Hybrid immunoglobulins with moving parts

Abstract

Hybrid immunoglobulins containing moving parts are provided as well as related compositions and methods of use and methods of production. In addition, analogous genetic devices are provided as well as related compositions and methods of use and methods of production.

1 . A compound comprising a first stretch of consecutive amino acids, each of which is joined to the preceding amino acid by a peptide bond and the sequence of which comprises a binding site for a target; and
 a second stretch of consecutive amino acids, each of which is joined to the preceding amino acid by a peptide bond and the sequence of which is identical to the sequence of the first stretch of consecutive amino acids and which comprises an identical binding site for the target;   wherein each of the first stretch of amino acids and the second stretch of amino acids has at a predefined end thereof a cysteine residue or a selenocysteine residue and such cysteine residues or such selenocysteine residues are joined by a bond having the structure:   
       
         
           
           
               
               
           
         
         wherein each X is the same and represents a sulfur (S) or a selenium (Se) and each C represents a beta-carbon of one of such cysteine or selenocysteine residues. 
       
     
     
         2 - 7 . (canceled) 
     
     
         8 . A compound comprising a first stretch of consecutive amino acids, each of which is joined to the preceding amino acid by a peptide bond and the sequence of which comprises a binding site for a target; and
 a second stretch of consecutive amino acids, each of which is joined to the preceding amino acid by a peptide bond and the sequence of which is different from the sequence of the first stretch of consecutive amino acids and which comprises a binding site for a different moiety;   wherein each of the first stretch of amino acids and the second stretch of amino acids has at a predefined end thereof a cysteine residue or a selenocysteine residue and such residues are joined by a bond having the structure:   
       
         
           
           
               
               
           
         
         wherein each X may be the same or different and represents a sulfur (S) or a selenium (Se) and each C represents a beta-carbon of one of such cysteine or selenocysteine residues. 
       
     
     
         9 - 17 . (canceled) 
     
     
         18 . A multimer comprising two or more identical compounds according to any one of  claims 1  or  8  joined together by at least one bond. 
     
     
         19 - 50 . (canceled) 
     
     
         51 . A method of affecting the activity of a target comprising contacting the target with a composition comprising the compound of  claim 1  under conditions such that the compound binds to and affects the activity of the target. 
     
     
         52 - 62 . (canceled) 
     
     
         63 . A process of making the compound of  claim 1 , comprising:
 (a) transfecting a cell with a recombinant nucleic acid which comprises (i) a first portion, the sequence of which is a N-terminal signal sequence, contiguous with (ii) a second portion, the sequence of which encodes a stretch of consecutive amino acids contiguous with (iii) a third portion, the sequence of which encodes a C-terminal intein-containing binding domain, under conditions permitting synthesis of a chimeric polypeptide comprising the stretch of consecutive amino acids contiguous with the C-terminal intein-containing binding domain;   (b) isolating the chimeric polypeptide produced in step (a);   (c) treating the chimeric polypeptide so as to cause thio-mediated cleavage of the C-terminal intein-containing binding domain from the stretch of consecutive amino acids and its replacement with a C-terminal thioester;   (d) treating the product of step (c) to permit the attachment of a cysteine residue to the product so as to form product with a C-terminal cysteine; and   (e) oxidizing the product of step (e) in the presence of another product of step (e) under conditions permitting formation of the compound.   
     
     
         64 - 69 . (canceled) 
     
     
         70 . A compound comprising an independently folding protein domain fused to a second independently folding protein domain by non-peptide bond. 
     
     
         71 - 74 . (canceled) 
     
     
         75 . A method of making a stretch of consecutive amino acids comprising an N-terminal cysteine comprising:
 (a) transfecting a cell with a recombinant nucleic acid which comprises (i) a first portion, the sequence of which encodes a N-terminal signal sequence contiguous with (ii) a second portion, the sequence of which encodes a stretch of consecutive amino acids comprising a N-terminal cysteine residue, under conditions permitting (i) synthesis of a chimeric polypeptide which comprises the N-terminal signal sequence joined by a peptide bond at its C-terminus to the N-terminal cysteine of the stretch of consecutive amino acids and (ii) cleavage of the N-terminal signal sequence from the chimeric polypeptide within the cell so as to produce a stretch of consecutive amino acids comprising an N-terminal cysteine;   (b) recovering the stretch of consecutive amino acids produced in step (a).   
     
     
         76 - 154 . (canceled) 
     
     
         155 . A process for making a compound comprising contacting a stretch of consecutive amino acids, each of which is joined to the preceding amino acid by a peptide bond and the sequence of which comprises a binding site for a target with a second stretch of consecutive amino acids, each of which is joined to the preceding amino acid by a peptide bond and the sequence of which is identical to the sequence of the first stretch of consecutive amino acids and which comprises an identical binding site for the target, wherein each of the first stretch of amino acids and the second stretch of amino acids has at a predefined end thereof a cysteine residue or a selenocysteine residue, under reducing conditions so as to make the compound. 
     
     
         156 . A process for making a compound comprising contacting a stretch of consecutive amino acids, each of which is joined to the preceding amino acid by a peptide bond and the sequence of which comprises a binding site for a target with a second stretch of consecutive amino acids, each of which is joined to the preceding amino acid by a peptide bond and the sequence of which is different to the sequence of the first stretch of consecutive amino acids and which comprises an identical binding site for the target, wherein each of the first stretch of amino acids and the second stretch of amino acids has at a predefined end thereof a cysteine residue or a selenocysteine residue, under reducing conditions so as to make the compound. 
     
