DE-IMMUNIZED, SHIGA TOXIN A SUBUNIT SCAFFOLDS AND CELL-TARGETING MOLECULES COMPRISING THE SAME
The present invention relates to Shiga toxin A Subunit derived polypeptides and cell-targeting molecules comprising amino acid substitutions which equip the polypeptides with 1) de-immunization; 2) reduced, protease-cleavage sensitivity; and/or 3) a heterologous epitope cargo(s) while retaining Shiga toxin function(s), such as, e.g., potent cytotoxicity. Certain polypeptides of the invention exhibit reduced immunogenic potential in mammals and/or are capable of delivering an epitope to an MHC class molecule of a cell in which the polypeptide is present. Certain molecules comprising a polypeptide of the invention are well-tolerated by mammals while retaining one or more of the features mentioned above. The Shiga toxin polypeptides of the invention have uses as components of cell-targeting molecules for selectively killing specific cells; for selectively delivering cargos to specific cells, and as therapeutic and/or diagnostic molecules for treating and diagnosing a variety of conditions, including cancers, immune disorders, and microbial infections.
1 . A cell-targeting molecule comprising:
i) a binding region capable of specifically binding an extracellular target biomolecule physically coupled to the cellular surface of a cell, wherein the extracellular target biomolecule is HER2/neu/ErbB2, PDL1, CD38, CD20, BCMA, SLAMF7, or CTLA-4, and
ii) a Shiga toxin effector polypeptide comprising an amino acid sequence having at least 90% identity to amino acids 1 to 251 of SEQ ID NO: 1, wherein the amino acid sequence comprises the amino acid substitutions: S45I, V54I, R55L, I57F, P59F, E60T, E61L, G110A, R188A, C242S, R248A and R251A according to SEQ ID NO: 1; and wherein the amino acid sequence comprises an asparagine at the amino acid residue corresponding to position 75 of SEQ ID NO: 1, a tyrosine at the amino acid residue corresponding to position 77 of SEQ ID NO: 1, a tyrosine at the amino acid residue corresponding to position 114 of SEQ ID NO: 1, a glutamate at the amino acid residue corresponding to position 167 of SEQ ID NO: 1, an arginine at the amino acid residue corresponding to position 170 of SEQ ID NO: 1, an arginine at the amino acid residue corresponding to position 176 of SEQ ID NO: 1, and a tryptophan at the amino acid residue corresponding to position 203 of SEQ ID NO: 1.
2 . The cell-targeting molecule of claim 1 , wherein the Shiga toxin effector polypeptide comprises or consists of an amino acid sequence having at least 95% sequence identity to amino acids 1 to 251 of SEQ ID NO: 1.
3 . The cell-targeting molecule of claim 1 , wherein the Shiga toxin effector polypeptide comprises or consists of an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 13.
4 . The cell-targeting molecule of claim 1 , wherein the binding region is fused to the carboxy terminus of the Shiga toxin effector polypeptide to form a single, continuous polypeptide.
5 . The cell-targeting molecule of claim 1 , wherein the binding region comprises at least one immunoglobulin-type binding region.
6 . The cell-targeting molecule of claim 5 , wherein the at least one immunoglobulin-type binding region comprises a polypeptide selected from: single-domain antibody fragment, single-chain variable fragment, antibody variable fragment, complementary determining region 3 fragment, constrained FR3-CDR3-FR4 polypeptide, Fd fragment, antigen-binding fragment, fibronectin-derived 10th fibronectin type III domain, tenascin type III domain, ankyrin repeat motif domain, low-density-lipoprotein-receptor-derived A-domain, lipocalin, Kunitz domain, Protein-A-derived Z domain, gamma-B crystallin-derived domain, ubiquitin-derived domain, Sac7d-derived polypeptide, Fyn-derived SH2 domain, miniprotein, C-type lectin-like domain scaffold, a heavy-chain antibody domain derived from a camelid V H H fragment, heavy-chain antibody domain derived from cartilaginous fish, immunoglobulin new antigen receptor (IgNAR), V NAR fragment, multimerizing scFv fragment, bivalent minibody, bispecific tandem scFv, bispecific tandem V H H, and bispecific minibody.
7 . The cell-targeting molecule of claim 6 , wherein the multimerizing scFv fragment comprises a diabody, a triabody, or a tetrabody.
8 . The cell-targeting molecule of claim 1 , which comprises the linker peptide shown in any one of SEQ ID NO: 540-543, 544-550, and 553-559.
9 . The cell-targeting molecule of claim 1 , which comprises the linker peptide shown in any one of SEQ ID NO: 540-543 and 553-557.
10 . A pharmaceutical composition comprising the cell-targeting molecule of claim 1 and a pharmaceutically acceptable excipient or carrier.
11 . A polynucleotide encoding the cell-targeting molecule of claim 1 , or a complement thereof.
12 . An expression vector comprising the polynucleotide of claim 11 .
13 . A host cell comprising the polynucleotide of claim 11 .
14 . A host cell comprising the expression vector of claim 12 .
15 . A method of treating a disease, disorder, or condition in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the cell-targeting molecule of claim 1 or a pharmaceutical composition thereof.
16 . The method of claim 15 , wherein treating the disease, disorder or condition comprises selectively killing target cells of the disease, disorder or condition, and wherein the target cells express the extracellular target biomolecule.
17 . The method of claim 15 , wherein the disease, disorder, or condition is a cancer, immune disorder, or microbial infection.
18 . The method of claim 17 , wherein the cancer is bone cancer, breast cancer, central/peripheral nervous system cancer, gastrointestinal cancer, germ cell cancer, glandular cancer, head-neck cancer, hematological cancer, kidney-urinary tract cancer, liver cancer, lung/pleura cancer, prostate cancer, sarcoma, skin cancer, or uterine cancer.
19 . The method of claim 15 , wherein said administering is via intravenous administration.