Bioconjugate Chem. 1999, 10, 231−240
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Labeling of Monoclonal Antibodies with Diethylenetriaminepentaacetic Acid-Appended Radioiodinated Peptides Containing D-Amino Acids† Serengulam V. Govindan,*,‡ M. Jules Mattes,§ Rhona Stein,§ Bill J. McBride,‡ Habibe Karacay,‡ David M. Goldenberg,§ Hans J. Hansen,‡ and Gary L. Griffiths‡ Immunomedics, Inc., 300 American Road, Morris Plains, New Jersey 07950, and Garden State Cancer Center, 520 Belleville Avenue, Belleville, New Jersey 07109. Received June 26, 1998; Revised Manuscript Received November 18, 1998
The optimal use of radioiodinated internalizing monoclonal antibodies (mAbs) for radioimmunotherapy necessitates the development of practical methods for increasing the level of retention of 131I in the tumor. Lysosomally trapped (“residualizing”) iodine radiolabels that have been previously designed are based mostly on carbohydrate-tyramine adducts, but these methods have drawbacks of low overall yields and/or high levels of mAb aggregation. We have developed a method using thiol-reactive diethylenetriaminepentaacetic acid (DTPA)-peptide adducts wherein the peptides are assembled with one or more D-amino acids, including D-tyrosine. Two such substrates, R-Gly-D-Tyr-D-Lys[1-(pthiocarbonylaminobenzyl)DTPA], referred to as IMP-R1, and [R-D-Ala-D-Tyr-D-Tyr-D-Lys]2(CA-DTPA), referred to as IMP-R2, wherein R is 4-(N-maleimidomethyl)cyclohexane-1-carbonyl, were synthesized by preparing functional group-protected peptides on a solid phase, selectively derivatizing the lysine side chain with 1-(p-isothiocyanatobenzyl)DTPA or DTPA dianhydride (CA-DTPA), deprotecting other functional groups, and finally derivatizing the peptide’s N-terminus so it contained a maleimide group. Radioiodinations of the peptides followed by conjugations to disulfide-reduced mAbs, carried out as a one-vial procedure, resulted in 32-89% overall yields, at specific activities of 1.8-11.1 mCi/mg, with less than 2% aggregation. Two internalizing mAbs, LL2 (anti-CD 22 B-cell lymphoma mAb) and RS7 (an anti-adenocarcinoma mAb which targets EGP-1 antigen), labeled with this procedure exhibited a 2-3-fold better cellular retention in Ramos and Calu-3 tumor cell lines, in vitro, respectively, compared to the same mAbs radioiodinated with the chloramine-T method. The rationale for the new approach, syntheses, radiochemistry and in vitro data are presented.
INTRODUCTION
The current status of radiolabeled mAbs1 in cancer diagnosis and therapy, and possible approaches for improved methods, have been discussed in recent reviews (1-3). Among the significant issues to consider are the role of mAb metabolism after internalization in tumor cells and the subsequent fate of the attached radionuclide, because this process affects tumor dosimetry (4). Radioiodine is released from the cell as iodotyrosine after lysosomal processing of radioiodinated mAbs, while a radiometal label on mAbs is trapped in lysosomes after such processing (4, 5). With radioiodinated mAbs, the residence times of the nuclide in tumor and tumor-to-nontumor discrimination are reduced, and in the case of 131I, the dosimetric advantage of the isotope’s 8 day half-life is diminished. This scenario potentially applies to most mAbs which bind to the tumor cell surface, since it has been shown in in vitro studies that most bound mAbs are catabolized with a half-life of 1-2 days (4, 6). The † Presented in part at the 215th American Chemical Society National Meeting, Dallas, TX, March 1998, 45th annual meeting of the Society of Nuclear Medicine, Toronto, ON, June 1998, and Seventh Conference on Radioimmunodetection and Radioimmunotherapy of Cancer, Princeton, NJ, October 1998. * To whom correspondence should be addressed. Phone: (973) 605-8200. Fax: (973) 605-1103. ‡ Immunomedics, Inc. § Garden State Cancer Center.
