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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
1 Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at University of California at Los Angeles; 2 Department of Pathology, Keck School of Medicine of the University of Southern California, Hoffman Medical Research Center, Los Angeles, California; 3 Division of Molecular Biology and 4 Division of Immunology, Beckman Research Institute of the City of Hope; 5 Department of Radioimmunotherapy, 6 Division of Radiology, and 7 Division of Radiation Oncology, City of Hope National Medical Center, Duarte, California; and 8 Molecular Imaging Program at Stanford, Department of Radiology and Bio-X Program, Stanford University School of Medicine, Stanford, California
Requests for reprints: Tove Olafsen, Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at University of California at Los Angeles, 700 Westwood Plaza, Los Angeles, CA 90095. Phone: 310-267-2819; E-mail: tolafsen{at}mednet.ulca.edu.
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Introduction |
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The principal determinants of the rate with which immunoglobulins are cleared from the circulation are their molecular size and the presence of the immunoglobulin Fc portion (CH2-CH3 region). Both diabodies and scFv fragments clear rapidly through the kidneys due to their low molecular weights. Increasing the size to above 60 kDa will bypass the kidneys and result in a slower clearance via the liver. One approach to produce larger, stable, and multivalent scFv fragments is the addition of COOH-terminal multimerization domains. Others and we have used individual constant immunoglobulin domains from human immunoglobulin G1 (CH3; refs. 5–7) and human immunoglobulin E (CH4; ref. 8) as dimerization domains to express intermediate-sized, bivalent scFv fragments of about 80 kDa in size. Our radiolabeled anti–carcinoembryonic antigen (CEA) T84.66 minibody (scFv-CH3 dimers; Fig. 1A) has shown excellent tumor uptake (21.4-32.9% ID/g at 6 hours) in vivo (2, 5, 9). In addition, high-resolution microPET images of xenografts in nude mice were achieved when this fragment was radiolabeled with the positron emitters 64Cu (T1/2 = 12.7 hours; ref. 10) and 124I (T1/2 = 4.2 days; ref. 11). Recently, in a pilot clinical study involving 10 patients with colorectal cancer, tumor imaging was observed with 123I-labeled anti-CEA T84.66 minibody in seven patients (12).
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Recently, we described the construction and characterization of a minibody specific for p185HER2, a transmembrane glycoprotein of 185 kDa encoded by the HER2/neu proto-oncogene (17). The overexpression of the tyrosine kinase receptor HER2/neu (c-erbB-2) in 20% to 30% of breast cancers and in a variety of other tumors of epithelial origin is often associated with poor prognosis (18). Herceptin (trastuzumab; Genentech, San Francisco, CA), version 8 of the humanized 4D5 monoclonal antibody (mAb; ref. 19), has been approved by Food and Drug Administration for the treatment of p185HER2-positive tumors (20). However, the overall objective response rate to trastuzumab monotherapy is only 15% to 20% (21, 22), necessitating the development of additional approaches for detection and treatment p185HER2-positive tumors. One such approach is to develop engineered antibody fragments as radiolabeled pharmaceuticals for diagnostic and therapeutic use.
In our previous study, a total of four variants of anti-p185HER2 minibodies were made (17) from the internalizing anti-p185HER2 10H8 mAb (23). The 10H8 minibodies showed high, specific binding to p185HER2-positive cells in vitro. One variant was radioiodinated and evaluated for its blood clearance, tumor targeting properties, and normal organ uptake of the radiolabel in nude mice bearing p185HER2-positive xenografts (17). The anti-p185HER2 10H8 minibody showed the expected blood clearance, but the tumor activity reached a maximum of only 5.6 ± 1.7% ID/g at 12 hours, which was substantially less than that previously observed with the radioiodinated anti-CEA minibody. The relatively low tumor uptake was thought to be partially due to dehalogenation on internalization of the fragment and/or metabolism of the label. In this work, we have therefore labeled the 10H8 minibody with radiometal to improve targeting to the tumor. In addition, a second anti-p185HER2 minibody was created from the humanized 4D5v8 mAb (19) and compared for its tumor targeting properties to that of the 10H8 minibody. Finally, a hu4D5v8 scFv-Fc H310A/H435Q [scFv-Fc double mutant (scFv-Fc DM)], was made and evaluated in nude mice to further optimize pharmacokinetics and normal tissue distribution.
