([*I]IB-Mal-d-GEEEK): A Radioiodinated Prosthetic Group Containing

-maleimido-Gly1-GEEEK ([125I]IB-Mal-D-GEEEK) was synthesized via iodo- ... results suggest that [125I]IB-Mal-D-GEEEK is a promising reagent for the ...
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Bioconjugate Chem. 2006, 17, 1085−1092

1085

NE-(3-[*I]Iodobenzoyl)-Lys5-Nr-maleimido-Gly1-GEEEK ([*I]IB-Mal-D-GEEEK): A Radioiodinated Prosthetic Group Containing Negatively Charged D-Glutamates for Labeling Internalizing Monoclonal Antibodies Ganesan Vaidyanathan,*,† Kevin L. Alston,† Darrel D. Bigner,‡ and Michael R. Zalutsky†,‡ Departments

of

Radiology

and

Pathology,

Duke

University

Medical

Center,

Durham,

North

Carolina

27710.

Received March 27, 2006; Revised Manuscript Received May 18, 2006

Novel methods are needed for the radiohalogenation of cell-internalizing proteins and peptides because rapid loss of label occurs after lysosomal processing when these molecules are labeled using conventional radioiodination methodologies. We have developed a radiolabeled prosthetic group that contains multiple negatively charged D-amino acids to facilitate trapping of the radioactivity in the cell after proteolysis of the labeled protein. N-(3[125I]iodobenzoyl)-Lys5-NR-maleimido-Gly1-GEEEK ([125I]IB-Mal-D-GEEEK) was synthesized via iododestannylation in 90.3 ( 3.9% radiochemical yields. This radioiodinated agent was conjugated to iminothiolanetreated L8A4, an anti-epidermal growth factor receptor variant III (EGFRvIII) specific monoclonal antibody (mAb) in 54.3 ( 17.7% conjugation yields. In vitro assays with the EGFRvIII-expressing U87MG∆EGFR glioma cell line demonstrated that the internalized radioactivity for the [125I]IB-Mal-D-GEEEK-L8A4 conjugate increased from 14.1% at 1 h to 44.7% at 24 h and was about 15-fold higher than that of directly radioiodinated L8A4 at 24 h. A commensurately increased tumor uptake in vivo in athymic mice bearing subcutaneous U87MG∆EGFR xenografts (52.6 ( 14.3% injected dose per gram versus 17.4 ( 3.5% ID/g at 72 h) also was observed. These results suggest that [125I]IB-Mal-D-GEEEK is a promising reagent for the radioiodination of internalizing mAbs.

INTRODUCTION Targeted radionuclide therapy is an attractive alternative cancer treatment to conventional external beam radiation, particularly when the molecule being targeted is truly tumor specific. An example of such a target is the epidermal growth factor receptor variant III (EGFRvIII), a mutant receptor found on glioma, and breast, lung, and ovarian carcinomas, and characterized by a deletion in the extracellular domain of EGFR (1). EGFRvIII-specific monoclonal antibodies (mAbs) have been generated and, as is generally the case with anti-receptor mAbs, undergo rapid internalization and extensive lysosomal degradation after cell surface receptor binding (2). Internalization is a necessary prelude for cytotoxic potency with immunotoxins (3). On the other hand, internalization creates a serious problem for radioimmunotherapy because of the rapid loss of radioactivity after tumor cell processing of mAbs radioiodinated by the direct electrophilic route (4, 5). This has provided motivation for the development of “residualizing” labeling methods which seek to overcome the rapid egress of radioiodine from tumor by labeling the mAb via a labeled prosthetic group that will remain trapped inside the cell after mAb degradation (6-9). The approach taken by our group for labeling internalizing mAbs has been to exploit the limited diffusion of charged species across cell and lysosomal membranes. We have evaluated a series of aromatic acylation agents bearing positively (4, 10) or negatively charged (11) substituents for this purpose. Indeed, in preclinical studies, substantially higher retention of radioiodine in tumor cells and human tumor xenografts have been achieved for anti-EGFRvIII mAbs radio* Correspondence to Ganesan Vaidyanathan, Box 3808, Radiology, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-7811. Fax: (919) 684-7122. e-mail: [email protected]. † Department of Radiology. ‡ Department of Pathology.

