In vitro and in vivo antileukemic activity of B43-pokeweed antiviral
From www.bloodjournal.org by guest on October 21, 2014. For personal use only. 1995 86: 4228-4233 In vitro and in vivo antileukemic activity of B43-pokeweed antiviral protein against radiation-resistant human B-cell precursor leukemia cells KG Waddick, DE Myers, R Gunther, LM Chelstrom, M Chandan-Langlie, JD Irvin, N Tumer and FM Uckun Updated information and services can be found at: http://www.bloodjournal.org/content/86/11/4228.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved. From www.bloodjournal.org by guest on October 21, 2014. For personal use only. In Vitro and In Vivo Antileukemic Activity of B43-Pokeweed Antiviral Protein Against Radiation-Resistant Human B-Cell Precursor Leukemia Cells BY Kevin G. Waddick, Dorothea E. Myers, Roland Gunther, Lisa M. Chelstrom, Mridula Chandan-Langlie, James D. Irvin, Nilgun Tumer, and Fatih M. Uckun B-cell precursor (BCP) leukemiathe is most commonform of childhood cancer and represents oneof the most radiationresistant forms of human malignancy.In this study, we examined the antileukemicefficacy of the B43 (anti-CD19)pokeweed antiviral protein (B43-PAPI immunotoxin against radiation-resistant BCP leukemiacells.B43-PAPcaused apoptosis of radiation-resistantprimary BCP leukemia cells, killed greater than 999’0 of radiation-resistantprimary leukemic progenitor cells from BCP leukemia patients, and con- ferred extended survival to severe combined immunodeficiency (SCID) mice xenograftedwith radiation-resistant human BCP leukemia. Furthermore, the combination of B43PAP and total body irradiation (TBI) was more effectivethan TB1 alone in two SCID mouse bone marrow transplantation models of radiation-resistant human BCP leukemia. Thus, B43-PAP may proveuseful in the treatment of radiationresistant BCP leukemia. 0 1995 by The American Society of Hematology. B ciency (SCID) mice xenografted with radiation-resistant human BCP leukemia. We also demonstrate that the combination of B43-PAP and TB1 is more effective than TB1 alone in two SCID mouse BMT models of radiation-resistant human BCP leukemia. -CELL PRECURSOR (BCP) leukemia is the most common form of childhood cancer“6 and represents one of the most radiation-resistant forms of human malignancy.”” Recent studies demonstrated that greater than 75% of clonogenic BCP leukemia cells from more than one third of the patients with newly diagnosed disease, and virtually all of the relapsed patients, are able to repair potentially lethal or sublethal DNA damage induced by radiation doses that correspond to the clinical total body irradiation (TBI) dose fractions (ie, 2 to 3 Gy).”” Consequently, the vast majority of high-risk BCP leukemia patients undergoing TB1 in the context of bone marrow transplantation (BMT) relapse within the first 12 months, and only 15%to 20% survive disease-free beyond the first 2 year^.^.".'^ Thus, the major challenge in BMT for BCP acute lymphoblastic leukemia (ALL) is the development of novel and more effective conditioning strategies. Here, we provide experimental evidence that B43-pokeweed antiviral protein (PAP), a potent anti-CD19 immunotoxin, causes apoptosis of radiation-resistant primary BCP leukemia cells, kills greater than 99% of radiation-resistant leukemic progenitor cells from patients with leukemia, and confers extended survival to severe combined immunodefiFrom the Biotherapy Program, Departmentsof Therapeutic Radiology-Radiation Oncology, Pediatrics, Pharmacology,and Comparative Medicine/ResearchAnimal Resources, and the Centralized lmmunoconjugate Reference Laboratory of the Childrens Cancer Group, University of Minnesota Health Sciences Center, Minneapolis,MN; the Department of Chemistry, Southwestern Texas Stare University, San Marcos, 7 X ; and the AgBiotech Center,Rutgers, Rutgers University, New Brunswick, NJ. Submitted April 25, 1995; accepted July 31, 1995. 