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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2001-12-0322.
TRANSPLANTATION
From the Clinical Research Division, Fred Hutchinson
Cancer Research Center, Seattle, WA; the Departments of Medicine and
Radiation Oncology, University of Washington, Seattle; the National
Cancer Institute, National Institutes of Health, Bethesda, MD; and
Pacific Northwest National Laboratory, Richland, WA.
To lower treatment-related mortality and toxicity of conventional
marrow transplantation, a nonmyeloablative regimen using 200 cGy
total-body irradiation (TBI) and mycophenolate mofetil (MMF) combined
with cyclosporine (CSP) for postgrafting immunosuppression was
developed. To circumvent possible toxic effects of external-beam Allogeneic marrow transplantation provides a
potential cure for a variety of hematologic and nonhematologic
diseases. To avoid mortality and toxic effects associated with
myeloablative marrow transplantation, a nonmyeloablative marrow
transplantation regimen was developed in a canine model. The regimen
uses 200 cGy total-body irradiation (TBI) before and administration of
mycophenolate mofetil (MMF) combined with cyclosporine (CSP) for
immunosuppression after transplantation.1 The combination
of MMF and CSP given after transplantation controlled not only
graft-versus-host disease (GVHD) but was also found to be essential for
maintenance of the donor graft (ie, control of host-versus-graft
reaction). The results of the preclinical studies have been
successfully translated into the clinic to treat elderly or medically
infirm patients with hematologic malignant diseases who were not
eligible to receive high-dose conventional grafts.2
Although the TBI dose used in these studies was low, there remains
concern about the possible late toxic effects of The In the study reported here, an Dogs
DLA-identical littermates were selected for transplantation on
the basis of identity for highly polymorphic major histocompatibility complex class I and class II microsatellite markers and identity for
DLA DRB1 alleles as determined by direct sequencing.23,24
The mAbs
Conjugation of the anti-CD45 antibody with benzyl-CHX-A"-DTPA The anti-CD45 mAb (CA12.10C12) was conjugated with the isothiocyanate form of the metal-binding chelate CHX-A"-DTPA after rigorous demetallation of the antibody, glassware, and solvents.22 In the demetallation, the anti-CD45 mAb (5 mg) was dialyzed by using a Slide-A-Lyzer 10K cassette (Pierce, Rockford, IL) against 1 L metal-free HEPES buffer, with a minimum of 5 buffer changes over 3 days at 4°C. All buffers used were prepared with metal-free (18 M ) water passed over a column of Chelex-100 resin
(250 g/12 L). Five grams of Chelex-100 resin was added to each buffer
change. The demetallated mAb was removed from dialysis and placed in an acid-washed microcentrifuge tube, and care was taken to ensure that no
metals were introduced.
To 1.2 mL (31 nM) of the metal-free mAb (3.87 mg/mL) was added 30 µL (464 nM) isothiocyanatobenzyl-CHX-A" solution (22.4 mg/mL in dimethyl sulfoxide), and the reaction mixture was stirred at room temperature for 18 hours. Subsequently, the reaction mixture was placed in a dialysis cassette, dialyzed against 3 × 1 L metal-free citrate buffer (50 mM sodium citrate, 150 mM sodium chloride [NaCl], and 0.05% sodium azide adjusted to pH 5.5) for 2 days and against 150 mM NaCl for 1 day and then stored at 4°C until used. The number of chelates was evaluated by a spectrophotometric assay using yttrium-arsenazo III complex at 652 nm (1-4 chelates per protein).32 A single batch of antibody conjugated with CHX-A"-DTPA was prepared for all the studies in dogs, and the number of chelates per mAb for that batch was 3.6. Flow cytometry was conducted to compare the mAb-CHX-A" with the