Radioimmunotherapy of CD22-Expressing Daudi Tumors in NudeMicewith a 90Y-Labeled Anti-CD22Monoclonal AntibodyDaniel A. Vallera,1Martin W. Brechbiel,5 Linda J. Burns,2 Angela Panoskaltsis-Mortari,3

Katie E. Dusenbery,1Dennis R. Clohisy,4 and Ellen S.Vitetta6

Abstract A study was undertaken to investigate the efficacy of a high affinity, rapidly internalizing anti-CD22 monoclonal antibody for selectively delivering high-energy 90Y radioactivity to B lympho-ma cells in vivo.The antibody, RFB4, was readily labeled with 90Yusing the highly stable chelate,1B4M-diethylenetriaminepentaacetic acid. Labeled RFB4 selectively bound to the CD22+

Burkitt’s lymphoma cell line Daudi, but not to CD22� control cells in vitro as compared with acontrol antibody, and was more significantly bound (P = 0.03) to Daudi solid tumors growing inathymic nudemice. Biodistribution data correlatedwell with the antitumor effect.The therapeuticeffect of 90Y-labeled anti-CD22 (Y22) was dose-dependent, irreversible, and the best resultswere achieved inmice receiving a single i.p. dose of196 ACi.These mice displayed a significantlybetter (P < 0.01) antitumor response than control mice and survived >200 days with no evidenceof tumor. Histology studies showed no significant injury to kidney, liver, or small intestine. Impor-tantly, tumor-bearing mice treated withY22 had no radiologic bone marrow damage comparedwith tumor-bearing mice treated with the control-labeled antibody arguing that the presence ofCD22+ tumor protected mice from bone marrow damage.When anti-CD22 radioimmunotherapywas compared to radioimmunotherapy with anti-CD19 and anti-CD45 antibodies, all three anti-bodies distributed significantly high levels of radioisotope to flank tumors in vivo compared withcontrols (P < 0.05), induced complete remission, and produced long-term, tumor-free survivors.These findings indicate that anti-CD22 radioimmunotherapy withY22 is highly effective in vivoagainst CD22-expressing malignancies and may be a useful therapy for drug-refractory B cellleukemia patients.

Radiolabeled monoclonal antibodies (MAb) represent prom-ising new therapies for drug-refractory hematopoietic malig-nancies such as leukemias and lymphomas. This may be dueto the radiosensitivity of lymphoid cells and the ability ofantibodies to more readily access hematopoietic tumorscompared to solid tumors. Radioimmunotherapy with 90Y-labeled or 131I-labeled CD20 are reaching the mainstream as

established therapies. The CD20-targeting 90Y-based radiophar-maceutical, Zevalin, was the first Food and Drug Administra-tion–approved radiolabeled radioimmunoconjugate. Althougheffective for lymphoma, CD20 is not broadly expressed onthe less differentiated B cell leukemias, the most commonchildhood form of acute lymphocytic leukemia. Thus, MAbsrecognizing other specificities must be considered. Anti-CD22MAbs are candidates because studies show that these MAbshave therapeutic effects when clinically administered (1). Anti-CD22 immunotoxins have been used to successfully treat rarehairy cell leukemia (2), and have been used to treat B cellleukemia in mice (3) and in humans (4). Because CD22 isexpressed in B cell leukemias, anti-CD22 is a logical choicefor radioimmunotherapy of B-acute lymphocytic leukemia.Alternative agents for therapy of B-acute lymphocytic leukemiatherapy are urgently needed because the American CancerSociety indicates that there are at least 2,000 new cases eachyear, and that chemotherapy-resistant disease is a frequentcause of treatment failure in leukemia patients (5). An anti-CD22–based radiopharmaceutical would be very useful.

CD22 is a 135-kDa B lymphocyte–specific glycoprotein anda member of the sialoadhesin family of molecules (6–8). It firstappears at the late pro-B cell stage of B cell differentiation andis a key regulatory cytoplasmic protein that is coexpressedsimultaneously with IgD on mature B cells (7). It is alsoexpressed on 60% to 70% of B cell lymphomas and leukemias.The major function of CD22 is to regulate B cell responses,

www.aacrjournals.orgClin Cancer Res 2005;11(21) November1, 2005 7920

Cancer Therapy: Preclinical

Authors’ Affiliations: Departments of 1Therapeutic Radiology-RadiationOncology, 2Medicine, 3Pediatrics, and 4Orthopedic Surgery, University ofMinnesota Cancer Center, Minneapolis, Minnesota; 5Radioimmune and InorganicChemistry Section, Radiation Oncology Branch, National Cancer Institute,Bethesda, Maryland; and 6Cancer Immunobiology Center, University of Texas,Southwestern Medical School, Dallas,TexasReceived 4/4/05; revised 7/21/05; accepted 8/8/05.Grant support: USPHS grant RO1-CA36725, awards from the National CancerInstitute, the National Institute of Allergy and Infectious Diseases, the Departmentof Health and Human Services and Children’s Cancer Research Fund, the JanieLymphoma Fund, and the Lion’s Fund. RFB4 was produced under a NationalCancer Institute Rapid Access to Intervention Development grant.The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Requests for reprints: Daniel A.Vallera, Department of Therapeutic Radiology-Radiation Oncology, University of Minnesota Cancer Center, MMC 367,Minneapolis, MN 55455. Phone: 612-626-6664; Fax: 612-624-3913; E-mail:valle001@@umn.edu.

F2005 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-05-0725

Research. on June 7, 2020. © 2005 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

which is likely accomplished by recruiting key signalingmolecules to the antigen receptor complex (9, 10). Experimentsin knockout mice have established the importance of CD22in modulating B cell responses in augmenting antibody re-sponses, expanding peritoneal B-1 cell populations, and inincreasing the levels of circulating autoantibodies (11–13).

For these studies, we chose 90Y, which is a powerful h-emitting radionuclide widely accepted as a therapeutic target-ing agent. 90Y has a favorable maximum h energy of 2.3 MeV,a half-life of 2.7 days, and a short path length of 5 mm (14).Together, these features contribute to the well-known cross-fireeffect of 90Y. The fact that 90Y, unlike 131I, has no gammacomponent means that the extensive precautions and isolationnecessary for 131I administration are not needed for 90Yadministration, and in some instances, 90Y-labeled antibodiesare given on an outpatient basis.

Perhaps the single most important component of theradiolabeled antibody is the chelate, the isotope-bindingmolecule that is conjugated to the antibody. A stable chelateconcentrates the therapy in the tumor site and prevents non-specific irradiation of nontarget organs. The 1B4M-diethylene-triaminepentaacetic acid (DTPA)–based chelate was chosen forour studies because it is highly stable in vivo in the studiesreported herein, and in our development of other radiophar-maceuticals (15, 16).