1I , Supported in part by GrantsNo. CA-42633, CA-21 737, CA-421 CA-61549, and CA-60437 from the National Cancer Institute, National Institutes of Health. F.M.U.isa Stohlman Scholar ofthe Leukemia Society of America. Address reprint requests to Fatih M. Uckun, MD, University oj Minnesota Biotherapy Program,2685 Parton Rd,Roseville, MN 55113. The publication costsof this article were defrayedin part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1995 by The American Society of Hematology. 0006-4971/95/861 l-0022$3.00/0 4228 MATERIALSANDMETHODS B43-PAP irnrnunotoxin. PAP was isolated from spring leaves of pokeweed and purified by ion-exchange chromatography, as previously rep~rted.’~”~ PAP amino groups were thiolated using2iminothiolane, and modified PAP was mixed with SPDP-modified monoclonal antibodies (MoAbs) using a 3 5 1 molar ratio of PAP to MoAb to generate PAP immunotoxins in a sulfhydryl-disulfide exchange reaction.ls The immunotoxins were initially purified by gel filtration high performance liquid chromatography toremove unreacted PAP and high-molecular-weight (greater than 300 !dl) conjugates/aggregates.I5CM-Sepharose ion-exchange chromatography was subsequently used to purify the immunotoxins from unconjugated MoAb, as previously described.15The procedures used for the large scale production of B43 MoAb in a dedicated ACUSYSTJr benchtop hollow-fiber celI culture system (Endotronics, Coon Rapids, MN), purification using the Affi-Prep Protein A MAPS system (Bio-Rad Laboratories, Hercules, CA), and conjugation to PAP have been previously described in detail.14.’5 Primay BCP leukemia cells. For the in vitro and in vivo analyses of the antileukemic activity of the B43-PAP immunotoxin, we used leukemia cell-enriched, cryopreserved bone marrow samples from six patients with relapsed BCP leukemia (provided by Dr K. Gajl-Peczalska, University of Minnesota Cell Marker Laboratory, Minneapolis, MN) and five patients with newly diagnosed t(4; ll)(q2l;q23)-canying BCP leukemia (provided by Dr W. Crist, St Jude Childrens Research Hospital, Memphis, TN) under the exemption category (45 CFR Part 46.101;b category 4 Existing data; Records review; Pathologic Specimens) in accordance with Department ofHealth and Human Services (DHHS) guidelines. These cryopreserved specimens were previously obtained from routine diagnosticbonemarrow aspirates before standard therapy, and informed consent for treatment was obtained from parents, patients, or both based on DHHS guidelines. In vitro treatment of primary BCP leukemia cells with B43-PAP immunotoxin or y-rays. Primary BCP leukemia cells (1 X 105/mL in alpha-minimal essential medium [MEM] supplemented with 5% [vol/vol] fetal bovine serum) were either ( 1 ) irradiated with 50 cGy, 100 cGy, 200 cGy, 400 cGy, and 800 cGy y-rays in a single exposure (100 cGy/min) using a “’CS irradiator, aspreviouslydescribed,’ I ’ or (2) treated for 8 hours at 37°C with1,000 ng/mL B43-PAP immunotoxin. Controls included unirradiateduntreated cells aswell as cells treated with 1,000 ng/mL G3.7 (anti-CD7)-PAP, a control imBlood, Vol 86, No 11 (December I), 1995: pp 4228-4233 From www.bloodjournal.org by guest on October 21, 2014. For personal use only. 4229 B43-PAP IMMUNOTOXIN munotoxin that does not react with BCP leukemia cells. After irradiation or treatment, 1 X IO5 cells per sample were assayed in duplicate for leukemic progenitor cell (LPC)-derived blast colony formation, as described.'"' The radiation survival curves were constructed, and the SF, (surviving fraction at 2 Gy) and alpha (a)values (initial slope reflecting the steepness of the linear component of cell killing in the linear-quadratic model of cell survival) were determined using computer programs for the analysis of cell survival data, as described.'"' According to radiation biology literature, a values 50.2 Cy" and SF2 values 20.5 are indicative of radiation resistance.'