the chimeric protein isolated in step (b). 
     
     
         3 . The method of  claim 1 , wherein the amino acid sequence of the binding region identified in step (c) is used to construct at least one chimeric cytotoxic protein comprising:
 (i) a binding region comprising, or derived from the amino acid sequence of the binding region identified in step (c) and capable of binding at least one target biomolecule; and   (ii) a Shiga toxin ribotoxic region comprising an amino acid sequence having at least 85% sequence identity to a naturally occurring Shiga toxin A Subunit sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or a Shiga toxin ribotoxic fragment thereof; wherein the Shiga toxin ribotoxin regions is capable of inactivating a ribosome and is more ribotoxic than a modified Shiga toxin ribotoxic region present in a chimeric protein provided in step a).   
     
     
         4 . The method of  claim 2 , wherein the amino acid sequence of the binding region identified in step (c) is used to construct at least one chimeric cytotoxic protein comprising:
 (i) a binding region comprising or derived from the amino acid sequence of the binding region identified in step (c) and capable of binding at least one target biomolecule;   (ii) a Shiga toxin ribotoxic region comprising an amino acid sequence having at least 85% sequence identity to a naturally occurring Shiga toxin A Subunit sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or a Shiga toxin ribotoxic fragment thereof;   wherein the Shiga toxin ribotoxin region is capable of inactivating a ribosome and is more ribotoxic than a modified Shiga toxin ribotoxic region present in a chimeric protein provided in step (a); and   wherein at least one of the additional polypeptide regions of the chimeric protein isolated in step b) and identified in step (c) is present in the constructed chimeric cytotoxic protein.   
     
     
         5 . The method of  claim 3  or  4 , wherein the Shiga toxin ribotoxic region of the chimeric cytotoxic protein that is capable of inactivating a ribosome comprises an amino acid sequence having at least 85% sequence identity to a Shiga toxin ribotoxic fragment selected from: amino acid residues 1-239 of SEQ ID NO: 1 or 2, amino acid residues 1-240 of SEQ ID NO: 1 or 2, and amino acid residues 75-247 of SEQ ID NO: 1 or 2. 
     
     
         6 . The method of  claim 3  or  4 , wherein the Shiga toxin ribotoxic region of the chimeric cytotoxic protein that is capable of inactivating a ribosome comprises a naturally occurring Shiga toxin A Subunit selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. 
     
     
         7 . The method of  claim 1 , wherein the modified Shiga toxin ribotoxic region comprises at least one amino acid substitution, deletion, insertion, or addition at the natively positioned amino acid residue of the Shiga toxin A Subunit selected from N75, Y77, Y114, E167, R170, R172, R176, R179, R188, V191, W203, or L233. 
     
     
         8 . The method of  claim 7 , wherein the modified Shiga toxin ribotoxic effector region comprises the A Subunit of Shiga-like toxin 1 (SLT-1) (SEQ ID NO:1) with a mutation selected from Y77S, E167D, or Y77S/E167D. 
     
     
         9 . The method of  claim 3  or  4 , wherein the at least one chimeric cytotoxic protein is produced by a method comprising:
 i) providing one or more polynucleotides encoding the at least one chimeric cytotoxic protein comprising the identified amino acid sequence of the binding region identified in step c), or optionally, encoding one or more chimeric cytotoxic protein consisting essentially of or derived from the amino acid sequence of the chimeric protein identified in step c); and 
 ii) expressing the one or more polynucleotides using a host cell or cell-free translation system. 
 
     
     
         10 . The method of  claim 3  or  4 , wherein the at least one chimeric cytotoxic protein is a fusion polypeptide comprising the Shiga toxin ribotoxic region fused, either directly or indirectly, to the binding region. 
     
     
         11 . The method of  claim 1 , wherein the plurality of chimeric proteins is a plurality of fusion polypeptides, each fusion polypeptide comprising the modified Shiga toxin ribotoxic region fused, either directly or indirectly, to a different binding region comprising a polypeptide and capable of binding at least one target biomolecule. 
     
     
         12 . The method of  claim 11 , further comprising before step a), the steps of:
 a′) providing an expression library of diverse nucleic acids constructed from a plurality of polynucleotides encoding a plurality of fusion polypeptides;   b′) expressing the expression library of diverse nucleic acids such that a plurality of chimeric proteins comprising fusion polypeptides is produced and wherein each fusion polypeptide comprises
 i) a modified Shiga toxin ribotoxic region comprising at least one amino acid substitution, deletion, insertion, or addition as compared to a corresponding unmodified Shiga toxin ribotoxic region, so as to reduce or eliminate ribotoxicity of the modified Shiga toxin ribotoxic region; and 
 ii) a different binding region comprising a polypeptide and capable of binding at least one target biomolecule; and 
   c′) providing the expressed plurality of chimeric proteins of (i) as the plurality of chimeric proteins for step a).   
     