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Materials and Methods |
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Expression, selection, and purification. All three anti-p185HER2 constructs were expressed in NS0 murine myeloma cells (27) that were selected in glutamine-deficient media (JHR Biosciences, Lenexa, KS; ref. 28), and supernatants were screened for expression by ELISA and analyzed by Western blot for size as described (17). The proteins of interest were purified from 450 to 800 mL dialyzed cell culture supernatants that had been pretreated with 5% AG1-X8, 100 to 200 mesh (Bio-Rad Laboratories, Hercules, CA), for removal of phenol red and cell debris, using a BioCAD 700E chromatography system (Applied Biosystems, Foster City, CA) as described (26). For 10H8 minibody, a two-step purification scheme with anion exchange followed by ceramic hydroxyapatite chromatography was employed as described (17). This scheme resulted in poor recovery and, as a result, an alternate three-step purification scheme was developed for the hu4D5v8 minibody and scFv-Fc DM. Here, the supernatant was dialyzed against 50 mmol/L acetic acid (pH 5.0) before being loaded onto a cation exchange column (Poros HS20, Perkin-Elmer, Foster City, CA). Bound proteins were eluted with a NaCl gradient from 0 to 0.4 mol/L in the presence of 50 mmol/L acetic acid (pH 5.0). Eluted fractions, containing the desired protein, were pooled, diluted 5x in 50 mmol/L MES (pH 6.5), and loaded onto the hydroxyapatite column (Macro-Prep Type 1, 20 µm, Bio-Rad Laboratories). Bound proteins were eluted with a KPi gradient from 0 to 0.15 mol/L in the presence of 50 mmol/L MES (pH 6.5). Eluted fractions containing the hu4D5v8 minibody or scFv-Fc DM were pooled, diluted 4x with 50 mmol/L HEPES (pH 7.4), and loaded onto the anion exchange column (Source 15Q, Amersham Biosciences Corp., Piscataway, NJ). Bound proteins were eluted with a NaCl gradient from 0 to 0.4 mol/L in the presence of 50 mmol/L HEPES (pH 7.4). SDS-PAGE analysis showed that the desired protein was in the flow-through, whereas the contaminants were bound to the column. The flow-through was concentrated using a Centricon 80 (Amicon, Inc., Beverly, MA) to 1 mL. The final concentration of purified protein was determined by A280 nm using an extinction coefficient (
) of 1.4 mg/mL.
Characterization of purified anti-p185HER2 antibody fragments. Aliquots of purified proteins were analyzed by SDS-PAGE under nonreducing or reducing (1 mmol/L DTT) conditions. Samples were also subjected to size-exclusion high-pressure liquid chromatography (HPLC) on a Superdex 200 HR 10/30 column (Amersham Biosciences) using a 0.5 mL/min flow rate and 50 mmol/L Na3PO4/0.15 mol/L NaCl (pH 7.0) buffer. Retention time was compared with standards of intact anti-CEA cT84.66 antibody, minibody, and diabody as described (15). Binding to p185HER2 was assessed by ELISA and by indirect immunofluorescence on the human breast tumor cell line MCF7/HER2 (gift of Dr. Dennis J. Slamon, University of California at Los Angeles School of Medicine; ref. 29) as described (17). Relative binding affinity was assessed by competition assays carried out in triplicate in ELISA microtiter plates as described (17).
