Poly(ethylene glycol)-Conjugated Anti-EGF Receptor Antibody C225

Several biological barriers, including significant liver uptake, limit the clinical application of radiolabeled antibodies in radioimmunoscintigraphy...
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Bioconjugate Chem. 2001, 12, 545−553

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Poly(ethylene glycol)-Conjugated Anti-EGF Receptor Antibody C225 with Radiometal Chelator Attached to the Termini of Polymer Chains Xiaoxia Wen,† Qing-Ping Wu,† Yang Lu,‡ Zhen Fan,‡ Chusilp Charnsangavej,† Sidney Wallace,† Diana Chow,§ and Chun Li*,† Department of Diagnostic Radiology and Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and Department of Pharmacology and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, Texas 77030. Received November 27, 2000; Revised Manuscript Received May 9, 2001

Several biological barriers, including significant liver uptake, limit the clinical application of radiolabeled antibodies in radioimmunoscintigraphy. Here, a general approach is described for radiolabeling of monoclonal antibodies conjugated with poly(ethylene glycol) (PEG). This strategy is demonstrated with C225, a monoclonal antibody directed against epidermal growth factor (EGF) receptor. We synthesized a heterofunctional PEG with one end attached to a radiometal chelator, diethylenetriaminepentaacetic acid (DTPA), and the other end to a protected thiol group, Sacetylthioacetate. After a deprotection step, the resulting DTPA-PEG-SH was conjugated to maleimideactivated C225 to yield DTPA-PEG-C225 conjugate. Characterization of DTPA-PEG-C225 with immunoprecipitation and Western blot analysis revealed that the conjugate was biologically active in binding to the EGF receptor in A431 cells. Competitive EGF receptor binding assay in MDA-MB-468 cells showed that DTPA-PEG-C225, with up to 60% of the amino groups in C225 substituted, retained 66% of C225’s binding affinity. Moreover, DTPA-PEG-C225 with increasing degrees of NH2 substitution from 20% to 70% retained the activity of C225 to induce apoptosis in DiFi cells. More importantly, DTPA-PEG-C225 demonstrated less nonspecific interaction than DTPA-C225. Pharmacokinetic analysis using 111In-labeled compounds revealed narrower steady-state distribution of 111In-DTPAPEG-C225 than 111In-DTPA-C225, probably due to reduced nonspecific binding of PEG-modified antibody to tissues. The terminal half-life (t1/2,γ) of 111In-DTPA-PEG-C225, 21.1 h, was shorter than that of 111In-DTPA-C225, 52.9 h. These data suggest that 111In-DTPA-PEG-C225 may provide better imaging characteristics than 111In-DTPA-C225, and that using PEG as a linker between the monoclonal antibody and DTPA may be a promising strategy in optimizing the imaging characteristics of immunoscintigraphic agents.

INTRODUCTION

Radioimmunodetection for diagnostic purposes has many potential applications in the management of cancer patients. These applications include presurgical staging of extent of disease, postsurgical evaluation of residual disease, confirmation of viable tumors known by other methods, disclosure of occult recurrence, confirmation of tumor targeting of antibody to be used for immunotherapy, and assessment of therapeutic response (1, 2). Although the potential usefulness of radiolabeled monoclonal antibody (mAb)1 is high, the actual progress concerning their clinical applications has not been satisfactory. Despite nearly 50 years of investigation and refinement, to date only one radiolabeled mAb has been approved by the FDA for use in humans (3). Limitations * Address correspondence to this author at the Department of Diagnostic Radiology, Box 59, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Phone: (713)792-5182. Fax: (713)794-5456. E-mail: [email protected]. † Department of Diagnostic Radiology, The University of Texas M. D. Anderson Cancer Center. ‡ Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center. § Department of Pharmacology and Pharmaceutical Sciences, University of Houston.

of radioimmunodetection have been multifactorial, and include low uptake by tumor confined by biological barriers, unfavorable pharmacokinetics, and poor tumorto-background ratio, etc. (1). Among a wide range of isotopes applied to the labeling of antibodies, the majority are transition metals. It is evident that in order to obtain optimal imaging characteristics, it is necessary to achieve a high ratio of radioactivity between the tumor and normal tissues. Unfortunately, although immunoconjugates labeled with radiometals such as yttrium and indium usually have greater retention in cancer than the corresponding iodinated radioimmunoconjugates, they also tend to be retained in normal tissues, particularly the liver (4, 5). To decrease the rate of radiometal detachment from the mAb and reduce the subsequent hepatic uptake of the 1 Abbreviations: mAb, monoclonal antibody; DTPA, diethylenetriaminepentaacetic acid; PEG, poly(ethylene glycol); EGF, epidermal growth factor; SATA, S-acetylthioacetate; GMBS, N-γ-maleimidobutyryloxysuccinimide ester; TNBS, 2,4,6-trinitrobenzenesulfonic acid; SDS, sodium dodecyl sulfate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TFA, trifluoroacetic acid; TEA, triethylamine; MWCO, molecular weight cutoff; DMF, dimethylformamide; GPC, gel permeation chromatography; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine albumin; BSA, bovine serum albumin.

