Targeting Human Cancer Xenografts with Monoclonal Antibodies Labeled Using Radioiodinated, Diethylenetriaminepentaacetic

A new nonmetabolizable peptide approach to the production of residualizing radioiodine was evaluated in nude mice bearing xenografts of human lung adenocarcinoma (Calu-3) and B-cell lymphoma (Ramos). Monoclonal antibodies (MAbs) RS7 (anti-epithelial glycoprotein-1) and LL2 (anti-CD22) were radioiodinated using the thiol-reactive diethylenetriaminepentaacet ic acid-D-peptide adducts IMP-R1 and IMP-R2. 125I-IMP-R1and 12sI-IMP-R2labeled MAbs were compared to the MAbs iodinated by the conventional chloramine-T approach, 111In, and 131Idilactitoltyramine (DLT). In vivo biodistribution studies demonstrated a significant improvement in the tumor accretion of radiolabel using the 12sI-IMP-R1 labeled MAbs compared with the conventionally iodinated antibodies. For example, at day 7, the percentage of injected dose per gram of tissue in Calu-3 was 7.9 4.1% and 18.1 -+ 7.9% (P < 0.05) for the conventional 131Iand 12sI-IMP-R1-RS7, respectively, and tumor :nontumor ratios were 2.6-4.5-fold higher with the 12sI-IMP-RI-RS7. It is estimated that J31IIMP-R1-RS7 would deliver a dose to tumor (at the estimated maximum tolerated dose) 3.9 times greater than conventional 131I-labeled RS7, 1.4 times greater than 9~ RS7, and 0.7 times that of 13~I-DLT-labeled RS7. Tumor accretion of ~2sI-IMP-R2-RS7 was also improved compared with conventionally iodinated antibody. However, this label also caused a large increase in kidney accretion. Similar improvements in tumor accretion and tumor :nontumor ratios were observed when 12sI-IMP-RI-LL2 was used in the Ramos model. IMP-R1 offers a practical and useful residualizing radioiodine label because labeling efficiency is at least 10 times greater than that of the residualizing label DLT, without MAb aggregation. Structural modifications can be envisioned for further improvements in radioiodine incorporation, specific activity, and tumor dosimetry, and efforts along these lines are under way. Introduct ion The catabolism of iodinated proteins results in the generation of iodotyrosine within lysosomes and its release from the cell (1, 2). This contrasts with the fate of radiometal chelatelabeled proteins, where the radiometal remains trapped inside the lysosomes after catabolism (3-5). To prevent the rapid loss of radioiodine from target cells, and thus increase the radiation dose delivered to tumors following radioimmunotherapy with 13 i i_labele d MAbs,3 novel methodologies for the incorporation of iodine to protein have been developed. In these approaches, the iodine residue is conjugated to antibody via adducts that become lysosomally trapped. These radioiodine constructs have been called "residualizing" labels. The relative size and hydrophilicity of the adduct restricts its diffusion out of the lysosomal compartment. Recent work with residualizing adducts has focused on nonmetabolizable carbohydrate-tyramine adducts, including DLT (6, 7) and tyramine-cellobiose (8). A substantial advantage in tumor retention, both in vitro and in vivo, has been observed when residualizing iodine is used. For example, when MAb RS7 was used in nude mice bearing human lung tumor xenografts, the %ID/g in tumor at day 7 increased 7-fold when the 131IDLT-labeled antibody was used (7). In addition, ~31I-DLTlabeled antibody was shown to be therapeutically more effective in the Calu-3 xenograft model than conventionally radioiodinated antibody (9). However, the low overall conjugation yields (overall radioiodine incorporation of 3-6%), and resulting lowspecific activity product (0.5-1.0 mCi/mg) obtained in the preparation of 131I-DLT-labeled antibody makes the development of this agent for clinical use impractical. We recently described the syntheses of two residualizing substrates that can be used to radioiodinate MAbs at greatly improved conjugation yields and specific activities (10). These substrates are thiol-reactive DTPA-peptide adducts that are capable of being iodinated. The peptides are assembled using D-amino acids. These substrates are referred to as IMP-R1, maleimide-X-Gly-D-Tyr-D-Lys(ITC-Bz-DTPA), and IMP-R2, [maleimide-X-I)-Ala-D-Tyr-D-Tyr-D-Lys]z(DTPA), where X is 4-(N-maleimidomethyl)cyclohexane-l-carbonyl. Radioiodination of the peptides followed by conjugation to disulfide-rePresented at the "Seventh Conference on Radioimmunodetection and Radioimmunotherapy of Cancer," October 15-17, 1998, Princeton, NJ. Supported in part by USPHS Grants CA60039 and CA39841, and Small Business Innovation Research Grant CA72324. 2 To whom requests for reprints should be addressed, at Garden State Cancer Center, 520 Belleville Avenue, Belleville, NJ 07109. Phone: 973-844-7012; Fax: 973-844-7020. 