." Apoptosisassays. To detect apoptotic changes, radiation-resistant primary BCP leukemia cells were harvested 16 to 18 hours after continuous exposure to the B43-PAP immunotoxin (0.01, 0.1, 1.0, or 10.0 pg/mL) or the control immunotoxin TXU-PAP (1 or 10 pg/ mL) or irradiation with y-rays (2, 4, or 8 Gy). DNA was prepared from Triton-X-100 lysates for analysis of fragmentati~n.'~.''In brief, equal numbers of cells were subjected to experimental treatments; lysed in hypotonic 10 mmol/L Tris-HC1 (pH 7.4). 1 mmoVL EDTA, 0.2% Triton-X-100 detergent; and subsequently centrifuged at 11,OOOg. This protocol allows the recovery of intact chromosomal DNAin the pellet and fragmented DNAinthe supernatant. To detect apoptosis-associated DNA fragmentation, supernatants were electropheresed on a 1.2% agarose gel, and the DNA fragments were visualized by ultraviolet light after staining with ethidium bromide. In some experiments, cells were pretreated with excess Leu 12 (antiCD19; 10 pg/mL) or control TXU (anti-CD7; 10 pg/mL) monoclonal antibodies before addition of B43-PAP to examine if the B43-PAPinduced apoptosis can be prevented specifically by occupation of the surface CD19 receptors with anti-CD19 antibody molecules.'6 LPC assays. Primary BCP leukemia cells (1 X lo5 cells/mL in a-MEM) were plated in duplicate 35-mm Petri dishes for a 7-day culture at 37°C in a humidified 5% CO2 atmosphere.'"' The medium was supplemented with 0.9% methylcellulose, 50 pmol/L 2-mercaptoethanol, 30% (voVvol) calf bovine serum (Hyclone Laboratories, Logan, UT), and 10% (voVvol) low-molecular-weight B-cell growth factor (Cellular Products, Buffalo, NY). On day 7, blast colonies containing greater than 20 cells were counted using an inverted phase microscope with high optical resolution. In each case, the whole Petri dish was counted to determine the number ofLPCderived blast colonies. Colony cells were then subjected to morphologic and immunophenotypic analyses, as previously described.''.'' Cell lines. The radiation-resistant BCP leukemia cell lines NALM-6-UMl2"." and LC1:1922were maintained by serial passages in RPM1 1640 medium (GIBCO Laboratories, Grand Island, NY) supplemented with 10% (vol/vol) heat-inactivated calf bovine serum (Hyclone Laboratories), 50 pglmL streptomycin, 50 IU/mL penicillin, 2 mmol/L L-glutamine, and 10 mmol/L Hepes buffer. Cells were cultured in tissue culture flasks at37°Cin a humidified 5% CO2 atmosphere. Before injection into SCID mice, cells were washed twice in phosphate-buffered saline (PBS) and resuspended in PBS at 25 X 106/mL.SCID mice were inoculated intravenously ([V) with 0.2 mL of these cell suspensions containing 5 X IO6 NALM-6-UMI or LC1;19 cells. SCID mice. All SCID mice were produced by specific pathogenfree (SPF) CB-l7 scidscid breeders (originally obtained from Dr Melvin Bosma, Fox Chase Cancer Center, Philadelphia, PA) and maintained in the American Association for Accreditation of Laboratory Animal Care (AAALAC)-accredited Research Animal Resources (RAR) SCID Mouse Facility of the Childrens Cancer Group ALL Biology Reference Laboratory at the University of Minnesota (Minneapolis, MN). SCID mice were maintained in a SPF environment in microisolator cages (Lab Products, Inc, Maywood, NY) containing autoclaved food, water, and bedding, as previously re(Bactrim; Lemmon CO, p ~ r t e d . ' ~Trimethoprim/sulfamethoxazole -~~ Sellersville, PA) was added to the drinking water of mice, which was changed three times a week. Mice were inoculated with 5 X IO6 leukemic cells via tail vein injections. Mice were observed daily for evidence of leukemia and killed when moribund or unable to obtain food or water. Event times were measured from the day of inoculation of leukemia cells to the day of paraplegia (which results from central nervous system [CNS] leukemia) or death. The probability of event-free survival was determined, and event-free interval curves were generated using the Kaplan-Meier product limit method, as previously rep~rted.~'-'~ We used the log-rank test to assess the effect of various treatment regimens on event-free survival of SCID mice, as previously Mice were killed12 at weeks or when they became moribund as a result of disseminated human BCP leukemia. Mice were necropsied at the time of death or euthanization, and histopathology, flow cytometry, and polymerase chain reaction (PCR) analyses were performed to assess their burden of human leukemia cells, as previously For each mouse, multiple tissues, including bone marrow, spleen, liver, brain, kidneys, lungs, heart, ovaries, and gut, were histologically evaluated. All histopathologic studies were performed by a veterinary pathologist (ie, R.G.). B43 (anti-CD19)-phycoerythrin (PE), 9.4 (anti-CD45)-PE, and 2C3 (anti-1gM)-fluorescein isothiocyanate (FITC) antibodies and multiparameter flow cytometry were used to detect human BCP leukemia cells in the SCID mousebonemarrow cell suspensions, asreHuman DNA was detected by amplifying a 1IO-bp fragment from thefirst exon of the human 0-globin gene, as described." B43 (anti-CD19)-PAP immunotoxin treatment of SCID mice inoculated with radiation-resistant primary BCP leukemia cells. At 1 day after inoculation with primary t(4; 11) ALL cells, SCID mice were treated on 3 consecutive days with intraperitoneal (IP) bolus injections of B43-PAP (total dose: 30 pg per mouse, 1.5 m a g ) , as previously described.'" TB1 and syngeneic BMT. Groups of five SCID mice challenged with 5 X IO6 NALM-6-UM1 or LC1 ;19 cells wereplaced in a circular plexiglass rotating jig and were subjected to 250 cGy TBI, as previously reported." The radiation dose was delivered at a rate of 63 cGylmin using a "'CS irradiator (Model Mark 1-68; J.L. Shephard and Assoc, Glendale, CA). At 24 hours after TBI, 15 X IO6 nucleated bonemarrow cells (in 0.5 mL PBS) fromhealthy syngeneic SCID mice were injected IV via the tail vein. Syngeneic donor marrow was collected by flushing it from the shafts of femurs and tibias of healthy SCID mice into 15-mL centrifuge tubes using a 27-gauge needle on a I - m L syringe filled with sterile PBS supplemented with 2.5%(vol/vol) fetal bovine serum. A single-cell suspension of syngeneic SCID mouse bone marrow was preparedby gentle pipetting. Controls consisted of irradiated healthy SCID micenot challenged with BCP leukemia cells. TBI plus B43-PAP radioimmunotherapy and syngeneic BMT. NALM-6-UM1 or LC1;19 cells ( 5 X IO6) were injected into the caudal vein of control mice (N = 15), which received PBS injections only, or test mice,whichwere subsequently treatedwith ( I ) 250 cGy TB1 (N = S ) , (2) 10 pg B43-PAP IP every day X 3 consecutive days (N = 51, or (3) a combination of (1) and (2), N = 5. All mice were transplanted IV via the tail vein with an inoculum of 15 x 1Oh