Covalent disulfide-linked diabodies and uses thereof

Abstract

The present invention provides recombinant antibody fragments which include a variable domain which has been modified by the addition of a tail sequence to its C-terminal end. The tail sequence comprises a terminal cysteine residue and an amino acid spacer and does not substantially affect the fragment's target-binding affinity. The present invention also provides pharmaceutical compositions comprising the described antibody fragments and a pharmaceutically acceptable ⋅ carrier and methods of delivering an agent to cells of interest in a subject using the fragments as delivery vehicles. The invention further provides compositions comprising the described antibody fragments for the in vitro detection and measurement of target molecules which bind to the fragments and method of determining the presence or amount of such targets in a biological sample by contacting the sample with such compositions.

1 - 47 . (canceled) 
     
     
         48 . A diabody comprising:
 a first single-chain polypeptide subunit that comprises:
 a first heavy chain variable domain polypeptide connected by a first linker sequence to a first light chain variable region polypeptide, and a first tail sequence that comprises a first amino acid spacer a first cysteine residue,
 wherein the first amino acid spacer comprises from 1 to about 10 amino acid residues, and wherein the first cysteine residue is at the C terminus of the first single-chain polypeptide subunit; and 
 
   a second single-chain polypeptide subunit that comprises:
 a second heavy chain variable domain polypeptide connected by a second linker sequence to a second light chain variable region polypeptide, and a second tail sequence that comprises a second amino acid spacer a second cysteine residue,
 wherein the second amino acid spacer comprises from 1 to about 10 amino acid residues, and wherein the second cysteine residue is at the C terminus of the second single-chain polypeptide subunit, 
 
   wherein the first cysteine residue forms a disulfide bond with the second cysteine residue.   
     
     
         49 . The diabody of  claim 48 , wherein the first heavy chain variable domain polypeptide and the second light chain variable region polypeptide together form a first target binding site, and wherein the second heavy chain variable domain polypeptide and the first light chain variable region polypeptide together form a second target binding site. 
     
     
         50 . The diabody of  claim 49 , wherein the first target binding site and the second target binding site bind the same target. 
     
     
         51 . The diabody of  claim 49 , wherein the first target binding site and the second target binding site bind different targets. 
     
     
         52 . The diabody of  claim 48 , wherein the first amino acid spacer comprises from about 5 to about 10 amino acid residues, and wherein the second amino acid spacer comprises from about 5 to about 10 amino acid residues. 
     
     
         53 . The diabody of  claim 48 , wherein the first amino acid spacer comprises from 2 to 5 amino acid residues, and wherein the second amino acid spacer comprises from 2 to 5 amino acid residues. 
     
     
         54 . The diabody of  claim 48 , wherein the first amino acid spacer comprises 6, 7, 8, or 9 amino acid residues, and wherein the second amino acid spacer comprises 6, 7, 8, or 9 amino acid residues. 
     
     
         55 . The diabody of  claim 48 , wherein the first amino acid spacer comprises 2 amino acid residues, and wherein the second amino acid spacer comprises 2 amino acid residues. 
     
     
         56 . The diabody of  claim 48 , wherein the amino acid residues of the first amino acid spacer are glycine residues. 
     
     
         57 . The diabody of  claim 56 , wherein the amino acid residues of the second amino acid spacer are glycine residues. 
     
     
         58 . The diabody of  claim 48 , wherein the first linker sequence comprises from about 5 to about 10 amino acid residues, and wherein the second linker sequence comprises from about 5 to about 10 amino acid residues. 
     
     
         59 . The diabody of  claim 58 , wherein the first linker sequence comprises 8 amino acid residues, and wherein the second linker sequence comprises 8 amino acid residues. 
     
     
         60 . The diabody of  claim 58 , wherein the residues of the first linker sequence are glycine residues and wherein the residues of the second linker sequence are glycine residues. 
     
     
         61 . The diabody of  claim 49 , the first target binding site and/or the second target binding site specifically binds a carcinoembryonic antigen (CEA). 
     
     
         62 . The diabody of  claim 61 , wherein the first heavy chain variable domain polypeptide corresponds to a heavy chain variable domain polypeptide sequence of the murine anti-CEA T84.66 antibody, and wherein the second light variable domain polypeptide corresponds to a light chain variable domain polypeptide sequence of the murine anti-CEA T84.66 antibody. 
     
     
         63 . The diabody of  claim 62 , wherein the second heavy chain variable domain polypeptide corresponds to a heavy chain variable domain polypeptide sequence of the murine anti-CEA T84.66 antibody, and wherein the first light variable domain polypeptide corresponds to a light chain variable domain polypeptide sequence of the murine anti-CEA T84.66 antibody.