iodinated using such templates compared to the same mAbs radioiodinated by the direct method (12-14). In an extension of this general strategy, the anti-EGFRvIII mAb L8A4 was radioiodinated using D-NR-Ac-KRYRR, a peptide containing three arginines to impart positive charge and composed of D-amino acids to inhibit proteolysis (15). The procedure involved three steps: peptide radioiodination, reaction with the bifunctional agent sSMCC, and finally, conjugation to mAb derivatized by reaction with iminothiolane. Tumor retentions of radioiodine, both in cells and xenografts, for L8A4 mAb labeled by this method compared favorably with those observed using other labeling approaches; however, kidney uptake was also higher, presumably due to the cationic nature of the D-amino acid peptide. In addition, the procedure involves radioiodination of the D-tyrosine, rendering the labeled mAb susceptible, albeit to a lesser degree (16), to in vivo deiodination. Furthermore, extension of this method for use with the heavier halogen 211At, of interest for R-particle radioimmunotherapy (17), would be problematic because introduction of 211At onto tyrosine residues has not been successful (18, 19). The objective of the current study was to refine our Damino acid peptide prosthetic group strategy by developing a labeling reagent with the following properties: (1) simpler chemistry, (2) potential applicability for both radioiodination and astatination, (3) structural dissimilarity to thyroid hormones, and (4) D-peptide sequence with net negative charge. To accomplish this, a pentapeptide consisting of a maleimidoglycine, three D-glutamines, and a D-lysine was synthesized, and the lysine -amino group was derivatized with a 3-(tri-nbutylstannyl)benzoyl moiety. Herein we present the results of synthesis of this precursor and its radioiodination to N-(3[*I]iodobenzoyl)-Lys5-NR-maleimido-Gly1-GEEEK ([*I]IB-MalD-GEEEK) and evaluation of the potential utility of this reagent for labeling the anti-EGFRvIII mAb L8A4.

10.1021/bc0600766 CCC: $33.50 © 2006 American Chemical Society Published on Web 06/24/2006

1086 Bioconjugate Chem., Vol. 17, No. 4, 2006

EXPERIMENTAL SECTION General. All chemicals were purchased from SigmaAldrich unless otherwise noted. Sodium [125I]iodide and sodium [131I]iodide with specific activities of 2200 Ci/mmol and 1200 Ci/mmol, respectively, were obtained from Perkin-Elmer Life and Analytical Sciences (Boston, MA). Fmoc-D-Lys(Boc) attached to Wang resin and Fmoc-D-Glu-(O-tBu) were obtained from Novabiochem (West Chester, PA). N-Succinimidyl 3-iodobenzoate (SIB) and N-succinimidyl 3-(tri-n-butyl)stannylbenzoate (STB) were prepared as reported (20). D-Amino acid peptides NR-Ac-KRYRR and KEYEE were obtained from Peptide Technologies Corp. (Gaithersburg, MD) and LSU Health Sciences Center Core Laboratories (New Orleans, LA), respectively. Solid-phase peptide synthesis was carried out on a PerkinElmer Applied Biosystems (Foster City, CA) Model 433A automated peptide synthesizer employing their Fast-Moc chemistry with HBTU activation of carboxyl groups. FAB mass spectra were obtained on a JEOL SX-102 high-resolution mass spectrometer. Two HPLC systems were used. For radiochemistry, a Beckman System Gold HPLC equipped with a Model 126 programmable solvent module, a Model 166 NM variable wavelength detector, a Model 170 radioisotope detector, and a Beckman System Gold remote interface module SS420X was employed; data were acquired in this system using 32 Karat software. For nonradioactive and preparative work, a Waters Model Delta 600 semiprep system with a Model 600 controller and a Model 2487 dual wavelength absorbance detector was used; data were acquired using Millenium software. A Waters XTerra C18 HPLC column (4.6 × 250 mm, 5 µm), which was eluted at a flow rate of 1 mL/min was used for analytical chromatography and a Waters XTerra C18 column (19 × 150 mm, 7 µm), which was eluted at 10 mL/min was used for semipreparative chromatography. In some cases, a YMC-basic column (S-5 micron, 4.6 × 250 mm; YMC Inc., Wilmington, NC) was used. For size-exclusion HPLC, a 7.5 × 600-mm TSK gel G3000 SW 113 (10 µm) gel filtration column (Tosohaas; Montgomeryville, PA) was used. The following different HPLC solvents and gradient conditions were employed: (I) XTerra analytical column; A ) 0.1% TFA/Water, B ) 0.1% TFA/ CH3CN; 90:10 (A/B) for 10 min and then to 10:90 over next 10 min; (II) XTerra analytical column; A ) 0.1% HOAc in water, B ) 0.1% HOAc in CH3CN; 96:4 (A/B) for 10 min and then to 0:100 over 30 min; (III) Semipreparative column; same solvents and gradient as in II; (IV) XTerra analytical column; same solvents as in II; 96:4 (A/B) for 10 min and then to 0:100 over 15 min and held at this composition for additional 15 min; (V) semipreparative column; same solvents and gradient as in IV; (VI) XTerra analytical column; same solvents as in IV; 96:4 (A/B) for 10 min, then to 0:100 over 5 min and held at this composition for additional 15 min; (VII) YMC column was eluted at a flow rate of 1 mL/min; same solvents as in I; 99:1 (A/B) for 1 min, to 35:65 in 25 min; and (VIII) Size-exclusion column; phosphate-buffered slaine (PBS) at 1 mL/min. Cells, mAb, and Culture Conditions. Murine IgG1 L8A4 is a mAb which reacts specifically with the 145 kDa deletion mutant EGFRvIII molecule found on multiple human malignancies including gliomas and breast carcinomas (21). The EGFRvIII-expressing cell line used for these studies was U87MG∆EGFR which was established from U87 MG human glioma cell line by transfection with EGFRvIII cDNA (22). In cell culture, this line expresses an average of 4-13 × 105 EGFRvIII molecules per cell (12). Cells were grown in Zinc Option media (Life Technologies, Inc., Grand Island, NY) containing 10% fetal calf serum and Geneticin sulfate (600 mg/ mL).