unconjugated antibody to determine whether the binding had been affected by conjugation. Radiolabeling of the anti-CD45 mAb-CHX-A" conjugate with 123I The mAb-CHX-A" conjugate was labeled with 123I in 2 portions under comparable conditions. In the labelings, 125 µL sodium phosphate (0.5 M; pH 7.4) was added to 600 µL of a 1.31 mg/mL solution of mAb-CHX-A" conjugate in phosphate-buffered saline (PBS). To the resultant solution were added 30 µL 123I (6.4 or 6.9 mCi [237 or 255 MBq] in 0.1 N sodium hydroxide) and 75 µL of a 1 mg/mL solution of Chloramine-T in water. After 5 minutes at room temperature, the reaction was stopped with 7.5 µL of a 10 mg/mL solution of sodium metabisulfite in water. The reaction mixture was then placed on a PD-10 column (Amersham Pharmacia Biotech, Piscataway, NJ) and eluted in 1-mL fractions with PBS. Protein fractions (2 and 3) were combined to yield 3.3 mCi (122 MBq; 52%) and 3.22 mCi (119 MBq; 47%). The 2 products were combined, and 4.91 mCi (182 MBq) of this mixture was injected.Radiolabeling of the mAb-CHX-A" conjugate with 213Bi The 225Ac nitrate was purchased on a column from the Department of Energy (Oak Ridge, TN). The 213Bi was obtained from 225Ac (t1/2, 10 days) by eluting the generator column with 1 M hydrochloric acid (HCl) with use of a dual-syringe pump system. As the elution proceeded, the 213Bi-HCl solution was mixed with water (in a plastic chamber) and run across a MP-50 cation exchange column. The 213Bi was trapped on the ion exchange column, and the column was removed from the generator system. The column was then eluted with 0.1 N hydrogen iodide, and the pH of the eluant was adjusted to 4.2 to 4.5 by using 3 M NH3OAc (Ultrex grade). A 200-µg quantity of the mAb-CHX-A" conjugate in saline was then mixed with the eluted 213Bi. After 2 to 5 minutes, the labeled antibody was purified on a size-exclusion column (PD-10). Labeling the mAb-CHX-A" with 213Bi was achieved in 80% to 95% (decay corrected) yields.To determine the radiochemical purity, a small drop of the
213Bi-labeled mAb-CHX-A" conjugate was analyzed by using
instant thin-layer chromatography (ITLC) on an ITLC SG strip (Gelman,
Ann Arbor, MI) and allowed to air dry. The dry ITLC strip was placed in
a development chamber containing a small amount of 80% methanol and
20% 10 mM DTPA in water. When the solvent had nearly reached the top
of the strip, it was air dried, cut in sections, and counted in a Flow cytometry To assess antigen saturation with the 213Bi-labeled antibody and to quantitate the leukocyte subsets, either whole blood or peripheral blood mononuclear cells (PBMCs) prepared by Ficoll-Hypaque density-gradient centrifugation (density, 1.074) were used for analyses by flow cytometry.28 Then, 10 µg/mL of the respective FITC-conjugated mAb was added to 1 × 106 cells resuspended in 50 µL 15% horse serum (HS) and Hanks buffered salt solution (HBSS) or 50 µL whole blood. The suspension was incubated at 4°C for 30 minutes and washed once with cold HBSS supplemented with 2% heat-inactivated HS. If whole blood was used, red blood cells were lysed with a hemolytic buffer containing EDTA. The cells were washed 2 times, resuspended in 1% paraformaldehyde, and analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA).Pharmacokinetic studies Saturation of the CD45 antigen by the injected mAb was assessed by flow cytometry. PBMCs were obtained from heparinized blood of study dogs before and at various intervals after mAb infusion and stained with anti-CD45 mAb directly conjugated to FITC or a goat antimouse (Fab')2-FITC. Saturation was evaluated by comparing the fluorescence intensity of cells incubated with anti-CD45-FITC with that of goat antimouse (Fab')2-FITC.Plasma levels of the mAb were measured with an enzyme-linked immunosorbent assay (ELISA).33 Briefly, 96-well