Shiga toxin A subunit effector polypeptides, Shiga toxin effector scaffolds, and cell-targeting molecules for site-specific conjugation

Patent metadata

Field	Value
Publication number	US-11857628-B2
Application number	US-202117533552-A
Country	US
Kind code	B2
Filing date	Nov 23, 2021
Priority date	Dec 7, 2016
Publication date	Jan 2, 2024
Grant date	Jan 2, 2024

Abstract

The present invention provides Shiga toxin A Subunit derived polypeptides, scaffolds, and cell-targeting molecules comprising amino acid substitutions which equip the molecules with site-specific positions (and often unique amino acid residues in the molecule) for linking other molecules while retaining Shiga toxin function(s), such as, e.g., efficient intracellular routing and/or potent cytotoxicity. The present invention also provides cell-targeting molecules, and/or components thereof, which comprise site-specific positions for linking other molecules, such as, e.g., agents that alters a property of the cell-targeting molecule or a cargo for delivery. Certain molecules comprising a polypeptide of the present invention exhibit reduced immunogenicity and/or are well-tolerated by mammals. The cell-targeting molecules of the present invention, and compositions thereof, have uses, e.g., for the selective delivery of cargos to target-expressing cells and as diagnostic and/or therapeutic molecules for the treatment of a variety of diseases, disorders, and conditions, which include genetic disorders, genetic predispositions, infections, cancers, tumors, growth abnormalities, and/or immune disorders.

First claim

The invention is claimed as follows:

1. A cell-targeting molecule comprising:
(a) a Shiga toxin effector polypeptide;
(b) a binding region capable of specifically binding at least one extracellular target biomolecule; and
(c) a linker peptide between the Shiga toxin effector polypeptide and the binding region,
wherein the Shiga toxin effector polypeptide comprises an amino acid sequence having at least 90% sequence identity to amino acids 1 to 251 of SEQ ID NO: 1,
wherein the Shiga toxin effector polypeptide comprises amino acid substitutions S45I, V54I, R55L, I57F, P59F, E60T, E61L, G110A, and R188A in SEQ ID NO: 1,
wherein the Shiga toxin effector polypeptide comprises one or more cysteines suitable for conjugation to a heterologous molecule, and wherein the Shiga toxin effector polypeptide comprises at least one of the following amino acid substitutions in SEQ ID NO: 1: S8C, S16C, S22C, S43C, S146C, and S186C.

2. The cell-targeting molecule of claim 1 , wherein the Shiga toxin effector polypeptide 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 114 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.

3. The cell-targeting molecule of claim 1 , wherein the Shiga toxin effector polypeptide comprises the amino acid substitutions R248A and R251A in SEQ ID NO: 1.

4. The cell-targeting molecule of claim 1 , wherein the Shiga toxin effector polypeptide comprises the amino acid substitution C242S in SEQ ID NO: 1.

5. The cell-targeting molecule of claim 1 , wherein the Shiga toxin effector polypeptide comprises at least one of the amino acid substitutions Y77S and E167D in SEQ ID NO: 1.

6. The cell-targeting molecule of claim 1 , wherein the linker peptide comprises one or more amino acid residues having a functional group suitable for conjugation to a heterologous molecule, and wherein the amino acid residue having the functional group is a cysteine, lysine, histidine, or a non-natural amino acid residue.

7. The cell-targeting molecule of claim 1 , further comprising one or more amino acid residues having a functional group suitable for conjugation to a second heterologous molecule in the linker peptide, and wherein the amino acid residue having the functional group is a cysteine, lysine, histidine, or a non-natural amino acid residue.

8. The cell-targeting molecule of claim 1 , wherein the binding region comprises one or more amino acid residues having a functional group suitable for conjugation to a heterologous molecule, and wherein the amino acid residue having the functional group is a cysteine, lysine, histidine, or a non-natural amino acid residue.

9. The cell-targeting molecule of claim 8 , wherein the binding region comprises a single-chain variable fragment comprising a heavy chain variable domain (V H ) and a light chain variable domain (V L ), and wherein a linker between the V H and the V L comprises the one or more amino acid residues having the functional group.

10. The cell-targeting molecule of claim 1 , wherein the Shiga toxin effector polypeptide comprises the amino acid substitution S8C in SEQ ID NO: 1.

11. The cell-targeting molecule of claim 6 , wherein the amino acid residue having the functional group is a lysine, a selenocysteine, or a pyrroline-carboxy-lysine.