Vaidyanathan et al.

Synthesis. Mal-D-GEEEK. Maleimido glycine (maleimido acetic acid) was synthesized following a literature procedure (23). Three D-glutamic acids (O-tBu) and the above glycine derivative were sequentially attached to D-lysine (Boc) supported on Wang resin (0.1 mmol) using the automated synthesizer. Cleavage from the resin and simultaneous removal of side-chain protecting groups were achieved by treatment with 100:95:2.5: 2.5 (v/v/v/v/) DCM:TFA:water:triisopropylsilane. The resin was filtered, and the peptide was precipitated from the concentrated filtrate with diethyl ether to yield 40 mg (60%) of NR-maleimidoGly1-GEEEK (Mal-D-GEEEK). HPLC conditions I: tR ) 5.4 min. MS (m/z) Calcd. for: C27H38N6O14 670.6. Found: 671.1 (MH+); 693.1 (M + Na)+. N-(3-Iodobenzoyl)-Lys5-NR-maleimido-Gly1-GEEEK (IBMal-D-GEEEK) (5). This compound was synthesized for use as an HPLC standard. A solution of Mal-GEEEK (1.2 mg; 1.8 µmol) in 30 µL DMF was added to a solution of SIB (7.2 mg; 21 µmol) in 70 µL of 1% (v/v) triethylamine in DMF over a period of 15 min, and the homogeneous mixture was stirred at room-temperature overnight. The peptide was precipitated with excess ether, pelleted, washed twice with ether, and purified by preparative HPLC using conditions III to obtain 5 (yield not determined). HPLC conditions II: tR ) 26.8 min. MS (m/z) Calcd. for: C34H41IN6O15 900.6. Found: 901.1 (MH+). N  -(3-(Tri-n-butyl)benzoyl)-Lys 5 -N R -maleimido-Gly 1 GEEEK (TB-Mal-D-GEEEK) (6). A solution of Mal-D-GEEEK (8 mg; 12 µmol) in 75 µL DMF was added over a period of 15 min to a solution of STB (24.4 mg; 48.75 µmol) in 125 µL of 1% triethylamine in DMF. The homogeneous mixture was stirred at room-temperature overnight, and the peptide was isolated as above and purified using HPLC conditions V to get 3 mg (23%). HPLC conditions IV: tR ) 30.2 min. MS (m/z) Calcd. for: C46H68N6O15Sn 1063.8. Found: cluster peaks around 1087.4 (M + Na)+; 1109.3 (M + 2Na - H)+; 1131.3 (M + 3Na - 2H)+; 1153.3 (M + 4Na - 3H)+. Radiochemistry. N-(3-[125I]Iodobenzoyl)-Lys5-NR-maleimidoGly1-GEEEK ([125I]IB-Mal-D-GEEEK) (5a). Acetic acid (20 µL) was added to a vial containing [125I]sodium iodide (0.5 mCi in 1 µL 0.1 N NaOH) followed by 6 (25 µg; 1 µg /µL AcOH) and NCS (0.4 mg/20µL AcOH). The mixture was incubated for 5 min, and the labeled peptide was isolated by reversed-phase HPLC (conditions VI; tR ) 19.7 min). The HPLC fractions containing the product was pooled and evaporated to dryness using an argon stream. D-NR-Ac-KR[125I]YRR and D-KE[131I]YEE. These peptides were prepared by electrophilic substitution of their tyrosine residues using the Iodogen method. For this, peptide in PBS (480 µg/160 µL and 400 µg/200 µL, respectively, for the positively and negatively charged peptides), was added to 1/ -dram vials coated with Iodogen (10 µg) and containing 558 2 µCi of 125I and 457 µCi of 131I, respectively. The mixtures were incubated at room temperature for 40 min, and the labeled peptides were isolated by reversed-phase HPLC using conditions VII. The retention times for D-NR-Ac-KR[125I]YRR and D-KE[131I]YEE were 10.3 and 7.5 min, respectively. The pooled HPLC fractions containing the peptide were concentrated by solid-phase extraction using a Sep-pak plus tC18 cartridge (Waters, Milford, MA). After the HPLC fractions were loaded on the cartridge, it was washed sequentially with 5 mL each of water and 5 mM hexanesulfonic acid. The peptides were eluted with 1% HOAc in methanol (4 × 1 mL). The solvents were evaporated with a stream of argon, and the residual radioactivity was reconstituted in PBS prior to use in the animal experiment. mAb Labeling. Direct. A solution of mAb L8A4 (250 µg; 2.5 mg/mL) in phosphate buffer, pH 7.4, was added to a 1/2dram vial coated with Iodogen (10 µg) followed by a solution