polyvinyl plates were coated with goat antimouse IgG and incubated with plasma from the infused dogs after blocking with 5% nonfat milk in PBS. Goat antimouse IgG horseradish peroxidase was used as the secondary antibody and 2,2' azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) as the color reagent. Plates were read with a Vmax microtiter plate reader (Molecular Devices, Menlo Park, CA) at 405 nm. Plasma obtained from dogs before infusion served as controls, and standard curves with known concentrations of mAb were established. Mixed leukocyte cultures To assess leukocyte function in mixed leukocyte cultures (MLCs) before and after transplantation,34 PBMCs from the dogs were resuspended in Waymouth medium supplemented with 1% nonessential amino acids, 1% sodium pyruvate, 1% L-glutamine and 20% heat-inactivated, pooled normal dog serum. Responder cells (1 × 105/well) and irradiated (2200 cGy) stimulator cells (1 × 105/well) were cocultured in triplicate in round-bottomed, 96-well plates for 6 days at 37°C in a humidified 5% carbon dioxide air atmosphere. In triplicates used as positive controls, 4 µg concanavalin was added to responder cells on day 3. On day 6, cultures were pulsed with 1 µCi (37 kBq) tritium-thymidine for 18 hours before harvesting. Tritium-thymidine uptake was measured as mean counts per minute for the 3 replicates by using a -scintillation counter (Packard BioScience Company, Meriden, CT).
Natural killer cell cytotoxicity assay To evaluate natural killer (NK) cell activity before and after transplantation, we conducted chromium-release assays.35 PBMCs served as effectors, and cells from a canine thyroid adenocarcinoma cell line served as targets. Effector-to-target ratios of 60:1, 30:1, and 15:1 in triplicate wells were used. The percentage of cytotoxicity (percentage of specific lysis) was calculated by using the mean value of triplicate cultures: % specific lysis = [(experimental release spontaneous release)/(maximum
release spontaneous release)] × 100. Spontaneous release was
determined in wells with target cells and medium alone. Maximum release
was determined in wells with target cells and 2% Triton X.
Biodistribution and dosimetry estimates To assess the biodistribution, retention, and clearance of 213Bi-labeled antibody and to estimate the radiation absorbed doses to major organs per unit of administered activity, one dog was injected with 4.9 mCi (181 MBq) 123I-labeled anti-CD45 mAb-CHX-A" conjugate. Because of difficulties in imaging 213Bi, we used 123I to determine the pharmacokinetic parameters of the antibody. This was possible because the CD45 antigen is not internalized or shed and 213Bi is not released from the CHX-A" chelate in vivo. Blood samples were obtained from the dog at various times after injection and counted in a counter to evaluate the blood concentration and clearance over time
of the 123I-anti-CD45 conjugate. These counts were
corrected for decay, and percentages of the injected immunoconjugate
dose per gram of blood were calculated.
Serial The radiation absorbed dose was then calculated according to the
following standard formula: D (absorbed dose, cGy)
= 0.0512 E f Ao/m Dose-escalation study of 213Bi A dose-escalation study was conducted in 7 dogs not given marrow grafts, after treatment with 213Bi-labeled anti-CD45 at increasing doses of radiation (0.1-5.9 mCi [3.7-218 MBq/kg] 213Bi/kg) and various doses of antibody (Table 1). The aim of the study was to evaluate hematopoietic and nonhematopoietic toxic effects of radiation. For
this purpose, daily peripheral blood samples were obtained to study
changes in blood cell counts, hematopoietic cell saturation, cell
subsets (flow cytometric analyses for CD3+,
CD4+, CD8+, TCR+,
CD14+, CD45+, and myeloid cells
[DM5+]), and results of kidney- and liver-function tests.
