Gene therapy for Fabry disease, a deficiency in α-galactosidase A (α-gal A) activity, has the potential to provide a cure for the disorder with a single treatment. Despite modifications to existing vectors, concerns have arisen regarding the risk of genotoxicity associated with the use of retroviruses. To address safety concerns, we propose that expression of a cell surface protein, human CD25 (huCD25) in a bicistronic format, with any therapeutic gene such as α-gal Acan provide a target that can be used to kill transduced cells selectively should transformative events occur. We show that an anti-CD25 antibody and immunotoxin can specifically target and eliminate transduced leukemia cells expressing CD25. In a murine leukemia model, antibody treatment reduced tumor burden 32-fold and increased survival compared with untreated mice. Furthermore, after a bone marrow transplant of therapeutically transduced cells into Fabry mice, antibody treatment reduced the number of retrovirally transduced huCD25-expressing cells in the peripheral blood. A systemic loss of transduced cells with functional consequences was also evident in the liver and spleen. This proof-of-principle study demonstrates that a targeted antibody can reduce tumor burden and selectively clear bicistronically transduced hematopoietic cells that express a target antigen, thus acting as a built-in safety mechanism. Gene therapy for Fabry disease, a deficiency in α-galactosidase A (α-gal A) activity, has the potential to provide a cure for the disorder with a single treatment. Despite modifications to existing vectors, concerns have arisen regarding the risk of genotoxicity associated with the use of retroviruses. To address safety concerns, we propose that expression of a cell surface protein, human CD25 (huCD25) in a bicistronic format, with any therapeutic gene such as α-gal Acan provide a target that can be used to kill transduced cells selectively should transformative events occur. We show that an anti-CD25 antibody and immunotoxin can specifically target and eliminate transduced leukemia cells expressing CD25. In a murine leukemia model, antibody treatment reduced tumor burden 32-fold and increased survival compared with untreated mice. Furthermore, after a bone marrow transplant of therapeutically transduced cells into Fabry mice, antibody treatment reduced the number of retrovirally transduced huCD25-expressing cells in the peripheral blood. A systemic loss of transduced cells with functional consequences was also evident in the liver and spleen. This proof-of-principle study demonstrates that a targeted antibody can reduce tumor burden and selectively clear bicistronically transduced hematopoietic cells that express a target antigen, thus acting as a built-in safety mechanism. Gene therapy has been used successfully to treat a number of inherited disorders.1Aiuti A Slavin S Aker M Ficara F Deola S Mortellaro A et al.Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning.Science. 2002; 296: 2410-2413Crossref PubMed Scopus (981) Google Scholar,2Gaspar HB Parsley KL Howe S King D Gilmour KC Sinclair J et al.Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector.Lancet. 2004; 364: 2181-2187Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar Although many viral and non-viral gene delivery alternatives exist, retroviral vectors offer the advantages of stable integration into host genomes, the ability to infect a wide variety of cell types, and relatively high levels of transgene expression.3Robbins PD Ghivizzani SC Viral vectors for gene therapy.Pharmacol Ther. 1998; 80: 35-47Crossref PubMed Scopus (415) Google Scholar Concerns regarding the safety of integrating vectors have been prompted, however, by the development of leukemia in three X-linked severe combined immunodeficiency patients in a recent clinical trial using an oncoretroviral vector.4Hacein-Bey-Abina S Von Kalle C Schmidt M McCormack MP Wulffraat N Leboulch P et al.LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.Science. 