nucleated bone marrow cells (in 0.5 mL PBS) from healthy syngeneic SCID mice. The scheduling of these treatments was as follows: day 0, inoculation of leukemia cells 1V; day I , TBI; day 2, BMT; days 3, 4, and 5, B43-PAP or PBS IP. RESULTS AND DISCUSSION B43-PAP induces apoptosis in radiation-resistantprimary BCP leukemia cells from patients with therapy-refractory ALL. Apoptosis is a common mode of eukaryotic cell death characterizedby distinct ultrastructural features and a ladderlike DNA fragmentation pattern produced by endonuclease- From www.bloodjournal.org by guest on October 21, 2014. For personal use only. WADDICK ET AL 4230 mediated cleavage ofDNA into oligonucleosome-length fragment^.'^"' Inhibitors of protein synthesis. including diphtheria toxin, ricin, and cycloheximide, havebeen demonstrated to cause apoptosis of human leukemia celllines." Therefore, we investigated whether the inhibition of protein synthesis effected by B43-PAP could trigger apoptosis in radiation-resistant BCP leukemia cells from four patients with therapy-refractory ALL. As shown in Fig 1, DNA from B43-PAP-treated primary BCP leukemia cells showed a ladder-like fragmentation pattern, consistent with apoptosis, whereas no DNA fragmentation was observed after exposure to 2 to 8 Gy y-irradiation. B43-PAP-induced apoptosis was mediated by the CD19-specific binding of the immunotoxin to leukemia cells because ( 1 ) prior incubation with excess unconjugated Leu12 (anti-CD19) antibody butnot excess unconjugated TXU (anti-CD7) antibody preventedB43PAP-associated DNA fragmentation in BCP leukemia cells, and (2) the anti-CD7 immunotoxin TXU-PAP did not cause apoptosis in these cells. Thus, B43-PAP induces apoptosis in BCP leukemia cells, and radiation resistance does not render CD19-positive leukemia cells resistant to the cytotoxicity of the B43-PAP immunotoxin. R43-PAP immutroto.xin kills radiation-resistant printan leukemic progenitor cells from patients with relapsed RCP leukemia. The activity of an agent against the bulk population of leukemia cells in apoptosis assays does not always predict its activity against the clonogenic self-renewing subpopulation of LPC. We next examined the in vitro antileukemic activity of the B43-PAP immunotoxin against primary LPC from six patients with relapsed BCP leukemia, including the four patients shown in Fig I . LPC from these four patients were highly resistant to y-rays, with SF2 values greater than 0.5 (range, 0.6 to 1 .O) and a-values 50.2 Gy" (range, 0 to 0.2 Gy"). B43-PAP killed 91 . l % to 99.9% of LPC regardless of their radiation sensitivity, whereas the control immunotoxin (33.7-PAP did not affect LPC-derived blast colony formation (Table l). R43-PAP irnmunotoxin prevents development of overt leukemia in SClD mice inoculated with radiation-resistant prim a n leukemic cellsfrompatients withnewlydiagnosed t(4; 11) RCP leukemia. We next examined the in vivo antileukemic activity of the B43-PAP immunotoxin against primary leukemia cells from fivepatients with newly diagnosed MLL-AF-4 fusion transcript-positive t(4; 1 1 ) leukemia. Leukemia cells from each case were radiation-resistant with avalues less than 0.1 Gy", and their CDIO-CDI9'sIgMimmunophenotype was consistent with BCP leukemia. When injected IV into SCID mice that did not receive any subsequent treatment with B43-PAP, leukemia cells from all five patients caused histopathologically detectable overt leukemia with extensive multiple organ involvement (Table 2). At the time of death, SCID mice with overt t(4; 1 1) leukemia were noted to have massive hepatosplenomegaly, lymphadenomegaly, enlarged kidneys, and enlarged ovaries. Histopathologically, densely packed leukemia-cell infiltrates werefound in multiple organs (Table 2). Multiparameter flow cytometric analyses of bone marrow, liver, and spleen lymphoid cells confirmed the abundance ofCD19'CD45' sIgM- human ALL cells (Table 2). Presence of human DNA, 0 Case 1 Case 3 Case 2 Case 4 Fig 1. 