The rates of decline and recovery of peripheral blood counts in the
dogs were compared with those in historical controls given 200 and 300 cGy external-beam TBI, respectively,36 to estimate the
dose equivalents of radiation delivered by 213Bi that
resulted in comparable hematologic cytotoxicity. Starting with the
fourth dog, a dose of unlabeled anti-CD45 mAb was injected first to
prevent nonspecific tissue binding of the radiolabeled mAb. Nonspecific
tissue binding was observed in previous studies with -emitting
radiolabeled mAbs and was easily corrected by adding unlabeled mAb,
which reduced early hepatic uptake by up to 80%.37
DLA-identical marrow grafts On the basis of the results of the dose-escalation studies, we chose a total dose of 0.5 mg/kg anti-CD45 mAb administered in 6 injections on days 3 to 2 before grafting, as appropriate for transplantation studies. Three dogs received total doses of 3.6, 4.6, and 8.8 mCi/kg (133, 170, and 326 MBq/kg) 213Bi-labeled anti-CD45 mAb. On day 0, the dogs were given marrow grafts intravenously (mean, 4.7 × 108 mononuclear cells/kg) from their DLA-identical littermates (Table 2). MMF (10 mg/kg given subcutaneously twice daily on days 0 to 27) and CSP (15 mg/kg given orally twice daily on the day before grafting until day 35 afterward) were administered to provide postgrafting immunosuppression.1 Supportive care was given after transplantation as described previously.38 Hematopoietic engraftment was determined by recovery of peripheral blood granulocyte and platelet counts after the postirradiation nadirs. Donor and host cell chimerism was assessed by using a polymerase chain reaction (PCR)-based assay of polymorphic (CA)n dinucleotide repeats.39 Digitalized images of the PCR gels were obtained by using the storage phosphorimaging technique.40 This allowed estimation of the proportion of donor-specific DNA among host DNA from digitalized gel pictures with use of image-analyzing software (ImageQuant; Molecular Dynamics, Sunnyvale, CA). The densities of the donor (D)-specific band and the host (H)-specific band were added as total (T) events (T = D + H). The percentage of donor-origin DNA was calculated as (D)/(T) × 100%. This technique allowed detection of 2.5% to 97.5% donor cell chimerism.1
At the completion of the study, the dogs were euthanized and complete autopsies, including histologic examinations, were performed to assess marrow engraftment, GVHD, hematopoietic recovery, and possible toxic effects.
Imaging, biodistribution, and dose estimation using 123I-anti-CD45 Dog E510 was injected with 123I-labeled anti-CD45 mAb-CHX-A" conjugate, and serial images were obtained with a camera. The blood clearance findings for the
123I-anti-CD45 conjugate are shown in Figure
1. A rapid clearance presumably due to
rapid binding of circulating mAb to cellular CD45 was noted in the
first 3 hours after injection. To standardize the quantification from
the -camera images, the dog was euthanized and samples from organs
were counted. As was observed in the images, the highest uptake was
obtained in blood, marrow, spleen, lymph nodes, and liver (Figure
2). From the data obtained,
time-activity curves were prepared for the radiolabeled mAb conjugate,
and the uptake, retention, and clearance of 213Bi-labeled
mAb conjugate were estimated. The absorbed radiation doses for
principal organs were calculated from the data. Among the nontarget
organs, the liver received the highest dose, which was estimated to be
3.3 cGy/mCi.