10.1021/bc0001443 CCC: $20.00 © 2001 American Chemical Society Published on Web 06/06/2001

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radiolabel, modified DTPA molecule (6) and also macrocyclic chelates, such as 1,4,7,10-tetraazacyclododecaneN,N′,N′′,N′′′-tetraacetic acid (DOTA) (7), have been used to increase the stability of radiometal-chelate complexes. Chelates with labile linkers can also exhibit such favorable characteristics as high tumor accumulation, rapid blood clearance, and low liver uptake in animal experiments (8, 9). For example, a peptide linker, triglycyl-Lphenylthiourea, susceptible to hepatic endopeptidase activity was designed and inserted between DOTA and chimeric mAb L6 (9). The resulting immunoconjugate exhibited reduced liver uptake due to increased hepatocyte cleavage and excretion of the radiochelate (10). A different conjugation strategy is to manipulate the biodistribution patterns of the antibodies by employing the uncharged, amphiphilic linear polymer poly(ethylene glycol) (PEG) as a linker between the monoclonal antibody and the metal chelators. PEG-modified proteins, antibodies, and liposomes have been shown to exhibit reduced liver uptake and increased blood circulation halflives, resulting in improved biological activity (11-13). In this report, we present the synthesis of a DTPA-PEG conjugate of an anti-epidermal growth factor (EGF) receptor antibody, C225, its radiolabeling with indium111, and the biological activities of the resulting immunoconjugate. EGF receptor is a transmembrane glycoprotein with an intracellular tyrosine kinase domain. EGF receptor is overexpressed on the cells of over onethird of all solid tumors, including bladder, breast, colon, ovarian, prostate, renal cell, and squamous cell (nonsmall cell lung and head and neck) carcinomas (14). C225 is a human-mouse chimeric monoclonal antibody directed against human EGF receptor. It specifically binds to the external domain of the receptor with an affinity comparable to the natural ligand. C225 has been demonstrated to inhibit the proliferation of a variety of human cancer cells stimulated by the transforming growth factor-R (TGF-R) and EGF receptor autocrine loop (15). EXPERIMENTAL PROCEDURES

Materials. C225 was kindly provided by ImClone Systems Inc. (New York, NY). t-Boc-NH-PEG-NH2 (MW 3400) was obtained from Shearwater Polymers, Inc. (Huntsville, AL). N-Succinimidyl S-acetylthioacetate (SATA), N-γ-maleimidobutyryloxysuccinimide ester (GMBS), 2,4,6-trinitrobenzenesulfonic acid (TNBS, 5% w/v aqueous solution), 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman’s Reagent), sodium dodecyl sulfate (SDS), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and PBS (0.01 M phosphate-buffered saline containing138 nM NaCl, 2.7 nM KCl, pH 7.4) were purchased from Sigma Chemicals (St. Louis, MO). DTPAdianhydride, trifluoroacetic acid (TFA, anhydrous), ninhydrin, triethylamine (TEA), and all the other solvents and reagents were purchased from Aldrich Chemical Co. (St. Louis, MO). All the chemicals and solvents were at least ACS grade and were used without further purification. Indium-111 radionuclide was obtained from DupontNEN (Boston, MA). PD-10 disposable column, Sephadex G-75 gel, and Sephacryl S-200 high-resolution gel were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Spectra/Pro 6 dialysis tubing [molecular weight cutoff (MWCO) 2000] and Centricon-YM-10 centrifugal filter devices (MWCO 10 000) were purchased from Fisher Scientific (Houston, TX). Silica gel 60 TLC plates were obtained from EM Sciences (Gibbstown, NJ). DTPA-PEG-NH2. To a stirred suspension of DTPAdianhydride (143 mg, 0.4 mmol) in 4 mL of chloroform

Wen et al.

were added TEA (81 mg, 0.8 mmol) and t-Boc-NH-PEGNH2 (340 mg, 0.1 mmol). The mixture was allowed to react at room temperature for 2 h. The reaction was followed by silica gel TLC using CHCl3-MeOH (4:1 v/v) as the mobile phase; the plates were visualized by both iodine vapor and ninhydrin spray (0.1% ninhydrin solution in ethanol). TLC showed complete conversion of NH2PEG-NH-t-Boc (Rf ) 0.55, purple in ninhydrin) to DTPAPEG-NH-t-Boc (Rf ) 0.4 with iodine vapor, negative in ninhydrin). After the reaction, the chloroform and TEA were removed under vacuum. The t-Boc protecting group was removed without purification by adding TFA (2 mL) to the resulting residue and stirring the mixture at room temperature for 4 h. The resulting DTPA-PEG-NH2 was purified by dialysis against PBS and deionized water using dialysis tubing (MWCO, 2000). Rf: 0.18 (chloroformmethanol, 4:1 v/v; ninhydrin spray); yield: 360 mg, 95%. DTPA-PEG-ATA. DTPA-PEG-NH2 (182 mg, 0.05 mmol) was reacted with SATA (14 mg, 0.06 mmol) in chloroform at room temperature for 1 h, and then purified by dialysis (MWCO, 2000) and by gel filtration on a PD10 column to afford DTPA-PEG-ATA, Rf ) 0.27 (chloroform-methanol, 4:1 v/v; iodine vapor). 1H NMR, 300 MHz, CDCl3: 2.34 (s, 3H, CH3COS-), 3.17-3.28 (m, 8H, -CH2CH2- in DTPA), 3.50 (s, 308H, -CH2CH2- in PEG with 77 repeating units). Anal Calcd for C169H337N5O5S: C, 52.54; H, 8.73; N, 1.81; S, 0.83. Found: C, 51.52; H, 8.90; N, 1.61; S, 0.81. Molecular mass determined by MALDI-TOF mass spectroscopy (BiFlexIII, Bruker Daltonics, Billerica, MA): 3750. No peaks of dimer or oligomers were found in the spectrum (data not shown). Yield: 180 mg, 92%. DTPA-PEG-C225. Maleimide-activated C225 with different ratios of C225 to maleimide was prepared according to the following general procedure. To an aqueous solution of C225 (2.4 mg/mL; 4.8 mg, 0.032 µmol) at room temperature were added aliquots of GMBS in dimethylformamide (DMF) (2.8 mg/mL). The mixture was stirred for 1 h, and then purified by gel filtration using a PD-10 column. Prior to conjugation with activated C225, the acetyl protecting group in DTPA-PEG-ATA was removed using hydroxylamine. For this purpose, an aliquot of NH2OH (50 µL) in 0.1 M Na2HPO4 (0.5 M) was added to a solution of DTPA-PEG-ATA (7.7 mg, 1.92 µmol) in 0.1 M Na2HPO4 (pH 8.5, 0.5 mL), and then incubated at room temperature for 30 min. The resulting DTPA-PEG-SH containing free sulfhydryl group was then mixed with maleimide-activated C225 with a DTPAPEG-SH-to-maleimide molar ratio of 2:1 and incubated at 4 °C overnight. The final product was separated from unreacted DTPA-PEG by gel filtration on a Sephacryl S-200 HR column (1.5 cm × 20 cm) with PBS as eluent. The presence of free sulfhydryl group was monitored using Ellman’s agent (16). DTPA-C225. DTPA-C225 was prepared using a previously described method (17). Briefly, DTPA-dianhydride (4.6 mg, 12.8 µmol) was added to an aqueous solution of C225 (2.4 mg, 0.016 µmol; 2.4 mg/mL). For reaction efficiency, the pH of the reaction solution was kept at 7-8 by adding 0.1 M Na2HPO4. After incubation at room temperature for 1 h, the solution was concentrated to half-volume on a Centricon-YM 10 centrifugal filter and purified from free DTPA by gel filtration on a PD-10 column. Radiolabeling. Generally, 40 µg of each antibody conjugate in 100 µL of PBS was incubated with 350400 µCi of 111InCl3 (in 20 µL of 1 M sodium acetate buffer, pH 5.5) at room temperature for 15 min. The resulting radioisotopic product was purified from free indium-111