Studies previously indicated that CD22 served as a usefultarget for radioactive metals, but these studies were lacking intheir evaluation of efficacy (17). In this report, we examined theanti-CD22 MAb, RFB4, as a vehicle for the delivery of theradionuclide 90Y to B cell malignancies growing in nude mice.Radiolabeled anti-CD22 (Y22) displayed impressive and sig-nificant anticancer effects in the flank tumor model. Bonemarrow studies indicated that the presence of CD22-expressingtumor might act to prevent radiolabeled antibody fromentering the bone marrow compartment and destroying bonemarrow. Comparisons of internalizing anti-CD22 to internal-izing anti-CD19 and noninternalizing anti-CD45 revealedthat all three 90Y-labeled antibodies induced complete remis-sions and tumor-free, long-term survivors, arguing that bothinternalizing and noninternalizing antibodies can be equallyeffective for 90Y radioimmunotherapy.

Materials andMethods

Monoclonal antibodies and cell lines. The anti-CD22 hybridoma,RFB4, which secretes mouse IgG1, has been previously described andused in clinical studies as a targeted immunotoxin conjugated to thericin toxin A chain (18, 19). 3A1e, a control IgG1, is a murine pan-T cellMAb recognizing human CD7. This hybridoma was provided byDr. Barton Haynes, Duke University, Durham, NC (20) and was usedas a negative control because it does not bind to Daudi cells. MAbswere purified from hybridoma supernatants by affinity chromato-graphy using a protein A column. Fractions of the eluted MAb werepooled, concentrated, diafiltered into buffer and stored at �70jC untiluse. For comparisons to anti-CD22, a noninternalizing anti-CD45(AHN-12), mouse IgG1 was similarly radiolabeled as reported byour group (15). Also, an internalizing anti-CD19 (HD37), mouse IgG1,was previously reported (16).

The CD22+, IgM+ human Burkitt’s lymphoma cell line, Daudi (21),the CD22+ Raji B cell line, the CD22� human T cell leukemia, HPBMLT(22), and the CD22� mouse C57BL/6 myeloid leukemia, C1498 (23)were obtained from the American Type Culture Collection, Rockville,

MD, and maintained in RPMI 1640 containing 10% fetal bovineserum, 100 units/mL penicillin, 100 Ag/mL streptomycin, and 100mmol/L L-glutamine. The cell lines were incubated at 37jC in ahumidified atmosphere of 5% CO2 in air. Viability was determinedby trypan blue exclusion and viabilities of z90% were required forusing cells in our experiments (24).

Chelation of the antibody. 1B4M is a modified DTPA chelate (25)obtained from Dr. Martin Brechbiel, NIH. We have used this IB4M-

DTPA crosslinker (referred to as IB4M) and have described its usein previous studies (15, 16). Briefly, in this anti-CD22 study, 1 to

10 mg/mL antibody, in a 0.05 mol/L carbonate buffer (pH 8.6), and

0.15 mol/L NaCl was conjugated with a 10-fold molar excess of 1B4Movernight at room temperature. The conjugate was separated from

unconjugated 1B4M and transferred to the chelation buffer, followedby six washes with 0.16 mol/L ammonium acetate buffer (pH 7.0). The

conjugation buffer and the labeling buffer were passed through a

Chelex 100 column to remove any extraneous metals and the finalprotein concentration was determined spectrophotometrically (absor-

bance = 280 nm). Previous studies showed that conjugated antibody

was robust and stable even after several days in human serum (15).Labeling efficiency of conjugated anti-CD22 monoclonal antibodies.

Conjugated antibody was labeled with 5 to 10 ACi of carrier-free111In Cl3 (MDS Nordion, Kanata, Ontario) in chelation buffer and

EDTA was added to the tube, vortexed, and incubated for 5 minutes.

The chelation mixture was then diluted with 1% bovine serumalbumin in PBS. The ratio of bound protein versus free radiometal

chelate was determined by TLC on silica gel–coated glass fiber paper

(ITLC-SG Pall Life Science, East Hills, NY) as previously described(15). Labeling efficiency was expressed as (cpm origin) / (cpm origin +

cpm front) � 100.Binding and immunoreactivity assessment. The immunoreactivity of

labeled RFB4 was evaluated using an established binding assay (26).Briefly, Daudi or C1498 cells were washed and plated with radiolabeledMAbs. After incubation, the total and the cell-bound radioactivitywere determined using a gamma counter. Data was plotted as thepercentage of binding versus increasing cell number. Immunoreactivity(immunoreactive fraction) was determined by nonlinear regressioncurve-fitting by plotting the inverse of the bound fraction comparedwith the inverse of the cell concentration, which is based on theassumption that the total antigen concentration (cell concentration)represents an accurate approximation of the concentration of freeantigen. This calculates the Bmax or immunoreactive fraction. GraphPadPrism software (San Diego, CA) was used for these calculations.

In vivo tumor studies. Female athymic nude mice were purchased

from the National Cancer Institute, Frederick Cancer Research andDevelopment Center, Animal Production Area (operated by Charles

River Laboratories, Hartford, CT) and housed in an Association for

Assessment and Accreditation of Laboratory Animal Care–accreditedspecific pathogen–free facility under the care of the Department of

Research Animal Resources, University of Minnesota. Animal research

protocols were approved by the University of Minnesota InstitutionalAnimal Care and Use Committee. Animals were housed in micro-

isolator cages to minimize the possibility of transmission of any

contaminating virus. Daudi cells (5 � 106), in 0.1 mL PBS, wereinjected s.c. into the right flank of the nude mice. For biodistribution

studies, mice with palpable tumors were given 7 ACi 111In-labeled

MAb (i.p.). On day 5, organs were harvested (15, 16). Blood, tumor,spleen, liver, lung, kidney, muscle and bone were counted in a Packard

Cobra 5002 Auto-Gamma well counter. Data was calculated as the

percentage of injected dose per gram of tissue.For therapy studies, tumors were grown in female athymic nude

mice in the same way. When the tumors could be visualized, twoperpendicular diameters and the height of the tumor were measuredusing calipers. Tumor volumes were estimated as a product of thethree measurements, using the formula for the volume of an ellipse(r1 � r2 � r3) (4/3) (p). Animals were randomly assigned to treat-ment groups and received i.p. injections of the specified doses of

Anti-CD22 Radiotherapy

www.aacrjournals.org Clin Cancer Res 2005;11(21) November1, 20057921

Research. on June 7, 2020. © 2005 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

90Y-labeled MAb or control MAb when the tumors were f0.4 to0.6 cm3. Mice were observed for visible toxic signs, weighed, andtumor dimensions were recorded every 2 days.

