Method employing bispecific protein complex

1. A method of identifying a bispecific protein complex that destroys a target cell or modulates a function of a target cell, comprising

(a) generating fusion proteins of formula A-X and B-Y and heterodimerically-tethered bispecific protein complexes of formula A-X:Y-B;

(b) contacting a population of cells comprising an effector cell and a target cell with:

i. a combination of the fusion proteins A-X and B-Y in an uncomplexed form, or

ii. A-X:Y-B in a heterodimerically-tethered bispecific protein complex form; and

(c) monitoring the population of cells for destruction of the target cell or modulation of the function of the target cell, thereby identifying the bispecific protein complex that destroys the target cell or modulates the function of the target cell,

wherein

X:Y is a heterodimeric-tether;

: is a binding interaction between X and Y;

A is a first protein component of the bispecific protein complex selected from an antibody or binding fragment thereof, and a protein, wherein A specifically binds a protein expressed on the surface of an effector cell;

B is a second protein component of the bispecific protein complex selected from an antibody or binding fragment or an antigen, wherein B specifically binds a target cell;

X is a scFv and Y is a peptide GCN4 or a fragment thereof (SEQ ID NO: 1 or amino acids 1-38 of SEQ ID NO: 1 or SEQ ID NO: 76 to 98), or

X is a peptide GCN4 or a fragment thereof (SEQ ID NO: 1 or amino acids 1-38 of SEQ ID NO: 1 or SEQ ID NO: 76 to 98) and Y is a scFv; and

wherein X or Y is specific to the peptide GCN4 or a fragment thereof (SEQ ID NO: 1 or amino acids 1-38 of SEQ ID NO: 1 or SEQ ID NO: 76 to 98), and wherein the scFv is 52SR4 (SEQ ID NO: 3 or amino acids 1-243 of SEQ ID NO: 3 or SEQ ID NO.99-100).

2. The method according to claim 1 , wherein X or Y is an antigen with no corresponding mammalian sequence.

3. The method according to claim 1 , wherein a binding affinity between X and Y is 5 nM, 900, 800, 700, 600, 500, 400 or 300 pM.

4. The method according to claim 1 where the bispecific protein complex comprises no more than two scFv and/or at least one Fab or Fab′ fragment.

5. The method according to claim 1 ,

i. wherein the effector cell is a cell capable of a cellular response, wherein the cellular response is phagocytosis, cytotoxicity, generating an antibody, release of a soluble molecule, or a combination thereof; and/or

ii. wherein A is independently selected from the group consisting of a full length antibody, a Fab fragment, a Fab′ fragment, a sdAb, a scFv, and an antigen.

6. The method according to claim 1 ,

wherein the effector cell is selected from the group consisting of a B cell, T cell, an NK cell, monocyte, macrophage, dendritic cell, mast cell, neutrophil, eosinophil and basophil.

7. The method according to claim 6 , wherein the effector cell is a B cell, wherein the B cell comprises a marker that is in a constant region of an antibody light chain or a constant region of an antibody heavy chain, expressed as part of an immunoglobulin on the surface of the cell.

8. The method according to claim 1 , wherein B is a full length antibody, a Fab fragment, a Fab′ fragment, a sdAb, or a scFv.

9. The method according to claim 1 , wherein the effector cell transgresses the blood brain barrier (BBB), wherein B binds a central nervous system expressed target.

10. The method according to claim 1 , wherein B binds a cell surface marker on the target cell, wherein the cell surface marker is:

i. a tumor antigen selected from erbB-2, CEA, NCAM, GD2, CD33, CD44, CD70, EpCAM, CD19, CD20, KDR, and Tag-72; or

ii. a HER receptor; or

iii. a B cell marker or T cell marker.

11. The method according to claim 1 , wherein:

i. X is fused to the N-terminal or C-terminal of A; or

ii. X and/or Y is fused to the C-terminal of the heavy chain of an antibody or an antigen binding fragment thereof; or

iii. Y is fused to the C-terminal of B.

12. The method of claim 1 , wherein A is an antibody, an antigen binding fragment thereof or an antigen, wherein B is an antibody or an antigen binding fragment or a protein ligand.

13. The method of claim 6 , wherein the protein expressed on the surface of the effector cell is CD45, CD2, CD3, CD4, CD5, CD7, CD8, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20, CD23, CD25, CD27, CD33, CD38, CD56, CD57, CD64, CD80, CD83, CD86, CD123, CD127, CD137, CD138, CD196, CD209, HLA-DR, Lin- 1 to -3, or a combination thereof.

14. The method of claim 6 , wherein the effector cell is a B cell comprising a marker, and the B cell marker is selected from the group consisting of CD19, CD20, CD21, CD22, CD23, CD24, CD27, CD35, CD38, CD40, CD45, CD43, CD81, CD138, CXCR4, BCMA and IL-6R.

15. The method of claim 6 , wherein the effector cell is a T cell comprising a marker and the T cell marker is selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD127 CD196 (CCR6), CD197 (CCR7), CD62L, CD69 and CD45.

16. The method of claim 5 , wherein the cellular response is release of an immunoglobulin, a cytokine, a chemokine, or a combination thereof.