Radiolabeling Internalizing Monoclonal Antibodies

of [131I]sodium iodide (0.5 mCi) in PBS (40 µL). The mixture was incubated for 10 min with occasional shaking, and the labeled mAb was isolated by gel filtration on a PD-10 column (GE Healthcare, Piscataway, NJ) eluted with PBS. [125I]IB-Mal-D-GEEEK. L8A4 was dialyzed overnight against PBS, pH 7.4, containing 1 mM EDTA. The above solution of L8A4 (95 µL; 2.6 mg/mL) was treated with a solution of 2-iminothiolane in 1 mM EDTA in PBS (1.5 mL; 2 mg/mL), and the mixture was incubated for 90 min at room temperature. The thiol-derivatized mAb was isolated using a Microspin G-25 column (GE Healthcare, Piscataway, NJ) and added to [125I]IB-Mal-D-GEEEK (0.4 mCi). The mixture was incubated at room temperature for 70 min with occasional mixing. Iodoacetamide (10 µL; 100 mg/mL PBS/EDTA) was added to quench the reaction. After a further incubation for 10 min, the labeled mAb was purified on a PD-10 column eluted with PBS. Characterization of Labeled mAbs. Protein-associated radioactivity was determined by coprecipitating the labeled mAbs with BSA in a paired-label format with cold methanol. The fraction of radioiodine activity with a retention time corresponding to IgG was measured by size-exclusion HPLC (conditions VIII). The immunoreactive fraction of the labeled mAbs was determined in a paired-label format using magnetic beads coated with EGFRvIII and analyzed according to the Lindmo method as described before (11). Internalization Assay. The retention of radioiodine activity inside the cell and on the cell surface for the L8A4 labeled by the two methods was determined as a function of time. The in vitro assay was performed in a paired-label format using the EGFRvIII-expressing U87MG∆EGFR cell line following previously described procedures (10). Briefly, cells at a density of 5 × 105 cells per well were incubated with 1 µCi (0.5-1.0 µg of each mAb) of the labeled mAbs for 1 h at 4 °C. The cells were washed and brought to 37 °C and then processed in triplicate at 0, 1, 2, 4, 8-10, 16, and 24 h as follows: The cell culture supernatant was withdrawn, and surface-bound radioactivity was removed by washing the cells with pH 2 Zinc Option medium. Finally, the cells were solubilized in 0.5 mL of 0.5 N NaOH to determine internalized radioactivity. The cell culture supernatants, acid washes (cell surface-bound), and the cell lysates (internalized) were counted and the results expressed as the percentage of radioactivity initially bound to the cells after the 1 h incubation at 4 °C. The experiment was performed twice. Biodistribution Measurements. Animal studies were performed under the guidelines established by the Duke University Institutional Animal Care and Use Committee. In these experiments, labeled compounds were injected intravenously via the tail vein, and groups of five animals were evaluated at each time point. Mice were killed by an overdose of halothane and dissected, and tissues of interest were harvested. Blood samples were obtained by retro-orbital bleeding. Tissues were blot-dried, weighed, and counted along with input standards for both 125I and 131I using a dual-channel automated gamma counter that applied corrections for radioactive decay and crossover. Results were expressed as the percent of injected dose per gram of tissue (% ID/g). The difference in the tissue radioactivity levels of the two compounds administered in each experiment was analyzed for statistical significance with a paired t-test using the Microsoft Excel program, with p < 0.05 considered to represent a significant difference. D-NR-Ac-KR[125I]YRR Versus D-KE[131I]YEE. The experiment was performed to evaluate the effect of charge (three negatively charged glutamates versus three positively charged arginine residues) on the tissue distribution of D-amino acid pentapeptides. Normal mice weighing approximately 25 g were obtained from Charles River Laboratories International Inc. (Wilmington, MA). Animals were injected with 5 µCi of each

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peptide, and groups of five mice were killed at 0.5, 1, 2, and 4 h postinjection. Paired-Label Biodistribution of L8A4 Labeled with [125I]IBMal-D-GEEEK and with 131I Using Iodogen. BALB/c nu/nu mice were obtained from a closed breeding colony at the Duke University Cancer Center Isolation Facility. Xenografts were established from the U87MG∆EGFR cell line (22). On the basis of the flow cytometry data, disaggregated U87MG∆EGFR xenografts express a mean of 2.5 × 105 EGFRvIII receptors per cell (12). The xenografts were generated by injecting the mice subcutaneously in the flank with 50 µL of U87MG∆EGFR tumor homogenate through a 16-gauge needle. Biodistribution measurements were initiated when the xenograft volume reached 200-400 mm3. Mice were injected with 5 µCi (18.5 µg) and 1.5 µCi (1.6 µg) of 125I- and 131I-labeled L8A4, respectively. Necropsy was performed at 12, 24, 48, 72, and 144 h postinjection.

