90Yttrium-Labeled Complementarity-Determining-Region-Grafted

Bioconjugate Chem. 1998, 9, 773−782

773

90Yttrium-Labeled

Complementarity-Determining-Region-Grafted Monoclonal Antibodies for Radioimmunotherapy: Radiolabeling and Animal Biodistribution Studies†

Serengulam V. Govindan,‡ Lisa B. Shih,‡ David M. Goldenberg,§ Robert M. Sharkey,§ Habibe Karacay,‡ Joseph E. Donnelly,‡ Michele J. Losman,‡ Hans J. Hansen,‡ and Gary L. Griffiths*,‡ Immunomedics, Inc., 300 American Road, Morris Plains, New Jersey 07950, and Garden State Cancer Center, Belleville, New Jersey 07109. Received April 21, 1998; Revised Manuscript Received July 21, 1998

90 Yttrium-labeled monoclonal antibodies (mAbs) are likely to be important to radioimmunotherapy (RAIT) of a variety of cancers. The goal of this study was to select and evaluate a form of [90Y]mAb suitable for RAIT and determine conditions for high-yield, reproducible radiolabelings. 90Y-Labelings, at 2-40 mCi levels, of cdr-grafted versions of anti-B-cell lymphoma (hLL2) and anti-CEA (hIMMU14) mAbs were optimized to >90% incorporations using the macrocyclic chelator DOTA as the metal carrier. In in vitro challenge assays, the stability of mAbs labeled with [90Y]DOTA was better than that of the corresponding [90Y]benzyl-DTPA conjugates. The retention of [90Y]DOTA-hLL2 on Raji tumor cells in vitro was similar to that of the same mAb labeled with [90Y]benzyl-DTPA and was about twice as much as with [125I]hLL2, indicating residualization of metalated mAb. Both [90Y]hLL2 conjugates, prepared using DOTA and Bz-DTPA, had similar maximum tolerated doses of 125 µCi in BALB/c mice and showed no discernible chelator-induced immune responses. Animal biodistribution studies in nude mice bearing Ramos human B-cell lymphoma xenografts revealed similar tumor and tissue uptake over a 10 day period, with the exception of bone uptake which was up to 50% lower for [88Y]DOTA-hLL2 compared to [88Y]Bz-DTPA-hLL2 at time points beyond 24 h. With [90Y]DOTA-hLL2 fragments, in vivo animal tumor dosimetries were inferior to those for the IgG, and kidney uptake was relatively high even with D-lysine administration. The ability of [111In]DOTA-hLL2 to accurately predict [90Y]DOTA-hLL2 biodistribution was established. These preclinical findings demonstrate that [90Y]DOTA-(CDR-grafted) mAbs are suitable for examination in clinical RAIT.

INTRODUCTION

RAIT1 using radiolabeled mAbs has shown considerable promise against radiosensitive cancers such as NHL (1-6). Its role as a treatment against solid tumors has † Presented in part at the 43rd Annual meeting of the Society of Nuclear Medicine, Denver, CO, June 1996 (abstract numbers 238 and 664) and at the 45th Annual meeting of the Society of Nuclear Medicine, Toronto, Ontario, Canada, June 1998 (abstract number 411). * Address correspondence to this author. Phone: 201-6058200. Fax: 973-605-1103. ‡ Immunomedics, Inc. § Garden State Cancer Center. 1 Abbreviations: ABS, 50 mM sodium acetate-150 mM sodium chloride-1% HSA, pH 6, buffer; anti-id, anti-idiotopic; aq, aqueous; BSA, bovine serum albumin; CDR, complementaritydetermining region; Bz-DTPA, benzyl DTPA; CEA, carcinoembryonic antigen; cpm, counts per minute; DOTA, 1,4,7,10tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid; DTPA, diethylenetriaminepentaacetic acid; EDC, 1-ethyl-(3,3-dimethylamino)propyl carbodiimide; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; hmAbs, humanized mAbs; HRP, horseradish peroxidase; HSA, human serum albumin; % ID/g, percent injected dose per gram of tissue; ITC-Bz-DTPA, isothiocyanatobenzyl DTPA; ITLC, instant thinlayer chromatography; mAbs, monoclonal antibodies; MSR, molar substitution ratio; MTD, maximum tolerated dose; MW, molecular weight; NHL, non-Hodgkin’s lymphoma; PBS, sodium phosphate buffer containing 150 mM sodium chloride; RAIT, radioimmunotherapy; SA, specific activity; SE-HPLC, sizeexclusion HPLC; sulfo-NHS, N-hydroxysulfosuccinimide.

yet to be clearly defined, but clinical and preclinical results obtained using unlabeled mAbs (7), radiolabeled mAbs alone (8), and radiolabeled mAbs along with chemotherapy (9) suggest that useful roles will also be identified in the treatment of at least some solid tumors. For adequate testing of RAIT and RAIT-related treatment modalities, high-quality radiolabeled mAbs must be made available. As far as the antibody part of any radioimmunoconjugate is concerned, because of the induction of human anti-mouse antibody (HAMA), particularly in patients treated with multiple doses of murine antibodies (10), CDR-grafted (humanized) mAbs (hmAbs), comprising over 90% human sequence homology, are preferred over murine or chimerized versions of mAbs. Two such hmAbs were prepared in our laboratories using the same human framework, and each was described in detail with regard to its binding properties compared to the original murine versions. They are hIMMU-14 (anti-carcinoembryonic antigen mAb, anti-CEA) (11) and hLL2 (antilymphoma mAb, anti-CD22) (12). 131Iodine has been the most widely used nuclide for RAIT studies, but its principal 364 keV, 81% abundant, γ emission and 8 day half-life will engender substantial levels of nonspecific toxic radioactivity being received by the recipient. Indeed, it has been estimated that up to one-half to two-thirds of the energy deposited from 131Ilabeled antibodies is due to penetrating γ emissions (13). The use of fragments (14) and antibody constructs (15) have been proposed to improve the therapeutic ratios

10.1021/bc980040g CCC: $15.00 © 1998 American Chemical Society Published on Web 10/24/1998