Immunohistochemistry and kidney lysate preparation. Immunohistochemical staining was done on frozen sections of MCF7/HER2 tumors as described (17), whereas staining of the kidneys was done on paraffin-embedded sections. Kidneys from a normal nude mouse were removed, and 5-µm sections were cut and mounted. The sections were deparaffinized, rehydrated, and antigen retrieval was achieved by steaming with 0.01 mol/L EDTA-Tris (pH 8.0) for 20 minutes (30). For staining, 10H8 and hu4D5v8 minibodies were used, as well as intact trastuzumab and a polyclonal rabbit anti–c-erbB2 antibody (A485, DAKO, Carpenteria, CA). The stain was developed by an avidin-biotin complex method using biotinylated goat antihuman (H + L) or goat anti-rabbit antibodies (H + L), respectively, included in the Vectastain ABC Elite Kits (Vector Laboratories, Inc., Burlingame, CA).
Kidney lysate was prepared as described (31). Briefly, kidneys from normal mice were removed, rinsed with PBS, and homogenized in an equal volume of PBS containing a cocktail of protease inhibitors (Complete tablets, Roche/Boehringer Mannheim, Indianapolis, IN) using a Kinematica homogenizer (Brinkman Instruments, Westbury, NY). The homogenized kidney was centrifuged at 14,000 rpm in a Brinkmann Eppendorf 5415C centrifuge for 60 minutes, passed through a 0.22 µm filter, and analyzed by Western blot for the presence of p185HER2.
Conjugation and radiolabeling with 111In. Purified proteins (except trastuzumab) were conjugated to 1,4,7,10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid (DOTA; Macrocyclics, Dallas, TX) by using the water-soluble N-hydroxysuccinimide method as described (9, 32). Trastuzumab was conjugated to p-isothiocyanatobenzyl-diethylenetriaminepentaacetic acid (MX-DTPA or 1B4M-DTPA; ref. 33). The molar ratio of the conjugate to antibody used was 15:1 and the reaction occurred at pH 7.2 over 18 hours at room temperature (13, 16). Following conjugation, the protein was extensively dialyzed in 0.9% NaCl (pH 7.2) and concentrated. The extent of modification was evaluated by isoelectric focusing.
10H8 and hu4D5v8 DOTA minibodies, as well as 10H8 DOTA mAb (ranging from 200 to 400 µg of protein), were incubated with 0.5 to 2.9 mCi of carrier-free 111In-chloride (Mallinckrodt, Inc., Hazelwood, MO) in 0.25 mol/L NH4OAc (pH 5.0) for 1 hour at 43°C, whereas MX-DTPA trastuzumab was incubated with 111In in 0.9% NaCl (pH 7.2) for 35 minutes at room temperature. The reactions were terminated and purified by HPLC size-exclusion chromatography and the labeling efficiency as well as the immunoreactivity was determined as previously described (9, 17). The 10H8 DOTA minibody was labeled twice and the labeling efficiency was 2% and 20% with its immunoreactivity being 81% and 62%, respectively. The labeling efficiency for the hu4D5v8 DOTA minibody was 48% and only 18% for the parental 10H8 DOTA mAb, and their immunoreactivities were 65% and 80%, respectively. For MX-DTPA trastuzumab, the labeling efficiency was 100% and the immunoreactivity was 77%.
Animal biodistribution studies. All animal handling was done in accordance with City of Hope Research Animal Care Committee and University of California at Los Angeles Chancellor's Animal Research Committee guidelines. The biodistribution of 111In-DOTA hu4D5v8 minibody (specific activity: 1.1 µCi/µg) was evaluated in normal, non-tumor-bearing female nude mice (activity administered per animal: 3 µCi), whereas 111In-DOTA 10H8 minibody and antibody as well as 111In-MX-DTPA trastuzumab were evaluated in tumor-bearing mice. MCF7/HER2 tumor xenografts were established as described (17). About 10 to 14 days postinoculation, mice bearing xenografts were injected with 111In-DOTA 10H8 minibody (specific activity: 0.2 µCi/µg), 111In-DOTA 10H8 mAb (specific activity: 0.9 µCi/µg), or 111In-MX-DTPA trastuzumab (specific activity: 0.5 µCi/µg) via the tail vein. The activity administered per animal was in the range of 1 to 4 µCi, and corresponded to 4 to 5 µg of protein. Time points for analysis of the minibodies were 0, 2, 6, 12, 24, and 48 hours whereas for the intact antibodies 0, 6, 12, 24, 48, 72 and 96 hours were used. At the selected time points, groups of five mice were euthanized and percent of injected dose per gram (% ID/g) tissue was determined as described (9). Animal blood curves were calculated using the ADAPT II software (34) and biostatistical analysis was done with two-way ANOVA and all significant testing was done at P < 0.01 level using SAS/STAT software (SAS, Inc., Cary, NC) as described (9). Stability of the radiolabeled minibodies in vivo was evaluated by size-exclusion HPLC analysis as previously described (5).