Histology. Tissue specimens of liver, kidney, and intestine wereobtained from mice, and histology studies were done as described (27).All samples were embedded in optimum cutting temperature com-pound (Miles, Elkhark, IN), snap-frozen in liquid nitrogen, and storedat �80jC until sectioned. Serial 4 Am sections were cut, thaw-mountedonto glass slides, and fixed for 5 minutes in acetone. The slides werethen stained with H&E.

For bone marrow studies, femora were decalcified, embedded inparaffin, and cut at 5 Am (28). Histologic sections involved full-lengthcoronal sections of each femur. Routine H&E staining was doneand each of four replicate sections were analyzed. Digital images wereacquired using a Spot 2 digital CCD mounted on an Olympusmodel BX51 microscope.

Statistical analysis. Groupwise comparisons of data were doneusing Student’s t test.

Results

Binding of radiolabeled anti-CD22 to Daudi cells as deter-mined by flow cytometry. To determine whether 1B4Mconjugation affected the ability of Y22 to bind to CD22+

Daudi cells, chelated and nonchelated RFB4 were analyzed byimmunofluorescence and flow cytometry. To perform thesestudies, RFB4 and RFB4-1B4M were adjusted to the samesaturating concentrations and then added to the Daudi cells.Prior testing of 1:25, 1:50, and 1:100 dilutions indicated thatthe correct saturating concentrations were chosen for fluores-cence-activated cell sorting studies. Next, an identical quantityof a FITC-labeled goat anti-mouse secondary IgG1 antibody wasadded to the cells and indirect immunofluorescent studiesshowed that the degree of binding (>95%) and the histogramswere identical for RFB4, RFB4-1B4M, and RFB4-1B4M plusnonradioactive indium, indicating that chemically alteringRFB4 by attaching 1B4M did not alter the binding site of theMAb to target cells (data not shown).

Binding analysis. Scatchard analysis previously showed thatthe affinity constant of anti-CD22 was 2.1 � 108 mol/L (29). Tofurther study the binding and determine the immunoreactivity,RFB4-1B4M was labeled with 111In and then reacted withDaudi cells or control CD22� C1498 cells. Indium-111 istypically used as a surrogate for 90Y showing differences of only10% to 15% in biodistribution (30). Figure 1 shows that whenthe percentage of binding was plotted against cell number thatlabeled RFB4 bound well to Daudi cells, but did not bind toCD22� C1498. The percentages of binding for 1, 2, 4, 8, 16, or32 million Daudi cells was 10%, 17%, 29%, 47%, 74%, and89%, respectively. Nonlinear regression analysis was doneand the fitted curve is shown in Fig. 1. The immunoreactivefraction calculated from these data and projected to infiniteantigen excess by the method of Lindmo et al. (26) indicatedthat >100% of the agent was immunoreactive with targetcells. In contrast, the negative control C1498, had no bindingactivity.

Biodistribution of 111In-anti-CD22 in nude mice with flankDaudi tumors. In order to determine whether 111In-labeledanti-CD22 was capable of reaching a tumor in vivo , biodis-tribution experiments were done. Figure 2 shows a significantly(P < 0.03) higher mean activity (23.5% injected dose/g) in thetumors of five mice treated with 111In-RFB4 after 5 days. In

contrast, only 2.8% activity of the injected dose reached tumorin a group of mice injected with an identical dose of the control111In-3A1e. Significantly less (P < 0.02) 111In-RFB4 activitycompared with 111In-3A1e activity was found in kidneyindicating that more of the specific agent was diverted tothe CD22-expressing tumor target. The level of activity wasstill high in the blood after 5 days (9.1%). A comparison of111In-RFB4 and 111In-3A1e in the other organs revealed nosignificant differences.

The in vivo efficacy of 90Y-anti-CD22 in mice with Daudiflank tumors. MAb was labeled with 90Y in an identicalmanner as described in the 111In experiments above. Thelabeled MAb was passed through a spin column and ITLCindicated that 98% of radioactivity was protein bound. Thespecific activities were 4.65 mCi/mg for Y22 and 7.8 mCi/mgfor 90Y3A1e. Nude mice (n = 6/group) were injected in theirflanks with Daudi cells and when tumors were about 0.4 to0.6 cm3, mice were given a single i.p. injection of either 157 ACiof Y22, 295 ACi of 90Y 3A1e or were not treated. In untreatedmice, Fig. 3 shows that tumors grew rapidly, exceeding 2 cm3 in12 days. In mice receiving 157 ACi Y22, all the tumors initially,completely regressed. However, two of the six mice relapsedbetween days 35 and 50. The other four mice remained tumor-free beyond day 200 when the experiment was terminated.Histologic examination of these mice showed no evidenceof tumor. Two of six control mice injected with 295 ACi of90Y 3A1e died of early toxicity, indicating that the maximumtolerated dosage had been exceeded. The remaining four miceall died with tumors by day 45. Also, we observed that 75 ACiY22 was not protective and that 310 ACi was too toxic (datanot shown).

In the group of six mice that were treated with high-dose Y22(310 ACi), four of six died between days 8 and 19 with noevidence of tumor, indicating that death was due to acutetoxicity. Tissues from one of the mice were collected when theanimal was preterminal and indicated extensive damage to liverdue to fatty degeneration and necrosis. Because we were onlyable to observe a single animal, it is difficult to assess whetherthis toxicity was directly or indirectly related to the Y22treatment and we were unable to assess bone marrow toxicity

Fig. 1. Selective binding of1BM4-conjugated antibody to CD22+ target cells.111In-labeled RFB4was added to increasing numbers of CD22+ Daudi cells orCD22�C1498 cells. Cells were washed and the radioactivity measured. A nonlinearregression curve was fitted to the Daudi data (r2 = 0.99) and used to estimate theimmunoreactive fraction (immunoreactivity) of labeled Daudi tumor cells by themethod of Lindmo et al. (26).

www.aacrjournals.orgClin Cancer Res 2005;11(21) November1, 2005 7922

Cancer Therapy: Preclinical

Research. on June 7, 2020. © 2005 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

at this time. Two of six of the mice receiving 310 ACi Y22 werelong-term survivors and survived >200 days. Together, thesefindings indicated that the antitumor effect of Y22 was specificand dose-dependent.