774 Bioconjugate Chem., Vol. 9, No. 6, 1998

obtained with 131I-based RAIT, although both approaches involve the adoption of radioimmunoconjugates with somewhat reduced tumor uptake, even when such fragments are chemically cross-linked to enhance stability (16). Another aspect to consider is the fact that certain mAbs are internalized, and when radioiodinated versions of these mAbs are used, radioiodine is not retained substantially in tumor after intracellular metabolism (17, 18). Of the available nuclides that can be reasonably considered as alternatives to 131I for RAIT, the pure β-particle-emitting 90Y is one of the most promising. Its lack of γ emissions will be advantageous in terms of target-localized radiation as well as general radiation safety. Its half-life could be suited for use with a F(ab′)2 fragment, but like all radiometals used with antibody fragments, 90Y tends to accumulate in kidney postinjection (19). Pretreatment with basic amino acids (20, 21) will reduce renal accretion substantially in animals and to some degree in humans, but there is still a significant portion of the injected fragment-associated-radioactivity retained. Among the bifunctional chelates useful for 90Y labeling of proteins, those based on the macrocycle DOTA produce the most stable complexes (22). However, a number of articles have pointed out the difficulties of obtaining reproducibly high yields of [90Y]DOTA-mAb conjugates (22-24), and for this reason, bifunctional chelates based on Bz-DTPA and its derivatives are still in widespread use (25). In addition, there have been reports of immune responses to both types of chelate in previously published works (26, 27), although use of a syngeneic mAb with either chelate has been predicted to be minimally immunogenic (28). The goal of this work was to choose a 90Y chelator and a form of CDR-grafted mAb (intact or fragment) in [90Y]mAb for eventual clinical application. For mAb labelings with radioyttrium, we set out to choose between DOTA and Bz-DTPA as preferred metal chelator on the basis of comparative in vitro and in vivo behavior of [90Y]DOTA-mAb and the corresponding [90Y]Bz-DTPA-mAb conjugates. The optimal conditions for the preparation and radiometal labelings of mAb-Bz-DTPA conjugates were already established (29). Optimization of mAbDOTA conjugate preparation and radiolabeling was examined in this study. DOTA conjugation and 90Y radiolabelings of two CDR-grafted mAbs were examined, and their in vitro stabilities and tumor cell bindings were compared with those of the corresponding [90Y]Bz-DTPAmAb conjugates. The 88Y-labeled DOTA and Bz-DTPA conjugates of a CDR-grafted mAb, hLL2, were also compared in in vivo animal studies, in terms of biodistribution in tumor-bearing mice, immune responses to chelates, and maximum tolerated dose in normal mice. Finally, we determined whether an [111In]mAb agent was predictive of the corresponding [90Y]mAb biodistribution (30) and whether 90Y-labeled fragments, used with Dlysine administration, might be superior to a comparably labeled IgG, using a humanized mAb. EXPERIMENTAL PROCEDURES

General Procedures. Intact and fragment forms of mAbs used in this study were obtained from Immunomedics’ antibody production laboratories. DOTA was purchased from Parish Chemical Co. (Orem, UT). All other chemicals, of high-purity grades, were obtained from commercial sources and used without further purification. Yttrium-90 chloride was purchased from

Govindan et al.

Pacific Northwest National Laboratory (Richland, WA) and, during the initial phase of the work, from NEN Life Science Products (Boston, MA). Indium-111 chloride was from NEN, while yttrium-88 chloride was from Los Alamos National Laboratory (Los Alamos, NM). HPLC analyses were performed on a Bio-Sil SEC-250 analytical column connected to a guard column (Bio-Rad Laboratories, Hercules, CA) and fitted with in-line UV and radiomatic detectors using 0.2 M sodium phosphate buffer, pH 6.8, as mobile phase at a flow rate of 1 mL/ min. Cell lines were obtained from the American Type Culture Collection (Rockville, MD), and were routinely tested for mycoplasma by the Mycotect assay (Life Technologies, Gaithersburg, MD). Preparation of DOTA Conjugates. A procedure based on the work of Lewis et al. (23) was adopted and modified. Briefly, antibodies were rendered metal-free, and exchanged with conjugation buffer in a sequence of dialyses or diafiltrations with 20 mM DTPA/0.25 M ammonium acetate, pH 7.0, 0.25 M ammonium acetate, pH 7.0, and 0.1 M potassium phosphate-0.1 M sodium bicarbonate, pH 8.5 buffers. Activated DOTA was prepared (4 °C, 45 min) using DOTA, sodium bicarbonate, sulfo-NHS, and EDC at 10:30:10:1 molar ratios. Conjugation was carried out by adding a 90-fold molar excess of activated DOTA to IgG, or a 30-50-fold molar excess to mAb fragments, adjusting pH to be in the 8.1-8.3 range, and incubating for 18 h at 4 °C. Purification was achieved by a series of dialyses or diafiltrations with 20 mM DTPA/0.25 M ammonium acetate, pH 7.0, and then with 0.25 M ammonium acetate, pH 5.4. The final conjugates, suitably concentrated, were filtered through 0.22 µm filters and stored under sterile conditions. A published procedure (31) involving conjugate labeling with a known excess of indium acetate, spiked with 111In, was used to determine DOTA MSRs. Variations in conjugation chemistry examined included coupling pH (7.5, 8.0, and 8.5) and molar excess of activated DOTA to mAb used (40, 100, or 500). Preparation of Bz-DTPAhLL2 conjugate was based on the procedure described previously (29). Comparison of Affinities of DOTA-Conjugated and Unmodified mAbs. Competitive binding assays were carried out using Raji cells (a human Burkitt lymphoma cell line) and CEA as the source of antigens for hLL2 and hIMMU-14, respectively. For hLL2, a 96well filtration plate fitted with a microporous (0.45 µm pore) hydrophilic membrane assembly (Millipore, Milford, MA) was used to incubate a 0.1 mL suspension of Raji cells (∼200 000) with a fixed concentration of [125I]murine LL2 (∼500 000 cpm) and varying concentrations (0.1100 µg/mL) of competitors (murine LL2 control or DOTA conjugate of hLL2). After gently shaking the assembly for 2.5 h at ambient temperature, the well contents were drained by vacuum, and the wells were washed three times with 0.2 mL of 10 mM PBS, pH 7.4, and 1%BSA. Membrane-bound residual 125I activity was determined in a γ counter, and the readings were corrected for nonspecific membrane binding. The latter was determined using a control consisting of [125I]mLL2 in the same final volume but without the antigen. Duplicate measurements were made, and the average residual bindings were plotted as a function of competitor concentrations. For hIMMU-14 substrates, an IMMU-14 blocking assay was performed as follows. ELISA plates were coated with CEA (Calbiochem, San Diego, CA) at a concentration of 2.5 µg/mL and incubated for 1 h at 20 °C. HRP-conjugated murine IMMU-14, at a concentration of 200 ng/mL, was mixed with varying concentrations