Dose-escalation study of 213Bi-anti-CD45 Seven dogs received injections of 0.1 to 5.9 (3.7-218 MBq/kg) mCi/kg 213Bi-labeled anti-CD45 (Table 1). The first 3 dogs received one injection with 0.1 mg/kg mAb labeled with 0.10 mCi/kg (3.7 MBq/kg), 0.17 mCi/kg (6.3 MBq/kg), and 0.68 mCi/kg (25 MBq/kg) 213Bi, respectively. No significant effects on peripheral blood counts were observed (data not shown). Because CD45 antigen saturation was not achieved at a dose of 0.1 mg/kg mAb, the fourth dog was given 1.0 mg/kg mAb divided into 6 injections on days 3 and 2 before transplantation with a total of 1.9 mCi/kg (70 MBq/kg) 213Bi. Saturation of the CD45 antigen on hematopoietic cells was observed by the fourth of the 6 injections (Figure 3A). Therefore, 3 other dogs were subsequently given 0.4, 0.5, and 0.6 mg/kg mAb divided in 6 injections and labeled with 2.1, 3.7, and 5.9 mCi/kg (78, 137, and 218 MBq/kg) 213Bi, respectively. Antigen saturation was not reached with 0.4 to 0.5 mg/kg mAb. However, with a dose of 0.6 mg/kg mAb, antigen saturation was again reached after 4 injections (Figure 3B). The injections were well tolerated, with no observable side effects. In these last 4 dogs, distinct declines in peripheral blood counts were observed.
To estimate the corresponding TBI dose equivalent to the administered
antibody-targeted radiation dose, blood cell changes in historic
control dogs treated with 200 and 300 cGy external-beam
DLA-identical marrow grafts On the basis of the results of the dose-escalation studies, we targeted a 213Bi dose of more than 4 mCi/kg (148 MBq/kg) labeled to 0.5 mg/kg anti-CD45 mAb for the marrow transplantation studies. Three dogs received 3.6, 4.6, and 8.8 mCi/kg (133, 170, and 326 MBq/kg) 213Bi-labeled anti-CD45 mAb, respectively, followed by DLA-identical marrow grafts. Table 2 shows the results of the allogeneic marrow transplantations. Antigen saturation was not reached, as indicated by flow cytometry and ELISA assays (data not shown). The granulocyte nadirs were reached by days 4 to 5 after transplantation, with 10 to 30 granulocytes/µL. Thrombocytopenia (< 20 × 109/L platelets) occurred between days 5 and 22, necessitating 1 to 3 platelet transfusions in each dog. The dogs had a rapid hematopoietic recovery, with neutrophil counts above 0.5 × 109/L on days 7 to 9 (Figure 5).
All 3 dogs showed mixed hematopoietic chimerism in peripheral blood as
early as 2 to 3 weeks after transplantation. Figure 6 illustrates the treatment regimen,
peripheral blood cell changes, and results of microsatellite marker
studies in one representative dog. The mixed chimerism was stable in
all dogs, with values for donor cells ranging from 30% to 60% through
the end of study. Quantitation of the T-cell, granulocyte, and monocyte
recoveries by flow cytometry showed rapid return to pretransplantation
levels by day 50. The dogs also had normal T-cell and NK cell functions as measured by MLCs and NK assays within 2 months after transplantation (Figure 7). There were no observable
acute toxic effects during injection of the radioimmunoconjugate.
No signs of GVHD were observed. The transplants were clinically well
tolerated, but reversible elevations in plasma levels of liver enzymes,
especially alkaline phosphatase (AP) and alanine aminotransferase
(ALT), occurred in 2 dogs (E889 and E805) between days 40 and 100, without any clinical abnormalities in liver function (Figure
8). In all dogs, the values for bilirubin
and creatinine remained normal throughout the transplantation (data not
shown). In the dog given the highest dose of 213Bi (E885),
a different pattern was observed, with marked elevations in AP, ALT,
and aspartate aminotransferase (AST) levels after day 120 and
development of ascites and signs of liver failure. On day 163, liver
ultrasonography and liver biopsy were performed. On ultrasound
assessment, the liver appeared very small, and a decreased portal
venous flow velocity suggested portal hypertension. The liver biopsy
showed no histologic abnormality apart from slight signs of sinusoidal
fibrosis. Because of intractable ascites, the dog was euthanized on day
191. The autopsy showed a very small, though macroscopically normal
liver. Histologic examination showed signs of marked periportal
fibrosis, irregular sinusoidal fibrosis, and Kupffer cell aggregates.