     
         157 - 164 . (canceled) 
     
     
         165 . A compound comprising a first stretch of consecutive amino acids, each of which is joined to the preceding amino acid by a peptide bond and the sequence of which comprises a binding site for a target; and
 a second stretch of consecutive amino acids, each of which is joined to the preceding amino acid by a peptide bond and the sequence of which is identical to the sequence of the first stretch of consecutive amino acids and which comprises an identical binding site for the target;   wherein each of the first stretch of amino acids and the second stretch of amino acids has at a predefined end thereof, independently, a natural amino acid or non-natural amino having a linear aliphatic side-chain acid comprising a sulfur (S) or a selenium (Se) and wherein such sulfur (S) or a selenium (Se) are joined by a bond having the structure:   
       
         
           
           
               
               
           
         
         wherein each X is a sulfur (S) or a selenium (Se) and each (C) represents a carbon of the linear aliphatic side-chain of one of such natural or non-natural amino acid and wherein n and m are, independently, 2, 3, 4, 5, 6, 7, 8, 9, or 10. 
       
     
     
         166 - 170 . (canceled) 
     
     
         171 . A compound comprising a first stretch of consecutive amino acids, each of which is joined to the preceding amino acid by a peptide bond and the sequence of which comprises a binding site for a target; and
 a second stretch of consecutive amino acids, each of which is joined to the preceding amino acid by a peptide bond and the sequence of which is different from the sequence of the first stretch of consecutive amino acids and which comprises a binding site for a different moiety;   wherein each of the first stretch of amino acids and the second stretch of amino acids has at a predefined end thereof, independently, a natural amino acid or non-natural amino having a linear aliphatic side-chain acid comprising a sulfur (S) or a selenium (Se) and wherein such sulfur (S) or a selenium (Se) are joined by a bond having the structure:   
       
         
           
           
               
               
           
         
         wherein each X may be the same or different and represents a sulfur (S) or a selenium (Se) and each (C) represents a carbon of the linear aliphatic side-chain of one of such natural or non-natural amino acid and wherein n and m are, independently, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. 
       
     
     
         172 - 176 . (canceled) 
     
     
         177 . A method of producing a protein which comprises a first polypeptide contiguous with an intein, which intein is contiguous with a second polypeptide comprising a binding domain, the method comprising transfecting an animal cell with a nucleic acid, which nucleic acid comprises (i) a first portion which encodes the polypeptide contiguous with (ii) a second portion which encodes the intein, contiguous with a and the third portion of which encodes the binding domain, under conditions such that the animal cell expresses and secretes the protein. 
     
     
         178 - 181 . (canceled) 
     
     
         182 . A compound comprising:
 a first stretch of consecutive amino acids each of which is joined to the preceding amino acid by a peptide bond and which first stretch of consecutive amino acids comprises an amino acid residue having a chalcogen functional group-containing side chain; and   a second stretch of consecutive amino acids, comprising at least 100 amino acids, each of which is joined to the preceding amino acid by a peptide bond, wherein at least 90 consecutive amino acids thereof of the second stretch of consecutive amino acids have a sequence identical to portion of a human immunoglobulin constant region polypeptide, and wherein the second stretch of consecutive amino acids comprises an amino acid residue having a chalcogen functional group-containing side chain at a predefined terminus thereof,   wherein said amino acid residue having a chalcogen functional group-containing side chain of the first stretch of consecutive amino acids and said amino acid residue having a chalcogen functional group-containing side chain of the second stretch of consecutive amino acids are joined by a bond having the structure:   
       
         
           
           
               
               
           
         
         wherein each X represents, independently, a chalcogen, and wherein C 1  represents a side chain carbon of the amino acid residue having a chalcogen functional group-containing side chain of the first stretch of consecutive amino acids and C 2  represents a side chain carbon of the second stretch of consecutive amino acids. 
       
     
     
         183 - 194 . (canceled) 
     
     
         195 . The compound of  claim 182 , wherein the human immunoglobulin constant region polypeptide is a human IgG1, human IgG2, human IgG3, or human IgG4. 
     
     
         196 . The compound of  claim 195 , wherein the side chain of at least one of amino acid residues having a chalcogen functional group-containing side chain comprises a C1-C10 alkylene. 
     
     
         197 . A composition comprising two of the compounds of  claim 182  bonded together via at least one disulfide bond between the second stretch of consecutive amino acids of each of the compounds. 
     
     
         198 . A polypeptide consisting of consecutive amino acids having the sequence set forth in one of SEQ ID NOS:35 through 46, or having the sequence set forth in one of SEQ ID NOS:53 through 67, or having the sequence set forth in one of SEQ ID NOS:74 through 82, or having the sequence set forth in one of SEQ ID NOS:89 through 97. 
     
     
         199 - 201 . (canceled) 
     
     
         202 . A polypeptide consisting of consecutive amino acids having a sequence identical to a portion of the sequence set forth in SEQ ID NO:44, SEQ ID NO:64, SEQ ID NO:81 or SEQ ID NO:96, wherein at least one of the terminal residues of the polypeptide has a chalcogen functional group-containing side chain. 
     
     
         203 . The polypeptide of  claim 202 , wherein the terminal residue having a chalcogen functional group-containing side chain is a cysteine or analog thereof