2003; 302: 415-419Crossref PubMed Scopus (3001) Google Scholar A variety of explanations for this outcome have been proposed, but the exact mechanism of leukemogenesis has remained unresolved, as no other clinical trials have reported this type of adverse event.5McCormack MP Rabbitts TH Activation of the T-cell oncogene LMO2 after gene therapy for X-linked severe combined immunodeficiency.N Engl J Med. 2004; 350: 913-922Crossref PubMed Scopus (241) Google Scholar,6Baum C von Kalle C Staal FJ Li Z Fehse B Schmidt M et al.Chance or necessity? Insertional mutagenesis in gene therapy and its consequences.Mol Ther. 2004; 9: 5-13Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar Despite this outcome, retroviral gene therapy continues because of the conceptual effectiveness of the treatment and the fact that gene therapy is the only potential cure available for many disorders such as X-linked severe combined immunodeficiency. Therefore, the development of improved vectors and viable alternative safety strategies is exceedingly important and timely. We have been developing various retrovirus-based gene therapy approaches for Fabry disease, a lysosomal storage disorder resulting from a deficiency of α-galactosidase A (α-gal A) activity.7Brady RO Gal AE Bradley RM Martensson E Warshaw AL Laster L Enzymatic defect in Fabry's disease. Ceramidetrihexosidase deficiency.N Engl J Med. 1967; 276: 1163-1167Crossref PubMed Scopus (844) Google Scholar Fabry disease is a good candidate for gene therapy because there is reduced neurological involvement in contrast to many other lysosomal storage disorders, and supra-physiological levels of α-gal A are well-tolerated.8Kase R Shimmoto M Itoh K Utsumi K Kotani M Taya C et al.Immunohistochemical characterization of transgenic mice highly expressing human lysosomal alpha-galactosidase.Biochim Biophys Acta. 1998; 1406: 260-266Crossref PubMed Scopus (12) Google Scholar Using retroviral gene transfer strategies, we have achieved long-term enzymatic correction and corresponding lipid reduction in a mouse model of Fabry disease by bone marrow transplantation (BMT) of transduced cells9Medin JA Tudor M Simovitch R Quirk JM Jacobson S Murray GJ et al.Correction in trans for Fabry disease: expression, secretion and uptake of alpha-galactosidase A in patient-derived cells driven by a high-titer recombinant retroviral vector.Proc Natl Acad Sci USA. 1996; 93: 7917-7922Crossref PubMed Scopus (65) Google Scholar,10Takenaka T Murray GJ Qin G Quirk JM Ohshima T Qasba P et al.Long-term enzyme correction and lipid reduction in multiple organs of primary and secondary transplanted Fabry mice receiving transduced bone marrow cells.Proc Natl Acad Sci USA. 2000; 97: 7515-7520Crossref PubMed Scopus (89) Google Scholar,11Yoshimitsu M Higuchi K Ramsubir S Nonaka T Rasaiah VI Siatskas C et al.Efficient correction of Fabry mice and patient cells mediated by lentiviral transduction of hematopoietic stem/progenitor cells.Gene Ther. 2007; 14: 256-265Crossref PubMed Scopus (40) Google Scholar and by direct delivery of lentivirus into neonates.12Yoshimitsu M Sato T Tao K Walia JS Rasaiah VI Sleep GT et al.Bioluminescent imaging of a marking transgene and correction of Fabry mice by neonatal injection of recombinant lentiviral vectors.Proc Natl Acad Sci USA. 2004; 101: 16909-16914Crossref PubMed Scopus (78) Google Scholar We have developed and utilized retroviral vectors that engineer expression of both α-gal A and human CD25 (huCD25) in a bicistronic format.13Qin G Takenaka T Telsch K Kelley L Howard T Levade T et al.Preselective gene therapy for Fabry disease.Proc Natl Acad Sci USA. 2001; 98: 3428-3433Crossref PubMed Scopus (66) Google Scholar CD25, also known as the T-cell activation antigen (Tac) and the interleukin (IL)-2 receptor alpha chain-α,14Taniguchi T Minami Y The IL-2/IL-2 receptor system: a current overview.Cell. 1993; 73: 5-8Abstract Full Text PDF PubMed Scopus (739) Google Scholar is incapable of mediating IL-2 internalization or signaling by itself; however, in tandem with the β-chain of the receptor and the γc-chain, it forms the “high-affinity” receptor for IL-2.15Gaffen SL Signaling domains of the interleukin 2 receptor.Cytokine. 