RESULTS AND DISCUSSSION Receptor-mediated internalization of radioligands generally results in their translocation to lysosomes where they are subject to degradation by proteases. A consequence of these processes for mAbs labeled by direct methods is the rapid loss of radioiodine from the cell in the form of mono-iodotyrosine (4). Because D-amino acids are poor substrates for endogenous proteases, prosthetic groups containing a D-peptide core are a “natural” choice for labeling internalizing mAbs. In this way, different modifications can be made at multiple positions along the peptide with the expectation that the molecule will remain intact in vivo. A series of D-peptides bearing one or two diethylenetriaminepentaacetic acid (DTPA) substituents have been evaluated for use in labeling internalizing mAbs (8, 24). The rationale for this particular approach is based on the fact that when radiometals are administered as DTPA-mAb conjugates, they are retained inside cells in the form of radiometal-DTPA-lysine. All D-peptide-DTPA reagents provided similar advantages in tumor retention of radioiodine activity for an internalizing mAb compared with directly labeled mAb. In contrast, considerable differences in normal tissue retention were observed, with less accumulation achieved when the hydrophilicity of the peptide was maximized by appending two DTPA moieties. We have been pursuing an alternative strategy in which the characteristics of the D-amino acid peptide itself are modulated in order to achieve trapping of the radionuclide inside the cell after mAb internalization. The feasibility of this approach was demonstrated in studies utilizing D-NR-Ac-KRYRR (15), a peptide with three positively charged arginine residues included in the sequence in order to inhibit egress of labeled catabolites across lysosome and cell membranes. Considerably higher tumor levels were observed for an internalizing mAb labeled with 125Ilabeled D-NR-Ac-KRYRR compared with 131I-labeled L-NR-AcKRYRR, confirming the role of D-amino acids in achieving these favorable results. One of the motivations for the current study was to determine whether similar results could be obtained with other D-amino acid peptides, in this case, bearing three negatively charged glutamic acids. In our D-NR-Ac-KRYRR-based conjugation labeling method (15), the radiolabeled peptide was conjugated first to iminothiolane-treated mAb via a bifunctional reagent containing a maleimido moiety. To avoid this extra step, we have included a maleimido-glycine at the N-terminus of the peptide. This was built-in during the solid-phase synthesis of the peptide (Scheme 1). The stability of maleimido moiety to coupling and cleavage conditions (peptide synthesis) was evaluated by thin-layer chromatography, and the maleimido glycine was shown to be inert under these conditions (data not shown). Other investigators

1088 Bioconjugate Chem., Vol. 17, No. 4, 2006 Scheme 1. Precursora

Syntheses of IB-Mal-D-GEEEK and Its Tin

Vaidyanathan et al. Scheme 2. Synthesis of [*I]IB-Mal-D-GEEEKa

a

a (a) NaHCO3; (b) 1; solid-phase peptide synthesis (SPPS); (c) TFA cocktail; (d) N-succinimidyl 3-iodo- or 3-(tri-n-butylstannyl)benzoate.

also have synthesized peptides with built-in maelimido functionalities (25). The molecular weights observed for the backbone peptide, as well as the SIB- and STB-derivatized peptides, were consistent with the theoretically calculated values. The tin precursor was radioiodinated to [125I]IB-Mal-DGEEEK (Scheme 2) in 90.3 ( 3.9% radiochemical yield and in more than 99% radiochemical purity. [125I]IB-Mal-D-GEEEK was coupled to thiolated mAb L8A4 in conjugation efficiencies of 54.3 ( 17.7%. Conjugation efficiencies were similar to those observed previously for D-NR-Ac-KRYRR to this same mAb (15) and comparable to those reported by Stein et al. (24) for D-peptides with one maleimido functionality. Although these investigators were able to increase coupling yield by inclusion of a second maleimido group, we chose not to pursue this tactic because of the potential for forming multimeric mAb species. Coprecipitation of the labeled protein with BSA using methanol indicated that 94 ( 6% of the radioactivity was protein-associated. This was corroborated by size-exclusion HPLC of the preparation, which showed a single radioactivity peak eluting with a retention time corresponding to monomeric IgG with minimal aggregation of the protein (data not shown). The immunoreactive fraction as determined by the Lindmo method was 83 ( 2% compared to 70% obtained for L8A4 labeled with Iodogen. The primary hypothesis of this work is that incorporation of multiple negatively charged amino acids in the D-peptide

(a) Radioiodine, NCS, acetic acid.