need to develop practical methods to enhance the residence time of radioiodine in tumors is therefore compelling. To increase the residence time of radioiodine inside cells, mAbs have been labeled with radioiodinated nonmetabolizable carbohydrate-tyramine adducts. Dilactitoltyramine (DLT; 7-9) and tyraminecellobiose (TCB; 10) are two substrates of this class which have been frequently utilized for this purpose. After mAb processing, 1 Abbreviations: Aloc, allyloxycarbonyl; BOC, tert-butyloxycarbonyl; tBu, tert-butyl; Bz-DTPA, benzyl-DTPA; CA-DTPA, cyclic DTPA dianhydride; CEA, carcinoembryonic antigen; cpm, counts per minute; CT, chloramine-T; DIC, diisopropylcarbodiimide; DIEA, diisopropylethylamine; DLT, dilactitoltyramine; DMF, dimethylformamide; DTPA, diethylenetriaminepentaacetic acid; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; EGP-1, epithelial glycoprotein-1; Et3N, triethylamine; Fmoc, fluorenylmethoxycarbonyl; HOAc, acetic acid; HOBt, 1-hydroxybenzotriazole; HPLC, high-performance liquid chromatography; IMP-R1, Immunomedics peptide-residualizing 1; IMP-R2, Immunomedics peptide-residualizing 2; IMR, immunoreactivity; ITC-Bz-DTPA, 1-(p-isothiocyanatobenzyl)DTPA; mAb, monoclonal antibody; MCC, 4-(N-maleimidomethyl)cyclohexane-1-carbonyl; MW, molecular mass; PBS, phosphate-buffered saline; RAIT, radioimmunotherapy; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SE-HPLC, size-exclusion HPLC; SIPC, succinimidyl 5-iodo-3-pyridinecarboxylate; sulfo-SMCC, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; TCA, trichloroacetic acid; TCB, tyraminecellobiose; TFA, trifluoroacetic acid.
10.1021/bc980075g CCC: $18.00 © 1999 American Chemical Society Published on Web 02/11/1999
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the radioiodinated carbohydrate metabolite is trapped (“residualized”) in lysosomes. Stein et al. (9) have observed a >3-fold advantage in the level of tumor cell retention of [125I]DLT-labeled mAbs RS7 and RS11 in vitro over a 69 h period, and a pronounced dosimetric (9) and therapeutic (11) advantage in animal experiments in which the Calu-3 lung carcinoma cell line was used, compared to data obtained using the same antibodies radioiodinated with the chloramine-T method. Similar advantages were reported by Mattes et al. (12) in the [125/131I]DLT labeling of anti-lymphoma antibody LL2. Ali et al. (10) have reported improved tumor retention of radioiodine when mAbs were labeled with radioiodinated TCB. However, these carbohydrate-based iodine radiolabels have certain practical drawbacks. In the case of DLT, the level of overall radioiodine incorporation into mAbs is only 3-6% (9, 12) which results in very low specific activities. With TCB, the conjugation to mAbs makes use of cyanuric chloride as the cross-linker, which produces 1025% mAb aggregation (10). In both cases, the mAb labeling is a multistep procedure. These limitations may impede further development of these agents for clinical use. Another reported method involves using N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC), as exemplified in the labeling of the anti-epidermal growth factor variant III mAb L8A4 (13). The major intracellularly trapped low-molecular mass catabolite, after internalization and processing of the radiolabeled mAb-antigen complex, was found to be a 5-iodonicotinic acid-lysine conjugate. The level of intracellular retention, in the HC2 20 d2 cell line, of L8A4 mAb radioiodinated by the SIPC method was 68% higher at 2 h than that of the same mAb radioiodinated with the iodogen method, although this difference was reduced at the 20 h time point. In vivo, the amounts of tumor-associated radioactivities in athymic mice bearing HC2 20 d2 xenografts were 25% higher for the mAb labeled with SIPC than for the iodogen mode of radioiodination at time points up to 48 h, but the levels were equalized for both the labels at the 72 h time point. However, the former label also cleared faster from normal organs and thus gave rise to better tumor-toorgan ratios (13). Radiometalated mAbs constitute another class of residualizing labels. The radionuclides from indium- and yttrium-labeled mAbs, for instance, are residualized in the lysosomes in the form of the lysine adducts of the respective metal chelates (14). The metal chelators in these cases are usually aminopolycarboxylates, such as EDTA or DTPA. The goal of this study was