12. The cell-targeting molecule of claim 6 , wherein the amino acid residue having the functional group comprises a reactive chemical group.

13. The cell-targeting molecule of claim 1 , wherein the Shiga toxin effector polypeptide comprises SEQ ID NO: 86 or SEQ ID NO: 106.

14. The cell-targeting molecule of claim 1 , wherein the linker peptide comprises the amino acid sequence of any one of SEQ ID NOs: 757-761 and 769-772.

15. The cell-targeting molecule of claim 1 , wherein the binding region comprises a: 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, Armadillo repeat polypeptide, fibronectin-derived 10 th 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, or C-type lectin-like domain scaffold.

16. The cell-targeting molecule of claim 1 , wherein the binding region comprises a single-chain variable fragment or single-domain antibody fragment.

17. The cell-targeting molecule of claim 1 , wherein the extracellular target biomolecule is: B7-H3, BCMA, CD20, PD-L1, CD22, CD40, CD45, CD79, CD25, CD30, HER2/neu/ErbB2, EGFR, EphB2, prostate-specific membrane antigen, Cripto, endoglin/CD105, fibroblast activation protein, Lewis-Y, CD19, CD21, CS1/SLAMF7, CD33, CD52, CD74, EpCAM, gpA33, mucin, TAG-72, carbonic anhydrase IX, folate binding protein, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside Lewis-Y2, VEGFR, Alpha Vbeta3, Alpha5beta1, ErbB1/EGFR, Erb3, c-MET, IGF1R, EphA3, LMP1, TRAIL-R1, TRAIL-R2, RANKL, tenascin, CD64, mesothelin, BRCA1, MART-1/MelanA, gp100, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, GAGE-1/2, BAGE, RAGE, NY-ESO-1, CDK-4, beta-catenin, MUM-1, caspase-8, KIAA0205, HPVE6, SART-1, PRAME, carcinoembryonic antigen, prostate specific antigen, prostate stem cell antigen, human aspartyl (asparaginyl) beta-hydroxylase, EphA2, HER3/ErbB-3, MUC1, tyrosinase associated antigen, human tyrosinase-related protein 1, HPV-E7, Epstein-Barr virus antigen, Bcr-Abl, alpha-fetoprotein antigen, 17-A1, bladder tumor antigen, CD38, CD15, CD23, CD53, CD88, CD129, CD183, CD191, CD193, CD244, CD294, CD305, C3AR, FceRIa, galectin-9, mrp-14, siglec-8, siglec-10, CD49d, CD13, CD44, CD54, CD63, CD69, CD123, TLR4, IgE, CD107a, CD203c, CD14, CD68, CD80, CD86, CD115, F4/80, ILT-3, galectin-3, CD11a-c, GITRL, MHC class II, CD284/TLR4, CD107/Mac3, CD195/CCR5, HLA-DR, CD16/32, or CD282/TLR2.

18. The cell-targeting molecule of claim 1 , wherein the cell-targeting molecule is covalently linked via the one or more cysteines to the heterologous molecule.

19. The cell-targeting molecule of claim 18 , wherein the heterologous molecule is an antibiotic, antigen, cytotoxic agent, radionuclide, solubility-altering agent, pharmacokinetic-altering agent, immunogenicity-altering agent, pharmacodynamics-altering agent, detection-promoting agent, dye, T-cell epitope, fluorophore, immunogen, enzyme, zymoxin, lipid, polymer, polyethylene glycol, serum albumin binding agent, small molecule chemotherapeutic agent, prodrug, peptide, protein, nucleic acid, or protein-nucleic acid complex.

20. The cell-targeting molecule of claim 18 , wherein the heterologous molecule is an auristatin, a pyrrolobenzodiazepine or a pyrrolobenzodiazepine dimer.

21. A pharmaceutical composition comprising the cell-targeting molecule of claim 1 and a pharmaceutically acceptable excipient or carrier.

22. A pharmaceutical composition comprising the cell-targeting molecule of claim 18 and a pharmaceutically acceptable excipient or carrier.

Assignees

• Molecular Templates Inc

Inventors

• Poma Eric
• Willert Erin

Classifications

• G01N2800/52

  Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis · CPC title

• G01N2333/25

  Shigella (G) · CPC title

• A61P31/04

  Antibacterial agents · CPC title

• G01N33/6893

  related to diseases not provided for elsewhere · CPC title

• G01N33/56916

  Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia · CPC title