Genetic engineering of an immunotoxin to eliminate pulmonary vascular leak in mice

Abstract

Vascular leak syndrome is a major and often dose-limiting side effect of immunotoxins and cytokines. We postulated that this syndrome is initiated by damage to vascular endothelial cells. Our earlier studies identified a three-amino acid motif that is shared by toxins, ribosome-inactivating proteins, and interleukin-2, all of which cause this problem. We have now generated a panel of recombinant ricin A chains with mutations in this sequence or in amino acids flanking it in the three-dimensional structure. These have been evaluated alone and as immunotoxins for activity, ability to induce pulmonary vascular leak in mice, pharmacokinetics, and activity in tumor-xenografted mice. One mutant was comparable to the ricin A chain used before in all respects except that it did not cause vascular leak at the same dose and, when used as an immunotoxin, was more effective in xenografted SCID mice.

Selective Apoptotic Killing of Malignant Hemopoietic Cells by Antibody-Targeted Delivery of an Amphipathic Peptide

“…Third, induction of vascular leak syndrome is often the dose-limiting factor in the therapeutic use of many natural toxins. A conserved three amino acid sequence has been identified as a cause of this syndrome (22). The absence of this motif from the amphipathic peptide suggests that its use in targeted strategies may avoid limitations resulting from vascular leakage.…”

Improved formulation of a recombinant ricin A-chain vaccine increases its stability and effective antigenicity

“…Because of the high intrinsic stability of the vaccine protein, adding stabilizing sugar excipients did not appear to be necessary. In comparison, an alternative ricin vaccine known as RiVax [19][20][21][22], containing substitution mutations at the active site, has a pronounced tendency towards aggregation. A study of required excipients by Peek et al [23] recommended formulation of that protein with 50% glycerol.…”

A Monoclonal Immunoglobulin G Antibody Directed against an Immunodominant Linear Epitope on the Ricin A Chain Confers Systemic and Mucosal Immunity to Ricin

“…Therefore, the current emphasis is on the development of attenuated subunit vaccines (6,16,32,45,52). One of the most promising candidates is a recombinant derivative of RTA containing two point mutations: one in the enzymatic active site (Y80A) and the other in a residue (V76M) involved in eliciting vascular leak syndrome (42)(43)(44)(45)52). This vaccine, known by the trade name RiVax, is safe and immunogenic in mice and rabbits and, when administered intramuscularly, elicits serum antitoxin IgG antibodies capable of protecting animals against a systemic ricin challenge of 10 50% lethal doses (LD 50 s) (42,44).…”