prosthetic group for mAb radiohalogenation would provide labeled catabolites that will be trapped inside cells after mAb internalization. The labeled reagent that was developed, [*I]IBMal-D-GEEEK, has three glutamic acid residues with carboxylate side chains (pK 4.25) that should be predominantly anionic at lysosomal pH. To determine whether labeling the antiEGFRvIII mAb L8A4 with [*I]IB-Mal-D-GEEEK increased cellular retention of radioactivity compared with direct iodination, a paired-label internalization experiment was performed in vitro using the U87MG∆EGFR cell line. As shown in Figure 1A, the percent of initially bound radioactivity that internalized increased with time throughout the 24 h observation period for L8A4 labeled with [125I]IB-Mal-D-GEEEK; on the other hand, not only was this amount considerably lower for L8A4 labeled by the direct method but intracellular activity decreased with the time. For example, internalized radioactivity for L8A4 labeled with [125I]IB-Mal-D-GEEEK at 24 h (44.7 ( 3.1%) was about 15-fold higher than that from directly labeled L8A4 (3.0 ( 0.3%). The surface-bound radioactivity (Figure 1B) was higher for L8A4 labeled with [125I]IB-Mal-D-GEEEK at each time point compared to the directly labeled mAb. As a result, the total cell-associated radioactivity was substantially higher for the mAb labeled using this novel method. The radioactivity pattern seen in cell culture supernatants (Figure 1C) was complementary to the above. The degree of enhancement in cell-associated radioactivity obtained with [*I]IB-Mal-D-GEEEK at 24 h was even more favorable than that obtained with D-NR-Ac-KRYRR (5-fold; same mAb and cell line) (15). The tumor delivery advantage obtained using the IB-Mal-GEEEK template also is higher than that obtained with other residualizing labels (10, 11). Although comparison to other mAbs and cell lines must be done with caution, it is worth noting that the increase in intracellular and total cell associated radioactivity seen with [*I]IB-Mal-DGEEEK was substantially higher than that reported for other D-amino acid peptide prosthetic groups (24). In addition, [*I]IBMal-D-GEEEK was the only reagent that resulted in an increase in intracellular activity with time. The reason for these differences is not known at this time. We speculate that a probable factor is the fact that the site of [*I]IB-Mal-D-GEEEK radioiodination is less susceptible to deiodination, with the result that a greater fraction of the label is still associated with the mAb at later time points. A drawback of the KRYRR method, which provided one of the motivations for the current study, was that the use of this reagent for labeling L8A4 increased renal uptake considerably

Radiolabeling Internalizing Monoclonal Antibodies

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Figure 1. Paired-label internalization by U87MG∆EGFR cells of L8A4 directly radioiodinated with 131I using Iodogen (open circles) and that labeled with [125I]IB-Mal-GEEEK (filled circles). Cells were allowed to take the tracers by incubating them together for 1 h at 4 °C. Medium containing unbound radioactivity was removed, and the cells were supplemented with fresh medium, brought up to 37 °C and processed as described under Experimental Section.

Figure 3. Paired-label tumor uptake of L8A4 directly radioiodinated with 131I using Iodogen (open bars) and that labeled with [125I]IB-MalGEEEK (filled bars) in athymic mice bearing U87MG∆EGFR xenografts.

Figure 2. Paired-label uptake of D-NR-Ac-KR[125I]YRR (open bars) and D-KE[131I]YEE (closed bars) in kidneys and liver of normal mice.

(15). It is well-known that positively charged molecules are retained to a greater degree by the kidneys because they are more likely to undergo tubular absorption due to the negative charge of the luminal membranes of kidney (26). In fact, lysine is often pre-administered to obviate the high renal accumulation of several radiopharmaceuticals (27). On the basis of this, we hypothesized that utilization of a peptide with multiple Dglutamates instead of D-lysines should allow enhancement of tumor retention of radioactivity without increasing uptake in kidneys. To determine whether a decrease in kidney uptake, and possibly other normal organ accumulation, could be achieved by substitution of negatively charged for positively charged amino acids, a tissue distribution experiment was performed in normal mice comparing the organ uptake of radioactivity from D-NR-Ac-KR[125I]YRR and D-KE[131I]YEE. The latter peptide was derived by substituting D-glutamates for D-lysines in D-NRAc-KRYRR; in addition, the N-terminus was not acetylated.

As shown in Figure 2, over a period of 4 h, the uptake of radioactivity in kidneys from D-KE[131I]YEE was more than 10-fold lower than that from the coadministered D-NR-AcKR[125I]YRR (p < 0.01). Accumulation of radioiodine in the liver with D-KE[131I]YEE also was at least four times less than observed for co-administered D-NR-Ac-KR[125I]YRR (p < 0.01). The substantial reduction in the renal uptake is consistent with the net negative charge of the KEYEE construct. In addition to negative charge, the lower lipophilicity of KEYEE might have been a contributory factor to lowering liver uptake. One would expect the cationic peptide to be more lipophilic due to capping of its N-terminus with an acetyl group, and indeed, under identical conditions, it had a retention time of 10.3 min in reversed-phase HPLC compared with 7.5 min for KEYEE. On the basis of these results, we embarked on the design and evaluation of IB-Mal-GEEEK as a reagent for labeling internalizing mAbs. The biodistribution of L8A4 labeled with [125I]IB-MalGEEEK and mAb labeled with 131I using Iodogen method were compared in a paired-label format in athymic mice bearing subcutaneous U87MG∆EGFR human glioma xenografts. In concert with the in vitro internalization data, the tumor uptake in vivo of [125I]IB-Mal-GEEEK-labeled L8A4 was substantially higher (p < 0.05 except at 144 h) than that of the directly labeled mAb (Figure 3). The tumor uptake of [125I]IB-Mal-GEEEKlabeled L8A4 increased with time, reaching a maximum of 52.6 ( 14.3% ID/g at 72 h. A 1.6, 2.3, 3.8, 3.0, and 4.0-fold higher

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Vaidyanathan et al.