111In-Labeled

PEG-Conjugated Antibody

by gel filtration on a PD-10 column using PBS as the eluent. Fractions of 0.5 mL each were collected. The radioactivity of each fraction was measured by a radioisotope calibrator (Capintec Instruments, Ramsey, NJ). The protein content in each fraction was determined using a Bio-Rad protein assay kit according to the manufacturer’s instruction (Bio-Rad Laboratories, Hercules, CA). The fractions containing the protein were combined. The radiochemical yield, expressed as a percentage of the radioactivity of the protein fractions to the total loaded radioactivity, was calculated. The radiochemical purity was determined by gel permeation chromatography (GPC). Determination of Degree of Modification. The degree of substitution of C225 by maleimide was determined by quantifying the free amino groups remaining in the antibody using TNBS assay according to the published protocol (18). Briefly, samples were dissolved in 0.1 M sodium bicarbonate (pH 8.5) at a concentration of 20-200 µg/mL. To 1 mL of each sample solution was added 0.5 mL of TNBS solution in 0.1 M sodium bicarbonate (0.01%, w/v). After incubation at 37 °C for 2 h, 0.5 mL of 10% SDS and 0.25 mL of 1 N HCl were sequentially added to each sample. The percentage of the reacted amino groups was determined by comparing the UV absorbance (335 nm) of the free amino groups in the modified antibody with that in the intact antibody. Gel Permeation Chromatography. Analytical GPC was performed with a Waters HPLC system (Waters Corp., Milford, MA) consisting of a 2410 refractive index detector and a 2487 dual λ UV detector applying a TSKG3000 PW 7.5 mm × 30 cm gel column (Tosoh Corp., Japan). Samples were eluted with PBS containing 0.1% LiBr at a flow rate of 1 mL/min, and the products were detected by the refractive index and UV absorbance at 254 nm. Radio-GPC was performed using an HPLC unit equipped with LDC pumps (Laboratory Data Control, Rivera Beach, FL), an LUDLUM radiometric detector (Measurement Inc., Sweetwater, TX), and an SP 8450 UV/VIS detector (Spectra-Physics, San Jose, CA). The samples were separated by a Phenomenex Biosep SECS3000 7.8 mm × 30 cm column, eluted with PBS containing 0.1% LiBr at a flow rate of 1 mL/min, and detected by radioactivity and UV absorbance at 254 nm. Cell Lines. Human breast adenocarcinoma MDA-MB468, human vulvar squamous carcinoma A431, and human colorectal carcinoma DiFi were maintained in 1:1 (v/v) Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 mixture supplemented with 10% fetal bovine serum (FBS) (Gibco Laboratories, Grand Island, NY) at 37 °C in 5% CO2/95% air. Competitive Binding Assay. MDA-MB-468 cells were seeded at 5 × 107 cells/well onto 12-well plates in 10% FBS medium and allowed to attach overnight. The medium was replaced by DMEM/F-12 medium plus 0.2% bovine serum albumin (BSA), and 1 µg/mL of 1:30 111InDTPA-PEG-C225 or 111In-DTPA-C225 plus native C225 mAb at the indicated concentrations was added to the wells. After incubation at 37 °C for 2 h, the cells were washed 5 times with PBS containing 0.2% BSA. The cells were then trypsinized and transferred to 5 mL disposable culture tubes. The level of radioactivity in each tube was measured with a Cobra Auto-gamma Counter (Packard Instrument Co., Downers Grove, IL). Immunoprecipitation and Western Blot Analysis. A431 cells were cultured with C225 or DTPA-PEG-C225 conjugates at 37 °C for 30 min, followed by washing the cells twice with cold PBS and lysis of the cells with a buffer containing 50 mM Tris-HCl, pH 7.4, 50 mM NaCl,