643-PAP induces apoptosisin radiation-resistant primaryBCP leukemia cellsfrom patients with therapy-refractory ALL. Primary leukemia cells from four patients with BCP leukemia in relapse were harvested 16 to 18 hours after continuous exposure to 643-PAP or irradiation with 2 to 8 Gy y-rays, and DNA from Triton-X-l00 lysates was analyzed for fragmentation, as described in Materials and Methods. as detected by PCR amplification of a I IO-bp DNA fragment fromthefirstexonofthehuman&globin gene, further confirmed the engraftment of t(4; 1 I ) ALL blasts in bone marrow, liver, spleen, and brain of SClD mice. Whereas all From www.bloodjournal.org by guest on October 21, 2014. For personal use only. B43-PAP IMMUNOTOXIN 4231 Table 1. Antileukemic Activity of B43-PAP lmmunotoxin Against Radiation-Resistant Primary LPC From Patients With BCP Leukemia Radiobiologic Mean Parameters Patient 843-PAP No. G3.7-PAP643-PAP SF, Control 0.60 0.69 0.71 1 77 0.06 0.36 1 2 3 4 Radiation-sensitive controls 5 6 (range) No. of Colonies % Kill a (Gv") .oo G3.7-PAP 0.000 500 (492-508) (247-263) 101 (80-121) 181 (169-193) 1 350 (344-356) 0 (0. 0 (0.0) 9 (8,9) 0 (0, 0) 0) 493 (489-497) 1.4 241 (238-244) 125 (120-129) 192 (179-205) 0.40 (0.0) >99.9 (1,137-1.160) 1,149 1 (0.2) 324 (313-335) >99.8 299.6 91.1 >99.4 7.4 5.5 0.0 0.0 99.7 Radiation sensitivity and 643-PAP sensitivity of primary LPC from patients with BCP leukemia were determined in colony assays, as described in Materials and Methods. t(1; 19)(q23;p13) translocation** cause disseminated and inof the untreated SCID mice inoculated with primary blasts variably fatal BCP leukemia in SCID mice. We used these from these five t(4; 11) ALL patients developed disseminated two SCID mouse modelsto examine the antileukemic efficacy leukemia, overt leukemia was not found in any of the B43of a TB1 plus B43-PAP radioimmunotherapy protocol. The PAP-treated SCID mice that were challenged with primary strategy underlying the combined TB1 plus B43-PAP protocol blasts from the same patients (Table 2). Immunophenotypic was to subject xenografted human BCP leukemia in SCID studies were consistent with this histopathologic finding, in mice first to the maximum tolerated dose of TB1 inthe context that the percentages of bone marrow cells expressing human CD19 or CD45 antigens were significantly lower in immunoof syngeneic BMT. After BMT to rescue the SCID mice from toxin-treated, nonleukemic mice than in untreated, leukemic possible radiation-induced mortality, the mice were treated with B43-PAP immunotoxin at a dose level that yields clinimice (Table 2). callyachievablesystemic exposure levels inan attempt to TBI plus B43-PAPimmunotoxin is more effective than TBI eradicate the remaining fraction of radiation-resistant leukealone against radiation-resistant human BCP leukemia in a SCID mouse B M T model system. We have previouslyremic cells. As detailed below, combined radioimmunotherapy ported that radiation-resistant NALM-6-UM1 cells" and E2A- with TB1 plus B43-PAP was superior to T B 1 alone or B43PBX1fusiontranscript-positive LC1; 19 cells carrying a PAP alone in both SCID mouse BMT models. Table 2. Antileukemic Activity of B43-PAP lmmunotoxin Against Radiation-Resistant Human BCP Leukemia Cells in SCID Mice lmmunophenotyping(% bone marrow lymphoidcells1 Histopathology