90Y-Labeled

Humanized mAbs for Cancer Therapy

of murine IMMU-14 (control) or hIMMU-14-DOTA conjugate, added to CEA-coated ELISA plates, and incubated for 1 h at 20 °C. CEA binding of HRP-conjugated IMMU14 was determined with a substrate solution containing 0.0125% of H2O2 and 0.004 M O-phenylenediamine dihydrochloride. Each measurement was the mean of duplicate experiments. Radiolabelings. In optimizing [90Y]mAb yields, the DOTA MSR, pH (5-6 range), incubation time (up to 2.5 h), use of ascorbic acid as a radioprotectant, and the effect of trace metal contaminants in commercial 90Y were examined. In an optimized radiolabeling, the DOTA conjugate, at a concentration of 3-10 mg/mL, was added to yttrium-90 acetate (obtained by buffering yttrium-90 chloride with 0.25 M ammonium acetate pH 5.4), so as to achieve a specific activity in the range 1-5 mCi/mg. Freshly prepared aq ascorbic acid (0.25 g of ascorbic acid dissolved in 1 mL of water) was added to the labeling mixture at 27 µL/mL of the labeling volume and heated at 45 °C (bath) for 2 h. The final ascorbic acid concentration was ∼6 mg/mL. The solution was made 10 mM in DTPA using aq 0.1 M DTPA (pH 6-7), and heated for 20-30 min at the same temperature. The radiolabeled mAb was diluted to ∼1 mCi/mL using ABS buffer. 111In and 88Y radiolabelings were carried out in a similar fashion, except the specific activities of [88Y]mAbs were ∼0.1 mCi/mg. Radiolabel incorporations in the latter categories were usually 90% or greater, but yields of less than 90% were observed in some 88Y labelings. For this reason, the radiolabeled mAbs for in vivo studies were routinely purified by centrifuged-size-exclusion column purification using Sephadex G50/80 equilibrated in 0.1 M sodium acetate, pH 6.5. These conjugates were at least 95% pure by ITLC and HPLC; the remainder was present as EDTA- or DTPA-bound isotope. In Vitro Stability. Purified [90Y]DOTA-mAb (∼1625 µCi, in ∼4-6 µL) was added to 0.55 mL of fresh human serum, which was made 0.02% in sodium azide, and incubated at 37 °C. The substrate concentration was 170 nM for hLL2 and 143 nM for hIMMU-14. In a similar fashion, radiolabeled preparations (∼14-18 µCi, in ∼3-6 µL) were added to 0.55 mL of 1 mM DTPA/1% HSA in 25 mM ammonium acetate/150 mM saline, pH 6.4, and incubated at 37 °C. The DTPA in the latter medium was in 10325-fold and 6244-fold molar excess with respect to hLL2 and hIMMU-14, respectively. Aliquots of serum and DTPA incubates were analyzed, on size-exclusion analytical HPLC, over a 10 day period. Serum aliquots were briefly challenged with DTPA (1 mM) prior to HPLC analyses. Analyses consisted of determining the percent of radioactivity associated with the antibody. This was accomplished by complexing the antibody with a 100-fold molar excess of either an antiidiotopic antibody, WN (32), for hLL2, or CEA (for hIMMU-14), either of which converted radiolabeled antibody to a higher MW component (hLL2:WN complex or hIMMU-14:CEA complex), appearing as an earlier eluting peak on SE-HPLC. This method separated, by HPLC retention time, [90Y]DOTA-mAb from serum proteins such as albumin and transferrin. Loss of label to serum proteins, as well as loss of label as free metal ion (analyzed as [90Y]DTPA), were then determined from integrations of all HPLC peaks. In a separate experiment, [90Y]DOTA-hLL2 was compared with [90Y]BzDTPA-hLL2 for stability in serum and 1 mM DTPA, over an 11 day period at 37 °C, using 5-7 µg of radiolabeled conjugate and 0.55 mL of serum or DTPA, as described above.

Bioconjugate Chem., Vol. 9, No. 6, 1998 775

In Vitro Retention of [90Y]hLL2 on Lymphoma Cells. For these studies, a DOTA conjugate, as well as a Bz-DTPA conjugate derived from ITC-Bz-DTPA, of hLL2-IgG were radiolabeled at over 90% incorporation. The radiolabeled conjugates were made 10 mM in DTPA (for DOTA conjugate) or EDTA (for Bz-DTPA conjugate) to chelate unbound radiometal. 125I-Labeled hLL2 (or [125I]mLL2) was prepared using a chloramine-T procedure (33). Raji cells (American Type Culture collection, Rockville, MD) were cultured in Dulbecco’s Eagle Medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum, 5% horse serum, and 1% pyruvate, penicillin/streptomycin and glutamine, in a 5% CO2 atmosphere. Cells were allowed to grow to confluence. For binding studies, cells (5 × 105 to 1 × 107) in cell culture media were treated with ∼10-50 µCi of [90Y]mAb conjugate (5-25 µg) and incubated at 37 °C for 2 h. The cells were separated from supernatant by centrifugation at 1500 rpm for 6 min, washed three times with cold media, resuspended in media, and incubated at 37 °C. At periodic intervals over 40 h, aliquots were withdrawn, centrifuged to separate cells from the supernatant, and the radioactivity associated with each was counted using appropriate windows set for 90Y and 125I. The percent of cell-associated radioactivity was thus calculated for each time point. In general, measurements reflect the mean of triplicate samples. To determine the percent of cellassociated radioactivity internalized into tumor cells, aliquots of cell suspension at various time points (as described in the above experiment) were first chilled at 4 °C and then centrifuged. The cell pellet was then treated with 0.1 M sodium acetate/0.1 M glycine at pH 3 for 30 min at 40 °C, then spun down and separated prior to counting. In these experiments, [125I]hLL2 was used as a comparative substrate and [90Y]IMMU-14 was used as nonspecific control. Animal Experiments. All animal experiments were carried out under approved IACUC protocols. For hLL2 conjugates, 3-4 week old nude mice (Taconic, Germantown, NY) were quarantined for 2 weeks, and then injected s.c. with 5 × 106 Ramos tumor cells grown in tissue culture. Tumor growth was visible after a further 2 week period, and animal experiments were started immediately thereafter. For biodistribution studies, 1-2 µCi of [88Y]mAb or 10-20 µCi of [111In]mAb was used for each tumor-bearing mouse. One experiment involved comparing [88Y]DOTA-hLL2-IgG (SA, 0.06 µCi/µg; 2.3% aggregation; protein dose per animal, 17.4 µg) with [88Y]Bz-DTPA-hLL2-IgG (SA, 0.08 µCi/µg; 0% aggregation; protein dose per animal, 14.2 µg). A second experiment was a paired-label experiment involving targeting of [88Y]DOTA-hLL2 (SA, 0.08 µCi/µg; 1.19 µCi/animal) and [111In]DOTA-hLL2 (SA, 1.1 µCi/µg, 16.4 µCi/animal), with a total protein dose of 15 µg/animal. Corrections for the backscatter of 88Y into the 111In window were made in determining 111In biodistributions. A third experiment consisted of two groups of animals administered with [88Y]DOTA-hLL2 F(ab′)2 or [88Y]DOTA-hLL2 Fab′, with D-lysine administration (40 mg/mouse) given intraperitoneally 15 min before and 1, 2, and 3 h after the i.v. injection of radiolabeled conjugates. Each animal in this experiment received a protein dose of 21.3 µg [F(ab′)2 fragment] or 20.7 µg [Fab′ fragment] and 1.0 µCi of 88Y activity. A fourth experiment was the repetition of radiolabeled fragment biodistributions, as before, but without D-lysine administration. Necropsy was performed at 1, 3, 5, 7, and 10 days after radiolabeled IgG injection or at 4, 24, and 48 h after radiolabeled fragment