Radiolabeling of hu4D5v8 minibody and scFv-Fc DM conjugates with 64Cu. The positron emitting isotope 64Cu (copper chloride in 0.1 mol/L HCl; radionuclide purity, >99%) was provided by Mallinckrodt Institute of Radiology (Washington University School of Medicine, St. Louis, WA). The hu4D5v8 DOTA-conjugated minibody and scFv-Fc DM (290-440 µg) were incubated with 0.7 to 3 mCi of 64Cu in 0.1 mol/L NH4 citrate (pH 5.5) for 50 minutes at 43°C. The reaction was stopped by addition of DTPA to 1 mmol/L. Labeled minibody was purified by HPLC size-exclusion chromatography using Superdex 75 (Amersham Biosciences). Labeling efficiency was determined by HPLC and immunoreactivity was determined by cell binding assay as described above. The hu4D5v8 DOTA minibody was labeled twice with 64Cu with a labeling efficiency essentially 100%, whereas the immunoreactivities were 75% and 39%. For the scFv-Fc DM, instant TLC using the monoclonal antibody instant TLC Strips Kit (Biodex Medical Systems, Shirley, NY) was used to determine the labeling efficiencies, which were 100% and 77%, with the immunoreactivities being 58% and 52% for these labelings.
MicroPET imaging. The human Burkitt lymphoma cell line Daudi (ATCC no. CLL 213) and the human breast cancer cell line MD-MBA-231 (ATCC no. HTB-26) were obtained from American Type Culture Collection (Manassas, VA) and maintained under standard conditions. MCF7/HER2 (p185HER2 positive) and Daudi (p185HER2 negative) or MD-MBA-231 (p185HER2 low expressing; ref. 35) xenografts were established as described above. Mice were imaged using a P4 microPET scanner (Concorde Microsystems, Inc., Knoxville, TN). Mice were injected in the tail vein with 128 to 165 µCi of 64Cu-DOTA hu4D5v8 minibody (specific activity: 5.3 µCi/µg) or with 128 to 140 µCi of 64Cu-DOTA hu4D5v8 scFv-Fc DM (specific activity: 1.8 µCi/µg). To enable imaging, mice were anesthetized using 2% isoflurance, positioned in a prone position along the long axis of the microPET scanner and imaged. Acquisition time was 10 minutes (1 bed position), and images were reconstructed using a filtered backprojection reconstruction algorithm (36, 37). After scanning, tumors were excised and either weighed and counted in a well counter (Cobra II AutoGamma, Packard, IL) or frozen for immunohistochemical analysis. Images were displayed and regions of interest (ROI) were drawn as described (11) and quantitated using AMIDE (38). ROIs from a cylinder with known weight and radioactivity were used to determine a calibration factor (µCi/voxel) for use in calculating %ID/g from the image ROIs.
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Results |
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Analysis of the purified proteins on SDS-PAGE (Fig. 1A) showed that the minibodies and scFv-Fc DM migrated as a monomer consistent with their predicted MW of 
40 kDa (lanes 2 and 4) and 53 kDa (lane 6), respectively, under reducing conditions, and as a covalent dimer of 80 kDa (lanes 1 and 3) and 105 kDa (lane 5) under nonreducing conditions. Size exclusion chromatography verified that the hu4D5v8 minibody and the scFv-Fc DM eluted at times corresponding to correctly folded dimers of expected molecular weights (Fig. 1B). The purity of the proteins was also determined from the size-exclusion chromatography to be above 95%.