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0.5% NP-40, 50 mM NaF, 1 mM Na3PO4, 1 mM phenylmethylsulfonyl fluoride, 25 µg/mL leupeptin, and 25 µg/ mL aprotinin. The lysates were centrifuged at the full speed of a microcentrifuge for 15 min, and the supernatants were collected for protein concentration determination. Immunoprecipitaiton was performed by incubation of 100 µg of cell lysate with 40 µL of Sepharose 4Bconjugated protein A at room temperature for 1 h, followed by washing the immunoprecipitates 3 times with the lysing buffer and separation of immunoprecipitates with 7% polyacrylamide-SDS electrophoresis. Western blot was carried out by electronically transferring the samples into a nitrocellulose membrane and incubation of the membrane for 1 h with an anti-EGF receptor antibody. The EGF receptor signals in the membrane were developed by the ECL chemoluminescence detection kit (Amersham, Arlington Heights, IL). MTT Assay. DiFi cells were seeded at 5 × 104 cells/ well onto 24-well culture plates. Cell viability after 72 h treatment of the cells with C225 or DTPA-PEG-C225 was assayed by adding 50 µL of 10 mg/mL MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma) into 0.5 mL of culture medium and incubating the cells for 3 h at a 37 °C in a CO2 incubator, followed by cell lysis with 500 µL of lysis buffer containing 20% SDS in dimethylformamide/H2O, pH 4.7, at 37 °C for more than 6 h. An optical absorbance of cell lysate was determined by measuring the cell lysate at a wavelength of 595 nm and normalizing the value with the corresponding control of untreated cells. Pharmacokinetics. Nude mice (Harlan Sprague Dawley, Indianapolis, IN) were divided into two groups of three mice each and administered i.v. with 1:30 111InDTPA-PEG-C225 at a dose of 1.5 µg/mouse or with 111 In-DTPA-C225 at a dose of 5 µg/mouse. At predetermined intervals, blood samples (30-60 µL) were taken from the tail vein, and the radioactivity of each sample was measured with a gamma counter. The pharmacokinetic parameters for 111In-DTPA-PEG-C225 and 111InDTPA-C225 were calculated from mean blood concentration values observed from the time of initial administration to 96 h after administration using WinNonlin 2.1 software (Scientific Consulting, Inc., Lexington, KY). RESULTS AND DISCUSSION

Synthesis and Characterization. Previous studies have shown that an 111In-labeled DTPA-mAb C225 specifically localizes in human cancer cell xenografts that overexpress EGF receptor (19). Phase I studies demonstrated that 111In-DTPA-C225 was able to image squamous cell lung carcinoma expressing high levels of EGF receptor and metastases greater than 1 cm in diameter. However, considerable hepatic radioactivity was seen as a result of nonspecific uptake (20). The significant liver uptake made it difficult to visualize tumors or lesions inside or close to the liver. To overcome this drawback and optimize the imaging properties of C225, we sought to introduce flexible, linear PEG molecules into mAb C225 to reduce nonspecific interaction. In the past, attempts have been made in altering the pharmacological characteristics of mAbs by shielding the antibody with PEG or other synthetic polymers (21-23). Kitamura et al., for instance, reported the attachment of PEG (MW 5000) to murine mAb A7 and its F(ab′)2 fragment (21). These antibodies were radiolabeled with iodine-125 by the chloramine-T method. Both PEG-modified mAb and its fragment exhibited lower tissue-to-blood ratios in all resected organs than the parent antibodies, suggesting

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Scheme 1. Synthesis of PEG-Modified C225 Monoclonal Antibody

reduced nonspecific interaction. Unfortunately, the tumorto-blood ratio was also decreased after PEG modification. A similar study by Pedley et al. with PEG-modified and 125I-labeled anti-CEA antibody A5B7 showed that the localization index was inferior to that of the unmodified antibody, and there was little increase in the tumor-toblood ratio with time (22). A major problem of radioiodinated antibodies is the in vivo instability due to circulating dehalogenases, with the released radioiodine accumulating in the thyroid. We were also concerned with avoiding excessive manipulation of the mAb by both PEG modification and radioiodination procedures. Therefore, we were especially interested in developing a simple, one-step method to introduce both PEG molecules and radiometal chelators to mAbs such as C225. Our approach was to conjugate the metal chelator DTPA through a PEG linker to C225 instead of attaching DTPA and PEG to the antibodies directly in a stepwise manner. The synthesis of a key intermediate, heterofunctional DTPA-PEG-ATA, with one end attached with a DTPA and the other with S-acetylthioacetate, is shown in Scheme 1. The sequence of reactions, i.e., the reaction with DTPA-dianhydride first, followed by deprotection and coupling with SATA, was necessary to achieve high yield. When t-Boc-NH-PEG-NH2 was first coupled to SATA, the S-acetyl group was found to be unstable in the presence of TFA in the deprotection step. The prematurely released SH group could be easily destroyed by DTPA-dianhydride in the subsequent step. The 1:1 ratio of DTAP and ATA in DTPA-PEG-ATA was established by 1H NMR analysis, elemental analysis, and by MALDI-TOF mass spectroscopic characterization. The theoretical ratio of integrals between -CH2CH2- in DTPA and CH3- in ATA is 2.67. The ratio derived from NMR was 2.57, suggesting that the molar ratio of DTPA to ATA in DTPA-PEG-ATA was close to 1. The percentage weight