     
         13 . The method of  claim 12 , further comprising before step a′), the steps of:
 a″) providing a library comprising a plurality of diverse polynucleotides encoding a plurality of binding regions, wherein at least two subsets of polynucleotides encode polypeptides with different binding regions capable of binding at least one target biomolecule; 
 b″) joining the polynucleotides of the library in an operable combination to a toxin template polynucleotide encoding a modified Shiga toxin ribotoxic region, wherein said modified Shiga toxin ribotoxic region comprises at least one amino acid substitution, deletion, insertion, or addition as compared to a corresponding unmodified Shiga toxin ribotoxic region, so as to reduce or eliminate ribotoxicity of the modified Shiga toxin ribotoxic region, to construct an expression library of diverse nucleic acids that encode a plurality of fusion polypeptides, each fusion polypeptide comprising a binding region fused with the modified Shiga toxin ribotoxic region, 
 and optionally recombining the polynucleotides of the library of joined polynucleotides to an expression polynucleotide template to construct an expression library of diverse nucleic acids capable of expressing a plurality of fusion polypeptides, each fusion polypeptide comprising a binding region fused to the modified Shiga toxin ribotoxic region; and 
 c″) providing the expression library as the expression library of diverse nucleic acids constructed from a plurality of polynucleotides encoding a plurality of fusion polypeptides for step a′). 
 
     
     
         14 . The method of  claim 1 , wherein the binding region comprises a polypeptide selected from the group consisting of: complementary determining region 3 fragment, constrained FR3-CDR3-FR4 polypeptide, single-domain antibody fragment, single-chain variable fragment, antibody variable fragment, antigen-binding fragment, Fd fragment, fibronectin-derived 10th fibronectin type III domain, tenascin type III domain, ankyrin repeat motif domain, low-density-lipoprotein-receptor-derived A-domain, lipocalin, Kunitz domain, Protein-A-derived Z domain, gamma-B crystallin-derived domain, ubiquitin-derived domain, Sac7d-derived polypeptide, Fyn-derived SH2 domain, and any genetically manipulated counterparts of any of the foregoing that retain binding functionality. 
     
     
         15 . The method of  claim 12 , wherein the expression library is operable using a protein display method selected from the group consisting of: bacteriophage display, RNA display, ribosome display, DNA display, bead surface display, virus display, microorganism display, and mammalian cell display. 
     
     
         16 . The method of  claim 1 , wherein at least one binding region is capable of binding to a target biomolecule found in physical association with at least one type of malignant cell. 
     
     
         17 . A method for producing a nucleic acid encoding a chimeric cytotoxic protein, wherein the chimeric cytotoxic protein comprises a Shiga toxin ribotoxic region, which is capable of inactivating a ribosome, fused either directly or indirectly to a binding region capable of binding at least one target biomolecule; the method comprising the steps of:
 a) identifying an amino acid sequence of a binding region of a chimeric protein isolated using a method according to  claim 1 ; and   b) generating a nucleic acid encoding the chimeric cytotoxic protein   wherein the binding region comprises, or is derived from the identified amino acid sequence of the binding region of the chimeric protein identified in step a),   and wherein the Shiga toxin ribotoxic region is more ribotoxic than a modified Shiga toxin ribotoxic region of a chimeric protein of step a) of  claim 1 .   
     
     
         18 . A method for isolating a protein for use in generating at least one engineered chimeric cytotoxic protein, the method comprising the steps of:
 a) providing a plurality of chimeric cytotoxic proteins, each protein comprising:
 i) a Shiga toxin ribotoxic region comprising an amino acid sequence having at least 85% sequence identity to a naturally occurring Shiga toxin A Subunit sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or a Shiga toxin ribotoxic fragment thereof; wherein the Shiga toxin ribotoxic region is capable of inactivating a ribosome, and 
 ii) a different 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 Shiga toxin ribotoxic region to isolate a chimeric cytotoxic 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, or expression level; and   c) identifying the amino acid sequence of a binding region of the chimeric cytotoxic protein isolated in step b), thereby isolating a protein for use in generating at least one engineered chimeric cytotoxic proteins.   
     
     
         19 . The method of  claim 18 , wherein an additional amino acid sequence(s) of the chimeric cytotoxic protein isolated in step b) is identified in step (c), such as the amino acid sequences of all the polypeptide regions of the chimeric cytotoxic protein isolated in step b). 
     
     
         20 . The method of  claim 18 , wherein the at least one engineered chimeric cytotoxic protein comprises:
 i) a Shiga toxin ribotoxic region comprising an amino acid sequence having at least 85% sequence identity to a naturally occurring Shiga toxin A Subunit sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or a Shiga toxin ribotoxic fragment thereof; wherein the Shiga toxin ribotoxic region is capable of inactivating a ribosome, and   ii) a binding region comprising a polypeptide and capable of binding at least one target biomolecule.   
     
     
         21 . The method of  claim 18 , wherein the Shiga toxin ribotoxic region comprises an amino acid sequence having at least 85% sequence identity to a Shiga toxin ribotoxic fragment selected from: amino acid residues 1-239 of SEQ ID NO: 1, amino acid residues 1-240 of SEQ ID NO: 2, and amino acid residues 75-247 of SEQ ID NO: 2. 
     
