Multispecific antibodies
The present invention relates to heterodimerically-tethered bispecific protein complexes (according to the general formula of A(A 1 )n-X:Y-B(B 1 )m and libraries/multiplexes thereof for use in research and therapy and in particular an in vitro/ex vivo method of detecting synergistic biological function of otherwise unknown pairs of targets. Such complexes may be used to capture soluble molecules secreted from a particular cell, in therapy, in research and for experimental purposes such as in assays to characterise patient populations by identifying cell populations relevant to a pathology or prognosis.
1. A multispecific protein complex having the formula A(A 1 )n-X:Y-B(B 1 )m wherein:
A(A 1 )n-X is a first fusion protein;
Y-B(B 1 )m is a second fusion protein;
X:Y is a heterodimeric-tether;
: is a binding interaction between X and Y;
wherein A is a Fab or sdAb;
B is a Fab;
A 1 and B 1 are independently a scFv or a sdAb,
n is 0 or 1,
m is 0 or 1,
with the proviso that at least one of n or m is 1;
wherein X is an antigen or an antibody or binding fragment thereof, and said X is appended to the N-terminal or to the C-terminal of A or of A 1 ; and
Y is an antigen or an antibody or a binding fragment thereof, and said Y is appended to the N-terminal or to the C-terminal of B or of B 1 ;
wherein when X is appended to A, A 1 is appended to the N-terminal or to the C-terminal of A which is not occupied by X;
wherein when Y is appended to B, B 1 is appended to the N-terminal or to the C-terminal of B which is not occupied by Y;
wherein X is a first binding partner of a binding pair selected from an antigen, an antibody, and a binding fragment of the antibody, wherein when X is an antigen, Y is an antibody or binding fragment thereof specific to the antigen represented by X; or wherein when Y is an antigen, X is an antibody or binding fragment thereof specific to the antigen represented by Y; and wherein X or Y is a scFv specific to the peptide GCN4 having SEQ ID NO: 1 or amino acids 1 to 38 of SEQ ID NO: 1 and wherein the scFv is 525R4 comprising SEQ ID NO:3 or amino adds 1 to 243 of SEQ ID NO:3; or wherein X or Y is a peptide GCN4 comprising SEQ ID NO:1 or amino acids 1 to 38 of SEQ ID NO: 1.
2. The multispecific protein complex according to claim 1 :
wherein A is a Fab comprising a heavy and light chain;
wherein B comprises a heavy and light chain;
wherein X is appended to the C-terminal of a light chain or heavy chain of the Fab of A; and
wherein Y is a second binding partner of the binding pair independently selected from an antigen, an antibody, and a binding fragment of the antibody, and said Y is appended to the C-terminal of a light chain or heavy chain of the Fab of B;
A 1 is a scFv or a sdAb appended from the C-terminal of the heavy or light chain of the Fab of A which is not occupied by X,
B 1 is a scFv or a sdAb appended from the C-terminal of the heavy or light chain of the Fab of B which is not occupied by Y.
3. The multispecific protein complex according to claim 1 , wherein A is a Fab.
4. The multispecific protein complex according to claim 1 , wherein A is a Fab and X is fused to the C-terminal of the heavy chain in the Fab of A.
5. The multispecific protein complex according to claim 1 , wherein Y is fused to the C-terminal of the heavy chain in the Fab of B.
6. The multispecific protein complex according to claim 1 , wherein X is a scFv specific to the peptide GCN4 having SEQ ID NO: 1 or amino acids 1 to 38 of SEQ ID NO: 1.
7. The multispecific protein complex according to claim 1 , wherein Y is a scFv the peptide GCN4 having SEQ ID NO: 1 or amino acids 1 to 38 of SEQ ID NO: 1.
8. The multispecific protein complex according to claim 1 , wherein the binding affinity between X and Y is from 5 nM to 300 pM.
9. The multispecific protein complex according to claim 8 , wherein the binding affinity between X and Y is 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM or 300 pM.
10. The multispecific protein complex according to claim 1 , wherein X or Y is a scFv specific to the peptide GCN4 (SEQ ID NO:1 or amino acids 1 to 38 of SEQ ID NO:1).
11. The multispecific protein complex according to claim 10 , wherein the scFv is 52SR4 (SEQ ID NO:3 or amino acids 1 to 243 of SEQ ID NO:3).
12. The multispecific protein complex according to claim 1 , wherein X or Y is a peptide GCN4 (SEQ ID NO:1 or amino acids 1 to 38 of SEQ ID NO:1).
13. The multispecific protein complex according to claim 1 , wherein A and/or B is specific for: a cell surface receptor.
14. The multispecific protein complex according to claim 1 , wherein A 1 and/or B 1 is specific for: a cell surface receptor.
15. The multispecific protein complex according to claim 1 , wherein at least one of A, A 1 , B or B 1 is specific to a cell marker.
16. The multispecific protein complex according to claim 15 , wherein the cell marker is selected from a B cell marker and T cell marker.
17. A composition comprising one or more multispecific protein complexes defined in claim 1 .