to devise a practical method for the production of residualizing radioiodine, based on what was known about the residualization of metal chelates. We hypothesized that if an aminopolycarboxylate, such as DTPA, were attached to the -amine of D-lysine, and the latter was further elaborated by sequential couplings to one or more D-amino acids, including D-tyrosine, the result would be a DTPA-attached peptide wherein the peptide bonds would be relatively resistant to the action of proteases in the lysosomes. The N-terminus of the peptide could be coupled to a crosslinker, such as sulfo-SMCC, carrying a thiol-reactive group. Radioiodination of such an entity, followed by conjugation to a thiol-containing mAb, could generate a residualizing iodine label, while concurrently enabling the desired yield and specific activity enhancements in radiolabeling. This work was carried out mainly using internalizing mAbs LL2 and RS7. LL2 is a pan-B-cell mAb, whose
Govindan et al.
binding is restricted to B cells, and is one of a number of mAbs that belong to the CD22 cluster (15, 16). Its internalization has been studied (12, 17). RS7, an internalizing mAb, targets epithelial glycoprotein EGP-1, which is expressed in a number of carcinoma cell lines (18, 19). LL2 belongs to the IgG2a isotype, while RS7 belongs to the IgG1 class. In this report, we describe the syntheses of two bifunctional substrates,2 IMP-R1 and IMP-R2, the radiochemistry of mAb labelings, and a proof of concept that radiolabeling internalizing mAbs using these radioiodinated peptides leads to enhanced retention of radioiodine in tumor cells. EXPERIMENTAL PROCEDURES
General Procedures. Monoclonal antibodies were obtained from Immunomedics’ and the Garden State Cancer Center’s antibody production laboratories. 2-Chlorotrityl chloride resin and protected D-amino acids were obtained from Advanced ChemTech (Louisville, KY) and used as received. Sulfo-SMCC was purchased from Pierce (Rockford, IL) and Molecular Biosciences, Inc. (Boulder, CO). All other chemicals and solvents (high-purity grade) were obtained from Advanced ChemTech, Aldrich Chemical Co. (Milwaukee, WI), Fisher Scientific (Pittsburgh, PA), and J. T. Baker (Phillipsburg, NJ). Na125I, Na131I, and 111InCl3 were purchased from NEN Life Science Products (Boston, MA). All nonaqueous reactions were carried out under argon or nitrogen. Electrospray mass spectral (ESMS) data were obtained from Mass Consortium (San Diego, CA) and were recorded using either a Hewlett-Packard 1100 MSD or an API 1 Perkin-Elmer SCIEX electrospray mass spectrometer. Exact mass determinations by high-resolution mass spectra were obtained using FAB on a VG ZAB-VSE mass spectrometer. Amino acid analyses were carried out at the Scripps Research Institute Core facility (La Jolla, CA). Analytical HPLC analyses of peptides were carried out using a Waters PrepLC 4000 system using a Nova Pak C-18 (4 µm, 8 mm × 100 mm) Radial Pak cartridge, equipped with a UV detector (220 nm), and using a gradient elution consisting of buffers A (0.1% aqueous TFA) and B (9:1 acetonitrile/water in 0.1% TFA). [The gradient was as follows: 100% A changing to 100% B over the course of 10 min (linear gradient) at a flow rate of 3 mL/min, changing to a flow rate of 5 mL/min at 10.1 min, and remaining isocratic at 100% B at this flow rate for the next 5 min.] Unless otherwise stated, purified peptides were found to be eluted essentially as single peaks under these HPLC analytical conditions. Preparative HPLC was performed on a Waters PrepLC 4000 system using a Delta Pak C-18 (15 µm, 100 Å, 4 cm × 31 cm) or Nova Pak C-18 (6 µm, 60 Å, 2.5 cm × 20 cm) preparative HPLC column. HPLC analyses of radiolabeled mAbs were carried out on an analytical Bio-Sil 250 size-exclusion column, in series with a guard column (both from BioRad Laboratories, Hercules, CA), using 0.2 M sodium phosphate buffer (pH 6.8) as the mobile phase at a flow rate of 1 mL/min, with in-line UV (280 nm) and radioactivity detection. Cell lines were obtained from American Type Culture Collection (Rockville, MD), cultured as previously described (9, 12), and were routinely tested for mycoplasma by the Mycotect assay (Life Technologies, Gaithersburg, MD). Synthesis of IMP-R1 (Scheme 1). Fmoc-D-lysine(Aloc) (0.325 g) was dissolved in anhydrous dichlo2 IMP-R1 and IMP-R2 were previously referred to as “DPep” and “DDipep”, respectively.