Table 1. Paired-Label Biodistribution of L8A4 Labeled with [125I]IB-Mal-GEEEK and with U87MG∆EGFR Xenografts

131I

Using Iodogen in Athymic Mice Bearing

%ID/grama tissue

a

12 h

liver spleen lungs heart kidneys stomach small intestine large intestine muscle blood brain

4.84 ( 1.10 3.34 ( 2.80 4.43 ( 0.64 3.49 ( 0.64 10.75 ( 1.27 0.54 ( 0.18 0.75 ( 0.17 0.66 ( 0.14b 0.61 ( 0.13 12.58 ( 2.16 0.34 ( 0.12

liver spleen lungs heart kidneys stomach small intestine large intestine muscle blood brain

4.23 ( 1.17 2.73 ( 0.73 4.90 ( 0.54 3.94 ( 0.71 4.26 ( 0.66 2.05 ( 0.70 0.90 ( 0.16 0.59 ( 0.09 0.73 ( 0.12 14.32 ( 2.54 0.39 ( 0.13

24 h

48 h Iodine-125

4.20 ( 0.98 2.94 ( 0.46b 2.80 ( 0.36 2.73 ( 0.42 3.69 ( 0.66 2.89 ( 0.63 2.95 ( 0.74 2.61 ( 0.45 9.75 ( 1.16 7.40 ( 0.75 0.64 ( 0.17 0.56 ( 0.07 0.75 ( 0.21 0.64 ( 0.14 0.56 ( 0.14b 0.55 ( 0.11 0.63 ( 0.17 0.56 ( 0.11 10.57 ( 2.59 8.39 ( 1.55 0.24 ( 0.05 0.23 ( 0.09 Iodine-131 3.60 ( 0.71 2.84 ( 0.47 2.06 ( 0.40 1.90 ( 0.38 4.27 ( 0.69 3.36 ( 0.67 3.45 ( 0.77 3.09 ( 0.37 3.80 ( 0.76 2.92 ( 0.47 2.55 ( 0.98 1.87 ( 0.83 0.91 ( 0.19 0.72 ( 0.14 0.58 ( 0.11 0.42 ( 0.08 0.77 ( 0.18 0.72 ( 0.15 12.50 ( 2.73 10.04 ( 1.47 0.29 ( 0.06 0.27 ( 0.10

72 h

144 h

4.52 ( 0.76b 4.07 ( 0.87 5.11 ( 1.25 4.11 ( 0.46 8.68 ( 0.73 0.67 ( 0.18 1.04 ( 0.15 0.73 ( 0.10 0.87 ( 0.18 14.07 ( 2.27 0.36 ( 0.10

1.08 ( 0.62b 1.33 ( 0.70 0.94 ( 0.84 0.82 ( 0.72 3.74 ( 0.69 0.15 ( 0.10 0.21 ( 0.15 0.26 ( 0.11 0.21 ( 0.11 2.65 ( 2.13 0.07 ( 0.06

4.35 ( 0.94 3.03 ( 0.75 5.73 ( 1.41 4.57 ( 0.43 4.66 ( 0.82 1.13 ( 0.34 1.10 ( 0.15 0.57 ( 0.07 1.01 ( 0.19 16.06 ( 2.41 0.41 ( 0.11

1.03 ( 0.66 0.66 ( 0.45 1.28 ( 1.04 1.20 ( 0.92 1.30 ( 0.79 0.56 (0.22 0.28 ( 0.17 0.19 ( 0.08 0.36 ( 0.15 4.04 ( 2.76 0.10 ( 0.07

Mean ( S.D (n ) 5; for 144 h, n ) 4) of %ID/gram. b The difference in uptake between the two methods not statistically significant (p > 0.05).

Figure 4. Uptake of radioiodine activity in mouse thyroid after administration of L8A4 labeled with 125I using [125I]IB-Mal-GEEEK (filled bars) and with 131I using the Iodogen method (open bars).

tumor uptake was observed for mAb labeled with [125I]IB-MalGEEEK compared with that labeled using Iodogen at 12, 24, 48, 72, and 144 h, respectively. The tumor delivery advantage using [125I]IB-Mal-GEEEK was about the same as observed previously with D-NR-Ac-KR[125I]YRR; however, the maximum tumor uptake was seen at 3 days rather than 1 day after injection (15). This suggests that [*I]IB-Mal-GEEEK may be a useful radiohalogenation method for increasing the tumor radiation dose achievable in radioimmunotherapy with internalizing mAbs such as L8A4 labeled with 8.1-day half-life 131I. Thyroid uptake is frequently used as an indicator of degree of in vivo deiodination of radioiodinated compounds. In Figure 4, the thyroid accumulation of 125I and 131I activity following injection of [125I]IB-Mal-GEEEK-labeled L8A4 and L8A4 labeled with 131I using Iodogen are compared. The % ID recovered from the thyroid for L8A4 labeled with [125I]IB-Mal-