ratio of N to S in DTPA-PEG-ATA should be 2.18. The ratio derived from elemental analysis was 1.99. These data again confirm the 1:1 ratio of DTPA and ATA in DTPA-PEG-ATA. Finally, the calculated molecular mass of DTPA-PEG-ATA is 3770 Da; the molecular mass of the conjugate determined by MALDI-TOF mass spectroscopy was 3750 Da. No peaks of dimer and oligomers were found in the spectrum. These data support the proposed structure of DTPA-PEG-ATA. DTAP-PEG-ATA is stable for more than 6 months when stored as lyophilized powder at 4 °C. DTPA-PEG-ATA was conveniently coupled to maleimide-activated C225 following a simple in situ deprotection step to release the free SH group (Scheme 1). Four DTPAPEG-C225 conjugates with different degrees of C225 modification were synthesized. These conjugates were designated as 1:10, 1:20, 1:30, and 1:40 DTPA-PEG-C225, with the numbers being the molar ratios of antibody to GMBS in the maleimide-activating reaction. The physicochemical properties of the newly synthesized conjugates and some of the 111In-labeled molecules are summarized in Table 1. Each C225 molecule contains approximately 50-60 free amino groups as measured by TNBS assay. In the 1:10, 1:20, 1:30, and 1:40 DTPA-PEGC225 conjugates, approximately 20-25%, 40%, 60%, and 70% of amino groups were substituted, respectively. DTPA-C225 with DTPA directly attached to C225 mAb was also synthesized for the purpose of comparison (Table 1). Because DTPA-anhydride was readily hydrolyzed in aqueous media, coupling of DTPA directly to C225 was an inefficient reaction. Only 10-20% of the amino groups in C225 were substituted by DTPA when the molar ratio of DTPA-dianhydride to C225 reached 800:1. Two PEG-modified antibody conjugates, 1:10 DTPAPEG-C225 and 1:30 DTPA-PEG-C225, as well as DTPAC225 were radiolabeled with indium-111. The radiochemical yields of the two 111In-DTPA-PEG-C225 conjugates were over 70%, whereas the yield of 111In-DTPA-C225 was only 40%. The lower yield with 111In-DTPA-C225 reflects the fact that a large amount of DTPA was introduced into the coupling reaction between DTPAdianhydride and C225. Although extensive purification procedures including ultracentrifugation and gel filtration on GPC column were used, DTPA-C225 could still be contaminated by trace amounts of DTPA molecules, leading to low labeling efficiency. GPC was used to monitor the purity of C225 conjugates and 111In-labeled C225 conjugates. As shown in Figures 1-3, coupling of PEG to C225 increased the hydrodynamic volume of C225. The retention time of intact C225 (6.0 min) on the TSK-G3000 column was shortened to 4.9 min for 1:30 DTPA-PEG-C225, suggesting that PEG molecules were chemically bound to the mAb (Figure 1). The GPC chromatogram of purified DTPA-PEG-C225 also indicated that gel filtration on the Sephacryl S-200 HR column adequately removed unconjugated C225. However, when 1:30 DTPA-PEG-C225 was labeled with indium-111, it gave two peaks in radio-GPC, with reten-

Table 1. Physicochemical Properties of DTPA-PEG-C225 Conjugates C225 conjugates

molar ratio of C225 to GMBS

degree of NH2 substitution (%)

retention time in GPC (min)

radiochemical yield (%)

radiopurity (%)

1:10 DTPA-PEG-C225 1:20 DTPA-PEG-C225 1:30 DTPA-PEG-C225 1:40 DTPA-PEG-C225 DTPA-C225

1:10 1:20 1:30 1:40 1:800a

20-25 40 60 70 10-20

6.1

>70

>97

5.7

>70

>99

6.7

40

>99

a

Molar ratio of C225 to DTPA-dianhydride.

111In-Labeled

PEG-Conjugated Antibody

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Figure 1. TSK-G3000 size-exclusion HPLC (UV absorbance at 254 nm) of native C225 mAb and 1:30 DTPA-PEG-C225 conjugate after purification on a Sephadex G-75 column. DTPA-PEG-C225 was eluted 1.1 min earlier than the native C225 mAb, revealing the conjugation of PEG molecules to the C225 mAb.

tion times of about 5.9 and 8.5 min, respectively (Figure 2A). The major peak at 5.9 min corresponded to 1:30 111In-DTPA-PEG-C225 while the minor peak at 8.5 min, which reflects a retention time identical to that of 111InDTPA-PEG (Figure 2B), was attributed to unconjugated DTPA-PEG. Thus, another gel filtration procedure was necessary to remove 111In-DTPA-PEG. Radio-GPC chromatograms of purified 1:30 111In-DTPA-PEG-C225, 1:10 111 In-DTPA-PEG-C225, and 111In-DTPA-C225 are presented in Figure 3. The 1:30 and 1:10 conjugates were eluted at 5.7 and 6.1 min, respectively, reflecting shorter retention times than 111In-DTPA-C225 (6.7 min). These results further confirmed that there were more PEG molecules attached to the 1:30 DTPA-PEG-C225 conjugate than to the 1:10 conjugate. The radiopurities of purified 1:30 111In-DTPA-PEG-C225, 1:10 111In-DTPAPEG-C225, and 111In-DTPA-C225 were >99%, >97%, and >99%, respectively (Table 1). In Vitro Biological Functions. To determine whether PEG-modified C225 retains the binding affinity of the

parent mAb, we evaluated the in vitro biological properties of DTPA-PEG-C225 conjugates using three methods: competitive binding to human breast cancer MDAMB-468 cells, immunoprecipitation of EGF receptor from human vulvar squamous carcinoma A431 cell lysates, and inhibition of growth of human colorectal carcinoma DiFi cells. All three cell lines express high levels of EGF receptor (24-26). Studies on the cellular uptake of 1:30 111In-DTPA-PEGC225 and 111In-DTPA-C225 in MDA-MB-468 cells have shown that cell-associated radioactivity increased with increasing concentrations of radiolabeled C225. A plateau was reached at 2 µg/mL for 1:30 111In-DTPA-PEG-C225 (data not shown). Therefore, a concentration of 1 µg/mL for the radiolabeled C225 was chosen for the competitive binding assay. The binding of both 1:30 111In-DTPA-PEGC225 and 111In-DTPA-C225 to MDA-MB-468 cells was displaced by C225 in a dose-dependent manner, suggesting that the binding is EGF receptor specific (Figure 4). Furthermore, 111In-DTPA-PEG-C225 was almost fully