SCID Mouse No. 6 Case No. 1778 1 B43-PAP 1 1780 1453 2 2 1720 1452 2 1719 2 1447 1449 6 1446 B43-PAP 6 1448 6 2782 8 2784 8 0166 8 1568 8 11 1533 1524 11 lrnmunotoxin BM Treatment None None None B43-PAP B43-PAP None None B43-PAP None None B43-PAP B43-PAP None 643-PAP SPL LIV BR KD LU + + + + + + - - - - - + + + + - + + + + + + - - - - - - - - - - - NE - - - + + + - + + + + - - - - - - - - - + + + + + - + + + N E + - + + + - - - - - - - - - - - + + + + + + - - - - - - HT OV - - 0.1 + + - N N GI + 2 E 39 + E + CD45+ 92 0.5 98 98 CD19' 91 63 0.4 0.6 0.5 - 0.8 - 0.90.4 27 35 + + 78 57 0.9 1 0.8 0.90.9 N E + 78 18 + N E 46 21 NE NE NE NE -1 2 1 + + + 45 97 52 1 1 CD19+, SlgM- 89 37 55 34 27 0.8 17 20 NE 0.2 Female SClD mice were innoculated Iv with 5 x 10' priman/ bone marrow blasts from patients with newly diagnosed t(4;ll) BCP leukemia. At 1 day after the innoculation with leukemia cells, half of the mice were treated with B43-pAp immunotoxin at a dose level (10 pg/mouse/d ~p X3 days, total dose. 30 kg/mouse) that was found to be safe in a recently completed phase I study in patients with relapsed ALL. Mice were electively killed at 12 weeks to assess the burden of human leukemia cells by histopathologic and flow cytometric examination, as described in Materials and Methods. Abbreviations: BM, bone marrow; SPL, spleen; LIV, liver; BR, brain; KD, kidney; LU, lungs; HT, heart; OV, ovan/; GI,stomach and intestine; NE, not evaluated. From www.bloodjournal.org by guest on October 21, 2014. For personal use only. 4232 WADDICK ET AL 1 l A 80 Time After Inoculation with Nalm-6 Cells (Days) 90 died of leukemia between 49 and 69 days, with a median survival of 62.9 days (Fig 2A), which is significantly longer than the median survival of PBS-treated mice ( P = .0001), B43-PAP-treated mice (P = .016), or mice exposed to TB1 only ( P = .022). In the second SCID mouse BMT model we used, 5 X lo6LCl; 19 cells killed 15 of 15 mice between 25 and 46 days (median survival, 36.6 days; Fig 2B). Treatment with B43-PAP (P = .001) or TB1 ( P = ,006) followed by syngeneic BMT extended survival, reminiscent of the NALM-6-UM1 results. Specifically, B43-PAP-treated SCID mice died of leukemia between 48 and 73 days (median survival, 59.4 days), whereas SCID mice exposed to TB1 died of leukemia between 21 and 50 days (median survival, 47.4 days). SCID mice treated with TB1 followed by B43-PAP died of leukemia between 69 and 86 days, with a median survival of 82.3 days (Fig 2B), which is significantly longer than the median survival of PBS-treated mice ( P = .0001), B43-PAP-treated mice ( P = .016), or mice exposed to TB1 only (P = .011). To our knowledge, this study is the first to examine the antileukemic efficacy of an immunotoxin against radiation-resistant BCP leukemia cells. Previous studies have demonstrated that B43-PAP is a potent antileukemic immunotoxin.20*21 At nontoxic doses, this immunotoxin kills greater than 99% of human BCP leukemia cells in SCID mice,17.20.21 Similartherapeutic efficacy could not be achieved by standard or investigational chemotherapeutic agents,includingcyclophosphamide,carmustine,etoposide, topotecan, cytarabine,taxol, vincristine, methylprednisone, doxorubicin, or L-asparaginase." Here, we provide experimental evidence that B43-PAP triggers apoptosisinradiation-resistant primary BCP leukemia cells, kills greater than 99%of radiation-resistant primary leukemic progenitor cells from patients with BCP leukemia, and confers extended survival to SCID mice xenografted with radiation-resistant MLL-AF4 fusion transcript-positive primary human BCP leukemia cells from Fig 2. Antileukemic efficacy of TB1 