Bispecific format suitable for use in high-through-put screening

Abstract

The present disclosure relates to a novel bispecific protein complex and a method of using the complexes to screen for synergistic or novel biological function. The bispecific format is particularly suitable for high-throughput screening because all of its components can be expressed from cells as individual units and the units can be assembled simply by mixing without employing conjugation or coupling chemistry.

1 . A bispecific protein complex having the formula A-X:Y-B wherein:
 A-X is a first fusion protein;   Y-B is a second fusion protein;   : is a binding interaction between X and Y to form a bispecific protein complex between the first and second fusion proteins;   A is a first protein component of the bispecific protein complex selected from a Fab or Fab′ fragment;   B is a second protein component of the bispecific protein complex selected from a Fab or Fab′ fragment;   X is a first binding partner of a binding pair independently selected from an antigen or an antibody or binding fragment thereof, wherein when X is an antigen, Y is an antibody or binding fragment thereof specific to the antigen represented by X; and   Y is a second binding partner of the binding pair independently selected from an antigen or an antibody or a binding fragment thereof, wherein when Y is an antigen, X is an antibody or binding fragment thereof specific to the antigen represented by Y;   wherein A and B bind to two different epitopes on an antigen, or bind two different antigens, and   wherein X or Y is a VHH specific to the peptide GCN4 having SEQ ID NO:1 or amino acids 1 to 38 of SEQ ID NO:1, and wherein X or Y is a peptide GCN4 having SEQ ID NO:1 or amino acids 1 to 38 of SEQ ID NO:1.   
     
     
         2 . A bispecific protein complex according to  claim 1 , wherein A is a Fab fragment. 
     
     
         3 . A bispecific protein complex according to  claim 1 , wherein B is a Fab fragment. 
     
     
         4 . A bispecific protein complex according to  claim 1 , wherein X is fused directly or via a linker, to the C-terminal of the heavy chain in the Fab or Fab′ fragment. 
     
     
         5 . A bispecific protein complex according to  claim 1 , wherein Y is fused directly or via a linker, to the C-terminal of the heavy chain in the Fab or Fab′ fragment. 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . A bispecific protein complex according to  claim 1 , wherein the binding affinity between X and Y is 5 nM or stronger. 
     
     
         10 . A bispecific protein complex according to  claim 9 , wherein the binding affinity between X and Y is 900 pM or stronger. 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . The bispecific protein complex according to  claim 1 , wherein A and/or B is specific for an antigen selected from the group comprising: cell surface receptors, co-stimulatory molecules, checkpoint inhibitors, natural killer cell receptors, Immunoglobulin receptors, TNFR family receptors, B7 family receptors, adhesion molecules, integrins, cytokine/chemokine receptors, GPCRs, growth factor receptors, kinase receptors, tissue-specific antigens, cancer antigens, pathogen recognition receptors, complement receptors, hormone receptors, cytokines, chemokines, leukotrienes, growth factors, hormones, enzymes, and ion channels. 
     
     
         15 . A composition comprising the bispecific protein complex defined in  claim 1 . 
     
     
         16 . A method of treating a subject in need thereof comprising administering a bispecific protein complex according to  claim 1  to the subject. 
     
     
         17 . A method of detecting synergistic biological function in a bispecific protein complex according to  claim 1  comprising testing the bispecific protein complex in one or more functional assays. 
     
     
         18 . A method for detecting synergistic biological function in a heterodimerically-tethered bispecific protein complex of formula A-X:Y-B,
 : is a binding interaction between X and Y to form a bispecific protein complex between the first and second fusion proteins,   A and B are protein components of the bispecific in the form of fusion proteins with X and Y respectively, said method comprising the steps of:
 (i) testing for activity in a functional assay for part or all of a multiplex comprising at least one heterodimerically-tethered bispecific protein complex; and 
 (ii) analysing the readout(s) from the functional assay to detect synergistic biological function in the heterodimerically-tethered bispecific protein complex; and 
   wherein A and B bind to two different epitopes on an antigen, or bind two different antigens, and wherein X or Y is a VHH specific to the peptide GCN4 having SEQ ID NO:1 or amino acids 1 to 38 of SEQ ID NO:1, and wherein X or Y is a peptide GCN4 having SEQ ID NO:1 or amino acids 1 to 38 of SEQ ID NO:1.   
     
     
         19 . A method according to  claim 18 , wherein the multiplex is in the form a grid. 
     
     
         20 . A method according to  claim 18  wherein the multiplex comprises at least two heterodimerically-tethered bispecific protein complexes. 
     
     
         21 . A method according to  claim 18 , wherein the heterodimerically tethered bispecific protein complex(es) do not contain an Fc region. 
     
     
         22 . A method according to  claim 18 , wherein A is independently selected from a Fab fragment, a Fab′ fragment, a VHH, a VH, a VL and a scFv. 
     
     
         23 . A method according to  claim 22 , wherein A is a Fab or Fab′ fragment. 
     
     
         24 . A method according to  claim 18 , wherein B is independently selected from an antibody, a Fab fragment, a Fab′ fragment, a VHH, a VH, a VL and a scFv. 
     
     
         25 . A method according to  claim 24 , wherein B is a Fab or Fab′ fragment. 
     
     
         26 . A method according to  claim 24 , wherein X is fused directly or via a linker, to the C-terminal of a heavy chain in the antibody, Fab or Fab′ fragment. 
     
     
         27 . A method according to  claim 24 , wherein Y is fused directly or via a linker to the C-terminal of a heavy chain in the antibody, Fab or Fab′ fragment. 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . (canceled) 
     
     
         31 . A method according to  claim 18  wherein the binding affinity between X and Y is 5 nM or stronger. 
     
     
         32 . A method according to  claim 31 , wherein the binding affinity of between X and Y is 900 pM or stronger. 
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         36 . A method according to  claim 18 , wherein A and/or B is specific for an antigen selected from the group comprising: cell surface receptors, co-stimulatory molecules, checkpoint inhibitors, natural killer cell receptors, Immunoglobulin receptors, TNFR family receptors, B7 family receptors, adhesion molecules, integrins, cytokine/chemokine receptors, GPCRs, growth factor receptors, kinase receptors, tissue-specific antigens, cancer antigens, pathogen recognition receptors, complement receptors, hormone receptors, cytokines, chemokines, leukotrienes, growth factors, hormones or enzymes or ion channels. 
     
     
         37 . A method according to  claim 18 , wherein the heterodimerically tethered bispecific protein complexes are not purified prior to testing. 
     
     
         38 . A method according to  claim 37 , wherein the A-X and Y-B fusion proteins are expressed transiently and not purified before being mixed in a 1:1 molar ratio to generate each heterodimerically tethered bispecific protein complex.