2001; 14: 63-77Crossref PubMed Scopus (152) Google Scholar Though it can be induced upon activation, expression of CD25 is absent on resting T cells, B cells, monocytes, and CD34+-enriched cells.16Hanisch UK Quirion R Interleukin-2 as a neuroregulatory cytokine.Brain Res Brain Res Rev. 1995; 21: 246-284Crossref PubMed Scopus (181) Google Scholar,17David D Bani L Moreau JL Demaison C Sun K Salvucci O et al.Further analysis of interleukin-2 receptor subunit expression on the different human peripheral blood mononuclear cell subsets.Blood. 1998; 91: 165-172PubMed Google Scholar Thus, its limited expression pattern and lack of ability to mediate signaling make it a good choice as a cell surface marking protein in bicistronic vectors. In our previous studies, huCD25 expression was used functionally to assess viral titers, for the enrichment of transgene-positive cells before BMT, and for tracking transduced cells after BMT.13Qin G Takenaka T Telsch K Kelley L Howard T Levade T et al.Preselective gene therapy for Fabry disease.Proc Natl Acad Sci USA. 2001; 98: 3428-3433Crossref PubMed Scopus (66) Google Scholar As it is cleaved from the IL-2 receptor complex on the cell surface and can be detected as soluble CD25 (sCD25) in the plasma,18Rubin LA Kurman CC Fritz ME Biddison WE Boutin B Yarchoan R et al.Soluble interleukin 2 receptors are released from activated human lymphoid cells in vitro.J Immunol. 1985; 135: 3172-3177PubMed Google Scholar we have also used sCD25 as a surrogate marker to evaluate the level of transgene expression in an experimental setting.12Yoshimitsu M Sato T Tao K Walia JS Rasaiah VI Sleep GT et al.Bioluminescent imaging of a marking transgene and correction of Fabry mice by neonatal injection of recombinant lentiviral vectors.Proc Natl Acad Sci USA. 2004; 101: 16909-16914Crossref PubMed Scopus (78) Google Scholar In this study, we extend the use of huCD25 expression from our bicistronic retroviral vector constructs into the development and application of a built-in safety mechanism within the gene therapy context. We propose that if an unwanted proliferative abnormality occurs following retroviral gene transfer, huCD25 can act as a target antigen to eliminate transduced cells selectively using either clinically approved anti-CD25 antibodies or newer, highly potent antibody–toxin conjugates (immunotoxins). We chose to use the murine anti-Tac (AT) monoclonal antibody19Uchiyama T Broder S Waldmann TA A monoclonal antibody (anti-Tac) reactive with activated and functionally mature human T cells. I. Production of anti-Tac monoclonal antibody and distribution of Tac (+) cells.J Immunol. 1981; 126: 1393-1397Crossref PubMed Google Scholar fused to saporin (SAP),20Lappi DA Esch FS Barbieri L Stirpe F Soria M Characterization of a Saponaria officinalis seed ribosome-inactivating protein: immunoreactivity and sequence homologies.Biochem Biophys Res Commun. 1985; 129: 934-942Crossref PubMed Scopus (71) Google Scholar a toxin that irreversibly damages ribosomes by cleaving adenine molecules from ribosomal RNA.21Barbieri L Valbonesi P Bonora E Gorini P Bolognesi A Stirpe F Polynucleotide:adenosine glycosidase activity of ribosome-inactivating proteins: effect on DNA, RNA and poly(A).Nucleic Acids Res. 1997; 25: 518-522Crossref PubMed Scopus (265) Google Scholar We have demonstrated both in vitro and in vivo that the AT–SAP (ATS) complex can specifically target and kill retrovirally transduced cells that express huCD25. Importantly, we have achieved enzymatic correction of a mouse model of Fabry disease using our bicistronic vector and were then able to remove transduced cells using both ATS and AT. Thus, we propose this model of using a cell surface antigen such as huCD25 in a bicistronic gene expression cassette as a novel safety mechanism for retroviral vectors. We first wanted to determine the specificity and efficacy of the huCD25-targeted immunotoxin ATS. A murine myeloid leukemia cell line, C1498, was infected with a lentiviral vector pHR′cPPT-EF-α-gal A-IRES-huCD25-W-SIN (LV/α-gal A/huCD25) that is engineered to express both human α-gal A and huCD25.12Yoshimitsu M Sato T Tao K Walia JS Rasaiah VI Sleep GT et al.Bioluminescent imaging of a marking transgene and correction of Fabry mice by neonatal injection of recombinant lentiviral vectors.Proc Natl Acad Sci USA. 2004; 101: 16909-16914Crossref PubMed Scopus (78) Google Scholar Infected pools were enriched for expression of huCD25 by magnetic