     
         22 . The method of  claim 18 , wherein the Shiga toxin ribotoxic region a naturally occurring Shiga toxin A Subunit selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or a Shiga toxin ribotoxic fragment selected from amino acid residues 1-239 of SEQ ID NO: 1, amino acid residues 1-240 of SEQ ID NO: 2, and amino acid residues 75-247 of SEQ ID NO: 2. 
     
     
         23 . The method of  claim 18 , wherein the at least one engineered chimeric cytotoxic protein is a fusion polypeptide comprising the Shiga toxin ribotoxic region fused, either directly or indirectly, to the binding region. 
     
     
         24 . The method of  claim 18 , wherein the plurality of chimeric cytotoxic proteins is a plurality of fusion polypeptides, each fusion polypeptide comprising the Shiga toxin ribotoxic region fused, either directly or indirectly, to the binding region. 
     
     
         25 . The method of  claim 18 , wherein the plurality of chimeric cytotoxic proteins is produced from an expression library of diverse nucleic acids operable using a protein display method selected from the group consisting of: bacteriophage display, RNA display, ribosome display, DNA display, bead surface display, virus display, microorganism display, and mammalian cell display. 
     
     
         26 . The method of  claim 19 , further comprising the step of:
 d) producing the at least one engineered chimeric cytotoxic protein comprising, or derived from the identified amino acid sequence of the chimeric cytotoxic protein; wherein the producing step comprises:   i) providing one or more polynucleotides encoding the at least one chimeric cytotoxic protein and   ii) expressing the one or more polynucleotides using a host cell or cell-free translation system.   
     
     
         27 . The method of  claim 18 , further comprising before step a), the steps of:
 a′) providing an expression library of diverse nucleic acids constructed from a plurality of polynucleotides encoding a plurality of fusion polypeptides, each fusion polypeptide comprising:
 i) a Shiga toxin ribotoxic region comprising an amino acid sequence having at least 85% sequence identity to a naturally occurring Shiga toxin A Subunit sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or a Shiga toxin ribotoxic fragment thereof; wherein the Shiga toxin ribotoxic region is capable of inactivating a ribosome; and 
 ii) a different binding region capable of binding at least one target biomolecule; 
   b′) expressing the expression library of diverse nucleic acids such that a plurality of fusion polypeptides is produced; and   c′) providing the plurality of fusion polypeptides as the plurality of chimeric cytotoxic proteins for step a).   
     
     
         28 . The method of  claim 27 , further comprising before step a′), the steps of:
 a″) providing a library comprising a plurality of diverse polynucleotides encoding a plurality of binding regions, wherein at least two subsets of polynucleotides encode polypeptides with different binding regions capable of binding at least one target biomolecule; 
 b″) joining the polynucleotides of the library to a toxin template polynucleotide encoding a Shiga toxin ribotoxic region in an operable combination to construct an expression library of diverse nucleic acids that encode a plurality of fusion polypeptides, each fusion polypeptide comprising the Shiga toxin ribotoxic region and one of the binding regions, 
 and optionally recombining the polynucleotides of the library of polynucleotides to an expression polynucleotide template to construct an expression library of diverse nucleic acids capable of expressing a plurality of fusion polypeptides, each fusion polypeptide comprising the Shiga toxin ribotoxic region and a binding region; and 
 c″) providing the expression library as the expression library of diverse nucleic acids constructed from a plurality of polynucleotides encoding a plurality of fusion polypeptides for step a′). 
 
     
     
         29 . The method of  claim 18 , wherein the binding region comprises a polypeptide selected from the group consisting of: complementary determining region 3 fragment, constrained FR3-CDR3-FR4 polypeptide, single-domain antibody fragment, single-chain variable fragment, antibody variable fragment, antigen-binding fragment, Fd fragment, fibronectin-derived 10th fibronectin type III domain, tenascin type III domain, ankyrin repeat motif domain, low-density-lipoprotein-receptor-derived A-domain, lipocalin, Kunitz domain, Protein-A-derived Z domain, gamma-B crystallin-derived domain, ubiquitin-derived domain, Sac7d-derived polypeptide, Fyn-derived SH2 domain, and any genetically manipulated counterparts of any of the foregoing that retain binding functionality. 
     
     
         30 . The method of  claim 18 , wherein at least one binding region is capable of binding to a target biomolecule found in physical association with at least one type of malignant cell. 
     
     
         31 . A method for producing a nucleic acid encoding a chimeric cytotoxic protein, the method comprising the steps of:
 a) identifying an amino acid sequence of a binding region of a chimeric cytotoxic protein, or optionally identifying all the amino acid sequences of a chimeric cytotoxic protein, isolated using a method according to  claim 1 ; and   b) generating a nucleic acid encoding a chimeric cytotoxic protein comprising
 i) a binding region comprising, or derived from the amino acid sequence of the binding region of the chimeric protein identified in step a) and capable of binding at least one target biomolecule; and 
 ii) a Shiga toxin ribotoxic region capable of inactivating a ribosome; 
   wherein the binding region and Shiga toxin ribotoxic region are fused, either directly or indirectly,   and optionally wherein the nucleic acid encodes a chimeric cytotoxic protein derived from, or comprising the chimeric cytotoxic protein identified in step a).