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Wen et al. Table 2. Pharmacokinetic Parameters of 1:30 111In-DTPA-PEG-C225 and 111In-DTPA-C225 parametersa

DTPA-C225

DTPA-PEG-C225 (1:30)

A (% ID/mL) B (% ID/mL) C (% ID/mL) R (h-1) β (h-1) γ (h-1) t1/2,R (h) t1/2,β (h) t1/2,γ (h) V1 (mL) Vss (mL) CL (mL/h) k10 (h-1) k12 (h-1) k21 (h-1) k13 (h-1) k31 (h-1)

11.7 14.8 13.3 1.74 0.08 0.01 0.40 9.11 52.9 2.52 5.41 0.08 0.03 0.48 1.24 0.03 0.04

11.3 37.3 7.6 2.82 0.10 0.03 0.24 6.82 21.1 1.78 2.94 0.16 0.09 0.53 2.27 0.02 0.04

a % ID/mL is the injected dose per milliliter of blood. A, B, C, R, β, and γ are hybrid constants. V1 and Vss are volume distributions in the central compartment and at steady state (Vss). CL is clearance. k10, k12, k21, k13, and k31 are microconstants.

Figure 2. Radio-gel permeation chromatography of (A) 111Inlabeled 1:30 DTPA-PEG-C225 prior to purification and (B) 111In-DTPA-PEG. In panel A, the peak at 5.9 min corresponds to the DTPA-PEG-C225 conjugate, and the peak at 8.5 min, which is identical to the peak in panel B, corresponds to unreacted DTPA-PEG.

displaced by a 16-fold excess of C225, whereas 111InDTPA-C225 was only 80% displaced by a 16-fold excess of C225 and could not be fully displaced even by 40-fold excess of C225. The remaining 20% of cell-associated 111 In-DTPA-C225 represent nonspecific binding to the cells. These results suggest that modification of C225 with PEG reduced the nonspecific interaction of the antibody. When the contribution of nonspecific binding is taken into consideration and is deducted from the cellassociated radioactivity, 44% of 111In-DTPA-C225 and 33% of 1:30 111In-DTPA-PEG-C225 remained bound to the cells when the concentration of native C225 mAb was equal to the concentration of the labeled molecules (1 µg/ mL). This means that 111In-DTPA-C225 retained about 88% of the receptor binding affinity of native C225, while 1:30 111In-DTPA-PEG-C225 retained about 66% of the binding affinity. To further assess the receptor binding affinity of DTPA-PEG-C225 conjugates, the ability of DTPA-PEGC225 conjugates to bind to EGF receptor was investigated in A431 cells, which express a very high level of EGF

receptors. The cells exposed to C225 or one of the three 1:10, 1:20, and 1:40 DTPA-PEG-C225 conjugates were lysed, followed by immunoprecipitation of the antibodybound EGF receptor with protein A-Sepharose beads and visualization of the EGF receptors with Western blot analysis. The results shown in Figure 5 indicate that all three DTPA-PEG-C225 conjugates, with 20%, 50%, and up to 70% of the amino groups in C225 substituted, retained their EGF receptor binding activities. However, the amounts of EGF receptor immunoprecipitated by C225 conjugates decreased with increasing degree of substitution, indicating that the binding affinity of the PEG-modified C225 decreased with the increasing number of PEG molecules attached to the mAb. The activities of DTPA-PEG-C225 conjugates were further assessed by an independent antiproliferation assay. We have previously reported that blocking EGF receptor tyrosine kinase activity with C225 leads to cell cycle arrest and subsequent cell death through apoptosis in DiFi cells (26). As shown in Figure 6, while the linker molecule PEG-DTPA itself had no effect on DiFi cell growth, all three conjugates, 1:10, 1:20, and 1:40 DTPAPEG-C225, inhibited the tumor cell growth to the same extent as native C225, indicating that all conjugates were capable of inducing apoptosis in the DiFi human colon cancer cells (Figure 6). Pharmacokinetics. The radioactivities of the blood samples obtained at different time intervals after i.v. injection of 1:30 111In-DTPA-PEG-C225 and 111In-DTPAC225 were measured with a gamma counter. Figure 7 plots blood radioactivity expressed as a percentage of injected dose per milliliter of blood (% ID/mL of blood) versus time following i.v. injection of radiolabeled C225. The profiles of both 111In-DTPA-PEG-C225 and 111InDTPA-C225 fit well into three-compartment models, and can be mathematically described by the triexponential equations: Ct ) 11.3e-2.82t + 37.3e-0.10t + 7.6e-0.03t and Ct ) 11.7e-1.74t + 14.8e-0.08t + 13.3e-0.01t, respectively, where Ct is % ID/mL of blood at any given time t. The pharmacokinetic parameters of volume distributions in the central compartment (V1) and at steady state (Vss), clearance (CL), hybrid constants (A, B, C, R, β, and γ), and microconstants (k10, k12, k21, k13, and k31) are summarized in Table 2. The volume distribution at steady state (Vss) of 1:30 111In-DTPA-PEG-C225, 2.94 mL,

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Figure 3. Radio-gel permeation chromatography of purified (A) 111In-DTPA-C225, (B) 1:10 111In-DTPA-PEG-C225, and (C) 1:30 111In-DTPA-PEG-C225. The three molecules were eluted through a Phenomenex Biosep SEC-S3000 column. The radiochemical purity of each conjugate was greater than 97%.