injection. At the indicated times, animals were anesthetized with sodium pentobarbital, bled by cardiac puncture, and then sacrificed. Tissues were weighed and the radioactivity was determined in a γ counter using appropriate windows. In each instance, counting standards were included for the determination of total injected radioactivity. The data were then calculated as a percentage per gram of the total injected activity. For MTD experiments, 11 mg each of hLL2-DOTA-IgG and hLL2-Bz-DTPA-IgG were radiolabeled with 30 mCi of 90Y activity. The radiochemical purity was 94% for the DOTA conjugate and 99% for the Bz-DTPA conjugate. Unbound 90Y was present as [90Y]DTPA in the DOTA conjugate and as [90Y]EDTA in the Bz-DTPA conjugate. In each case, the radiolabeled mAb was diluted serially with ABS buffer to obtain the desired microcurie amount in a final volume of 0.15 mL. Radioactivity contained in the injectate volume in each case was determined by counting suitably diluted samples in a β-counter. Eightyfour normal BALB/c mice were sorted into six groups of seven animals, with animals in each group given 125, 150, 175, 200, 225, or 250 µCi of [90Y]DOTA-hLL2 or [90Y]Bz-DTPA-hLL2. Animal weights and mortality, postinjection of radiolabeled conjugates, were recorded over time. To examine the immunogenicity of DOTA and BzDTPA, forty-eight normal BALB/c mice were divided into four groups of 12 animals each. Animals in each group received either murine LL2-IgG or LL2 F(ab′)2 conjugates of DOTA (containing 3-4 chelates/mAb) or Bz-DTPA (containing 1-2 chelates/mAb), administered i.v. at 33 µg/mouse. After 3 weeks, four mice in each group were anesthetized, bled by cardiac puncture, and sacrificed by cervical dislocation. Serum samples were collected and stored frozen for subsequent analyses. The remaining mice in each group were readministered the respective substrates at the same dose of 33 µg/mouse. After a further 3 week period, again, four mice from each group were taken and serum samples collected and stored, while remaining mice in each group received a third dose of the conjugates. These mice were taken in turn after an additional 3 week period. Serum samples (50 µL) from individual mice were incubated with 100 ng (10 µL) of [125I]mLL2-Bz-DTPA and [125I]mLL2-DOTA, in a total volume made up to 100 µL with 5% HSA, at 37 °C for 1 h. The samples were then analyzed, by SE-HPLC, for their ability to form immune complexes with added radiolabeled conjugates. RESULTS AND DISCUSSION

Development of suitable bifunctional chelating agents for 90Y has evolved from using DTPA cyclic anhydride (34), to backbone-substituted bifunctional DTPAs (BzDTPA, MX-DTPA, and cyclohexyl-DTPA) (35, 36) and to chelates based on the macrocyclic chelating agent DOTA (37). The exceptional kinetic inertness of DOTA-type chelates has been documented (38). Such stability portends well for RAIT applications, as any dissociation of 90Y from mAb-chelate conjugates can result in a portion of this radioactivity being bound in cortical bone, which could increase the level of myelotoxicity associated with these agents. The routine use of DOTA-mAb conjugates has been hampered by required lengthy syntheses of DOTA bifunctional chelates and subsequent difficulties in 90Y radiolabeling of DOTA-mAb conjugates. Lewis et al. (23) addressed these problems by using in situgenerated monoactivated DOTA for mAb conjugation, which obviated a long synthesis, and by employing a

Govindan et al.

Figure 1. Comparisons of immunoreactivities of hLL2-DOTA conjugates with that of unmodified antibody, determined in competitive binding assays using Raji cells as the source of antigen. The competitive binding of hLL2 conjugate with a DOTA MSR of 3.7 (2) is similar to that of unmodified hLL2 (0). The conjugate with a DOTA MSR of 6.7 (O) exhibits a somewhat reduced competitive binding.

higher temperature 90Y-labeling of a mAb-DOTA conjugate. By conducting the radiolabelings at 43 °C, a 59% 90Y incorporation was obtained for a cT84.66-DOTA mAb conjugate. We have optimized this methodology for our own humanized DOTA-mAbs, using a 90-100-fold molar excess of activated DOTA for IgGs and a 40-50-fold molar excess of activated DOTA for F(ab′)2 and Fab′ fragments. The DOTA:mAb MSRs for our conjugates were determined by the method of Meares et al. (31) using 111In-spiked indium. At a given molar excess of DOTA-NHS used in conjugations, variations of pH in the 7.5-8.5 range did not affect the final DOTA MSR. The MSR was influenced by the molar excess of activated DOTA used in conjugations. At pH 8.0-8.3, a 100-fold molar excess of DOTA gave a substitution ratio of 3.73.9 for hLL2, while a 500-fold excess of DOTA gave a MSR of 9.0. With hIMMU-14, typically 4-5 DOTAs were introduced under the same conditions, using 100-fold excess of monoactivated DOTA. For hLL2-F(ab′)2 and hLL2 Fab′, DOTA substitutions of 3.6 and 1.5, respectively, were obtained using a 40-50-fold molar excess of activated DOTA. By comparison, the average number of Bz-DTPA groups on hLL2, in the hLL2-Bz-DTPA conjugate, was 1.5. Competitive Binding Assays. The effect of MSR on immunoreactivity was examined for conjugates of hLL2 and hIMMU-14 by competitive binding assays on Raji cells and CEA, respectively. Figure 1 shows competitive binding to Raji cells of hLL2 (control) and two hLL2DOTA conjugates differing in the DOTA MSR. Figure 2 shows results from an ELISA assay involving residual binding, to CEA, of HRP-conjugated IMMU-14 as a function of competitor concentration against either hIMMU-14-DOTA substituted at a 4.4:1 ratio or murine IMMU-14 as control. For the hLL2 conjugate, the binding profiles for a conjugate with 3.7 DOTA is similar to that of unmodified hLL2, whereas a DOTA substitution of 6.7 leads to a somewhat reduced competitive inhibition (Figure 1). The CEA-blocking experiment (Figure 2) showed that a hIMMU-14-IgG-DOTA conjugate, with a DOTA MSR of 4.4, had a target binding profile identical to that of control IMMU-14. For a murine IMMU-14-DOTA conjugate containing 6.8 DOTAs, competitive binding to CEA was similar to that of unsubstituted IMMU-14 (not shown). While the limit of DOTA substitution beyond which a reduction in immunoreactivity is observed is seen to be antibody dependent,

90Y-Labeled



Figure 2. Comparison of immunoreactivity of hIMMU-14DOTA conjugate with DOTA MSR of 4.4 (2) with that of unmodified IMMU-14 (O), determined in a CEA-blocking assay. The competitive binding profiles of the two are very similar. Table 1. Representative Incorporations of IgG-DOTA Conjugates

conjugate

mCi

hIMMU-14-IgG

2.3a

hLL2-IgG

2.8a 4.0a 4.9a 13.0b 22.0b 23.5b 30.0b 32.2b 45.0b

specific activity (mCi/mg) 4.6 2.8 4.7 3.3 3.4 2.2 2.1 2.7 2.9 4.1

90Y

into

incorporation (%) by HPLC IgG

aggregate

94.7 93.6 93.2 91.3 94.3c 99.5d 90.0 94.4e 99.4f 98.6g

5.0 5.2 3.6 6.5 0.7 0 1.0 1.6 0 0

a Ascorbic acid was not used. b Ascorbic acid was used at a concentration of ∼6 mg/mL, in these labelings. c ITLC incorporations of 96.3% were observed in these findings. d ITLC incorporations of 97.0% were observed in these findings. e ITLC incorporations of 94.5% were observed in these findings. f ITLC incorporations of 97.9% were observed in these findings. g ITLC incorporations of 97.6% were observed in these findings.

