CTLA-4 BINDING MOLECULES COMPRISING SHIGA TOXIN A SUBUNIT SCAFFOLDS AND USES THEREOF
Provided herein are binding molecules that each comprise (1) a Shiga toxin A subunit effector polypeptide and (2) a binding region capable of specifically binding CTLA-4 on the surface of cell, such as a tumor cell or an immunosuppressive immune cell. Further provided are methods of using such binding molecules to treat diseases and disorders, such as cancer.
1 . A CTLA-4 binding molecule comprising a Shiga toxin A subunit effector polypeptide and a binding region capable of specifically binding an extracellular part of CTLA-4; wherein the binding region comprises a VHH domain comprising a HCDR1, a HCDR2, and a HCDR3.
2 . The CTLA-4 binding molecule of claim 1 , wherein
(a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 23, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 24, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 25; or
(b) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 26, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 27, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 28.
3 . The CTLA-4 binding molecule of claim 1 , wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 22.
4 . A CTLA-4 binding molecule comprising a Shiga toxin A subunit effector polypeptide and a binding region capable of specifically binding an extracellular part of CTLA-4; wherein the binding region comprises a first VHH domain comprising a first HCDR1, a first HCDR2, and a first HCDR3 and a second VHH domain comprising a second HCDR1, a second HCDR2, and a second HCDR3.
5 . The CTLA-4 binding molecule of claim 4 , comprising a linker that links the first VHH domain and the second VHH domain.
6 . The CTLA-4 binding molecule of claim 4 , wherein the first HCDR1 comprises the amino acid sequence of SEQ ID NO: 23, the first HCDR2 comprises the amino acid sequence of SEQ ID NO: 24, and the first HCDR3 comprises the amino acid sequence of SEQ ID NO: 25.
7 . The CTLA-4 binding molecule of claim 4 , wherein the first VHH domain comprises the amino acid sequence of SEQ ID NO: 21.
8 . The CTLA-4 binding molecule of claims 4 , wherein the second HCDR1 comprises the amino acid sequence of SEQ ID NO: 26, the second HCDR2 comprises the amino acid sequence of SEQ ID NO: 27, and the second HCDR3 comprises the amino acid sequence of SEQ ID NO: 28.
9 . The CTLA-4 binding molecule of claim 4 , wherein the second VHH domain comprises the amino acid sequence of SEQ ID NO: 22.
10 . The CTLA-4 binding molecule of claim 5 , wherein the linker comprises the amino acid sequence of SEQ ID NO: 29.
11 . The CTLA-4 binding molecule of claim 4 , wherein the Shiga toxin A subunit effector polypeptide comprises a polypeptide having the sequence of:
(i) amino acids 1 to 251 of any one of SEQ ID NOs: 1-18; or
(ii) amino acids 1 to 261 of any one of SEQ ID NOs: 1-18;
or a polypeptide having a sequence that is at least 90% or at least 95% identical thereto.
12 . The CTLA-4 binding molecule of claim 4 , wherein the Shiga toxin A subunit effector polypeptide comprises or consists of a polypeptide having the sequence of any one of SEQ ID NO: 40 to 68.
13 . The CTLA-4 binding molecule of claims 4 , wherein the Shiga toxin A subunit effector polypeptide comprises the amino acid sequence of SEQ ID NO: 41.
14 . The CTLA-4 binding molecule of claim 4 , wherein the CTLA-4 binding molecule comprises a linker that links the Shiga toxin A subunit effector polypeptide and the binding region.
15 . The CTLA-4 binding molecule of claim 14 , wherein the linker that links the Shiga toxin A subunit effector polypeptide and the binding region comprises the amino acid sequence of SEQ ID NO: 218.
16 . The CTLA-4 binding molecule of claim 14 , wherein the CTLA-4 binding molecule comprises, from N-terminus to C-terminus or from C-terminus to N-terminus, the Shiga toxin A subunit effector polypeptide, the linker, and the binding region.
17 . The CTLA-4 binding molecule of claim 14 , wherein the CTLA-4 binding molecule comprises, from N-terminus to C-terminus, the Shiga toxin A subunit effector polypeptide, the binding region linker, the first VHH domain, the linker, and the second VHH domain.
18 . The CTLA-4 binding molecule of claim 17 , wherein the CTLA-4 binding molecule comprises the amino acid sequence of SEQ ID NO: 329, or an amino acid sequence at least 95% identical to SEQ ID NO: 329.
19 . The CTLA-4 binding molecule of claim 4 , wherein the CTLA-4 binding molecule is a single continuous polypeptide.
20 . The CTLA-4 binding molecule of claim 4 , wherein the CTLA-4 binding molecule comprises two polypeptides.
21 . The CTLA-4 binding molecule of claim 20 , wherein the polypeptides are non-covalently linked.
22 . The CTLA-4 binding molecule of claim 20 , wherein the polypeptides are covalently linked.
23 . The CTLA-4 binding molecule of claim 4 , wherein the CTLA-4 binding molecule is cytotoxic.
24 . The CTLA-4 binding molecule of claim 4 , wherein the CTLA-4 binding molecule is non-cytotoxic.
25 . A pharmaceutical composition comprising the CTLA-4 binding molecule of claim 4 , and at least one pharmaceutically acceptable excipient or carrier.
26 - 38 . (canceled)
39 . A polynucleotide encoding the CTLA-4 binding molecule of claim 4 , or a complement thereof.
40 . An expression vector comprising a polynucleotide according to claim 39 .
41 . A host cell comprising a polynucleotide according to claim 39 .
42 . A method of treating cancer, the method comprising administering to a subject in need thereof an effective amount of the CTLA-4 binding molecule of claim 4 .
43 - 106 . (canceled)
107 . A CTLA-4 binding molecule comprising a Shiga toxin A subunit effector polypeptide and a binding region capable of specifically binding an extracellular part of CTLA-4; wherein the CTLA-4 binding molecule comprises the amino acid sequence of SEQ ID NO: 329, or an amino acid sequence at least 95% identical thereto.
108 - 110 . (canceled)