PD-L1 -binding molecules comprising Shiga toxin A subunit scaffolds
Provided herein are PD-L1 binding molecules comprising or conjugated to a toxin, e.g. a Shiga toxin A Subunit derived polypeptide. In some embodiments, the PD-L1 binding molecules are cytotoxic. In some embodiments, the PD-L1 binding molecules are capable of delivering a CD8+ T-cell epitope to an MHC class molecule inside a PD-L1 positive cell. The PD-L1 binding molecules described herein have uses for selectively killing specific cells (e.g., PD-L1 positive tumor cells and/or immune cells); for selectively delivering cargos to specific cells (e.g., PD-L1 positive tumor cells or immune cells), and as therapeutic and/or diagnostic molecules for treating and diagnosing a variety of conditions, including cancers and tumors involving PD-L1 expressing cells (e.g., PD-L1 positive tumor cells or immune cells).
1. A PD-L1 binding molecule comprising:
a Shiga toxin A subunit effector polypeptide comprising an amino acid sequence at least 98% identical to SEQ ID NO: 41;
a single chain variable fragment (scFv) capable of specifically binding an extracellular part of human PD-L1;
wherein the scFv comprises:
(a) a heavy chain variable region (V H ) comprising:
(i) a CDR1 comprising the amino acid sequence EYTMH (SEQ ID NO:27),
(ii) a CDR2 comprising the amino acid sequence GINPNNGGTWYNQKFKG (SEQ ID NO:29), and
(iii) a CDR3 comprising the amino acid sequence PYYYGSREDYFDY (SEQ ID NO:32);
and
(b) a light chain variable region (V L ) comprising:
(i) a CDR1 comprising the amino acid sequence SASSSVSYMY (SEQ ID NO:19),
(ii) a CDR2 comprising the amino acid sequence LTSNLAS (SEQ ID NO:20), and
(iii) a CDR3 comprising the amino acid sequence QQWSSNPPT (SEQ ID NO:26);
and a scFv linker between the V H and the V L which is 3 to 12 amino acids in length; and
a human CD8+ T-cell epitope.
2. The PD-L1 binding molecule of claim 1 , wherein the V H comprises the sequence of SEQ ID NO: 34, or a sequence at least 90% identical thereto.
3. The PD-L1 binding molecule of claim 1 , wherein the V L comprises the sequence of SEQ ID NO: 35, or a sequence at least 90% identical thereto.
4. The PD-L1 binding molecule of claim 1 , wherein the V H comprises the sequence of SEQ ID NO: 34 and the V L comprises the sequence of SEQ ID NO: 35.
5. The PD-L1 binding molecule of claim 1 , wherein the scFv linker comprises the sequence of SEQ ID NO: 72.
6. The PD-L1 binding molecule of claim 1 , wherein the scFv comprises the sequence of SEQ ID NO: 106, or a sequence at least 90% identical thereto.
7. The PD-L1 binding molecule of claim 1 , wherein the PD-L1 binding molecule comprises a binding domain linker which links the Shiga toxin A subunit effector polypeptide and the scFv.
8. The PD-L1 binding molecule of claim 7 , wherein the binding domain linker is a polypeptide and comprises the sequence of SEQ ID NO: 73.
9. The PD-L1 binding molecule of claim 1 , wherein the CD8+ T-cell epitope comprises or consists of the sequence NLVPMVATV (SEQ ID NO: 78).
10. The PD-L1 binding molecule claim 1 , wherein the CD8+ T-cell epitope is linked to the scFv via a polypeptide spacer.
11. The PD-L1 binding molecule of claim 10 , wherein the spacer has the sequence HHAA (SEQ ID NO: 265).
12. The PD-L1 binding molecule of claim 1 , wherein the PD-L1 binding molecule comprises the sequence of SEQ ID NO: 128, or a sequence at least 90% identical thereto.
13. The PD-L1 binding molecule of claim 1 , wherein the PD-L1 binding molecule is a single continuous polypeptide.
14. The PD-L1 binding molecule of claim 13 , wherein the PD-L1 binding molecule is a single continuous polypeptide comprising, in order from N-terminus to C-terminus, the Shiga toxin A subunit effector polypeptide, the scFv, and the CD8+ T-cell epitope.
15. The PD-L1 binding molecule of claim 13 , wherein the binding molecule is a single continuous polypeptide comprising, from N-terminus to C-terminus, the Shiga toxin A subunit effector polypeptide, a binding domain linker, the scFv, and the CD8+ T-cell epitope.
16. The PD-L1 binding molecule of claim 13 , wherein the binding molecule is a single continuous polypeptide comprising, in order from N-terminus to C-terminus, the Shiga toxin A subunit effector polypeptide, a binding domain linker, the V H , the scFv linker, the V L , and the CD8+ T-cell epitope.
17. The PD-L1 binding molecule of claim 13 , wherein the binding molecule is a single continuous polypeptide comprising, from N-terminus to C-terminus, the Shiga toxin A subunit effector polypeptide, a binding domain linker, the scFv, a spacer, and the CD8+ T-cell epitope.
18. The PD-L1 binding molecule of claim 1 , wherein the PD-L1 binding molecule comprises two polypeptides.
19. The PD-L1 binding molecule of claim 18 , wherein the two polypeptides are non-covalently linked to each other.
20. The PD-L1 binding molecule of claim 18 , wherein the two polypeptides are linked to each other via the scFv.
21. The PD-L1 binding molecule of claim 18 , wherein each of the two polypeptides comprises, in order from N-terminus to C-terminus, the Shiga toxin A subunit effector polypeptide, the scFv, and the CD8+ T-cell epitope.
22. The PD-L1 binding molecule of claim 18 , wherein each of the two polypeptides comprises, from N-terminus to C-terminus, the Shiga toxin A subunit effector polypeptide, a polypeptide binding domain linker, the scFv, and the CD8+ T-cell epitope.
23. The PD-L1 binding molecule of claim 18 , wherein each of the two polypeptides comprises, in order from N-terminus to C-terminus, the Shiga toxin A subunit effector polypeptide, a polypeptide binding domain linker, the V H , the scFv linker, the V L , and the CD8+ T-cell epitope.
24. The PD-L1 binding molecule of claim 18 , wherein each of the two polypeptides comprises, from N-terminus to C-terminus, the Shiga toxin A subunit effector polypeptide, a polypeptide binding domain linker, the scFv, a polypeptide spacer, and the CD8+ T-cell epitope.
25. The PD-L1 binding molecule of claim 18 , wherein each of the two polypeptides comprises the sequence of SEQ ID NO: 128.
26. The PD-L1 binding molecule of claim 25 , wherein each of the two polypeptides consists of SEQ ID NO: 128.
27. A PD-L1 binding molecule consisting of the amino acid sequence of SEQ ID NO: 128.
28. A pharmaceutical composition comprising the PD-L1 binding molecule of claim 1 , and at least one pharmaceutically acceptable excipient or carrier.
29. A pharmaceutical composition comprising the PD-L1 binding molecule of claim 27 , and at least one pharmaceutically acceptable excipient or carrier.
30. The PD-L1 binding molecule of claim 1 , wherein the molecule is cytotoxic.