A second experiment was done based on the findings on thefirst experiment (Fig. 4). Groups of nine mice with tumorsf0.4 to 0.6 cm3 were treated with either 196 ACi 90Y-RFB4(Y22) or 197 ACi of 90Y 3A1e. No toxic deaths were observed inmice treated in this experiment. Figure 4A shows the meantumor growth for the mice. In mice treated with 90Y 3A1e, themean tumor growth initially slowed, but the tumor quicklygrew again and all mice were withdrawn from the experimentby day 30. In contrast, tumors initially regressed in all micetreated with Y22. The bump in the curve around day 70represents the mean tumor growths of two mice that relapsedand were removed from the experiment. The remaining sevenmice were tumor-free for the remainder of the experiment,which was terminated on day 195. Values between the 90Y 3A1eand the Y22 mice varied significantly (P < 0.01) whencompared by Student’s t test on days 18 to 24. Figure 4Bshows the individual data for the mice. Together, the data fromthese two experiments showed that Y22 was highly effectiveand selective in treating Daudi flank tumors.

Histology. To provide some insight into whether there wereearly toxic effects of the 196 ACi Y22 or 197 ACi 90Y 3A1e dose

regimen, two randomly selected mice were removed from eachtreatment group from the second tumor study (experiment 2)on day 12 and histology studies were done. Thus, theexperiment proceeded with nine mice per group. Histologicexamination of kidney tissue sections from treated micerevealed no major damage. The glomeruli were intact (Fig. 5A)and there was no overt damage to the proximal tubules and nosigns of infiltration. The kidney responds late to radiationdamage and one might not expect to see early damage.However, there was no evidence of renal toxicity in the long-term survivors in the Y22 group, even when the experiment wasterminated on day 200. In the liver, there was evidence of onlymild fatty changes, but no perivascular infiltrate, no hepatocytedamage, and bile ducts were intact and nonoccluded (Fig. 5B).Histology studies were done on small intestines and villi werenormal, elongated, with a normal complement of goblet cells(Fig. 5C). Necropsies revealed no gross abnormalities andtherefore correlated with our histology findings. These dataindicated that the 196 ACi dose regimen did not damage criticalnontarget organs.

In contrast, pronounced differences were apparent in coronalcross-sections of femora examined from the Y22-treated and90Y 3A1e-treated mice that were given nearly identical dosesof labeled antibody. Figure 6A shows a day 12 bone sectionfrom one of the two Y22-treated mice. The histology was

Fig. 2. Biodistribution of 111In-labeled antibodies in varioustissues of nudemice xenograftedwith CD22+ Daudi tumors.Mice (n = 5/group) with palpable Daudi tumors wereinjected with 5 ACi of 111In-anti-CD22 (RFB4), 111In-labeledanti-CD19 (HD37), anti-CD45 (AHN-12), or negative control111In-3A1e. Five days later, various organs including bloodand tumor were removed and counted to determine thelocalization of the radiolabeled agent. Columns, meaninjected dose/gram tissue; bars,F1SD; *, P < 0.05,significant when compared with 111In-3A1e ^ treated groupby Student’s t test.

Fig. 3. The effect ofY22 on the mean growth of establishedDaudi flank tumors in nude mice. Groups of mice were injected inthe flank with CD22+ Daudi cells.When tumors were established(0.4-0.5 cm3), micewere given a single i.p. injectionof either157 ACiofY22, 295 ACi of 90Y 3A1e, or were untreated as controls.Tumordata are represented as a tumor volume (cm3) where the volumeswere averaged for six mice per group. Points, mean tumor growthover time; bars,F1SD.

Anti-CD22 Radiotherapy

www.aacrjournals.org Clin Cancer Res 2005;11(21) November1, 20057923

Research. on June 7, 2020. © 2005 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

indistinguishable from that of a normal nonirradiated mouse.The most striking feature of the Y22 section was that the hema-topoietic cells including megakaryocytes and erythrocytes werenormal. Healthy endothelial cells were clearly visible. Findingswere similar for the second mouse. On the other hand, onesection from the 90Y 3A1e-treated mouse shown in Fig. 6Brevealed a complete absence of hematopoietic cells includingmegakaryocytes after 12 days. Only a background of adipo-cytes, histiocytes, and endothelial cells were visible indicatingpronounced radiologic injury with resulting bone marrowaplasia. The second 90Y 3A1e-treated mouse showed similarresults, but there was some hematopoiesis in the proximalregion of the bone.

Comparison of radioimmunotherapy with anti-CD22, anti-CD19, and anti-CD45. In Fig. 2, biodistribution studies weretried with 111In-anti-CD22. In the same experiment, anti-CD19(HD37) and anti-CD45 (AHN-12) were also labeled with 111Inand tested for tumor distribution in vivo . Anti-CD22 and anti-CD19 are established internalizing antibodies and anti-CD45does not internalize (31). Interestingly, findings were similarwith all three MAbs in that all showed significant tumorlocalization after 5 days. Only the negative 3A1e controlantibody did not localize to tumor. The slight differences intumor accumulation of radioisotope that were observed directlycorrelated with the known avidities of the antibodies (RFB4 >AHN-12 > HD37).

To determine the comparative ability of the three radio-labeled antibodies to produce long-term tumor-free survivors,mice were given lethal flank tumors. Experiments (n = 5) were

comparable because all consistently induced large (2 cm3) flanktumors 10 to 20 days after an injection of 5 million Daudi cells(Table 1). Also, the negative controls produced identical resultsin all experiments in that either untreated mice or mice givennegative control 90Y 3A1e had no antitumor effect. In contrast,all three labeled antibodies, anti-CD22, anti-CD19, and anti-CD45, induced complete regressions of all tumors at mid andhigh doses. These treatments all produced tumor-free survivorsin the majority of treated mice. No single antibody treatmentproduced antitumor results that were remarkably different fromany of the others. Taken together, the data indicate thatsignificant antitumor effects were obtained using any of theantibodies that reacted with Daudi, but not antibodies thatwere nonreactive. Despite whether the antibodies could orcould not internalize, all three antibodies showed a similarability to target tumor in vivo and induce antitumor responses.Because at least one toxic death occurred in the high-dose groupfor all the antibodies, it seemed that a maximum tolerated dosewas reached.