apparently even in mAbs having the same human framework, we conclude that DOTA substitution in the 3-5 range would generally be well tolerated. 90Y Radiolabelings. Maximizing the DOTA:mAb substitution ratios, consistent with the preservation of tumor targeting properties, provided the best opportunity for near-quantitative 90Y incorporations under optimized labeling conditions. Optimal radiolabeling conditions involved adding the desired radioactivity of yttrium-90 chloride buffered with 0.25 M ammonium acetate, pH 5.4) to the conjugate containing aq ascorbic acid (∼6 mg/mL) and incubating at 45 °C for 2 h. The final conjugate concentration was 3-12 mg/mL (the range of concentrations examined), and the pH was approximately in the 5.0-5.4 range. Table 1 shows typical yields for 90Y labelings of hIMMU-14- and hLL2-DOTA, using 2.3-45 mCi of 90Y. Initially, ascorbic acid was not used during radiolabeling, but we found that addition of ascorbic acid during labeling significantly reduced aggregation (Table 1). During the course of our work, Chakrabarti et al. (39) reported on the use of ascorbic acid as a radioprotectant during 90Y labelings. These workers found that the immunoreactivity of a 100 µg sample of a 125I-labeled murine mAb was reduced by about 65-90% in the presence of 320-640 Gy doses of [90Y]DTPA (1.1-2.2 mCi), but the immunoreactivity was completely restored when the same experiment was conducted in the presence of ∼0.2-11 mg/mL concentration of ascorbic acid. Incorporation of 90Y into DOTA-mAb conjugates was

Figure 3. In vitro stabilities of [90Y]DOTA-hIMMU-14 in serum (0) and in 1 mM DTPA/1% HSA (4) and of [90Y]DOTA-hLL2 in serum (2) and in 1 mM DTPA/1% HSA (O). The y-axis scale is displayed in a narrow range of 95-101%.

found to be affected by trace metal contaminants in the yttrium-90 chloride used. Stimmel et al. (24) have studied the role of ferrous, zinc, and calcium ions on the efficiency of 90Y incorporation into free macrocyclic and acyclic 90Y chelators. Their quantitative analyses showed that 90Y chelation is most sensitive to the presence of trace levels of zinc and, to a lesser extent, ferrous and calcium ions. We found the same connection between 90Y yields into DOTA-mAb conjugates and trace metal contaminants in 90 Y shipments, with >10-50 ppm levels of contaminating trace metals, particularly zinc, being detrimental. In Vitro Stability of [90Y]hLL2-DOTA and hIMMU14-DOTA. The stability of radiolabeled conjugates to serum and to DTPA challenge was examined using substrate concentations of 140-170 nM in serum and DTPA/conjugate molar ratios of ∼6000-10000:1. Incubations were kept at 37 °C for 10 days. At regular intervals, aliquots were analyzed by SE-HPLC. To separate radiolabeled IgG from other serum proteins, as well as from HSA in DTPA-challenge assays, aliquots of challenged material were complexed with CEA (in the case of hIMMU-14) or WN (anti-id mAb to hLL2; ref 32) prior to HPLC analyses. As seen from Figure 3, the percentage of 90Y associated with antibody was >98% over this time period, which affirmed the kinetic stability of these [90Y]DOTA-mAb conjugates. In a separate experiment, 0, 0, and 0.4% of the radioactivity (analyzed as the [90Y]DTPA by HPLC) were found to be dissociated from the [90Y]DOTA-hLL2 conjugate when incubated in serum at 37 °C and analyzed after 18 h, 5 d, and 11 d, respectively. Under the same conditions, 3.0, 3.6, and 10.7% of the radioactivity (analyzed as the [90Y]EDTA by HPLC) were found dissociated from the [90Y]Bz-DTPAhLL2 conjugate after serum challenge. When incubated in 1 mM DTPA under the same conditions and analyzed over the same period of time, 0, 1, and 0.3% of the radioactivity of the DOTA conjugate and 1.6, 5.2, and 8.7% of the radioactivity of the Bz-DTPA conjugate of hLL2, respectively, were found unbound. In Vitro Binding Studies of Radiolabeled hLL2 Conjugates. In vitro binding studies were carried out using 90Y-labeled murine and humanized LL2 mAbs, along with the respective mAbs radioiodiated by the chloramine-T method (33), to compare binding, retention, turnover, and metabolism by Raji tumor cells (40). At time points out to 40 h, a higher percentage of the [90Y]hLL2 (12-70% higher, increasing with time), compared to radioiodinated hLL2, remained associated with the cells (Figure 4A). A small reduction in the percentage of retained activity was observed for [90Y]DOTA versus [90Y]Bz-DTPA conjugates of hLL2 (Figure 4A) in some


Govindan et al.

Figure 4. In vitro cell bindings of 90Y-[Bz-DTPA]-hLL2 (9), [90Y]DOTA-hLL2 (4) and [125I]hLL2 (O) on Raji cells. (A) Percent of initially bound radioactivity still retained on cells at various times, and (B) percent of cell-bound radioactivity internalized into tumor cells at various times.

earlier experiments, but this difference was not noticed subsequently when the radiolabeling procedure included the use of ascorbic acid as radioprotectant (not shown). Percent of cell-bound mAb internalized at various times is shown in Figure 4B. At later time points, cells treated with [90Y]DOTA-hLL2 and [90Y]Bz-DTPA-hLL2 had between 50 and 60% of nonremovable cell-bound radioactivity, while those treated with [125I]hLL2 had between 30 and 40% nonremovable activity. In a cell-binding experiment using [90Y]Bz-DTPA-hLL2, the SE-HPLC of cell-culture supernatant showed a peak eluting with a retention time of 13.4 min, indicating that 90Y was released from cells in a low MW form. This retention time was similar to that of [90Y]DTPA, although the SEHPLC would not distinguish it from [90Y]DTPA-lysine adduct. Lysine adducts of metal chelates had been inferred to be the end products of antibody-chelate catabolism in the lysosomes (41). The bindings of [90Y]-

DOTA-hLL2 and [90Y]Bz-DTPA-hLL2 were similar when continuously exposed to Raji, Daudi, or Ramos cell lines, as were the bindings observed for each of these conjugates, in the Raji cell line, using ∼30 mCi level radiolabeling preparations (not shown). These findings implied that higher temperature DOTA conjugate labeling with high-energy 90Y did not impair its cell-binding ability. Other experiments involving divalent fragments of murine and humanized LL2 showed similar trends of higher binding for 90Y versus 125I-labeled conjugates, while nonspecific controls ([90Y]Bz-DTPA-murIMMU-14 and other IMMU-14 derivatives) did not bind to Raji cells (not shown). This is the first extensive study involving radiolabeled conjugates of intact and fragment forms of hmAb, including comparisons with the murine counterparts and using three different cell lines (40). The results obtained are similar to in vitro tumor cell binding data reported for murine LL2 labeled with either a residualizing form of radioiodine or 111In (42). Internalizations observed in Raji cell binding of [90Y]hLL2 conjugates suggest that they should be preferred agents compared to iodinated hLL2 in targeting B-cell lymphoma. In Vivo Experiments. For biodistributions involving radioyttrium, conjugates labeled with γ-emitting 88Y were used to render determination of tissue-bound radioactivity more accurate. Tumor targeting and biodistribution of [88Y]DOTA-hLL2 and [88Y]Bz-DTPA-hLL2 were compared in s.c. Ramos tumor xenograft-bearing nude mice. Table 2 shows comparative biodistribution out to 10 days, which is 3.75 times 90Y physical half-life. [88Y]DOTAhLL2 had 2.4, 1.8, 1.6, 1.4, and 1.0% injected dose/g (% ID/g) uptake in bone at 1, 3, 5, 7, and 10 days postinjection and the [88Y]Bz-DTPA-hLL2 conjugate had 2.8, 3.4, 3.4, 2.7, and 3.6% ID/g in bone at the same times. As discussed by DeNardo et al. (43), these differences may be particularly significant when one considers clinical RAIT since human marrow volume is much larger than that of mouse, and the differences seen in these animals may be reflective of a larger toxicity difference in a clinical situation. Dosimetrically (44), per millicurie numbers derived from these data were 10 564, 8274, 6866, and 23 921 for [90Y]DOTA-hLL2-IgG and 9901, 9579, 7403, and 27 124 for [90Y]Bz-DTPA-hLL2 for blood, liver, bone, and Ramos tumor, respectively. A second experiment compared biodistribution of [111In]DOTA-hLL2 to [88Y]DOTA-hLL2 (Table 3). This was a paired-label experiment which maintained identi-

