Insertable variable fragments of antibodies and modified a1-a2 domains of nkg2d ligands

Abstract

This application relates generally to the production of polypeptides having specific antigen-binding properties of Fv domains, for example, insertable variable fragments of antibodies, and modified α1-α2 domains of NKG2D ligands.

What is claimed is: 
     
         1 . An insertable Fv (iFv) polypeptide exhibiting specific antigen-binding properties comprising a variable fragment (Fv) of an antibody, wherein the specific antigen-binding domain of the Fv comprises two antibody variable domains, one variable domain from the light chain (VL) and one variable domain from the heavy chain (VH), each with 1-3 complementarity determining regions (CDRs), and each with a C-terminus and an N-terminus; and wherein the VL and VH domains are joined by two linker regions connecting both canonical termini of the VL domain to both canonical termini of the VH domain. 
     
     
         2 . The iFv of  claim 1 , wherein a non-natural pair of N- and C-termini have been created within either the VL or the VH domain to provide an attachment site for conjugation to or insertion into a recipient molecule. 
     
     
         3 . The iFv of  claim 2 , wherein the non-natural N- and C-termini are spatially positioned proximal to each other. 
     
     
         4 . The iFv of  claim 2 , further comprising a recipient polypeptide or protein containing one or more solvent exposed loops, wherein the iFv is inserted into one or more of the loops. 
     
     
         5 . The iFv of  claim 2 , further comprising a recipient polypeptide or protein containing one or more solvent exposed loops, wherein the iFv is inserted into one or more of the loops, such that the non-natural N- and C-termini within the VL or VH domains are spatially positioned proximal to each other. 
     
     
         6 . The iFv of  claim 1 , wherein either the C-terminus of the VL domain is connected to the N-terminus of the VH domain and the N-terminus of the VL domain is connected to the C-terminus of the VH domain; or wherein the N-terminus of the VL domain is connected to the C-terminus of the VH domain and the C-terminus of the VL domain is connected to the N-terminus of the VH domain. 
     
     
         7 . The iFv of  claim 5 , wherein the distance between the non-natural N- and C-termini is between about 0.2 and about 2.0 nm. 
     
     
         8 . The iFv of  claim 7 , wherein the distance between the termini is between about 0.5 nm and about 1.5 nm. 
     
     
         9 . The iFv of  claim 1 , wherein the two linker regions comprise identical or non-identical peptides of about 10 to 25 amino acids. 
     
     
         10 . The iFv of  claim 5 , wherein the recipient polypeptide or protein is an antibody, Ig fold, Ig domain, globulin, albumen, fibronectin, fibronectin domain, integrin, fluorescent protein, enzyme, outer membrane protein, receptor protein, T-cell receptor, chimeric antigen receptor, viral antigen, virus capsid, viral ligand for cell receptor, high molecular weight bacteriocin, histone, hormone, knottin, cyclic peptide, ULB protein, lectin, or a ligand for a lectin. 
     
     
         11 . The iFv of  claim 1 , further comprising a non-protein molecule to which the iFv is inserted into, or attached. 
     
     
         12 . The iFv of  claim 11 , wherein the non-protein molecule is a polysaccharide, dendrimer, polyglycol, peptidoglycan, chemotherapeutic agent, toxin, fluorophore, chromophore, pharmacophore, antibiotic or polyketide. 
     
     
         13 . The iFv of  claim 2 , further comprising a non-protein molecule or an atom conjugated to one or both of the non-natural N-terminus and the non-natural C-terminus. 
     
     
         14 . The iFv of  claim 5 , wherein all or a portion of the one or more solvent-exposed loops of the recipient molecule is deleted and replaced with the iFv.