Improved Internalizing [131I]mAbs for Therapy
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Scheme 1a
a (1) 2-Chlorotrityl chloride resin, DIEA, CH Cl , 18 h; (2) 20% piperidine/DMF; (3) Fmoc-D-Tyr(tBu), activated with HOBt and 2 2 DIC, DIEA, step 2; (4) BOC-Gly, activated with HOBt and DIC, DIEA; (5) [(C6H5)3P]4Pd, Bu3SnH; (6) 1:1:8 HOAc/CF3CH2OH/CH2Cl2, 3% TFA; (7) ITC-Bz-DTPA, pH 8.5; (8) TFA; (9) sulfo-SMCC, pH 7.0-7.2, 1 h.
romethane (5 mL) and mixed with DIEA (0.55 mL). The solution was mixed with 0.5 g of 2-chlorotrityl chloride resin at room temperature for 18 h. The resin was filtered, washed with solvents, and dried in a stream of nitrogen. The rest of the steps for the synthesis of resinbound BOC-Gly-D-Tyr(tBu)-D-Lys(Aloc) followed standard Fmoc protocol (20, 21) utilizing, sequentially, Fmoc-Dtyrosine(tBu) and BOC-glycine. The Aloc protecting group was removed by adding a solution of 0.1547 g of tetrakis(triphenylphosphine)palladium(0) in a mixture consisting of dichloromethane (40 mL), acetic acid (2 mL), and DIEA (5 mL), followed by the addition of tributyltin hydride (5 mL). After 1 h, the filtered resin was washed with solvents, dried, and treated with 10 mL of 1:1:8 acetic acid/trifluoroethanol/dichloromethane, containing 3% TFA, for 1 h. The peptide, cleaved from the resin, was collected by filtration of the reaction mixture. Solvent removal furnished the protected peptide III as a gummy material which was essentially pure as determined by HPLC (>95%), and was used as such. It had a retention time (HPLC) of 7.10 min. The electrospray mass spectrum exhibited an M + H peak at m/e 523 (positive ion mode) and an M - H peak at m/e 521 (negative ion mode). This product (53 mg, 0.1 mmol) was derivatized with ITC-BzDTPA (0.2 mmol) in water (pH 8.5) at 37 °C for 4 h. HPLC analysis indicated ∼65% conversion. Preparative purification via reverse-phase HPLC furnished 30 mg of the precursor of IV as a colorless solid. It had a retention time (HPLC) of 7.54 min. The mass spectrum exhibited an M + H peak at m/e 1063 (positive ion mode) and an M - H peak at m/e 1061 (negative ion mode). This material was then treated with 0.8 mL of a mixture consisting of TFA (2 mL), dichloromethane (0.5 mL), 0.12 mL of 1:3 (v/v) ethanedithiol/anisole, and 0.06 mL of water and the mixture stirred for 1 h at room temperature, and the product (IV, >98% pure as determined by
HPLC) was precipitated with diethyl ether. It had a retention time (HPLC) of 5.31 min. Mass spectrum: M + H at m/e 907, M - H at m/e 905. Exact mass: calcd for M + Na 929.3327, found 929.3370 (mass spectrum). Amino acid analysis, found (expected): Gly, 1.05 (1); Tyr, 0.92 (1); Lys, 0.95 (1). The intermediate IV (25 mg) was reacted with a 5-fold molar excess of sulfo-SMCC in 0.1 M sodium phosphate (pH periodically adjusted to be in the range of 7.0-7.2) for 1 h. The reaction proceeded with ∼50% efficiency (HPLC). Preparative reverse-phase HPLC yielded IMP-R1 (V). It had a retention time (HPLC) of 6.36 min. Electrospray mass spectrum: M+H at m/e 1126 (positive ion mode) and M-H at m/e 1124 (negative ion mode). Additionally, the mass spectra exhibited peaks at m/e 1186 and 1184 in the two ion modes, respectively, which were about 30% of the intensity of the main mass peak (see Results and Discussion). Exact mass: calcd for M + H 1126.4423, found 1126.4381 (mass spectrum). A repetition of the last step using >95% pure sulfo-SMCC gave IMP-R1 whose mass spectrum showed a negligible peak at m/e M + H + 60. Synthesis of IMP-R2 (Scheme 2). The resin-bound Fmoc-D-Lys(Aloc) was elaborated to VI