Figure 4. Competitive binding of 111In-DTPA-C225 and 1:30 111In-DTPA-PEG-C225 with native C225 to MDA-MB-468 cells. The cells were incubated with 1 µg/mL of each 111In-labeled C225 conjugate plus unlabeled C225 at different concentrations. After incubation at 37 °C for 2 h, the cell-associated radioactivity was measured with a gamma counter. The data are expressed as counts per minute (CPM) as a percentage of control and presented as the means of triplicates with standard deviations.

was smaller than that of 111In-DTPA-C225, 5.41 mL. The narrower distribution of PEG-modified molecules might be due to the reduced nonspecific binding of these molecules to tissues and the faster returning rate constant from tissues to the central compartment, reflected by the larger k21. The elimination rate constant from the central compartment (k10) and the clearance (CL) of 1:30 111In-DTPA-PEG-C225 were 0.09 h-1 and 0.16 mL/h, respectively, higher than those of 111In-DTPAC225, 0.03 h-1 and 0.08 mL/h, respectively. The terminal half-life (t1/2,γ) of 111In-DTPA-PEG-C225, 21.1 h, was shorter than that of 111In-DTPA-C225, 52.9 h, resulting from its smaller volume distribution and faster clearance.

Figure 5. Immunoprecipitation of EGF receptor from A431 cells by C225 and its PEG conjugates. Western blotting with anti-EGF receptor antibody revealed the binding of DTPA-PEGC225 molecules to EGF receptor in A431 cells. In 1:10, 1:20, and 1:40 DTPA-PEG-C225s, PEG molecules were attached to 25%, 50%, and 70% of the amino groups in C225, respectively. The asterisks represent different batches of DTPA-PEG-C225 with the same degree of substitution (1:20).

Because the molecular weight of C225 is relatively high (150 000), its modification with PEG (34 000) was not expected to have profound effects on its blood circulation time. Nevertheless, our finding that the terminal halflife of 1:30 111In-DTPA-PEG-C225 was actually shorter than that of 111In-DTPA-C225 is somewhat unexpected. A possible explanation is in vivo cleavage of 111In-DTPAPEG from 111In-DTPA-PEG-C225. The shortened blood retention of 111In-DTPA-PEG-C225 could be advantageous in obtaining improved images of target organs (810). SUMMARY

The procedures for synthesizing and radiolabeling DTPA-PEG-C225 are efficient and reproducible. DTPAPEG-C225 retained its binding affinity to EGF receptor with reduced nonspecific interaction with cells, although

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Wen et al. LITERATURE CITED

Figure 6. Blockage of DiFi cell growth by C225, DTPA-PEGC225 conjugates, and free DTPA-PEG. The DiFi cells were exposed to the indicated reagents at concentrations of 20 nM. After 3 days of incubation, the numbers of surviving cells were measured by MTT assay. The results are presented as the means of triplicates with standard deviations.

Figure 7. Pharmacokinetics of 111In-DTPA-C225 and 1:30 111In-DTPA-PEG-C225. Each mouse was given 1.5 µg of a radiotracer i.v. The blood samples were collected at different time intervals, and the radioactivity of each sample was measured. The data are expressed as percentages of injected dose per milliliter of blood (% ID/mL) and presented as the means of triplicates. The standard derivation for each time point is less than 10%. The profiles of both 111In-DTPA-C225 and 1:30 111In-DTPA-PEG-C225 fit well into the three-compartment model.

a high degree of PEG modification appeared to interfere with the binding activity of the antibody. Modification of C225 with PEG (MW 3400), even at a high degree of substitution, did not appear to increase blood circulation time of the antibody. PEG-modified C225 is less widely distributed to normal tissues than C225 without PEG, suggesting reduced nonspecific binding of PEG-modified C225 to tissues. Taken together, our results suggest that this method, introducing DTPA through a PEG linker, may be a useful strategy in developing radioimmunodetection imaging agents. This approach should be applicable to the labeling of other mAbs as well as their F(ab′)2, Fab′, and ScFv fragments. ACKNOWLEDGMENT

This work was supported in part by the John S. Dunn Foundation. We thank Dr. Terry Marriott of Rice University for helping with MALDI-TOF mass spectroscopic analysis and Kathryn Hale for editorial assistance.