Table 2. Biodistributions of [88Y]DOTA-hLL2 (A) and 88Y-Bz-DTPA-hLL2 (B) in Nude Mice Bearing s.c. Ramos Human Tumor Xenografts (Weight in Grams) at Times Indicated percent injected dose per gram ( SD, n ) 4-5 tissue Ramos tumor (g)

A B

liver spleen kidney lungs blood bone washed bone

A B A B A B A B A B A B A B

24 h

72 h

120 h

168 h

240 h

16.0 ( 4.5 (0.5 ( 0.2) 19.0 ( 3.4 (0.2 ( 0.1) 9.6 ( 2.0 8.3 ( 1.1 5.9 ( 1.6 6.4 ( 0.9 5.9 ( 1.7 6.0 ( 0.9 6.4 ( 1.4 6.7 ( 1.1 11.4 ( 3.1 12.7 ( 1.2 2.4 ( 0.8 2.8 ( 0.1 1.7 ( 0.3 1.7 ( 0.2

27.6 ( 5.1 (0.3 ( 0.1) 25.6 ( 3.9 (0.4 ( 0.1) 6.4 ( 0.9 8.8 ( 1.3 4.4 ( 0.9 6.6 ( 1.2 4.6 ( 0.7 6.5 ( 0.8 5.2 ( 0.4 5.2 ( 1.4 7.4 ( 4.3 9.3 ( 1.3 1.8 ( 0.7 3.4 ( 0.8 1.7 ( 0.7 2.3 ( 0.7

18.5 ( 3.8 (0.7 ( 0.4) 26.3 ( 15.9 (0.9 ( 0.5) 6.3 ( 2.8 7.2 ( 2.6 4.9 ( 1.0 5.5 ( 1.5 3.6 ( 0.3 4.6 ( 1.3 3.8 ( 0.5 4.0 ( 0.5 5.5 ( 1.4 4.2 ( 0.9 1.6 ( 0.3 3.4 ( 0.6 1.0 ( 0.2 3.0 ( 0.7

15.5 ( 5.6 (0.8 ( 0.5) 16.4 ( 5.5 (0.8 ( 0.3) 3.9 ( 0.6 6.0 ( 1.3 4.2 ( 0.5 4.7 ( 1.0 3.3 ( 0.5 4.1 ( 0.4 2.9 ( 0.6 3.6 ( 0.4 5.2 ( 1.1 4.2 ( 0.6 1.4 ( 0.3 2.7 ( 0.2 1.1 ( 0.3 2.7 ( 0.7

12.6 ( 17.2 (1.6 ( 1.0) 11.4 ( 10.1 (1.9 ( 1.3) 2.4 ( 0.6 4.8 ( 1.2 3.4 ( 1.1 4.0 ( 1.4 2.0 ( 0.4 2.6 ( 0.6 1.6 ( 0.6 2.4 ( 1.1 2.5 ( 0.9 2.2 ( 1.6 1.0 ( 0.4 3.6 ( 0.6 0.6 ( 0.2 2.7 ( 0.6

90Y-Labeled



Table 3. Biodistributions of [88Y]DOTA-hLL2 (A) and [111In]DOTA-hLL2 (B) in Nude Mice Bearing s.c Ramos Human Tumor Xenografts (Weight in Grams) at Times Indicated percent injected dose per gram ( SD, n ) 4-5 tissue Ramos tumor (g)

A B

liver

A B A B A B A B A B A B A B A B

spleen kidney lungs blood small intestine large intestine bone

24 h

72 h

120 h

168 h

240 h

17.8 ( 3.7 (0.2 ( 0.1) 16.7 ( 3.2 (0.2 ( 0.1) 7.7 ( 2.1 6.2 ( 1.4 5.3 ( 1.0 5.1 (1.0 3.6 ( 0.6 3.8 ( 0.6 5.5 ( 0.6 5.5 ( 0.5 11.7( 1.8 11.3 ( 1.6 1.4 ( 0.1 1.3 ( 0.1 2.3 ( 1.1 2.1 ( 1.0 2.3 ( 0.4 2.1 ( 0.3

17.6 ( 6.1 (0.2 ( 0.1) 17.3 ( 6.6 (0.2 ( 0.1) 6.0 ( 1.2 5.7 ( 1.1 4.7 ( 0.7 4.3 ( 0.7 3.4 ( 0.8 3.4 ( 0.7 4.8 ( 0.8 4.6 ( 0.9 9.3 ( 1.3 8.9 ( 1.2 1.1 ( 0.2 1.1 ( 0.1 1.2 ( 0.2 1.2 ( 0.2 2.5 ( 0.8 2.1 ( 0.9

18.9 ( 12.6 (0.2( 0.1) 18.2 ( 11.4 (0.2 ( 0.1) 4.1 ( 1.3 4.0 ( 1.2 3.5 ( 1.0 3.3 ( 0.9 2.0 ( 0.4 2.0 ( 0.4 3.1 ( 0.6 3.0 ( 0.7 5.0( 1.5 4.9 ( 1.4 0.6 ( 0.1 0.6 ( 0.1 0.8 ( 0.3 0.7 ( 0.3 1.4 ( 0.4 1.1 ( 0.3

11.0 ( 6.0 (0.4 ( 0.2) 11.0 ( 5.7 (0.4 ( 0.2) 3.7 ( 0.7 3.8 ( 0.7 3.9 ( 0.3 3.5 ( 0.3 2.5 ( 0.5 2.4 ( 0.5 2.8 ( 0.5 2.8 ( 0.5 5.7 ( 1.3 5.6 ( 1.3 0.8( 0.2 0.7 ( 0.2 0.7 ( 0.1 0.7( 0.1 1.5 ( 0.1 1.3 ( 0.2

7.7 ( 4.9 (1.0 ( 1.3) 7.3 ( 4.4 (1.0 ( 1.3) 2.8 ( 0.9 2.7 ( 0.9 3.2 ( 1.4 2.6 ( 1.4 1.5 ( 0.5 1.3 ( 0.5 2.4 ( 1.2 2.1( 1.0 3.2 ( 1.7 3.0 ( 1.6 0.5 ( 0.3 0.5 ( 0.3 0.6 ( 0.3 0.5 ( 0.3 1.4 ( 0.7 1.0 ( 0.7