via treatment with two successive D-tyrosine(tBu) units followed by BOC-D-alanine. After the Aloc group removal, the -amine at the C-terminus of the resin-bound peptide was reacted with a 6-fold molar excess of CA-DTPA in DMF, added in portions over the course of 1 h. After workup involving filtering and washing the resin with solvents, the final peptide was cleaved from the resin under mild acidic conditions. The product was purified by preparative reverse-phase HPLC. Cleavage of BOC and tBu protecting groups using TFA/dichloromethane/anisole produced the precursor to the title compound. It had a retention time (HPLC) of 5.44 min. Electrospray mass spectrum: m/e 1445 (M + H). Exact mass: calcd for M + Na
234 Bioconjugate Chem., Vol. 10, No. 2, 1999
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Scheme 2a
a (1-3) Steps 1-3 of Scheme 1; (4) step 3 of Scheme 1; (5) BOC-D-Ala, activated with HOBt and DIC, DIEA; (6 and 7) steps 5 and 6 of Scheme 1; (8) DTPA dianhydride, DMF, Et3N; (9 and 10) steps 8 and 9 of Scheme 1.
1466.6456, found 1466.6489 (mass spectrum). Amino acid analysis, found (expected): Ala, 2.10 (2); Tyr, 3.69 (4); Lys, 2.22 (2). This intermediate was further derivatized with a 10-fold excess of sulfo-SMCC as described previously. Purification by preparative reverse-phase HPLC yielded IMP-R2 (VII). It had a retention time (HPLC) of 6.99 min. Electrospray mass spectrum: m/e 1883 (M + H). In addition, a peak at m/e 1943 (M + H + 60) was observed (see Results and Discussion). Reaction yields were estimated by HPLC analyses of reaction mixtures. The precursor to the DTPA-coupled peptide was obtained in >95% yield, and the coupling to CA-DTPA gave ∼80% yield; the TFA deprotection proceeded in >95% yield, and the final derivatization with sulfo-SMCC resulted in ∼40% conversion. Stock aqueous solutions (0.75-1.24 mM, pH 5.6) of the peptides were aliquoted in several vials and stored frozen (-80 °C). For radioiodinations, 5-7 µL (∼6 nmol) of the peptide solution was used. Radioiodination and Conjugation to mAb-SH. DTT Reduction of mAbs. Intact IgG of LL2 (0.55 mL, 13 mg/mL) or RS7 (0.55 mL, 9 mg/mL) was mixed with 0.55 mL of 0.1 M sodium phosphate (pH 7.0), 0.11 mL of 0.5 M sodium borate buffer (pH 8.5), and 6 µL of a freshly prepared DTT solution (2.6 M). The solution was purged with argon to exclude the presence of air, incubated at room temperature for 30 min, and purified with centrifugation SEC (22) on two successive 3 mL columns of Sephadex G50/80 in 0.1 M sodium phosphate (pH 6.6). Another method involved mixing 0.1 mL of IgG (8-10 mg/mL) with 6 µL of 0.1 M aqueous EDTA (pH 7) and 6 µL of 40 mM DTT, purging with an inert gas such as argon, incubating at 37 °C in a water bath for 40-50 min, and purifying as described above. Iodogen Method. Twenty microliters of a diluted solution of PBS [0.8 mL of water and 0.02 mL of 0.5 M PBS (pH 7.5)] was added to 3-5 µL of Na125I (2 mCi) in the
activity vial. Buffered Na125I was transferred into a septum-sealed 1 mL vial containing the peptide solution (5-7 µL, 6 nmol) and a coating of iodogen (60 µg). The vial was shaken every 5 min for 20-30 min, and the solution was syringed out into a second septum-sealed vial containing a (4-hydroxyphenyl)acetic acid solution (40 mM, 1.5 µL). After 2-5 min, DTT-reduced antibody (0.5 mg) was added and the vial contents were incubated for a further 40 min. [Although DTT reduction was carried out on ∼1-7 mg scale for convenience, only a small portion was used for conjugating to radioiodinated IMPR1 or -R2.] The rest of the processing and purification were