At the end of the study (days 277 and 279), the remaining 2 dogs (E889 and E805) were euthanized and extensive autopsies performed. No abnormalities, including no signs of GVHD, were found.
The aim of these studies was to replace external-beam Several We chose to investigate the use of We selected CD45 as the target antigen because it is the most widely expressed antigen on malignant and nonmalignant hematopoietic tissues and it has a high density on hematopoietic cell surfaces.47,48 About 200 000 copies of the CD45 antigen are expressed on the average circulating leukocyte. This high antigen density is important because it permits the use of readily achievable specific activities of antibodies labeled with the short half-lived 213Bi. CD45 is a tyrosine phosphatase that is critical for activation mediated by T- and B-cell antigen receptors, and it is expressed on all leukocytes, including their precursors in the marrow.21,49 Our data on the biodistribution of CD45-targeted radiation in dogs when using an 123I-CD45 radioimmunoconjugate are consistent with the results of Matthews et al5,41 showing a very high uptake of the radioimmunoconjugate in blood, lymph nodes, bone marrow, and spleen. Among nontarget organs, the liver received the highest dose, probably because of nonspecific uptake of the radioimmunoconjugate by Kupffer cells. The efficacy and toxicity of the radioimmunoconjugate are highly
influenced by the labeling technique used. The conjugates must be
thermodynamically stable and kinetically inert to avoid deposition of
radiobismuth in the kidney or other nontarget organs. The very short
t1/2 of 213Bi and its highly energetic
Multiple factors, including antigen distribution, antibody dose, labeled radiation activity, and nonspecific tissue binding influence the in vivo pharmacokinetics and pharmacodynamics. To best account for the numerous variables present, we conducted in vivo dose-escalation studies. First, an antibody dose targeting enough antigens to deliver the necessary irradiation effectively without reaching antigen saturation (to prevent the circulation of unbound radiolabeled antibody) had to be determined. After dose-escalation studies, we chose a dose of 0.5 mg/kg CD45 mAb for further experiments. With this amount of mAb, a radiation dose corresponding to 200 to 300 cGy TBI (determined by comparing hematology data from treated dogs with findings in historic controls36 treated with TBI) could be administered. Of note, there were steeper declines in blood counts in the 213Bi-treated dogs, presumably because of the immediate elimination of all targeted cells without sparing already committed peripheral progenitor cells. These results are in contrast to those with external-beam photon irradiation, after which certain hematopoietic precursor cells might still be able to mature and appear in the circulating blood, thereby delaying the decline in peripheral blood counts. To determine whether radioimmunotherapy with the described 213Bi-anti-CD45 conjugate can replace TBI in the nonmyeloablative conditioning regimen, we performed marrow-transplantation studies in 3 dogs. Stable and rapid engraftment occurred in all 3 dogs, with mixed chimerism levels ranging from 30% to 70% donor cells after 1 month. In all dogs, this level of chimerism was sustained through the end of study, although only a short course of immunosuppression therapy with MMF and CSP (28 and 37 days, respectively) was administered. The nadirs of platelet and neutrophil counts were lower than observed in dogs treated with 200 cGy TBI.1 The immune reconstitution evaluated by peripheral blood CD4+, CD8+, TCR+, granulocyte, and monocyte counts was rapid, with neutrophil levels above 0.5 × 109/L by day 8, platelet levels above 20 × 109/L by day 17, and pretransplantation T-cell levels reached after one month. The treatment was clinically well tolerated, with no signs of hypersensitivity or other immediate side effects. The only possible toxic effects we observed were a transient elevation in liver-enzyme levels in 2 dogs and an elevation in liver-enzyme levels in conjunction with ascites in one dog. In dogs given transplants after conditioning with 200 cGy TBI only, no similar pattern of liver-enzyme alteration was observed, suggesting that this