Cell Targeting of a Pore-Forming Toxin, CytA δ-Endotoxin fromBacillus thuringiensisSubspeciesisraelensis, by Conjugating CytA with Anti-Thy 1 Monoclonal Antibodies and Insulin

Abstract

The cytolytic protein toxin CytA was linked to two monoclonal antibodies (mAb) directed against the mouse or the rat Thy 1 antigen. The purified CytA-mAb conjugates were not toxic to either target or nontarget cells. The conjugates did bind specifically to target cells since they agglutinated the target cells but not nontarget cells. When the conjugates were treated with dithiothreitol, the released CytA was toxic to all cells tested. These results suggested that the attachment of CytA to a molecule such as the mAb prevented it from forming a pore. Another conjugate was made by linking CytA to insulin. The purified insulin-CytA conjugate bound to and intoxicated cells bearing a high number of insulin receptors. Furthermore, the conjugate was far less toxic to cells expressing a low number of insulin receptors and not toxic to a known CytA target cell line from Aedes aegypti. However, CytA released from the conjugate by reduction was toxic to all cells tested. These results suggested that the cytotoxicity exhibited by CytA in the conjugate form against cells bearing insulin receptors was mediated through insulin and that, in the conjugate form, CytA no longer shows its broad in vitro cytolytic activity. The difference in toxicity between CytA-mAb conjugates and insulin-CytA conjugate is discussed in relation to size of the ligands, the number, distribution, and mobility of the target molecules, and intracellular trafficking.

Partial Restoration of Antibacterial Activity of the Protein Encoded by a Cryptic Open Reading Frame ( cyt1Ca ) from Bacillus thuringiensis subsp. israelensis by Site-Directed Mutagenesis

“…kurstaki (41) or Escherichia coli (11,24) causes loss of viability, accompanied in the latter by pronounced nucleoid compaction (25). The cytotoxicity of Cyt1Aa against eukaryotic cells may render it useful for cancer treatment if specificity to tumor cells is raised by, for example, linking it chemically to targeting ligands (2). Due to potential important biological role for Cyt toxins, screening for new cyt genes is on the current agenda (18).…”

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

“…Linking Cyt1Aa via Cys-190 to the insulin B-chain elevated its specificity to NIH 3T3 NIR3.5 cells overexpressing the insulin receptor (26), but the cytotoxicity of the conjugate was lower than that of the free Cyt1Aa. The structure of Cyt1Aa has still not been solved, but comparing its amino acid sequence to that of Cyt2Aa (38) one can see that Cys-190 is located in the middle of ␤6 strand that is thought to be important for channel formation (38,39).…”

“…Some unsuccessful attempts have been made to exploit this activity. A Cyt1Aa-anti-Thy1.1 fusion protein, for example, lacked cytolytic specificity against a Thy 1.1-expressing mouse AKR-A/2 lymphoma cell line (26). In the same study, a Cyt1Aa-insulin conjugate displayed specificity for target cells overexpressing insulin receptors, but the activity level and response rate were lower than for the free Cyt1Aa.…”

A Single Chain Fv Fragment of P-glycoprotein-specific Monoclonal Antibody C219

“…charged peptide sequences (45)) or by targeting cell surface receptors (e.g. the insulin receptor (46)). An alternative approach relevant to gene therapy protocols is to express products intracellularly (47,48).…”