as described below for the chloramine-T method. Chloramine-T (CT) Method. To Na125I (2 mCi) in the activity vial were added PBS (0.5 M, pH 7.5, 20 µL), a peptide solution (5-7 µL, 6 nmol), and aqueous chloramine-T (4.4 mM, 20 µL). After 1-2 min, piperidine (14 mM, 20 µL), (4-hydroxyphenyl)acetic acid (5 mM, 20 µL), and potassium iodide (5 mM, 20 µL), each in water or 50 mM sodium phosphate (pH 7.5), were added sequentially, and the mixture was incubated for 1 min. Reduced IgG (0.20.5 mg) was then added, and the mixture was incubated for 20-30 min. Finally, aqueous sodium tetrathionate (50 mM, 20 µL) was added and the mixture incubated for 5 min. The iodination mixture was diluted with 0.25 mL of a solution of 40 mM PBS/10 mM sodium iodide, containing 0.02% sodium azide and 0.1% gelatin (pH 7.0), and transferred onto a 3 mL size-exclusion column of Sephadex G50/80 equilibrated in 50 mM PBS (pH 7.0). The vial was further rinsed, and the rinse was also transferred. Purification was carried out via centrifugation SEC. Radiolabelings of mAbs with 111In, [125I]DLT, and 125/131 I (CT Method). These labelings were carried out as reported (9, and references therein). For 111In labelings, conjugates of RS7 and LL2 intact mAbs with ITC-BzDTPA were used, unless otherwise stated.
Improved Internalizing [131I]mAbs for Therapy
Immunoreactivity (IMR). Comparisons of IMRs of the various RS7 preparations were carried out via a direct cell binding assay as described previously (18). Antigen-expressing cells (typically ME180 cells) were washed with PBS (Dulbecco’s PBS without calcium and magnesium), and the cell suspension (100 µL) was mixed with labeled mAb (∼50000 cpm) and 25 µL of either an unlabeled irrelevant mAb or an unlabeled specific mAb (RS7). A minimum of a 100-fold excess of unlabeled mAb was used. After incubation for 1 h at 4 °C with shaking, the mixture was diluted with PBS containing 1% horse serum, cells were pelleted for 5 min at 400g, and the amount of radioactivity associated with the pellet was counted. Data from triplicate experiments were computed as average percentages of specific bindings. Results obtained at two or three cell concentrations were compared to determine if a plateau of mAb excess had been reached. In Vitro Antibody Retention Experiments. These studies were carried out as described in detail in published work (6, 9). Raji, Calu-3, and ME180 cell lines were grown in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 5% fetal bovine serum, 5% horse serum, penicillin, streptomycin, glutamine, and sodium pyruvate (Gibco). All incubations were carried out in tissue culture medium at 37 °C. Confluent cells in 96well plates were incubated with 5 × 105 cpm of labeled antibody for 2 h and then washed four times with media by centrifugation. Tissue culture medium (0.2 mL) was added, and incubation was continued for specified times as indicated in the Results. Cells remained viable for at least 3 days. At indicated times, 0.1 mL of the medium was removed, and after washing via centrifugation, the cells were solubilized with 2.0 M NaOH to determine the amounts of cell-bound radioactivity. After the total counts per minute were determined in the culture medium, iodinated mAbs were precipitated with 5 mL of cold 10% trichloroacetic acid, the indium samples were precipitated with 10 mL of methanol with the use of 1.0 mg of bovine IgG as a carrier protein, and the precipitates were collected by centrifugation. The amounts of precipitable radioactivity and soluble radioactivity in the supernatant were thus determined. The extents of antigen-specific bindings, determined by adding a large excess of unlabeled specific mAbs (50 µg/mL) in control wells, were >90%. Each mAb processing experiment was carried out in triplicate unless otherwise stated. RESULTS
