Therapeutic compositions and methods for antibody and Fc-containing targeting molecule-based targeted delivery of bioactive molecules by bacterial minicells
The present application relates to the use of bacterial minicells as targeted delivery agents in vivo and in vitro. Described herein are genetically engineered eubacterial minicells designed to preferentially target and deliver therapeutically relevant agents using a minicell surface coupling molecule capable of binding and displaying antibodies or other Fc-containing targeting moiety fusions and conjugates.
1. A bacterial minicell, comprising:
(i) an Fc-binding fusion protein displayed on the surface of the minicell, wherein the Fc-binding fusion protein comprises a) an outer membrane anchoring domain and b) an Fc binding portion of Protein A, wherein the Fc-binding portion of Protein A has no F(ab') 2 binding capability and comprises no cleavage sites by OmpT protease;
(ii) one or more bioactive molecules; and
(iii) one or more Fc-containing targeting molecules bound to said Fc binding portion, wherein said one or more Fc-containing targeting molecules recognize a eukaryotic antigen.
2. The minicell of claim 1 , wherein at least one of the one or more bioactive molecules is a protein toxin.
3. The minicell of claim 2 , wherein said protein toxin is selected from the group consisting of gelonin, diphtheria toxin fragment A, diphtheria toxin fragment A/B, tetanus toxin, E. coli heat labile toxin LTI, E. coli heat labile toxin LTII, cholera toxin, C. perfringes iota toxin, Pseudomonas exotoxin A, shiga toxin, anthrax toxin, MTX ( B. sphaericus mosquilicidal toxin), perfringolysin O, streptolysin, barley toxin, mellitin, anthrax toxins LF and EF, adenylate cyclase toxin, botulinolysin B, botulinolysin E3, botulinolysin C, botulinum toxin A, cholera toxin, clostridium toxins A, B, and alpha, ricin, shiga A toxin, shiga-like A toxin, cholera A toxin, pertussis S1 toxin, E. coli heat labile toxin (LTB), pH stable variants of listeriolysin O, thermostable variants of listeriolysin O, pH and thermostable variants of listeriolysin O, streptolysin O, streptolysin O c, streptolysin O e, sphaericolysin, anthrolysin O, cereolysin, thuringiensilysin O, weihenstephanensilysin, alveolysin, brevilysin, butyriculysin, tetanolysin O, novyilysin, lectinolysin, pneumolysin, mitilysin, pseudopneumolysin, suilysin, intermedilysin, ivanolysin, seeligeriolysin O, vaginolysin, pyolysin, and any combination thereof.
4. The minicell of claim 1 , wherein at least one of the one or more bioactive molecules is a therapeutic small molecule drug.
5. The minicell of claim 4 , wherein said therapeutic small molecule drug is selected from the group consisting of DNA damaging agents, agents that inhibit DNA synthesis, microtubule and tubulin binding agents, anti-metabolites, inducers of oxidative damage, anti-angiogenics, endocrine therapies, anti-estrogens, immuno-modulators, histone deacetylase inhibitors, inhibitors of signal transduction, inhibitors of heat shock proteins, retinoids, inhibitors of growth factor receptors, anti-mitotic compounds, anti-inflammatories, cell cycle regulators, transcription factor inhibitors, and apoptosis inducers, and any combination thereof.
6. The minicell of claim 1 , wherein at least one of the one or more bioactive molecules is a therapeutic nucleic acid.
7. The minicell of claim 1 , wherein at least one of the one or more bioactive molecules is a therapeutic polypeptide.
8. The minicell of claim 1 , wherein at least one of the one or more bioactive molecules is a combination of a small molecule drug and a therapeutic nucleic acid.
9. The minicell of claim 1 , wherein at least one of the one or more Fc-containing targeting molecules is specific for a tumor cell surface molecule.
10. The minicell of claim 1 , wherein at least one of the one or more Fc-containing targeting molecules is specific for an endothelial cell surface molecule.
11. The minicell of claim 1 , wherein at least one of the one or more Fc-containing targeting molecules is specific for a target common to both a tumor cell and an endothelial cell.
12. The minicell of claim 1 , wherein said minicell further comprises an endosomal escape agent.
13. A composition, comprising the minicell of claim 1 and a pharmaceutically acceptable carrier.
14. The minicell of claim 1 , wherein at least one of the one or more bioactive molecules is a protein from an infectious agent.
15. The minicell of claim 14 , wherein at least one of the one or more Fc-containing targeting molecules is specific for a professional antigen presenting cell.
16. The minicell of claim 15 , wherein said professional antigen presenting cell is a eukaryotic dendritic cell or macrophage.
17. The minicell of claim 1 , wherein at least one of the one or more bioactive molecules is a protein antigen from a tumor.
18. The minicell of claim 17 , wherein at least one of the one or more Fc-containing targeting molecules is specific for a eukaryotic dendritic cell, eosinophil, neutrophil, basophil, T-cell, B-cell, mast cell, or macrophage.
19. The minicell of claim 14 , wherein said minicell further comprises an endosomal escape agent, or an immunomodulatory adjuvant.
20. A composition, comprising the minicell of claim 14 and a pharmaceutically acceptable carrier.
21. The minicell of claim 1 , wherein the Fc-binding fusion protein further comprises an autotransporter of type 5a secretion system.
22. The minicell of claim 1 , wherein the minicell is fully intact.
23. The minicell of claim 1 , wherein the Fc-binding portion of Protein A comprises an amino acid substitution of glycine to alanine at glycine 29.
24. The minicell of claim 5 , wherein the immune-modulators are Toll-like receptor agonists or antagonists.