GEEEK ranged from 0.18 ( 0.02% at 12 h to 0.47 ( 0.07% at 72 h and were 4- to 10-fold lower than that seen for mAb labeled using Iodogen, but not significantly different from those observed for L8A4 labeled with D-NR-Ac-KR[125I]YRR (15). Thus, labeling on an iodobenzoyl moiety offered no advantage compared with a D-tyrosine site in minimizing the deiodination in vivo of this internalizing mAb. These results are consistent with the stability of D-tyrosine toward deiodination (16) but also may reflect other factors as well. Thyroid accumulation of a mAb that does not undergo internalization, 81C6, labeled by coupling to SIB was about an order of magnitude lower than that seen in the current study (28). Interestingly, the thyroid uptake observed after injection of this mAb labeled with using N-succinimidyl iodobenzoates bearing positively (10) or negatively (11) charged polar substituents was somewhat lower than that seen with the two D-amino acid peptide reagents. In addition differences related to the processing of the two mAbs and the nature of the linkage between the prosthetic group and the mAb also may play a role. The tissue distribution of radioiodine labels in other normal tissues was similar for L8A4 mAb labeled by the two methods with two notable exceptions (Table 1). Uptake of 131I was higher at all time points in the stomach, another tissue known to take up free iodide avidly, and this is consistent with the observed higher thyroid uptake of 131I. Contrary to the expectations, however, the accumulation of radioactivity in the kidneys for L8A4 labeled with [125I]IB-Mal-GEEEK was about 2- to 3-fold higher than that for directly labeled mAb. In fact, the renal uptake of [125I]IB-Mal-GEEEK-labeled L8A4 was similar to that seen for the same mAb labeled with the cationic D-peptide reagent (15), suggesting that changing the net charge of the D-peptide from positive to negative was not an effective strategy for reducing kidney uptake of intact mAbs labeled using this type of prosthetic group. On the other hand, the uptake in liver for L8A4 labeled with [125I]IB-Mal-GEEEK was quite similar to that for coadministered mAb labeled using Iodogen and about 3-fold lower compared to L8A4 labeled with D-NR-AcKR[125I]YRR (15). Increased kidney and liver retention of radioiodine has been observed, compared with direct labeling,

Bioconjugate Chem., Vol. 17, No. 4, 2006 1091

Radiolabeling Internalizing Monoclonal Antibodies

Figure 5. Tumor-to-normal tissue ratios of 125I (filled circles) and [125I]IB-Mal-GEEEK and with 131I using the Iodogen method.

131I

for mAb RS7 after labeling with six different D-peptide labeling reagents, with the effect most prominent for the IMP-R2 peptide bearing four tyrosine residues, a DTPA, and two maleimido moieties (24). Taken together, these results suggest that decreasing the hydrophobicity of the D-peptide results in decreased liver accumulation; however, it is difficult to ascertain the best strategy for minimizing renal accumulation with this class of radiohalogenation agents. Tumor:normal tissue ratios for L8A4 labeled with [125I]IBMal-GEEEK and with 131I using Iodogen are shown in Figure 5. Except for the kidney at some time points, significantly higher tumor:normal tissue ratios were observed for the D-peptide conjugate. With the exception of the kidney, selectivity of radioiodine increased with time as did the selectivity advantage for mAb labeled using the D-peptide reagent. At 6 days after injection, tumor:normal tissue ratios for L8A4 labeled with [125I]IB-Mal-GEEEK reached 5.3 ( 2.7, 8.9 ( 2.7, 15.2 ( 3.1, 19.6 ( 5.1, 25.7 ( 7.0, and 145 ( 32 in kidney, blood, spleen, liver, lung and stomach, respectively. These values were 1.5to 17-fold higher than those for coadministered L8A4 labeled using Iodogen and reported previously for this mAb labeled with D-NR-Ac-KR[125I]YRR (15). In summary, a new prosthetic group, [*I]IB-Mal-GEEEK, consisting of three D-glutamates, a D-lysine carrying an [*I]iodobenzoyl moiety, and a maleimide function was synthesized from a tin precursor and conjugated to an internalizing anti-EGFRvIII mAb with preservation of immunoreactivity. Compared with mAb labeled using Iodogen, labeling L8A4 using [*I]IB-MalGEEEK enhanced intracellular retention of radioiodine in EGFRvIII-expressing tumor cells in vitro and in tumor xenografts and resulted in significantly higher tumor:normal organ uptake ratios. We conclude that [*I]IB-Mal-GEEEK is a promising reagent for the radioiodination of mAbs that undergo internalization. Further work is in progress to evaluate the potential of this approach for labeling mAbs with the R-emitter astatine-211.

ACKNOWLEDGMENT This work was supported by Grants CA42324, NS20003, and CA93371 from National Institutes of Health. The authors want

(open circles) in athymic mice after paired injection of L8A4 labeled with

thank Philip Welsh, Katia Peixoto, and Susan Slade for excellent technical assistance.

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