activated cell sorting. We tested two populations of cells that have a broad spectrum of huCD25 expression with a 5 nM concentration of each reagent: ATS, AT, control immunogloblin (Ig)G Ab conjugated to SAP (IgG-SAP), or SAP only. These populations, shown in Figure 1a and b, were 93% and 45% positive for huCD25 expression, respectively. MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assays confirmed that both populations of cells treated with ATS showed reduced proliferation (Figure 1c and d) and increased cell death as measured by lactate dehydrogenase release (Figure 1e and f) compared with cells treated with other reagents. Non-transduced cells did not show any inhibition of proliferation or increased cytotoxicity when treated with ATS (data not shown). Next, we tested the ability of ATS to clear a clonal population of transduced cells. A single-cell clone expressing huCD25 (C1498/huCD25) was isolated from the infected pool of cells by flow cytometry–based sorting (Figure 2a). Both C1498/huCD25 and C1498 non-transduced (C1498 NT) cells were incubated with increasing concentrations of each reagent. We then measured the effects on cellular proliferation and cell killing. As shown in Figure 2b, inhibition of cellular proliferation was significantly higher (P < 0.001) when cells were treated with ATS than when cells were treated with control reagents. This effect was specific to cells expressing huCD25, as C1498 NT cells treated with ATS did not show the same level of impaired growth. Similar results were obtained from a lactate dehydrogenase release assay. At low doses (<1 nM), cell killing was higher in cells treated with ATS than in cells treated with control reagents (P < 0.001) (Figure 2c). Leukemia model. As an initial step toward determining whether treatment with a CD25 antibody or immunotoxin could clear huCD25-expressing leukemic cells in our mouse model of Fabry disease, we first optimized the dose of C1498 leukemia cells to use in this strain. Increasing doses (1 × 103 to 1 × 106) of C1498 NT cells were injected into Fabry mice and the effects were monitored. Although leukemic cells were not present in the peripheral blood, we found systemic subcutaneous invasion, splenomegaly, and lymphoadenopathy, which mimics some leukemic phenotypes (data not shown). For cell doses of 1 × 103 and 1 × 104 cells/mouse, it was found that 100% and 70% of mice, respectively, survived the challenge (data not shown). For higher cell doses of 1 × 105 and 1 × 106 cells/mouse, 100% of the mice succumbed to the leukemia within 60 days and 30 days, respectively (data not shown). To obtain a more clinically relevant leukemia model, we chose to use a cell dose of 1 × 106 cells/mouse for future studies as at this higher cell dose the phenotype of the transplanted mice progressed to the disease state more quickly and aggressively. As no previous in vivo studies have been carried out with murine ATS and most studies using other AT derivatives use receptor-saturating doses of antibody,22Zhang Z Zhang M Garmestani K Talanov VS Plascjak PS Beck B et al.Effective treatment of a murine model of adult T-cell leukemia using 211At-7G7/B6 and its combination with unmodified anti-Tac (daclizumab) directed toward CD25.Blood. 2006; 108: 1007-1012Crossref PubMed Scopus (28) Google Scholar it was next necessary to determine an effective dose of immunotoxin. We tested the ability of two different doses of ATS to eliminate huCD25-expressing cells in Fabry mice challenged with C1498/huCD25 leukemia. Mice were lethally irradiated and injected with 1 × 106 C1498/huCD25 cells and supportive syngeneic BM cells. At days 2, 4, and 6 after leukemic transplant, animals were injected with either 5 μg ATS or 20 μg ATS, injected with SAP only, or left untreated (n = 3 per group). Eleven days after challenge, blood was sampled and plasma analyzed for levels of sCD25 by enzyme-linked immunosorbent assay (ELISA). Evaluation of sCD25 levels is a common method used in the clinical setting to monitor tumor burden and treatment response in patients with CD25-expressing lymphoma and leukemia.23Perez-Encinas M Villamayor M Campos A Gonzalez S Bello JL Tumor burden and serum level of soluble CD25, CD8, CD23, CD54 and CD44 in non-Hodgkin's lymphoma.Haematologica. 