Table 4. Biodistributions of [88Y]DOTA-hLL2-F(ab′)2 with Administration of 4 × 2 mg of D-Lysine per Gram of Animal Weight (A) and without D-Lysine Administration (B) in Nude Mice Bearing s.c. Ramos Human Tumor Xenografts (Weight in Grams) at Times Indicated

Table 5. Biodistributions of [88Y]DOTA-hLL2-Fab′ with Administration of 4 × 2 mg of D-Lysine per Gram of Animal Weight (A) and without D-Lysine Administration (B) in Nude Mice Bearing s.c. Ramos Human Tumor Xenografts

percent injected dose per gram ( SD, n ) 4 tissue Ramos tumor (g)

4h A B

liver spleen kidney lungs blood bone

A B A B A B A B A B A B

24 h

percent injected dose per gram ( SD, n ) 4 48 h

2.5 ( 0.7 2.6 ( 0.7 1.7 ( 0.3 (0.2 ( 0.1) (0.3 ( 0.1) (0.23 ( 0.21) 2.0 ( 0.8 2.7 ( 0.8 3.1 ( 1.0 (0.19 ( 0.15) (0.3 + 0.1) (0.13 ( 0.07) 8.7 ( 1.4 5.8 ( 0.8 5.2 ( 1.4 7.7 ( 3.2 8.0 ( 0.9 7.7 ( 1.1 5.3 ( 0.8 4.9 ( 0.6 4.8 ( 1.3 6.4 ( 3.1 6.9 ( 2.1 13.0 ( 4.1 19.9 ( 4.6 39.3 ( 4.3 28.4 ( 6.2 69.9 ( 27.6 73.7 ( 6.9 76.0 ( 18.4 4.9 ( 0.8 1.1 ( 0.2 1.2 ( 0.8 3.4 ( 1.0 1.4 ( 0.1 1.3 ( 0.3 10.2 ( 5.6 0.5 ( 0.1 0.4 ( 0.1 5.6 ( 1.2 0.4 ( 0.0 0.4 ( 0.1 2.5 ( 0.3 1.7 ( 0.4 1.2 ( 0.3 2.9 ( 1.5 2.7 ( 0.6 5.9 ( 1.9

cal animal and tumor weight conditions for both the labels. The 111In radioconjugate very accurately predicted the blood clearance of the 88Y conjugate with 11.3, 8.9, 4.9, 5.6, and 3.0% ID/g and 11.7, 9.3, 5.0, 5.7 and 3.2% ID/g at 1, 3, 5, 7, and 10 days postinjection, for the 111In and 88Y conjugates, respectively. As can be seen, the percent injected dose per gram and standard deviations for all other organs, particularly bone, at all time points were also remarkably similar. This finding is important in that at least one other study, with other mAb conjugates, has suggested that 111In is not predictive of 90Y biodistribution (45). Tables 4 and 5 show animal data for [88Y]DOTA-hLL2F(ab′)2 and [88Y]DOTA-hLL2-Fab′, respectively, and in each case, biodistributions were determined with and without D-lysine administration. With radiometalated fragments, kidney uptake of the radiometal is a serious problem, and Behr et al. (20) and others (21) have shown that kidney radioactivity levels can be reduced by administering basic amino acids, such as lysine, with D-lysine recommended (20). Lysine dose selection and timing are in themselves problematic, as discussed by DePalatis et al. (21), in that timing of the lysine admin-

tissue Ramos tumor (g)

A B

liver spleen kidney lungs blood bone

A B A B A B A B A B A B

4h

24 h

48 h

1.9 ( 0.9 (0.3 ( 0.3) 1.9 ( 1.2 (0.2 ( 0.1) 2.5 ( 0.5 1.8 ( 0.8 1.3 ( 0.3 2.0 ( 1.2 19.9 ( 11.5 104 ( 24.7 1.5 ( 0.4 1.6 ( 0.1 2.0 ( 0.4 1.4 ( 0.3 0.8 ( 0.2 1.0 ( 0.6

1.6 ( 0.2 (0.2 ( 0.1) 2.1 ( 0.4 (0.2 ( 0.1) 1.8 ( 0.2 2.1 ( 0.4 1.6 ( 0.2 2.8 ( 0.5 29.1 ( 11.8 105 ( 15.2 0.6 ( 0.1 1.0 ( 0.2 0.3 ( 0.02 0.3 ( 0.03 0.6 ( 0.3 1.4 ( 0.5

1.8 ( 0.7 (0.1 ( 0.03) 1.1 ( 0.2 (0.3 ( 0.1) 1.4 ( 0.2 2.0 ( 0.4 1.1 ( 0.3 2.1 ( 0.7 15.9 ( 4.8 81.4 ( 4.4 0.4 ( 0.1 3.1 ( 4.6 0.2 ( 0.01 0.2 ( 0.03 0.4 ( 0.1 1.0 ( 0.5

istration for Fab′ and F(ab′)2 species is expected to be different. In our work, kidney levels decreased up to 5-fold in the case of the [88Y]Fab′ and somewhat less in the case of F(ab′)2, although kidney percent injected dose per gram then rose sharply between 4 and 24 h time points, from 19.89 to 29.11% ID/g for the Fab′ and 19.93 to 39.31% ID/g for the F(ab′)2. The tendency to increased kidney percent injected dose per gram seems indicative of a need to continuously administer D-lysine beyond 3 h postinjection of the labels. For clinical applications, these D-lysine levels appear impractical. Even with lysine administration, dosimetry numbers remained adverse for both fragments at 1200, 5443, 17 129, and 1162 rads/mCi for [90Y]DOTA-F(ab′)2 and 1536, 1497, 10 959, and 350 rads/mCi for [90Y]DOTA-Fab′ for Ramos tumor, liver, kidney and blood, respectively. A MTD study was performed with 90Y-labeled DOTAand Bz-DTPA-IgG conjugates in normal BALB/c mice, using six increasing doses in the range 125-250 µCi. The MTD for both reagents was identical since all animals survived at a dose of 125 µCi, while with each reagent, three of seven died at the 150 µCi dose level. Two points are of significance here. First, these data differ from that