Discussion

The major findings to emerge from these studies is that Y22given i.p. is highly effective in eliminating lethal tumors fromDaudi-infected nude mice. A control-labeled antibody was not.Previous studies indicated that MAb labeled with radioactiveyttrium showed potential (17, 32), but these were primarilybiodistribution and not radioimmunotherapy studies. In thisstudy, we analyzed efficacy and despite the fact that these were

Fig. 4. The effect ofY22 on the growth of established Daudi flanktumors in individual nude mice. A second experiment (experiment 2)was done in a manner similar to experiment1in Fig. 3. A, groups oftumormice (n =9/group)were giveneither196 ACiY22or197 ACi 90Y3A1e. Mean tumor growthwas plotted. Comparisons were analyzedby Student’s t test on days18 to 24. B, tumor growth graphed againsttime for the individual mice in experiment 2.

www.aacrjournals.orgClin Cancer Res 2005;11(21) November1, 2005 7924

Cancer Therapy: Preclinical

Research. on June 7, 2020. © 2005 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

aggressive, established Daudi flank tumors, all tumorscompletely regressed after treatment with mid to high dosagesof Y22 and most of the mice were long-term, tumor-freesurvivors. Although our results were improved by increasingthe dose, we were approaching toxic dosages at or near the 300ACi dose level and the therapeutic index was narrow in thesemice. One must consider the limitations of the model in theseevaluations because xenograft models are highly artificial and weare administering Y22 in a murine environment where humanCD22 is not expressed on normal B cells. Despite this, the resultswere impressive against such a rapidly growing tumor.

These studies were also novel in that simultaneous bonestudies were done because of the concerns of using 90Y whichhas historically been considered a bone-seeking isotope(33, 34). Importantly, a clear difference in bone histologyof Daudi tumor–bearing mice was observed in mice thatreceived Y22 in comparison with the mice that received control90Y 3A1e. Bone marrow damage in the form of a completeabsence of hematopoietic cells was severe in the control 90Y3A1e-treated mice and nearly nonexistent in the Y22-treatedmice. Because the outcome of the tumor experiments revealedthat Y22 selectively targets CD22-positive tumor cells andprotects, and 90Y 3A1e does not, it is likely that the CD22+

tumor load reduced bone marrow–related radiation damage.This may argue in favor of using a high avidity antibody suchas RFB4 with 90Y for selectively targeting tumor cells in humansbecause less radiolabeled antibody may nonspecifically collect

in the bone marrow. This also implies that CD22+ leukemiacells, as well as CD22+ normal B cells, will bind Y22 and reducebone marrow damage. However, a fraction of cells in the bonemarrow are CD22+ cells (35) and leukemia cells are commonlyfound in the bone marrow in afflicted patients. Thus, the effectsof these cells will have to be considered. Also notable fromthese studies is the fact that 90Y 3A1e treatment caused moreinjury in the distal region of the bone as compared with theproximal region where the blood flow is greater.

Another unique aspect of these studies was our comparisonof anti-CD22 radioimmunotherapy to radioimmunotherapywith anti-CD19 and anti-CD45 MAbs. When labeled in anidentical manner and compared in the same experiment, allthree antibodies similarly localized in tumor. Significantlymore radiolabel was selectively delivered to tumor in compar-ison to the negative control 3A1e antibody. There was acorrelation between antibody avidity and the amount ofradioisotope delivered to tumor, although all antibodiesdelivered well. All antibodies were remarkable in producingcomplete remission at mid and high doses and most of thesemice became tumor-free, long-term survivors. The clinical useof 90Y-anti-CD45 will be much different than Y22. The anti-CD45 agent targets all hematopoietic cells including themajority of leukemias/lymphomas. Although more broadlyreactive, CD45 is also expressed on stem cell progenitor cellsand thus this approach must include stem cell infusion/bonemarrow transplantation. The Y22 approach presented in thisstudy offers an alternative therapy that does not have to be usedin combination with a bone marrow transplant. This wouldoffer treatment options to those patients who are not eligiblefor aggressive bone marrow transplant protocols.

Fig. 5. Histology of kidneys, livers, and intestines of treated mice.The experimentshown in Fig. 4 originally had11miceper group, but 2miceper groupwere randomlyremoved on day12 for histology studies. Kidney (A), liver (B), and small intestine(C)were removed, cryosectioned, and stainedwithH&E to visualize organdamage.The animals were examined with identical results. Magnification is�200 (kidney),and�100 (liver), and small intestine (�40). glom, glomerulus; prox, proximal tubule;gob, goblet cell.

Fig. 6. Histologic analysis of bone. Bone from the samemice as Fig. 5 wasexamined. (A) bone fromY22-treated mouse; (B) bone from 90Y 3A1e-treatedmouse. Magnification is�200. adi, adipocyte; meg, megakaryocyte.

Anti-CD22 Radiotherapy

www.aacrjournals.org Clin Cancer Res 2005;11(21) November1, 20057925

Research. on June 7, 2020. © 2005 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Some believe that internalizing determinants are not asdesirable for radiotherapy, particularly because earlier studieswith iodinated antibodies showed that they are metabolizedquickly with a subsequent release of low molecular mass tar-gets from the cell (36, 37). Studies by Press et al. comparingradioiodinated noninternalizing anti-CD45 and internalizinganti-CD19 indicate that noninternalizing antibodies such asanti-CD45 are excellent choices for 131I radioimmunotherapybecause they are resistant to intracellular degradation anddeiodination whereas internalizing antibodies are not (31).Our data indicate that regardless of whether we usedinternalizing anti-CD22 and anti-CD19 or noninternalizinganti-CD45, 90Y radioimmunotherapy was highly effective ininducing an anticancer effect. Our data support the contentionthat radioactive metals are retained intracellularly (38–41) andthat radiometals such as 90Y that are known to be residualized,particularly when combined with an internalizing target, resultin a beneficial clinical effect. Our visual analysis of bone, inconjunction with the impressive efficacy observed in thesestudies, further support the fact that 90Y delivered withinternalizing antibodies localized in its intended target (tumor)as opposed to nontarget tissue (bone, liver, kidney).

Our group is most interested in alternative therapies for drug-refractory leukemia. CD20 is ontogenically expressed later thanCD22 (42). Whereas CD20 is expressed on only a subpopu-lation of precursor B cells, CD22 is expressed on all precursor Bcells. We already know that RFB4 reactivity is highly B lineage–restricted because in a panel of >40 human tissues, it recog-nized only B cells (29, 43, 44), and is highly expressed onleukemias. Thus, anti-CD22 is a better candidate for treatingleukemia than anti-CD20. Several successful studies nowindicate that CD20 targeting is highly effective for lymphoma.Unfortunately, diseases such as pre–B cell leukemia are lesslikely to express CD20, hence, alternative radiolabeled anti-bodies are urgently needed.