1998; 83: 752-754PubMed Google Scholar This method also allows sensitive detection of the presence of CD25-positive cells for such studies as it can reflect contributions from concealed populations. As shown in Figure 3, treatment with ATS significantly reduced sCD25 (P < 0.05) levels compared with animals treated with the control reagent, SAP, and with those animals left untreated. As treatment with the lower dose of 57 μg of ATS had a similar effect to treatment with the 20 μg dose (Figure 3), we chose to use the lower dose of ATS in future experiments because this was more cost-effective and may lower the risk of secondary or non-specific toxicities. To test the efficacy of our CD25-targeting approach further, a larger experiment using 5 μg doses of ATS was then performed. Mice were lethally irradiated and injected with 1 × 106 C1498/huCD25 or C1498 NT cells along with supportive syngeneic BM cells via the tail vein. Mice transplanted with C1498/huCD25 cells were then treated with equimolar amounts of ATS, AT, or IgG-SAP. Mice transplanted with C1498 NT cells were treated with 57 μg ATS as a control. All animals were bled on days 7, 11, and 18 after transplantation, and levels of sCD25 in the plasma were measured by ELISA. As shown in Figure 4a, in mice challenged with C1498/huCD25 cells, at 18 days after transplantation average plasma sCD25 levels were significantly lower in animals treated with ATS (474 pg/ml) and AT (848 pg/ml) than in mice treated with IgG-SAP (4,762 pg/ml; P < 0.01) or not treated (15,450 pg/ml; P < 0.05). This indicates a lower tumor burden in mice treated with both CD25-targeted reagents, ATS and AT. The inherent α-gal A deficiency of Fabry mice and the fact that the transplanted tumor cells were engineered to express α-gal A meant that differences in α-gal A activity itself could be used as another surrogate marker of tumor burden. Therefore, plasma α-gal A activity was measured and was found to be lowest in mice treated with ATS (16 nmol/hour/ml) and AT (21 nmol/hour/ml) (Figure 4b). These levels were significantly lower than those in mice that received IgG-SAP (47 nmol/hour/ml; P < 0.001 and P < 0.01, versus ATS and AT, respectively) or that were left untreated (77 nmol/hour/ml; P < 0.05). Therefore, both ATS and AT are able to de-bulk tumor burden in this huCD25-expressing leukemia model. To determine the ability of anti-CD25 antibodies to affect survival, animals were monitored daily; a Kaplan–Meier representation of survival is shown in Figure 4c. In mice treated with ATS, the median survival duration was 29 days. This was significantly higher (P < 0.01) than that seen in mice that were not treated (median survival = 23 days). Increased survival was also seen in mice treated with AT (median survival of 30 days, P <0.05 versus untreated mice). Therefore, even in the context of a very high leukemic burden, treatment with CD25-targeted antibodies increased survival compared with control treatments. Note that these results are representative of two independent experiments. BMT model. We next wanted to test our clearance strategy in the context of a therapeutic BMT model. BMT is a common gene therapy approach,24Boelens JJ Trends in haematopoietic cell transplantation for inborn errors of metabolism.J Inherit Metab Dis. 2006; 29: 413-420Crossref PubMed Scopus (95) Google Scholar and incorporation of a cell surface protein that can be targeted can improve the safety of the system. Murine bone marrow mononuclear cells (BMMNCs) were isolated and infected twice with one of two ecotropic oncoretroviral vectors, either E86/pMFG/α-gal A/IRES/huCD25 clone 21 (RV/α-gal A/huCD25) or E86/pUMFG/enYFP (RV/enYFP).13Qin G Takenaka T Telsch K Kelley L Howard T Levade T et al.Preselective gene therapy for Fabry disease.Proc Natl Acad Sci USA. 2001; 98: 3428-3433Crossref PubMed Scopus (66) Google Scholar Flow cytometry analysis of these transduced BMMNCs showed that cells infected with RV/α-gal A/huCD25 were approximately 20% positive for expression of huCD25 (Figure 5a) and cells infected with RV/enYFP were approximately 20% positive for enhanced yellow fluorescent protein (enYFP) expression (Figure 5b). Cells were