3 The abbreviations used are: MAb, monoclonal antibody; DLT, dilactitoltyramine; %ID, percentage of injected dose; DTPA, diethylenetriaminepentaacetic acid; IMP-R1, maleimide-X-Gly-D-Tyr-D-Lys(ITC-Bz-DTPA); IMP-R2, [maleimide-X-D-Ala-D-Tyr-D-Tyr-D-Lys]2(DTPA); CT, chloramine-T; MTD, maximum tolerated dose. Research. on July 16, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 3080s MAbs Labeled with Iodinated, DTPA-appended Peptides Table I Details of labeled MAb preparations Overall Specific activity Biodistribution T/NT data Specific Label MAb incorporation (%) (mCi/mg) data shown in F ig . shown in F ig . b~Ci/animal binding (%) 125I-IMP-R1 RS7 40 1.8 1A 2A 10 58.5 131I(CT) RS7 84 15.3 1 B 2A 25 52.7 ~ eSI-DLT RS7 4 0.9 1C 2B 10 72.3 ~3'I-IMP-R1 RS7 31 0.8 Not shown 2B 20 57.6 1 ~ lin_DTP A RS7 99 6.7 1D 2C 25 52.1 125I-IMP-R2 RS7 75 6.0 1E 2D 10 73.1 13 JI(CT) RS7 91 9.1 Not shown 2D 25 75.3 J 25I-IMP-R 1 LL2 34 1.0 4A 5A 10 ND a 31I(CT) LL2 81 12.9 4B 5A 10 ND ~zsI-IMP-R2 LL2 86 5.4 4C 5B 10 ND 131I(CT) LL2 71 12.3 Not shown 5B 20 ND a T/NT, tumor/nontumor; ND, not determined. duced MAbs resulted in 40-90% overall yields, with specific activities of 2-11 mCi/mg and less than 3% aggregation. Two internalizing MAbs, RS7 (anti-epithelial glycoprotein-1) and LL2 (anti-CD22), labeled by this procedure exhibited a 2-3-fold better retention in Calu-3 and Ramos cell lines, respectively, in vitro compared with the same MAbs radioiodinated by the CT method. The rationale behind the development of these substrates was that DTPA chelate conjugates of MAbs are known to residualize after internalization and catabolism. In addition, the bond between DTPA and the e-amine group of lysine is known to be stable within lysosomes (11), and the peptide bonds between D-amino acids are relatively resistant to proteases. Taken together, these properties should enable the iodinated substrates to remain trapped in their target cells inside the lysosomal compartment in undigested forms following catabolism of the antibody. In this study we investigated the effect of using MAbs labeled with radioiodinated IMP-R1 and IMP-R2 for targeting human cancer xenografts. We show that IMP-R1 offers a practical and useful residualizing radioiodine label because it provides a significant improvement in the tumor accretion of radiolabel in comparison to the conventional ~31I-labeled antibodies and because labeling efficiency is at least 10-fold greater than with the residualizing label DLT, without MAb aggregation. Materials and Methods MAbs and Cell Lines. The production and characterization of MAbs RS7 and LL2 have been described previously (12-15). The antibodies were purified from ascites fluid by passage through a protein A-immunoadsorbent column. Calu-3, a human adenocarcinoma of the lung cell line, and Ramos, a human Burkitt's lymphoma cell line, were purchased from the American Type Culture Collection (Rockville, MD). The cells were grown in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 Ixg/ml), and L-glutamine (2 raM). Radioiodinations. Synthesis and radioiodination of IMP-R1 and IMP-R2, and conjugation to disulfide-reduced MAbs have been described (10). Briefly, the peptides were synthesized on the solid phase by the standard fluorenylmethyloxycarbonyl strategy (16), using differential amine protections for the side chain amino group of D-lysine and the amino group at the peptide's NH 2 terminus. Deprotections of these amino groups under different conditions enabled the introduction of DTPA to the I>lysine and the maleimide group at the NH 2 terminus. The peptides were radioiodinated by the conventional CT method, excess unreacted iodine was quenched with 4-hydroxyphenylacetic acid, and the radioiodinated peptides were conjugated to dithiothreitol-reduced MAbs. For comparison, MAbs were also directly radioiodinated with 131I and 125I (New England Nuclear, North Billerica, MA) by the CT method (17) and with DLT (7). As expected from previous studies (7), overall incorporation of 125I was low, 4%, when the DLT labeling methodology was used. lllIn-labeled MAbs were prepared as described previously (4), using indium purchased from New England Nuclear. The bifunctional chelate used in the preparation of the 11 lln radioimmunoconjugates was the p-isothiocyanatobenzyl derivative of DTPA. Specific activities and overall incorporation yields of the radiolabeled MAb preparations used in the biodistribution studies are listed in Table 1. Assessment of immunoreactivity after radiolabeling was performed with a direct cell binding assay (14). The percentages of specific binding for the 125I-, 131I-, and lllln-labeled MAbs are shown in Table 1. Values ranged from 52% to 75% for all preparations. In Vivo Studies. Tumors were propagated in female nu/nu mice (Taconic Farms, Germantown, NY) at 6 8 weeks of age by s.c. injection of Calu-3 or Ramos cells, which had been propagated in tissue culture. Radioiodinated antibodies were injected i.v., via the lateral tail vein, into the tumorbearing animals. Details on the quantities of radioisotope injected for each experiment are indicated in Table 1. Typically, 10-1xCi doses of lzsI-labeled MAbs were injected into each nude mouse. Because of the shorter half-life of 131[, 13~I-MAbs were used at 20-25 btCi/nude mouse, except in the paired label study of ~25I-IMP-R1-L2 and 13JI-LL2(CT), where 10 ixCi/nude mouse of each label was used. The animals were sacrificed at the times indicated, and the radioactivity in the tumor, liver, spleen, kidneys, lungs, small and large intestines, muscle, bone (whole femur), and blood was determined in a gamma-scintillation counter. Results were Research. on July 16, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from A 50 125-1-1MP-R1-RS7