toxic effect was due to the radioimmunotherapy.1 Liver biopsies in the dog with ascites showed subtle signs of sinusoidal fibrosis, findings that were not sufficient to explain portal hypertension. On autopsy, the liver was found to be very small but macroscopically normal, with marked periportal and sinusoidal fibrosis on histologic examination. In the preclinical and clinical studies with 213Bi radioimmunoconjugates reported thus far, mild liver toxicity with transient increases in levels of transaminases was observed.54 In contrast, studies in mice showed renal and bone marrow toxic effects to be dose limiting.44,52 We did not observe any signs of renal toxicity, despite the potentially high avidity of free, unbound bismuth for the kidneys in the dogs treated with the radioimmunoconjugate, a result that reflects the high in vivo stability of the construct. This is consistent with findings by other investigators who used this bifunctional chelating agent for in vivo sequestration of bismuth isotopes.22,50,52,55 Because the liver was the nontarget organ that received the highest dose of radiation, the elevation in liver-enzyme levels could have been a sign of transient radiation damage to this organ. CD45 is not expressed on hepatocytes, but abundant cells of hematopoietic origin in the liver do express CD45, for example, Kupffer cells56 (monocyte lineage) and circulating leukocytes, which comprise up to 20% of nonparenchymal cells.57 In addition, circulating immunoglobulins are nonspecifically taken up in the liver by Kupffer cells and endothelial cells, and this could lead to an increased radiation dose.58 Recently, a sinusoidal obstruction syndrome characterized by portal hypertension and intense sinusoidal fibrosis has been observed in patients after treatment with gemtuzumab ozogamicin (Mylotarg; Wyeth-Ayerst Pharmaceuticals, Madison, NJ), an anti-CD33-toxin conjugate.59,60 The histologic findings in the liver in these clinical cases were similar to the sinusoidal fibrosis in the dog in our study that was given the highest dose of radiation. The time from radiation exposure to signs of liver injury in our dogs was more than 30 days, suggesting not direct hepatocyte toxicity but activation of stellate cells, sinusoidal obstruction, and ischemic hepatocyte injury.61 Mechanisms possibly involved in this pathologic process are increased activity of matrix metalloproteinases and glutathione depletion.62 Studies in animals demonstrated successful use of metalloproteinase inhibitors and glutathione to prevent such hepatic veno-occlusive disease.63-65 These agents could be of use in preventing the liver toxicity observed in the dogs in our study. Previous studies have shown that the efficacy of radioimmunotherapy is
influenced largely by the radionuclide used, the affinity of the
antibody to the target antigen, the tissue distribution of the antigen,
and the vascularization of the target tissue.11,66,67 Given these prerequisites, radioimmunotherapy of hematopoietic tissue
using an In summary, we demonstrated that the 213Bi-anti-CD45 radioimmunoconjugate can be used to deliver selective radiation to hematopoietic tissue and, in combination with postgrafting immunosuppression therapy using MMF and CSP, allows stable engraftment of DLA-identical marrow grafts. This is the first report of successful marrow allografting after conditioning with radioimmunotherapy alone.
We thank George McDonald, MD, for reviewing the manuscript; Howard Shulman, MD, for performing the pathology studies; Michele Spector, DVM, and the technicians in the canine facilities of the Fred Hutchinson Cancer Research Center; Drs. Nash, Kiem, Zaucha, Georges, Junghanss, and Little, who participated in the weekend treatments; Marie-Terese Little, PhD, Stacy Zellmer, and Serina Gisburne for the DLA typing; the technicians of the hematology and pathology laboratories of the Fred Hutchinson Cancer Research Center; Dr Elizabeth Squires, Sangstat Medical Corporation, Menlo Park, CA, for the gift of oral CSP; Dr Sabine Hadulco, Roche Bioscience, Nutley, NJ, for the gift of MMF; Peter F. Moore, PhD, DVM, for providing the CA17.6B3 antibody; and MDS Nordion, Vancouver, British Columbia, for the gift of 123I.
Submitted December 21, 2001; accepted February 19, 2002.