Binding to target antigen was initially shown by ELISA and by indirect immunofluoroscence using flow cytometry of MCF7/HER2 cells with crude supernatants (data not shown). Affinity of purified proteins was measured by ELISA in the presence of competitors at different concentrations. As shown in Fig. 1C, by competition assay the affinities of hu4D5v8 minibody and scFv-Fc DM are essentially the same with their relative KD estimated to be 6.7 nmol/L, whereas the relative KD for the intact trastuzumab antibody is estimated to be 2 nmol/L. The relative KD for 10H8 mAb and minibody were estimated to be 1.6 and 4.2 nmol/L, respectively (17). These results suggest about a 3-fold reduction in apparent affinity when the variable genes of the parental antibodies are rearranged into these dimeric scFv fragments.
In vivo biodistribution and targeting of 111In-DOTA conjugated proteins. Biodistribution studies of 111In-DOTA 10H8 mAb, 111In-MX-DTPA trastuzumab, and 111In-DOTA 10H8 minibody were conducted in athymic mice bearing MCF7/HER2 xenografts. The intact antibodies showed excellent tumor targeting with the 10H8 mAb reaching a maximum of 39.8 ± 9.0% ID/g at 96 hours, and trastuzumab a maximum of 33.9 ± 5.1% ID/g at 72 hours (see Supplementary data). The nonspecific accumulation of the intact antibody in normal organs (liver, spleen, kidney, and lung) was as expected for intact radiolabeled antibodies.
The 111In-DOTA 10H8 minibody reached a maximum tumor uptake at 5.7 ± 0.1% ID/g at 24 hours as the uptake persisted from 6 hours (4.5 ± 1.3% ID/g) through 48 hours (4.7 ± 1.5% ID/g; Table 1). However, unexpectedly, the 10H8 minibody showed high localization in the kidneys, with 27.6 ± 2.4% ID/g at 2 hours and reaching a maximum of 34.0 ± 4.0% ID/g at 24 hours. We examined the biodistribution in non-tumor-bearing animals to rule out the effect of shed p185HER2 extracellular domain–forming complexes that could get trapped in the kidney. The 111In-DOTA hu4D5v8 minibody, however, also showed elevated activity in the kidneys in non-tumor-bearing mice with the uptake being 16.9 ± 1.8% ID/g at 2 hours, which was increased to a maximum of 28.4 ± 6.5% ID/g at 24 hours (Table 1).
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) being 0.55 and 0.75 hours and their terminal half-lives (T1/2ß) being 6.3 and 7.7 hours. For comparison, the 111In-DOTA anti-CEA T84.66 minibody (9) and scFv-Fc DM half-lives are also shown. According to Fig. 2A and C, the terminal half-lives seem slightly shorter for the T84.66 minibody and slightly longer for the T84.66 scFv-Fc DM, relative to the anti-p185HER2 minibodies. However, there is no significant difference between the three minibodies (P = 0.01), whereas a significant difference is observed between the minibodies and the scFv-Fc DM at each time point (P < 0.0001), except at 48 hours.