The primary purpose of these studies was to assess theefficacy of 90Y-labeled anti-CD22 in an established animaltherapy model because only a small number of efficacy studiesregarding 90Y-labeled anti-CD22 administration have been

published. A secondary goal was to compare 90Y-labeled anti-CD22 to 90Y-labeled anti-C19 because CD19, another highlyinternalized B cell marker, is also widely expressed on Bleukemia cells. Previous studies from our laboratory showedthat 90Y-labeled anti-CD19 antibodies were highly effectivefor targeting Daudi cells in this same animal model (16). Noclinical studies using anti-CD19 90Y radioimmunotherapy havebeen published, so whether it would be better to target CD22 orCD19 is unknown.

Interestingly, studies argue in favor of targeting CD22 andCD19 simultaneously (19). Preclinical studies with a combi-nation of both anti-CD19 and anti-CD22 antibodies labeledwith toxins or with radionuclide indicate that the combineduse of these agents may have pronounced advantages over theuse of single-agent therapy (3, 45). Recent studies with recom-binant antibody fragments in which anti-CD22 and anti-CD19 sFv are engineered on the same molecule indicate thatthe future may yield important molecules which can be usedfor bispecific targeting, and can be bioengineered to addressimportant shortcomings such as toxicity, immunogenicity,and rapid clearance (46).

Others are using MAbs of murine origin for radioimmuno-therapy. For example, 131I-labeled Tositumomab is a murineanti-CD20 MAb and used for therapy of follicular lymphoma(reviewed in ref. 47). Short-term toxic effects are mild,including immediate infusion reactions, moderate myelosup-pression, and an influenza-like reaction, which are managedon an outpatient basis. Although radiation from the conjugatemay pose risks to those in physical contact with the patientimmediately posttreatment, straightforward protocols for thesafe outpatient administration of drug can be followed. Long-term toxic effects such as hypothyroidism occurring in f10%of patients are easily managed. No cases of myelodysplasiahave been noted, but only a small number of patients havebeen treated thus far. No serious infections have been observed.Normal B lymphocytes were only temporarily depleted, andthere was no evidence of an effect on overall antibody levels.The approach is promising because an effective systemictreatment for disseminated follicular lymphoma can be

Table 1. Comparison of 90Y-labeled anti-CD22 (Y22), anti-CD19 (HD37), and anti-CD45 (YAHN-12) to generatetumor-free, long-term survivors in nudemice given lethal injections of Daudi tumor cells

Long-term tumor-free survivors/total number ofmice treated

Y22 Y22 Repeat YAHN-12 Y19 Y19 Repeat

No treatment 0 of 6 L 0 of 5 0 of 5 L90Y 3A1e treatment 0 of 6 0 of 9 0 of 5 0 of 5 0 of 6Low dose 2 of 6 L 2 of 5 1of 5 LMid dose 4 of 6 7 of 9 5 of 5 1of 6 LHigh dose 2 of 6 L 3 of 5 3 of 6 5 of 6Days of experiment 140* 150* 135 70 119Untreated tumors reach 2.0 cm3 10 ND 20 10 ND

NOTE: Mice were given flank injections of 5 million Daudi cells and then treated with low dose, mid dose, or high dose radiolabeled antibody. Data is derived from fiveexperiments.The experiments are comparable because tumor reached 2.0 cm in10 to 20 days in all experiments and the two negative controls (the no-treatment controland the 90Y 3A1e-treatedmice) showed identical results in all experiments. Data are presented as the number of tumor-free survivors out of the total number ofmice thatwere originally treated as part of the experiment. Low dose is 69 to 77 ACi. Mid dose is137 to197 ACi. High dose is 254 to 310 ACi.* Y22 experiments are the same experiments shown in Figs. 3 and 4.They were not terminated at this time and the results were the same at 200 days posttreatment.

www.aacrjournals.orgClin Cancer Res 2005;11(21) November1, 2005 7926

Cancer Therapy: Preclinical

Research. on June 7, 2020. © 2005 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

completed entirely within a few weeks on a convenient out-patient basis with modest toxicity. The clinical results obtainedwith this anti–B cell murine monoclonal antibody encouragethe clinical testing of Y22 which would have the addedadvantage of use against less differentiated B cell malignanciessuch as B-acute lymphocytic leukemia.

In the future, if these molecules are to become mainstreamtherapies, important issues will need to be addressed. Anti-CD22 is a mouse antibody and this will likely mean a rapidclearance when administered to humans. If they do show clin-ical promise, then chimerization or humanization to improveclearance may be a desirable option. This will also reduce theirimmunogenicity. However, it is important to keep in mind thatimmunogenicity may not be a problem in patients becausea highly effective radiolabeled antibody against B cells couldsuppress the production of neutralizing antibodies by killinghighly radiosensitive B cells. Also, by the very nature of theirtreatment, leukemia patients will likely be highly immunosup-pressed from prior chemotherapy. Regardless, RFB4, has beenchimerized and is available for testing.

The 1B4M chelate was chosen for these studies because,in the case of metals such as 90Y, it is critical that the chelateretain the radiometal and not permit its release from the com-plex in vivo (reviewed in refs. 48, 49). Earlier generation ofC-functionalized DTPA compounds (50) were improved byadding a methyl group to the structure of the base compoundproducing the M-DTPA producing a more stable con-figuration (25). This agent has already done well in preclinical

and clinical radioimmunotherapy trials using 90Y (51–54)and is similar to the chelate used in Zevalin. Brechbiel et al.have developed a simple, dependable, and reproducible pro-cess for the preparation of 1B4M-DTPA in large clinical scalebatches (25). Currently, an Investigational New Drug–ap-proved study of an 90Y-labeled anti-CD45 antibody is underway at our institution using this chelate and performedoptimally, consistently yielding high labeling efficiencies andimmunoreactive fractions in clinical labelings. In our studies,as in others (30), 111In was used as a surrogate marker for90Y because studies by Carrasquillo et al. showed that thedifferences in biodistribution were only 10% to 15%.

1,4,7,10-tetra-azacylododecane �N,NV,N 00,NV00-Tetraacetic(DOTA)-based linkers have been successfully used in otherstudies (55). Interestingly, these studies also showed that anti-CD22 MAbs can have independent lymphomacidal propertiesand complement the activity of radiolabeled antibodies.

In summary, the high affinity, internalizing Y22 is highlyeffective against human tumors in a mouse xenograft model.These studies indicate that when a residualizing radiometal iscombined with a highly internalizing antibody on the sameradioconjugate, the result is a high level of distribution ofthe drug to tumor and an impressive antitumor response. Thiswarrants further consideration of Y22 for clinical trials.

Acknowledgments

We thankMichael Elson for excellent technical assistance in these studies.

