Inducible monovalent antigen binding protein
Disclosed herein are inducible monovalent target-binding proteins which are activated upon protease cleavage. Pharmaceutical compositions comprising the binding proteins disclosed herein and methods of using such formulations are further provided.
1. An inducible monovalent target-binding protein, wherein the protein comprises a first polypeptide chain and a second polypeptide chain,
the first polypeptide chain comprising: a VH target-binding domain (VH), an inactive VL domain (iVL) that binds to the VH domain, a linker (L1), and a first monomeric Fc domain (Fcl) comprising a CH3 and a CH2 domain;
the second polypeptide chain comprising: a VL target-binding domain (VL), an inactive VH domain (iVH) that binds to the VL domain, a linker (L6), a second monomeric Fc domain (Fc2) comprising a CH3 and a CH2 domain;
wherein the iVL and the iVH each comprise at least one protease cleavage site, wherein upon activation by protease cleavage of the at least one protease cleavage site of both the iVL and the iVH, the VH and the VL associate to form an active target-binding domain, and wherein the CH3 domains of the Fcl and Fc2 form a heterodimer.
2. The inducible monovalent target-binding protein of claim 1 , wherein the iVL comprises a CDRL1, CDRL2, and CDRL3, and the at least one protease cleavage site of the iVL is located within the CDRL1, CDRL2, or CDRL3.
3. The inducible monovalent target-binding protein of claim 1 , wherein the iVH comprises a CDRH1, CDRH2, and CDRH3, and the at least one protease cleavage site of the iVH is located within the CDRH1, CDRH2, or CDRH3.
4. The inducible monovalent target-binding protein of claim 1 , wherein the first polypeptide chain comprises a further protease cleavage site, and wherein the second polypeptide comprises a further protease cleavage site.
5. The inducible monovalent target-binding protein of claim 4 , wherein the further protease cleavage site of the first polypeptide chain is located within L1, and the further protease cleavage site of the second polypeptide chain is located within L6.
6. The inducible monovalent target-binding protein of claim 1 , wherein the Fcl and the Fc2 each independently comprise one or more amino acid substitutions favoring formation of a heterodimeric Fc region.
7. The inducible monovalent target-binding protein of claim 1 , wherein the iVH and the iVL each are derived from an anti-respiratory syncytial virus (RSV) F-protein antibody.
8. The inducible monovalent target-binding protein of claim 1 , wherein the VH, the iVL, and the L1 of the first polypeptide form a scFv.
9. The inducible monovalent target-binding protein of claim 1 , wherein the VL, the iVH, and the L6 of the second polypeptide form a scFv.
10. The inducible monovalent target-binding protein of claim 1 , wherein the active target-binding domain binds to a target expressed on a tumor cell.
11. The inducible monovalent target-binding protein of claim 1 , wherein the active target-binding domain binds to a tumor antigen.
12. The inducible monovalent target-binding protein of claim 1 , wherein the active target-binding domain binds to an immune checkpoint protein.
13. The inducible monovalent target-binding protein of claim 1 , wherein the active target-binding domain binds to CD27, CD40, OX40, GITR, CD137, B7, CD28, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, PD-L1, TIM-3, VISTA.
14. The inducible monovalent target-binding protein of claim 1 , wherein the at least one protease cleavage site of the first polypeptide and the at least one protease cleavage site is independently recognized by at least one of a serine protease, a cysteine protease, an aspartate protease, a threonine protease, a glutamic acid protease, a metalloproteinase, a gelatinase, and a asparagine peptide lyase.
15. An inducible monovalent target-binding protein, wherein the protein comprises a first polypeptide chain and a second polypeptide chain,
the first polypeptide chain comprising: a VH target-binding domain (VH), an inactive VL domain (iVL) that binds to the VH domain, a linker (L1), and a first monomeric Fc domain (Fcl) comprising a CH3 and a CH2 domain;
the second polypeptide chain comprising: a VL target-binding domain (VL), an inactive VH domain (iVH) that binds to the VL domain, a linker (L6), a second monomeric Fc domain (Fc2) comprising a CH3 and a CH2 domain;
wherein the L1 and L6 each comprise at least one protease cleavage site, wherein upon activation by protease cleavage of the at least one protease cleavage site of both L1 and L6, the VH and the VL associate to form an active target-binding domain, and wherein the CH3 domains of the Fcl and Fc2 form a heterodimer.
16. The inducible monovalent target-binding protein of claim 15 , wherein the iVH and the iVL each are derived from an anti-respiratory syncytial virus (RSV) F-protein antibody.
17. The inducible monovalent target-binding protein of claim 15 , wherein the VH, the iVL, and the L1 of the first polypeptide form a scFv.
18. The inducible monovalent target-binding protein of claim 15 , wherein the VL, the iVH, and the L6 of the second polypeptide form a scFv.
19. The inducible monovalent target-binding protein of claim 1 , wherein the VH comprises a sequence selected from the group consisting of SEQ ID Nos. 43, 45, 47, and 49.
20. The inducible monovalent target-binding protein of claim 1 , wherein the VL comprises a sequence selected from the group consisting of SEQ ID Nos. 44, 46, 48, and 50.