then injected into lethally irradiated Fabry mice, which were monitored monthly for engraftment. At 8 weeks after transplantation, plasma from recipient Fabry mice was analyzed for α-gal A activity and for levels of sCD25. Average plasma α-gal A activity in mice transplanted with BMMNCs infected with RV/α-gal A/huCD25 was 65 nmol/hour/ml, approximately sixfold higher than in both control Fabry mice and mice transplanted with RV/enYFP-infected BMMNCs (Figure 5c). This indicates that we achieved therapeutic correction of α-gal A activity in Fabry animals at levels approximately twofold higher than in wild-type C57BL/6 mice (Figure 5c). At this time, the average level of sCD25 in the plasma of Fabry mice transplanted with BMMNCs infected with RV/α-gal A/huCD25 was 1212 ± 370 pg/ml. In contrast, sCD25 was undetectable in mice transplanted with RV/enYFP-infected cells, in wild-type C57BL/6 mice, and in untreated Fabry mice (data not shown). Mice were then treated with either ATS, AT, or IgG-SAP, as in our leukemia model (see above). Seven days after the third dose of immunotoxin, plasma was sampled to determine the effect of treatment. Comparisons were made with pre-treatment values collected for each mouse at 8 weeks after transplantation. As shown in Figure 6a, treatment with ATS resulted in lower plasma sCD25 levels than in mice that were treated with IgG-SAP or mice that were not treated (P < 0.05). In addition, analysis of huCD25 expression on peripheral blood (PB)mononuclear cells by flow cytometry showed that mice treated with ATS had significantly reduced numbers of huCD25-expressing PB mononuclear cells than mice treated with IgG-SAP (P < 0.01) or untreated mice (P < 0.05) (Figure 6b). Similar effects were observed in mice treated with AT, further supporting the conceptual ability of targeted anti-CD25 antibodies to eliminate retrovirally transduced donor hematopoietic cells in vivo. Expression of enYFP was monitored before and after treatment with ATS and it was found that levels remained stable over the course of the experiment (Figure 6c), demonstrating the specificity of the immunotoxin for cells expressing huCD25. To examine the effect of a later administration of antibody or immunotoxin, one final dose was administered and then mice were killed. Enzyme activity was measured in various tissues to determine the systemic effect of each reagent. PB mononuclear cells from mice that were treated with ATS showed significantly lower (P < 0.05) α-gal A activity than mice treated with IgG-SAP (Figure 7a). Similarly, α-gal A activity in the livers of mice treated with ATS was significantly lower (P < 0.05) than enzyme activity in the livers of mice treated with AT or IgG-SAP or untreated mice (Figure 7b). Likewise, in the spleens of mice treated with ATS, there was significantly lower (P< 0.01) α-gal A activity than in IgG-SAP-treated or untreated mice (Figure 7c). Gene therapy is the most promising curative treatment for monogenetic diseases such as lysosomal storage disorders.25Futerman AH van Meer G The cell biology of lysosomal storage disorders.Nat Rev Mol Cell Biol. 2004; 5: 554-565Crossref PubMed Scopus (628) Google Scholar Although our laboratory has made considerable advances toward the development of retrovirus-based gene therapy strategies for Fabry disease, concerns remain regarding the safety of integrating vectors. To address this issue, we propose that a cell surface marker such as huCD25 can act as an effective built-in safety mechanism in the event of insertional genotoxicity by facilitating the clearance of transduced cells with a specifically targeted immunotoxin. Our laboratory has previously used huCD25 in combination with α-gal A in studies evaluating the efficacy of retroviral gene therapy for Fabry disease.12Yoshimitsu M Sato T Tao K Walia JS Rasaiah VI Sleep GT et al.Bioluminescent imaging of a marking transgene and correction of Fabry mice by neonatal injection of recombinant lentiviral vectors.Proc Natl Acad Sci USA. 2004; 101: 16909-16914Crossref PubMed Scopus (78) Google Scholar,13Qin G Takenaka T Telsch K Kelley L Howard T Levade T et al.Preselective gene therapy for Fabry disease.Proc Natl Acad Sci USA. 2001; 98: 3428-3433Crossref PubMed Scopus (66) Google Scholar We have not observed any untoward effects of exogenously expressing this protein nor have we observed altered therapeutic effects of this surface antigen on α-gal A–mediated correction in vivo. Monoclonal antibodies have been successfully used in the clinic for many years to treat hematological malignancies, with minimal toxicity.26Dillman RO Monoclonal antibody therapy for lymphoma: an update.Cancer Pract. 