Anti-CD22 Radiotherapy

www.aacrjournals.org Clin Cancer Res 2005;11(21) November1, 20057927

References1. Coleman M, Goldenberg DM, Siegel AB, et al.Epratuzumab: targeting B-cell malignancies throughCD22. Clin Cancer Res 2003;9:3991^4S.

2. Kreitman RJ, WilsonWH, Bergeron K, et al. Efficacyof the anti-CD22 recombinant immunotoxin BL22 inchemotherapy-resistant hairy-cell leukemia. N Engl JMed 2001;345:241^7.

3. Herrera L,Yarbrough S, Ghetie V, et al. Treatment ofSCID/human B cell precursorALL with anti-CD19 andanti-CD22 immunotoxins. Leukemia 2003;17:334^8.

4. Herrera L, Farah RA, Pellegrini VA, et al. Immuno-toxins against CD19 and CD22 are effective in killingprecursor-B acute lymphoblastic leukemia cellsin vitro. Leukemia 2000;14:853^8.

5. Minden M, Imrie K, Keating A. Acute leukemia inadults. Curr Opin Hematol1996;3:259^65.

6. Tedder TF, Tuscano J, Sato S, et al. CD22, A Blymphocyte-specific adhesionmolecule that regulatesantigen receptor signaling. Annu Rev Immunol 1997;15:481^504.

7. Law CL, Sidorenko SP, Clark EA. Regulation oflymphocyte activation by the cell surface moleculeCD22. ImmunolToday1994;15:442^9.

8. Powell LD,Varki A. I-type lectins. J Biol Chem1995;270:14243^6.

9. Pezzutto A, Rabinovitch PS, Dorken B, et al. Role ofthe CD22 human B cell antigen in B cell triggering byanti-immunoglobulin. J Immunol1988;140:1791^5.

10. Peaker CJG, Neuberger MS. Association of CD22with the B cell antigen receptor. Eur J Immunol 1993;23:1358^63.

11. O’KeefeTL,Williams GT, Davies SL, et al. Hyperre-sponsive B cells in CD22-deficient mice. Science1996;274:798^801.

12. Otipoby KL, Andersson KB, Draves KE, et al. CD22regulates thymus-independent responses and the life-span of B cells. Nature1996;384:634^7.

13. Sato S, Miller AS, Inaoki M, et al. CD22 is both apositive and negative regulator of B lymphocyte anti-

gen receptor signal transduction: altered signaling inCD22-deficient mice. Immunity1996;5:551^62.

14. Berger MJ. Distribution of absorbed dose aroundpoint sources of electrons and h particles in waterand other media. JNucl Med1971;5:5^23.

15.Vallera DA, Elson M, Brechbiel MW, et al. Preclinicalstudies targeting normal and leukemic hematopoieticcells with yttrium-90-labeled anti-CD45 antibodyin vitro and in vivo in nude mice. Cancer BiotherRadiopharm 2003;18:133^45.

16.Vallera DA, ElsonM, Brechbiel MW, et al. Radiother-apy of CD19 expressing Daudi tumors in nude micewith yttrium-90-labeled anti-CD19 antibody. CancerBiother Radiopharm 2004;19:11^23.

17. Griffiths GL, Govindan SV, Sharkey RM, et al. 90Y-DOTA-hLL2: an agent for radioimmunotherapyof non-Hodgkin’s lymphoma. J Nucl Med 2003;44:77^84.

18.Vitetta ES, StoneM, Amlot P, et al. Phase I immuno-toxin trial in patients with B-cell lymphoma. CancerRes1991;51:4052^8.

19.Messmann RA,Vitetta ES, Headlee D, et al. A phaseI study of combination therapy with immunotoxinsIgG-HD37-deglycosylated ricin A chain (dgA) andIgG-RFB4-dgA (Combotox) in patients with refracto-ry CD19(+), CD22(+) B cell lymphoma. Clin CancerRes 2000;6:1302^13.

20. Haynes BF, Eisenbarth GS, Fauci AS. Human lym-phocyte antigens : production of a monoclonalantibody that defines functional thymus-derived lym-phocyte subsets. Proc Natl Acad Sci U S A1979;76:5829^33.

21. Klein E, Klein G, Nadkarni JS, et al. Surface IgM-nspecificity on a Burkitt lymphoma cell in vivo and inderived culture lines. Cancer Res1968;28:1300^10.

22.Vallera DA,Todhunter D, Kuroki DW, et al. Molecularmodification of a recombinant, bivalent anti-humanCD3 immunotoxin (Bic3) results in reduced in vivotoxicity inmice. Leuk Res 2005;29:331^41.

23.ValleraDA, JinN, BaldricaJM, et al. Retroviral immu-notoxin gene therapy of acute myelogenous leukemiainmiceusing cytotoxicTcells transducedwith an inter-leukin 4/diphtheria toxin gene. Cancer Res 2000;60:976^84.

24. Blazar BR,Taylor PA,Vallera DA. In vivo or in vitroanti-CD3 epsilon chain monoclonal antibody therapyfor the prevention of lethal murine graft-versus-hostdisease across the major histocompatibility barrier inmice. J Immunol1994;152:3665^74.

25. Brechbiel MW, Gansow OA. Backbone-substitutedDTPA ligands for 90Yradioimmunotherapy. BioconjugChem1991;2:187^94.

26. Lindmo T, Boven E, Cuttitta F, et al. Determinationof the immunoreactive fraction of radiolabeled mono-clonal antibodies by linear extrapolation to binding atinfinite antigen excess. JImmunMed1984;72:77^89.

27.Vallera DA, Panoskaltsis-Mortari A, Blazar BR. Renaldysfunction accounts for the dose limiting toxicity ofDT390anti-CD3sFv, a potential new recombinant antiGVHD immunotoxin. Protein Eng1997;10:1071^6.

28. Goblirsch M, MathewsW, Lynch C, et al. Radiationtreatment decreases bone cancer pain, osteolysis andtumor size. Radiat Res 2004;161:228^34.

29. Ghetie MA, May RD,Till M, et al. Evaluation of ricinA chain-containing immunotoxins directed againstCD19 and CD22 antigens on normal and malignanthumanB-cells as potential reagents for in vivo therapy.Cancer Res1988;48:2610^7.

30. CarrasquilloJA,WhiteJD, Paik CH, et al. Similaritiesand differences in 111In- and 90Y-labeled 1B4M-DTPAantiTac monoclonal antibody distribution. JNucl Med1999;40:268^76.

31. Press OW, Howell-Clark J, Anderson S, Bernstein I.Retention of B-cell-specific monoclonal antibodies byhuman lymphoma cells. Blood1994;83:1390^7.

