Specific Targeting to Murine Myeloma Cells of Cyt1Aa Toxin from Bacillus thuringiensis Subspecies israelensis

Abstract

Multiple myeloma is currently an incurable cancer of plasma B cells often characterized by overproduction of abnormally high quantities of a patient-specific, clonotypic immunoglobulin "M-protein." The M-protein is expressed on the cell membrane and secreted into the blood. We previously showed that ligand-toxin conjugates (LTC) incorporating the ribosome-inactivating Ricin-A toxin were very effective in specific cytolysis of the anti-ligand antibody-bearing target cells used as models for multiple myeloma. Here, we report on the incorporation of the membrane-disruptive Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis into LTCs targeted to murine myeloma cells. Proteolytically activated Cyt1Aa was conjugated chemically or genetically through either its amino or carboxyl termini to the major peptidic epitope VHFFKNIVTPRTP (p87-99) of the myelin basic protein. The recombinant fusion-encoding genes were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis through the shuttle vector pHT315. Both chemically conjugated and genetically fused LTCs were toxic to anti-myelin basic protein-expressing murine hybridoma cells, but the recombinant conjugates were more active. LTCs comprising the Cyt1Aa toxin might be useful anticancer agents. As a membrane-acting toxin, Cyt1Aa is not likely to induce development of resistant cell lines.Chemotherapy has for many years been an important component of the cancer therapeutic arsenal, but lack of target cell specificity of most anticancer drugs leads to concentration-dependent toxicity of normal cells. The resultant use of suboptimal therapeutic doses precludes the elimination of all the cancer cells, a situation that encourages clinical recurrence of the tumor (1) as well as emergence of resistant clones (2). This scenario has stimulated development of a variety of targeted drug delivery systems, the advantages of which include, aside from target cell specificity, reduced drug dosage and increased cytotoxic efficacy (reviewed in Refs. 3, 4).As components of the targeting system, delivery moieties with a wide range of biological functions have been employed, such as monoclonal antibodies, receptor binding peptides, liposomes, and nucleotide aptamers (4 -6). Most of the cytotoxic components currently used are designed to interfere with intracellular biochemical processes (7-10). For instance, we recently demonstrated that delivery of the ribosome-inactivating protein Ricin-A efficiently destroys murine myeloma cells (11) and that targeted, chemiluminescent activation of the photodynamic process results in endomembrane destruction (12). The activity of these types of cytotoxic systems requires endocytosis, and this process often induces intracellular activation of resistance mechanisms (2). There is therefore a need to expand the portfolio of cytotoxic agents, for instance to those whose mode of action is endocytosis-independent.Cyt1Aa is a plasma membrane-acting cytotoxin isolated from the paracrystalline ␦-endotoxin of the bacterium Bacillus thurin...

Cyt1Aa Toxin: Crystal Structure Reveals Implications for Its Membrane-Perforating Function

“…57,58 The crystal structure of Cyt1Aa and its unique segments described here broaden our understanding of the mechanisms underlying its toxicity and hence significantly contribute to the achievement of this goal.…”

Construction and Application in Plasmid Vectors of Bacillus cereus Group

“…All of these have increased expression of Cry1Ac, which increases insecticidal activity in the recombinant strains. The product of the expression of the myelin basic protease gene and cyt1Aa fusion expression vector is active in mouse myeloma cells, and it can be developed into effective anti-cancer drugs (Cohen et al 2007).…”

Expression in Escherichia coli of the Native cyt1Aa from Bacillus thuringiensis subsp. israelensis

“…In addition, Cyt1Aa exerts, a general cytolytic effect on a variety of prokaryotic (5,20) and eukaryotic (16) cells by a detergent-like mode of action (12). This property makes it potentially useful for treating bacterial infections and even cancerous growths if specificity to tumor cells is raised by genetically fusing Cyt1Aa to targeting ligands (6).…”

“…On the other hand, the unaffected bactericidal level against its host E. coli implies that the above-described modification is probably too small to impair proper folding for antibacterial activity of Cyt1Aa. This fact can be used to engineer genetically modified versions of Cyt1Aa fused to short ligand-peptides that target its cytolytic activity to specific cell types as desired (6 …”