(1) Potamianos, S., Varvarigou, A. D., and Archimandritis, S. C. (2000) Radioimmunoscintigraphy and radioimmunotherapy in cancer: principles and application. Anticancer Res. 20, 925-948. (2) Goldenberg, D. M. (1993) Monoclonal antibodies in cancer detection and therapy. Am. J. Med. 94, 297-312. (3) Bohdiewicz, P. J. (1998) Indium-111 satumomab pendetide: the first FDA-approved monoclonal antibody for tumor imaging. J. Nucl. Med. Technol. 26, 155-163. (4) Halpern, S. E., Hagan, P. L., Garver, P. R., Kozol, J. A., Chen, A. W. N., Frincke, J. M., Bartholomew, R. M., David, G. S., and Adams, T. H. (1983) Stability, characterization, and kinetics of 111In-labeled monoclonal antitumor antibodies in normal animals and nude mouse human tumor models. Cancer Res. 43, 5347-5355. (5) Pimm, M. V., Perkins, A. C., and Baldwin, R. W. (1985) Differences in tumor and normal tissue concentrations of iodine and indium labeled monoclonal antibody II. Biodistribution studies in mice with human tumor xenografts. Eur. J. Nucl. Med. 11, 300-304. (6) Brechbiel, M. W., Gansow, O. A., Atcher, R. W., Schlom, J., Esteban, J., Simpson, D. E., and Colcher, D. (1986) Synthesis of 1-(p-Isothiocyanatobenzyl) derivatives of DTPA and EDTA. Antibody labeling and tumor-imaging studies. Inorg. Chem. 25, 2772-2781. (7) Meares, C. F., Moi, M. K., Diril, H., Kukis, D. L., McCall, M. J., Deshpande, S. V., DeNardo, S. J., Snook, D., and Epenetos, A. A. (1990) Macrocyclic chelates of radiometals for diagnosis and therapy. Br. J. Cancer (Suppl.) 10, 21-26. (8) Quadri, S. M., and Vriesendorf, H. M. (1998) Effects of linker chemistry on the pharmacokinetics of radioimmunoconjugates. Q. J. Nucl. Med. 42, 250-261. (9) Li, M., and Meares, C. F. (1993) Synthesis, metal chelate stability studies, and enzyme digestion of a peptide-linked DOTA derivative and its corresponding radiolabeled immunoconjugates. Bioconjugate Chem. 4, 275-283. (10) DeNardo, S. J., Zhong, G. R., Salako, Q., Li, M., DeNardo, G. L., and Meares, C. F. (1995) Pharmacokinetics of chimeric L6 conjugated to indium-111- and yttrium-90-DOTA-peptide in tumor-bearing mice. J. Nucl. Med. 36, 829-836. (11) Fuertges, F., and Abuchowski, A. (1990) The clinical efficacy of poly(ethylene glycol)-modified proteins. J. Controlled Release 11, 139-148. (12) Chapman, A. P., Antoniw, P., Spitali, M., West, S., Stephens, S., and King, D. J. (1999) Therapeutic antibody fragments with prolonged in vivo half-lives. Nat. Biotechnol. 17, 780-783. (13) Gabizon, A. (1992) Selective tumor localization and improved therapeutic index of anthracyclines encapsulated in long-circulating liposomes. Cancer Res. 52, 891-896. (14) Modjahedi, H., and Dean, C. (1994) The receptor for EGF and its ligands: expression, prognostic value and target for therapy in cancer (Review). Int. J. Oncol. 4, 277-296. (15) Mendelsohn, J. (1997) Epidermal growth factor receptor inhibition by a monoclonal antibody as anticancer therapy. Clin. Cancer Res. 3, 2703-2707. (16) Hermanson, G. T. (1996) Ellman’s assay for the determination of sulfhydryls. in Bioconjugate Techniques (G. T. Hermanson, Ed.) pp 88-90, Academic Press, San Diego. (17) Hnatowich, D. J., Layne, W. W., Childs, R. L., Lanteigne, D., Davis, M. A., Griffin, T. W., and Doherty, P. W. (1983) Radioactive labeling of antibody: A simple and efficient method. Science 220, 613-615. (18) Hermanson, G. T. (1996) Amine detection reagents. in Bioconjugate Techniques (G. T. Hermanson, Ed.) pp 112-114, Academic Press, San Diego. (19) Goldenberg, A., Masui, H., Divgi, C., Kamrath, H., Pentlow, K., and Mendelshon, J. (1989) Imaging of human tumor xenografts with an indium-111-labeled anti-epidermal growth factor receptor monoclonal antibody. J. Natl. Cancer Inst. 82, 1616-1625.

111In-Labeled

PEG-Conjugated Antibody

(20) Divgi, C. R., Welt, S., Kris, M., Real, F. X., Yeh, S. D. J., Gralla, R., Merchant, B., Schweighart, S., Unger, M., Larson, S. M., and Mendelsohn, J. (1991) Phase I and imaging trial of indium 111-labeled anti-epidermal growth factor receptor monoclonal antibody 225 in patients with squamous cell lung carcinoma. J. Natl. Cancer Inst. 83, 97-104. (21) Kitamura, K., Takahashi, T., Yamaguchi, T., Noguchi, A., Noguchi, A., Takashina, K., Tsurumi, H., Inagake, M., Toyokuni, T., and Hakomori, S. (1991) Chemical engineering of the monoclonal antibody A7 by poly(ethylene glycol) for targeting cancer chemotherapy. Cancer Res. 51, 4310-4315. (22) Pedley, R. B., Boden, J. A., Boden, R., Begent, R. H. J., Turner, A., Haines, A. M. R., and King, D. J. (1994) The potential for enhanced tumor localization by poly(ethylene glycol) modification of anti-CEA antibody. Br. J. Cancer 70, 1126-1130. (23) Torchilin, V. P., Trubetskoy, V. S., Narula, J., Khaw, B. A., Klibanov, A. L., and Slinkin, M. A. (1993) Chelating

Bioconjugate Chem., Vol. 12, No. 4, 2001 553 polymer modified monoclonal antibodies for radioimmunodiagnostics and radioimmunotherapy. J. Controlled Release 24, 111-118. (24) Fang, M., Liu, B., Schmidt, M., Lu, Y., Mendelsohn, J., and Fan, Z. (2000) Involvement of p21Waf1 in mediating inhibition of paclitaxel-induced apoptosis by epidermal growth factor in MDA-MB-468 human breast cancer cells. Anticancer Res. 20, 103-112. (25) Ozanne, B., Richards, C. S., Hendler, F., Burns, D., and Gusterson, B. (1986) Overexpression of the EGF receptor is a hallmark of squamous cell carcinomas. J. Pathol. 149, 9-14. (26) Liu, B., Fang, M., Schmidt, M., Lu, Y., Mendelsohn, J., and Fan Z. (2000) Apoptosis and activation of caspase cascade induced by anti-EGF receptor monoclonal antibodies in DiFi human colon cancer cells do not involve the c-jun N-terminal kinase activity. Br. J. Cancer 82, 1991-1999.

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