32. Milenic DE, Brady ED, Brechbiel MW. Antibody-targeted radiation cancer therapy. Nat Rev 2004;3:489^98.

Research. on June 7, 2020. © 2005 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

www.aacrjournals.orgClin Cancer Res 2005;11(21) November1, 2005 7928

Cancer Therapy: Preclinical

33. BurlinTE, Simmons R. The dosimetry of radioiso-topes incorporated inboneusing cavity ionization the-ory. PhysMed Biol1976;21:1^15.

34. Kutzner J, Becker M, GrimmW. Bone-seeking be-havior of rhenium and yttrium complexes. Nuklearme-dizin1983;22:162^5.

35. Chapple MR, MacLennan IC, Johnson GD. Aphenotypic study of B lymphocyte subpopulationsin human bone marrow. Clin Exp Immunol 1990;81:166^72.

36. Geissler F, Anderson SK,Venkatesan P, et al. Intra-cellular catabolism of radiolabeled anti-A antibodiesby malignant B-cells. Cancer Res1992;52:2907^15.

37. Naruki Y, Carrasquillo JA, Reynolds JC, et al. Differ-ential cellular catabolism of 111In, 90Yand125I radiola-beled T101anti-CD5 monoclonal antibody. Int J RadAppl Instrum B1990;17:201^7.

38. Motta-Hennessy C, Sharkey RM, Goldenberg DM.Metabolism of indium-111-labeled murine monoclonalantibody in tumor and normal tissue of the athymicmouse. JNucl Med1990;31:1510^9.

39. Press OW, Shan D, Howell-Clark J, et al. Compara-tive metabolism and retention of iodine-125, yttrium-90, and indium-111radioimmunoconjugates by cancercells. Cancer Res1996;56:2123^9.

40. Duncan JR,Welch MJ. Intracellular metabolism ofindium-111-DTPA-labeled receptor targeted proteins.JNucl Med1993;34:1728^38.

41. Franano FN, EdwardsWB,WelchMJ, et al.Metabo-lism of receptor targeted 111In-DTPA-glycoproteins:

identification of 111In-DTPA-epsilon-lysine as the pri-mary metabolic and excretory product. Nucl Med Biol1994;21:1023^34.

42. van DongenJJM, Adriaansen HJ. Immobiology ofLeukemia. Leukemia. In: Henderson ES, ListerTA,GreavesM,editors.WBSaundersCo.;1996. p. 85^90.

43. Shen GL, LiJL, Ghetie MA, et al. Evaluation of fourCD22 antibodies as ricin A chain-containing immuno-toxins for the in vivo therapy of human B-cell leuke-mias and lymphomas. IntJCancer1988;42:792^7.

44. Anderson KC, Bates MP, Slaughenhoupt BL, et al.Expression of human B cell-associated antigens onleukemias and lymphomas: a model of human B celldifferentiation. Blood1984;63:1424^33.

45.Wei BR, GhetieMA,Vitetta ES.The combineduse ofan immunotoxin and a radioimmunoconjugate to treatdisseminated human B-cell lymphoma in immunodefi-cient mice. Clin Cancer Res 2000;6:631^42.

46.Vallera DA,Todhunter DA, Kuroki DW, et al. Abispe-cific recombinant immunotoxin (DT2219) targetinghuman CD19 and CD22 receptors in a mouse xeno-graft model of B cell leukemia/lymphoma. ClinCancerRes 2005;11:3879^88.

47. Connors JM. Radioimmunotherapy: hot new treat-ment for lymphoma. NEnglJMed 2005;352:496^8.

48. KozakRW, Raubitschek A,Mirzadeh S, et al. Natureof the bifunctional chelating agent used for radioim-munotherapy with yttrium-90 monoclonal antibodies:critical factors indetermining in vivo survival andorgantoxicity. Cancer Res1989;49:2639^44.

49. Khazaeli MB. Quality control of radiolabeledmono-clonal antibodies. Cancer Biother Radiopharm 2000;15:529^30.

50. Roselli M, Schlom J, Gansow OA, et al. Com-parative biodistribution studies of DTPA-derivativebifunctional chelates for radiometal labeled monoclo-nal antibodies. Int J Rad Appl Instrum B 1991;18:389^94.

51.Wiseman GA,White CA, Stabin M, et al. Phase I/II90Y-Zevalin (yttrium-90 ibritumomab tiuxetan, IDEC-Y2B8) radioimmunotherapy dosimetry results in re-lapsed or refractory non-Hodgkin’s lymphoma. Eur JNucl Med 2000;27:766^77.

52. Gordon LI,WitzigTE,Wiseman GA, et al.Yttrium 90ibritumomab tiuxetan radioimmunotherapy for re-lapsed or refractory low-grade non-Hodgkin’s lym-phoma. Semin Oncol 2002;29:87^92.

53.WitzigTE,White CA,Wiseman GA, et al. Phase I/IItrial of IDEC-Y2B8 radioimmunotherapy for treatmentof relapsed or refractory CD20(+) B-cell non-Hodg-kin’s lymphoma. JClin Oncol1999;17:3793^803.

54.Waldmann TA, White JD, Carrasquillo JA, et al.Radioimmunotherapy of interleukin-2R a-expressingadultT-cell leukemia withYttrium-90-labeled anti-Tac.Blood1995;86:4063^75.

55. Tuscano JM, O’Donnell RT, Miers LA, et al. Anti-CD22 ligand-blocking antibody HB22.7 has indepen-dent lymphomacidal properties and augments theefficacy of 90Y-DOTA-peptide-Lym-1 in lymphomaxenografts. Blood 2003;101:3641^7.

Research. on June 7, 2020. © 2005 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

2005;11:7920-7928. Clin Cancer Res   Daniel A. Vallera, Martin W. Brechbiel, Linda J. Burns, et al.   Antibody

Y-Labeled Anti-CD22 Monoclonal90Nude Mice with a Radioimmunotherapy of CD22-Expressing Daudi Tumors in

  Updated version

  http://clincancerres.aacrjournals.org/content/11/21/7920

Access the most recent version of this article at:

   

   

  Cited articles

  http://clincancerres.aacrjournals.org/content/11/21/7920.full#ref-list-1

This article cites 51 articles, 23 of which you can access for free at:

  Citing articles

  http://clincancerres.aacrjournals.org/content/11/21/7920.full#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://clincancerres.aacrjournals.org/content/11/21/7920To request permission to re-use all or part of this article, use this link

Research. on June 7, 2020. © 2005 American Association for Cancerclincancerres.aacrjournals.org Downloaded from