plus 643-PAP egainstradiationchildren with t(4; 1 1 ) ALL. B43-PAP, when administered resistant human BCP leukemia in SClD mice. SCIDmice were inocuon days 3 through 5 after leukemic cell inoculation, was lated with NAL"CUM1 cells (A) or LC1;19 cells (B), as described in unable to prevent the development of fatal leukemia, but Materials and Methods. Mice were subsequently treated with 250 cGyTB1 IN = 51 (AI, 10 p g B43-PAPIPeveryday x 3 consecutive extended survival in SCID mice challenged with NALMdays (N = 5) ( V ) , or a combination of TB1 and "PAP (N = 51 (0). 6 or LC1; 19 cell lines, which have a higher in vivo cloning Control mice were treated wlth PBS (01 instead of 843-PAP. All mice efficiency than primary leukemic cells. Furthermore, the were transplanted with 15 x lo6 nucleated bone marrow cells (in 0.5 combination of B43-PAP and TBI was more effective than mL PBS) from healthy syngeneic SClDmice. The scheduling of these TB1 alone in two SCID mouse BMT models of radiationtreatments was as follows: day 0, IV inoculation of leukemia cells; day 1, TBI; day 2, BMT; days 3,4. and 5, IP 843-PAP or PBS. resistant human BCP leukemia. In the present study, B43PAP was used at a total dose level of 1.5 mg/kg, which is lower than the maximum tolerated dose identified in a In the first model system, 15 of 15 SCID mice injected phase I study in patients with relapsed BCP leukemia.*' IV with 5 X lo6 NALM-6-UM1 cells died of disseminated Thus, B43-PAPmay prove useful in the treatment of radiahuman BCP leukemia between 30 and 45 days (median surtion-resistant BCP leukemia. Despite the clinicalradiation vival, 37.8 days) (Fig 2A). Treatments with a cumulative 30 resistance of BCP leukemias, BMT remains the best prospg IP dose of B43-PAP on days 3 through 5 ( P = .001) or pect for survival of high-risk patients with BCP leuke250 cGy T B 1 on day 1 ( P = .02) followed by syngeneic ~ n i a . ~Combined ' or adjunctive therapies that exploit diBMT on day 2 significantly extended survival of SCID mice. verse cytotoxicmechanismsoffered by biotherapy and chemotherapy may assist in the elimination of radiationSpecifically, B43-PAP-treated SCID mice died of leukemia resistant BCP leukemia cells.The results presented herein between 41 and 50 days (median survival, 43.9 days), indicate that immunotoxins such asB43-PAP might be whereas SCID mice exposed to TB1 died of leukemia beused in vivo in addition to TB1 as part of pre-BMT conditween 36 and 53 days (median survival, 45.5 days). By comtioning. parison, SCID mice treated with TB1 followed by B43-PAP From www.bloodjournal.org by guest on October 21, 2014. For personal use only. 4233 B43-PAP IMMUNOTOXIN REFERENCES 1. Champlin R, Gale RP. Acute lymphoblastic leukemia: Recent advances in biology and therapy. Blood 73:2051, 1989 2. Greaves MF: Differentiation linked leukemogenesis in lymphocytes. Science 234:697, 1986 3. Bleyer WA, Sather H, Coccia P, Lukens J, Siege1 S, Hammond D: The staging of childhood acute lymphoblastic leukemia: Strategies of the Childrens Cancer Study Group and a three dimensional technique of multivariate analysis. Med Pediatr Oncol 14:271, 1986 4. Rivera GK, Pinkel D, Simone JV, Hancock ML, Crist WM: Treatment of acute lymphoblastic leukemia. N Engl J Med 329: 1289, 1993 5. Gaynon P, Bleyer WA, Steinherz P, Finkelstein J, Littman P, Miller D, Reaman G, Sather H, Hammond D. Modified BFM therapy for children with previously untreated acute lymphoblastic leukemia and unfavorable features. Am J Pediatr Hematol Oncol 10:42, 1988 6. Poplack DG, Reaman G : Acute lymphoblastic leukemia in childhood. 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