Regulated genetic suicide mechanism compositions and methods

Abstract

Embodiments of the present invention relates to the incorporation and use of a regulated genetic suicide mechanism for use in the improved purification of biologics, including adjunct use in various eubacterial minicell production and purification methodologies. Described herein are high-yield eubacterial minicell-producing strains with genetic modifications that comprise a regulated genetic suicide mechanism that irreparably destroys the parent cell chromosome such that live parental cells in a culture can be functionally eliminated at any time during the course of a minicell production and purification run. Embodiments of the present invention also describe methods useful in the elimination of live parental cells during the production of other cell-based biologics.

1 .- 59 . (canceled) 
     
     
         60 . A eubacterial minicell comprising an outer membrane, wherein the outer membrane comprises a lipopolysaccharide that comprises a Lipid A component having no myristic acid moiety, wherein the eubacterial minicell comprises one or more bioactive payloads for delivery to a target tissue, organ, or cell. 
     
     
         61 . The eubacterial minicell of  claim 60 , wherein the outer membrane has a composition that results in the reduction of pro-inflammatory immune responses in a mammalian host compared to the outer membrane of eubacterial minicells that are derived from a corresponding wild-type bacteria. 
     
     
         62 . The eubacterial minicell of  claim 60 , wherein at least one of the one or more bioactive payloads is a radioisotope, a polypeptide, a nucleic acid, or a small molecule. 
     
     
         63 . The eubacterial minicell of  claim 60 , wherein at least one of the one or more bioactive payloads is a small molecule drug. 
     
     
         64 . The eubacterial minicell of  claim 60 , wherein at least one of the one or more bioactive payloads is a small molecule imaging agent. 
     
     
         65 . The eubacterial minicell of  claim 60 , wherein at least one of the one or more bioactive payloads is a chemotherapeutic agent. 
     
     
         66 . The eubacterial minicell of  claim 60 , wherein at least one of the one or more bioactive payloads is a nucleic acid. 
     
     
         67 . The eubacterial minicell of  claim 60 , wherein at least one of the one or more bioactive payloads is a therapeutic polypeptide. 
     
     
         68 . The eubacterial minicell of  claim 60 , wherein at least one of the one or more bioactive payloads is a pro-drug converting enzyme. 
     
     
         69 . The eubacterial minicell of  claim 60 , wherein the one or more bioactive payloads comprises a combination of a nucleic acid and a small molecule. 
     
     
         70 . The eubacterial minicell of  claim 60 , wherein the one or more bioactive payloads comprises a combination of a small molecule imaging agent and a small molecule drug. 
     
     
         71 . The eubacterial minicell of  claim 60 , wherein the one or more bioactive payloads comprises a combination of a small molecule drug, a small molecule imaging agent, and a nucleic acid. 
     
     
         72 . The eubacterial minicell of  claim 60 , wherein the one or more bioactive payloads comprises a combination of a nucleic acid and a polypeptide. 
     
     
         73 . The eubacterial minicell of  claim 60 , further comprising a cell-surface localized targeting moiety, wherein the targeting moiety is specific for a surface antigen of a eukaryotic cell. 
     
     
         74 . The eubacterial minicell of  claim 73 , wherein the cell-surface localized targeting moiety is an antibody. 
     
     
         75 . The eubacterial minicell of  claim 73 , wherein the cell-surface localized targeting moiety is a fusion protein, wherein the fusion protein is a fusion of an autotransporter protein and a binding moiety. 
     
     
         76 . The eubacterial minicell of  claim 73 , wherein the surface antigen is a tumor selective antigen. 
     
     
         77 . The eubacterial minicell of  claim 76 , wherein the tumor selective antigen is selected from the group consisting of adipophilin, AIM-2, BCLX (L), BING-4, cleavage and polyadenylation specificity factor (CPSF), Cyclin D1, Dickkopf-1(DKK1), ENAH, Epithelial cell adhesion molecule (Ep-CAM), EPH receptor A3 (EphA3), fibroblast growth factor-5 (FGF5), G250/MN/CAIX, HER-2/neu, IL-13R alpha 2, Intestinal carboxyl esterase, alpha-foetoprotein, macrophage colony stimulating factor (M-CSF), melanoma chondroitin sulfate proteoglycan (MCSP), mdm-2, matrix metalloproteinase-2 (MMP-2), MUC-1, p53, PBF, FRAME, prostate-specific membrane antigen (PSMA), RAGE-1, RGS5, RNF43, RU2AS, secernin 1, SOX10, six-transmembrane epithelial antigen of prostate 1 (STEAP1), survivin, Telomerase, Wilms' tumor gene 1 (WT1), Cdc27, cyclin-dependent kinase 4 (CDK4), CDKN2a, BCR-ABL, BAGE-1, GAGE1-8, GnTV, HERV-K-MEL, KK-LC-1, LAGE-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A9, mucin, NA-88, NY-ESO-1, LAGE-2, SAGE, Sp17, SSX-2, SSX-4, TRAG-3, CD-166, and TRP2-INT2.