BACKGROUND Cellular therapies could play a role in cancer treatment and regenerative medicine if it were possible to quickly eliminate the infused cells in case of adverse events. We devised an inducible T-cell safety switch that is based on the fusion of human caspase 9 to a modified human FK-binding protein, allowing conditional dimerization. When exposed to a synthetic dimerizing drug, the inducible caspase 9 (iCasp9) becomes activated and leads to the rapid death of cells expressing this construct. METHODS We tested the activity of our safety switch by introducing the gene into donor T cells given to enhance immune reconstitution in recipients of haploidentical stem-cell transplants. Patients received AP1903, an otherwise bioinert small-molecule dimerizing drug, if graft-versus-host disease (GVHD) developed. We measured the effects of AP1903 on GVHD and on the function and persistence of the cells containing the iCasp9 safety switch. RESULTS Five patients between the ages of 3 and 17 years who had undergone stem-cell transplantation for relapsed acute leukemia were treated with the genetically modified T cells. The cells were detected in peripheral blood from all five patients and increased in number over time, despite their constitutive transgene expression. A single dose of dimerizing drug, given to four patients in whom GVHD developed, eliminated more than 90% of the modified T cells within 30 minutes after administration and ended the GVHD without recurrence. CONCLUSIONS The iCasp9 cell-suicide system may increase the safety of cellular therapies and expand their clinical applications. (Funded by the National Heart, Lung, and Blood Institute and the National Cancer Institute; ClinicalTrials.gov number, NCT00710892.)

Article abstract-We treated 18 clinically definite relapsing-remitting MS patients with recombinant gamma interferon in a pilot study designed to evaluate toxicity and dosage. Patients received low (1 pg), intermediate (30 pg) dence that a viral infection, an immunoregulatory defect, or both, may be implicated in disease In a double-blind, placebo-controlled crossover trial of natural alpha IFN given systemically,* the number of acute exacerbations in patients with relapsing-remitting MS was reduced, but the study was complicated by the crossover design and a marked placebo effect. Natural beta IFN administered intrathecally to 10 patients5 appeared to prevent exacerbations; however, the study lacked a placebo-treated control group. Recently, a multicenter double-blind controlled trial of intrathecal beta IFN6 confirmed the favorable results of the earlier study. The first clinical trial of recombinant IFN in MS, in which 2 million units of alpha, IFN were given subcutaneously three times per week for 1 year, was recently ~ompleted.~ There was no therapeutic effect, possibly because the dose was too low. Other studies, using higher systemic doses of recombinant IFN, are in progress.Gamma or immune IFN has many of the antiviral and immunoregulatory properties of other interferonss; in addition, it can activate macrophages and induce class I1 histocompatibility antigens on monocytes,9 endothelial cells,1° and astrocytes." These are properties that could stimulate an autoimmune process. On the other hand, recent reports of deficient gamma IFN production in MS patients12J3 suggested the possibility of a therapeutic trial. We therefore performed a pilot study of recombinant gamma IFN to determine toxicity and dose, and to judge whether a larger, long-term clinical trial would be feasible. The results have been partially reported elsewhere as a preliminary comm~nication.'~ Methods. Patients and treatment protocol. Eighteen patients with clinically definite relapsing-remitting MS participated. There were 3 men and 15 women ranging in age from 19 to 42 years. All patients had had at least two exacerbations in the preceding 2 years, and were in remission at the time of entry. The study was approved by the University of Maryland Human Volunteers Research Committee. Recombinant gamma IFN (Immuneron, Biogen Research, Cambridge, MA) was given in doses of 1, 30, or 1,000 pg, corresponding approximately to 1.5 X lo4, 4.5 X lo5, and 1.5 X lo7 IU of IFN, respectively. Patients were selected at random to receive the low, intermediate, or high doses twice a week for 4 weeks. One half of each dose was administered as an intravenous bolus; the remainder was given as an intravenous infusion over 2 hours. At the beginning and end of the trial, patients were scored according to the

and Epstein-Barr virus (EBV)-specific responses in 4 of 6 evaluable patients at dose level 2 as early as 2 to 4 months after transplantation, whereas such responses were not observed until 6 to 12 months in dose-level 1 patients. The incidence of significant acute (2 of 16) and chronic graft-versus-host disease (GVHD; 2 of 15) was low. These data demonstrate that allodepleted donor T cells can be safely used to improve T-cell recovery after haploidentical SCT and may broaden the applicability of this approach.

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We have selectively depleted host-reactive donor T cells from peripheral blood stem cell (PBSC) transplant allografts ex vivo using an anti-CD25 immunotoxin.

Ricin, a highly potent toxin produced by castor beans, is classified by the Centers for Disease Control and Prevention as a level B biothreat because it is easily produced, readily available, and highly stable. There have been >750 cases of documented ricin intoxication in humans. There is no approved vaccine for ricin. Ricin contains a lectin-binding B chain and a ribotoxic A chain (RTA). In addition to its ribotoxic site, we have identified a separate site on RTA that is responsible for inducing vascular leak syndrome (VLS) in humans. We have generated a recombinant RTA with two amino acid substitutions that disrupt its ribotoxic site (Y80A) and its VLS-inducing site (V76M). This mutant recombinant RTA (named RiVax) was expressed and produced in Escherichia coli and purified. When RiVax was injected i.m. into mice it protected them against a ricin challenge of 10 LD 50 s. Preclinical studies in both mice and rabbits demonstrated that RiVax was safe. Based on these results, we have now conducted a pilot clinical trial in humans under an investigational new drug application submitted to the Food and Drug Administration. In this study, three groups of five normal volunteers were injected three times at monthly intervals with 10, 33, or 100 μg of RiVax. The vaccine was safe and elicited ricin-neutralizing Abs in one of five individuals in the low-dose group, four of five in the intermediate-dose group, and five of five in the high-dose group. These results justify further development of the vaccine.

The anti-pan T cell monoclonal antibody OKT3 was administered daily for 2 weeks in four human renal allograft recipients. The antibody induced a dramatic and immediate depletion of peripheral T cells followed by an in vivo antigenic modulation of the OKT3-defined membrane antigen: after three injections, OKT3-treated patients showed a limited but significant number of OKT3- cells of T cell nature (as defined by OKT4 and OKT8) which recovered the OKT3 receptor after an overnight in vitro incubation in the absence of the monoclonal antibody.

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