2001; 9: 71-80Crossref PubMed Scopus (40) Google Scholar,27Gokbuget N Hoelzer D Treatment with monoclonal antibodies in acute lymphoblastic leukemia: current knowledge and future prospects.Ann Hematol. 2004; 83: 201-205Crossref PubMed Scopus (72) Google Scholar For instance, rituximab, an anti-CD20 antibody, has been used to treat a variety of lymphoid malignancies.26Dillman RO Monoclonal antibody therapy for lymphoma: an update.Cancer Pract. 2001; 9: 71-80Crossref PubMed Scopus (40) Google Scholar, 28McLaughlin P Grillo-Lopez AJ Link BK Levy R Czuczman MS Williams ME et al.Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program.J Clin Oncol. 1998; 16: 2825-2833Crossref PubMed Scopus (2570) Google Scholar, 29Grillo-Lopez AJ Hedrick E Rashford M Benyunes M Rituximab: ongoing and future clinical development.Semin Oncol. 2002; 29: 105-112Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 30Cvetkovic RS Perry CM Rituximab: a review of its use in non-Hodgkin's lymphoma and chronic lymphocytic leukaemia.Drugs. 2006; 66: 791-820Crossref PubMed Scopus (95) Google Scholar In addition, a strategy for clearing transduced hematopoietic cells in vivo using an anti-CD20 Ab was proposed for the treatment of graft-versus-host disease.31Introna M Barbui AM Bambacioni F Casati C Gaipa G Borleri G et al.Genetic modification of human T cells with CD20: a strategy to purify and lyse transduced cells with anti-CD20 antibodies.Hum Gene Ther. 2000; 11: 611-620Crossref PubMed Scopus (114) Google Scholar The premise is that T cells can be transduced with a viral vector carrying the complementary DNA for CD20 before BMT, and if graft-versus-host disease occurs, then anti-CD20 antibodies can be used to eliminate the donor T cells. These studies have shown promising results in vitro; however, no studies have been carried out to demonstrate efficacy in vivo.32van Meerten T Claessen MJ Hagenbeek A Ebeling SB The CD20/alphaCD20 'suicide' system: novel vectors with improved safety and expression profiles and efficient elimination of CD20-transgenic T cells.Gene Ther. 2006; 13: 789-797Crossref PubMed Scopus (30) Google Scholar,33Serafini M Manganini M Borleri G Bonamino M Imberti L Biondi A et al.Characterization of CD20-transduced T lymphocytes as an alternative suicide gene therapy approach for the treatment of graft-versus-host disease.Hum Gene Ther. 2004; 15: 63-76Crossref PubMed Scopus (79) Google Scholar Aberrant levels of CD25 expression characterize numerous disorders such as adult T-cell leukemia/lymphoma, Hodgkin's lymphoma, hairy cell leukemias, and true histiocytic lymphomas.34Kreitman RJ Wilson WH White JD Stetler-Stevenson M Jaffe ES Giardina S et al.Phase I trial of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) in patients with hematologic malignancies.J Clin Oncol. 2000; 18: 1622-1636Crossref PubMed Scopus (391) Google Scholar Treatment of these diseases using antibodies against CD25, as well as newer recombinant immunotoxins, has resulted in complete and partial remissions in patients.34Kreitman RJ Wilson WH White JD Stetler-Stevenson M Jaffe ES Giardina S et al.Phase I trial of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) in patients with hematologic malignancies.J Clin Oncol. 2000; 18: 1622-1636Crossref PubMed Scopus (391) Google Scholar,35Kreitman RJ Wilson WH Robbins D Margulies I Stetler-Stevenson M Waldmann TA et al.Responses in refractory hairy cell leukemia to a recombinant immunotoxin.Blood. 1999; 94: 3340-3348Crossref PubMed Google Scholar Currently, anti-CD25 antibodies are widely used for the prevention of renal graft rejection and in some cases for prophylactic treatment against graft-versus-host disease.36Lin M Ming A Zhao M Two-dose basiliximab compared with two-dose daclizumab in renal transplantation: a clinical study.Clin