Prepublished online as Blood First Edition Paper, April 17, 2002; DOI 10.1182/blood-2001-12-0322.
Supported by a grant from the Gabrielle Rich Leukemia Foundation and in part by grants HL36444, CA78902, and CA15704 from the National Institutes of Health, Department of Health and Human Services, Bethesda, MD. W.A.B. was supported by a fellowship from Deutsche Krebshilfe, Dr Mildred-Scheel-Stiftung für Krebsforschung.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Brenda M. Sandmaier, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave North, D1-100, PO Box 19024, Seattle, WA 98109-1024; e-mail: bsandmai{at}fhcrc.org.
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© 2002 by The American Society of Hematology.
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  H. Nakamae, D. S. Wilbur, D. K. Hamlin, M. S. Thakar, E. B. Santos, D. R. Fisher, A. L. Kenoyer, J. M. Pagel, O. W. Press, R. Storb, et al. Biodistributions, Myelosuppression, and Toxicities in Mice Treated with an Anti-CD45 Antibody Labeled with the {alpha}-Emitting Radionuclides Bismuth-213 or Astatine-211 Cancer Res., March 15, 2009; 69(6): 2408 - 2415. [Abstract] [Full Text] [PDF]
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 C. U. Louis, K. Straathof, C. M. Bollard, C. Gerken, M. H. Huls, M. V. Gresik, M.-F. Wu, H. L. Weiss, A. P. Gee, M. K. Brenner, et al. Enhancing the in vivo expansion of adoptively transferred EBV-specific CTL with lymphodepleting CD45 monoclonal antibodies in NPC patients Blood, March 12, 2009; 113(11): 2442 - 2450. [Abstract] [Full Text] [PDF]
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 P. Kletting, D. Bunjes, S. N. Reske, and G. Glatting Improving Anti-CD45 Antibody Radioimmunotherapy Using a Physiologically Based Pharmacokinetic Model J. Nucl. Med., February 1, 2009; 50(2): 296 - 302. [Abstract] [Full Text] [PDF]
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 E. R. Nemecek, D. K. Hamlin, D. R. Fisher, K. A. Krohn, J. M. Pagel, F. R. Appelbaum, O. W. Press, and D. C. Matthews Biodistribution of Yttrium-90-Labeled Anti-CD45 Antibody in a Nonhuman Primate Model Clin. Cancer Res., January 15, 2005; 11(2): 787 - 794. [Abstract] [Full Text] [PDF]
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D. A. Mulford, D. A. Scheinberg, and J. G. Jurcic The Promise of Targeted {alpha}-Particle Therapy J. Nucl. Med., January 1, 2005; 46(1_suppl): 199S - 204S. [Abstract] [Full Text] [PDF]
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C. E. Muller-Sieburg, R. H. Cho, L. Karlsson, J.-F. Huang, and H. B. Sieburg Myeloid-biased hematopoietic stem cells have extensive self-renewal capacity but generate diminished lymphoid progeny with impaired IL-7 responsiveness Blood, June 1, 2004; 103(11): 4111 - 4118. [Abstract] [Full Text] [PDF]
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W. A. Bethge, D. S. Wilbur, R. Storb, D. K. Hamlin, E. B. Santos, M. W. Brechbiel, D. R. Fisher, and B. M. Sandmaier Selective T-cell ablation with bismuth-213-labeled anti-TCR{alpha}{beta} as nonmyeloablative conditioning for allogeneic canine marrow transplantation Blood, June 15, 2003; 101(12): 5068 - 5075. [Abstract] [Full Text] [PDF]
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D. G. Maloney, B. M. Sandmaier, S. Mackinnon, and J. A. Shizuru Non-Myeloablative Transplantation Hematology, January 1, 2002; 2002(1): 392 - 421. [Abstract] [Full Text]
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Top
Abstract
Introduction
B(t) dt, where E is the average 
0.28 to 1.0 mCi/kg (10-37 MBq/kg)