of DeNardo et al. (43) using radiolabeled Lym-1 antibody in that they found a significantly higher toxicity for their mAb when labeled with DTPA versus DOTA chelates. Second, the microcurie MTD amount of [90Y]mAb using hLL2 in nude mice is much less than reported with other mAbs, which are usually in the 250 µCi/animal range (43). The occurrence of immune response to DOTA chelate was first noticed (26) in the form of serum sickness in some patients in a phase I/II trial of i.p. therapy of ovarian cancer using a [90Y]DOTA conjugate of murine mAb HMFG1 [[90Y]2-IT-(s)-BAD-HMFG1]. In a detailed study of this phenomenon, Watanabe et al. (28) found that the carrier protein of the immunoconjugate played a significant role in eliciting anti-chelate responses. They found that when a rabbit IgG conjugate of the same bifunctional DOTA, in the form of 111In-labeled conjugate, was injected into rabbits, no hapten immunogenicity was observed even when using a protein dose equivalent of 233 mg in 70 kg patient and a chelate/protein substitution ratio of 4.5:1. However, their study found the corresponding mouse IgG conjugate to be immunogenic when administered in rabbits. Watanabe et al. (28) concluded that the induction of chelate immunogenicity is observed when a conjugate of a foreign protein is administered, but not when the conjugate of same species mAb is used, and they further predicted that conjugates of humanized antibodies would be considerably less antigenic in humans than the murine forms. In an experiment designed to test whether DOTA and/ or Bz-DTPA chelators are immunogenic, we repeatedly (up to three times) injected BALB/c mice with conjugates using the respective murine LL2-IgG and murine-LL2 F(ab′)2 and tested serum for the presence of anti-chelate mAbs. There was no evidence of immune complex formation by HPLC after the addition of serum from these mice to trace amounts of radiolabeled mAbs. In terms of sensitivity, 100 ng of iodinated probe would be sufficient to form a 1:1 immune complex with antichelate-mAb entity, if present. We concluded that no immune responses were found, in mice, to murine mAb conjugate of either chelate, given three times over 9 weeks at protein doses equivalent to 100 mg/70 kg in humans. This finding supports the contention of Watanabe et al. (28) that chelates are substantially more immunogenic when attached to foreign antibodies compared to syngeneic immunoglobulins. It is encouraging that no immune response is seen in mice with murine mAb substituted with as much as four DOTA chelates per IgG. CONCLUSIONS

We have optimized a procedure for simple DOTA conjugation of CDR-grafted monoclonal antibodies, leading to consistently >90% 90Y incorporation in conjugate radiolabelings. Factors governing DOTA molar substitution ratios and optimized labelings were addressed. [88Y]DOTA-hLL2 was more stable to challenge and had substantially lower bone uptake in Ramos lymphomabearing animals than [88Y]Bz-DTPA-hLL2. In other properties such as cell binding and retention, MTD, and induction of anti-chelate response in animals, the reagents appeared to be similar. Whether DOTA conjugates will prove more useful than Bz-DTPA conjugates for 90Y RAIT in humans can only be answered by a direct comparison of both in patients with similar disease burdens. It is our intention to develop [90Y]DOTA-hLL2

Govindan et al.

and [90Y]DOTA-hIMMU-14 for clinical RAIT applications against NHL and CEA-expressing solid tumors, respectively, due to their better stabilities, evidenced by lower bone uptake, in these preclinical studies. ACKNOWLEDGMENT

We are grateful to Ms. Rosarito Aninipot for expert technical assistance. This work was supported in part by U.S. Public Health Service (PHS) Outstanding Investigator Grant CA 39841 from the National Institutes of Health (D.M.G.) and Grant CA 66348 under the SBIR program of the NIH (G.L.G.). LITERATURE CITED (1) Corcoran, M. C., Eary, J. F., Bernstein, I., and Press, O. W. (1997) Radioimmunotherapy strategies for Non-Hodgkin’s Lymphoma. Ann. Oncol. (Suppl. 1) 8, 133-138. (2) Kaminski, M. S., Zasadny, K. R., Francis, I. R., Fenner, M. C., Ross, C. W., Milik, A. W., Estes, J., Tuck, M., Regan, D., Fisher, S., Glenn, S. D., and Wahl, R. (1996) Iodine-131-antiB1 radioimmunotherapy for B-cell lymphoma. J. Clin. Oncol. 14, 1974-1981. (3) Knox, S. J., Goris, M. L., Trisler, K., Negrin, R., Davis, T., Liles, T.-M., Grillo-Lo´pez, A., Chinn, P., Varns, C., Ning, S.C., Fowler, S., Deb, N., Becker, M., Marquez, C., and Levy, R. (1996) Yttrium-90-labeled anti-CD20 monoclonal antibody therapy of recurrent B-cell lymphoma. Clin. Cancer Res. 2, 457-470. (4) Press, O. W., Eary, J. F., Applebaum, F. R., Martin, P. J., Nelp, W. B., Glenn, S., Fisher, D. R., Porter, B., Matthews, D. C., Gooley, T., and Bernstein, I. D. (1995) Phase II trial of 131I-B1 (anti-CD20) antibody therapy with autologous stem cell transplantation for relapsed B cell lymphomas. Lancet 346, 336-340. (5) DeNardo, G. L., Lamborn, K. R., Denardo, S. J., Goldstein, D. S., Dolber-Smith, E. G., Kroger, L. A., Larkin, E. C., and Shen, S. (1995) Prognostic factors for radioimmunotherapy in patients with B-lymphocytic malignancies. Cancer Res. 55, 5893s-5898s. (6) Juweid, M., Sharkey, R. M., Markowitz, A., Behr, T., Swayne, L. C., Dunn, R., Hansen, H. J., Shevitz, J., Leung, S.-O., Rubin, A. D., Herskovic, T., Hanley, D., and Goldenberg, D. M. (1995) Treatment of non-Hodgkin’s lymphoma with radiolabeled murine, chimeric or humanized LL2, an anti-CD22 monoclonal antibody. Cancer Res. 55, 5899s-5907s. (7) Riethmu¨ller, G., Schneider-Ga¨dicke, E., Schlimok, G., Schmiegel, W., Raab, R., Ho¨ffken, K., Gruber, R., Pichlmaier, H., Hirche, H., Pichlmaier, R., Buggisch, P., and Witte, J.; German Cancer Aid 17-1A study group (1994) Randomized trial of monoclonal antibody for adjuvant therapy of resected Dukes C colorectal carcinoma. Lancet 343, 1177-1183. (8) Blumenthal, R. B., Sharkey, R. M., Natale, A. M., Kashi, R., Wong, G., and Goldenberg, D. M. (1994) Comparison of equitoxic radioimmunotherapy and chemotherapy in the treatment of human colonic cancer xenografts. Cancer Res. 54, 142-151. (9) Tschemelitsch, J., Barendswaard, E., Williams, Jr., C., Yao, T.-J., Cohen, A. M., Old, L. J., and Welt, S. (1997) Enhanced antitumor activity of combination radioimmunotherapy (131I-labeled monoclonal antibody A33) with chemotherapy (fluorouracil). Cancer Res. 57, 2181-2186. (10) Khazaeli, M. B., Conry, R. M., and LoBuglio, A. F. (1994) Human immune response to monoclonal antibodies. J. Immunother. 15, 42-52. (11) Sharkey, R. M., Juweid, M., Shevitz, J., Behr, T., Dunn, R., Swayne, L. C., Wong, G. Y., Blumentahl, R. D., Griffiths, G. L., Siegal, J. A., Leung, S.-O., Hansen, H. J., and Goldenberg, D. M. (1995) Evaluation of a complementaritydetermining region-grafted (humanized) anti-carcinoembryonic antigen monoclonal antibody in preclinical and clinical studies. Cancer Res. 55, 5935s-5945s. (12) Leung, S.-O., Goldenberg, D. M., Dion, A. S., Pellegrini, M. C., Shevitz, J., Shih, L. B., and Hansen, H. J. (1995) Construction and characterization of a humanized, internal-

90Y-Labeled


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BC980040G

90Yttrium-Labeled Complementarity-Determining-Region-Grafted

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