Solid-phase peptide syntheses were carried out utilizing a standard Fmoc strategy (20, 21). The functional group-protected peptide was assembled on the 2-chlorotrityl chloride resin; the Aloc group was selectively deprotected by a Pd(0)-mediated reaction (23), and the peptide was cleaved from the resin under mild acidic condition. This was followed by the derivatization of the lysine side chain, the cleavage of BOC and tBu groups, and finally the reaction of the amine terminus with sulfoSMCC. Differential protections of the -amine of D-lysine and the peptide’s N-terminus, with Aloc and BOC protecting groups, respectively, which were cleaved under different conditions, enabled differential derivatizations of the amines. The methods used for peptide bond formation are known to cause negligible racemization (21). The intermediates prior to SMCC derivatization, in both IMP-R1 and IMP-R2, were characterized by electrospray mass spectra, by exact mass analyses, by highresolution mass spectrometry, and by amino acid analyses. After derivatization with the cross-linker, both the
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bifunctional substrates exhibited M + H + 60 peaks (estimated to be 30%) in the electrospray mass spectra in addition to the expected mass peaks, as a result of an impurity (M + 60) present at the same level in the particular lot of sulfo-SMCC used.3 This suggested that in the particular lot of the cross-linker used, perhaps the maleimide group’s functional purity was compromised by a 1,4-addition of acetic acid, or some other entity with a molecular mass of 60 Da, to the same.3 The consequence of the presence of a M + 60 side product is in the lowering of the observed overall radioiodination-conjugation yield, but does not otherwise affect the utility aspect of the radiolabeled conjugates. The preparation of IMP-R1 was repeated when >95% pure sulfo-SMCC (including maleimide purity) was made available from a different commercial source. As expected, the mass spectrum of this preparation of IMP-R1 exhibited only a negligible level of the previously seen impurity. Disulfide reduction was carried out using DTT. Reduced IgGs (3.8-5.6 mg/mL) were estimated to contain 8.84 thiols (for LL2) and 8.95 thiols (for RS7) per IgG molecule by Ellman’s assay (24), corresponding to the breakage of four disulfide bonds. Under a variety of conditions, using up to 250 molar equiv of DTT, 8-9 thiols per IgG were produced (not shown). Using 2-mercaptoethanol, the number of thiol groups produced per IgG was 4-5, but the final radioiodinated mAbs derived thereof were not different from those derived from the DTT-reduced IgGs in terms of in vitro and in vivo characteristics (not shown). Radiolabeling consisted of peptide radioiodination and subsequent conjugation to reduced IgG. Only the combined yield of the radioiodination and the conjugation steps (overall yield) was determined routinely. Overall yields were based on the amount of radioactivity recovered after purification by centrifugation SEC. The radioactive material obtained after purification was determined to be the relevant mAb, based on the retention time on analytical SE-HPLC. In a few cases where the HPLC analyses showed the presence of a slower-eluting component (low-molecular mass entity, such as a labeled peptide not completely removed by the purification method, usually