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MicroPET imaging of 64Cu-DOTA hu4D5v8 minibody and scFv-Fc DM. The tumor targeting of 64Cu-DOTA hu4D5v8 minibody and scFv-Fc DM was evaluated in nude mice carrying antigen-positive (MCF7/HER2; breast cancer cells) and antigen-negative (Daudi; Burkitt lymphoma cells) or low antigen expressing (MDA-MB-231; breast cancer cells) tumors established by s.c. inoculation on the shoulders. A whole-body microPET scan was done at 3 to 4 and 18 to 21 hours. The image of 64Cu-DOTA hu4D5v8 minibody at 18 hours shows uptake in the positive tumor (arrow) and low activity in the antigen negative control tumor (arrowhead), whereas the kidney and liver regions have relatively higher activity (Fig. 3A). The positive tumor to control tumor uptake ratio deduced from quantitative microPET analysis was 1.8:1 at 18 hours, demonstrating specificity. The uptake in the tumors and normal tissues calculated from the scans is shown in Fig. 3C. The positive tumor uptake is similar to the tumor uptake observed with the 111In-DOTA 10H8 minibody in the biodistribution study, being 4.2% ID/g at both 4 and 18 hours. In this study, the animals were sacrificed at 48 hours and the activities in tissues at the time of death (Fig. 3C) resemble those observed for the 111In-DOTA hu4D5v8 minibody in the non-tumor-bearing mice (Table 1).
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Anti-p185HER2 versus anti–carcinoembryonic antigen antibody fragments. The tumor activity of the anti-p185HER2 minibodies and scFv-Fc DM in the MCF7/HER2 tumors was compared with that observed with the anti-CEA minibody (10) and scFv-Fc DM10 in the LS174T tumors (Fig. 4A). Only about one fourth of the activity observed with the anti-CEA minibody is obtained with the anti-p185HER2 minibodies, whereas the anti-p185HER2 scFv-Fc DM shows almost equal tumor activity to that of the anti-CEA minibody and scFv-Fc DM in the different animal models at the late time. When the renal and hepatic activities of the anti-p185HER2 minibodies and the anti-CEA T84.66 minibody (9) are compared, the anti-p185HER2 minibodies exhibit a significantly higher activity in the kidneys (P < 0.0001; Fig. 4B), whereas the anti-CEA minibody has a significantly higher activity in the liver (P < 0.0001; Fig. 4C). A higher activity in the liver is expected as these molecules have a molecular mass above the threshold for renal filtration. The anti-CEA scFv-Fc DM has uptakes in the kidney and liver similar to that of the anti-CEA minibody. The intact, parental anti-p185HER2 antibodies have been included for comparison and behave as expected for intact antibodies, with moderate to low levels of activity in both organs.
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Discussion |
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Another explanation for the modest tumor uptake and the elevated kidney uptake could be shed antigen, which would form antigen-antibody complexes in blood that can be trapped in the kidneys, thus preventing efficient tumor targeting. However, the presence of HER2 extracellular domain in sera could not be detected by ELISA (17). Furthermore, biodistribution in non-tumor-bearing animals revealed the same elevated kidney uptake, which suggests that circulating antigen is not responsible for this uptake.
Because the minibody has a molecular weight above the renal threshold, another explanation might be that the minibody dissociates or is cleaved in the serum, resulting in molecules of lower molecular mass that are subjected to renal filtration. However, serum samples taken from mice at different times show that only the intact minibody was detected. It is known that p185HER2 is expressed in the kidneys (40) and that renal tumors frequently overexpress this antigen (41). The distinct staining by the minibodies in the proximal tubules in the kidneys from normal mouse and the more general staining by polyclonal anti-p185HER2 antibodies suggest the presence of a cross-reacting antigen. Moreover, Western blots of normal kidney lysate, probed with anti-p185HER2 antibodies and minibodies, resulted in detection of distinct bands, verifying the presence of a cross-reactive antigen.
Because the presence of antigen in the kidneys presents a problem for efficient tumor targeting of the minibodies and not so for intact antibodies, we hypothesized that by increasing the size of the antibody format we would also be able to increase the tumor uptake. Hence, we proceeded to generate a larger fragment in which the CH2 domain was included to produce a scFv-Fc fragment. However, this fragment clears almost as slowly as an intact antibody, resulting in high background, thus lowering the sensitivity in imaging studies. It has been shown that one mutation in the Fc region, affecting the interaction with the neonatal Fc receptor (FcRn, Brambell receptor; refs. 42, 43), is enough to affect the half-life of the chimeric TNT-3 antibody (44). Other sites that interfere with the FcRn receptor binding have also been identified (13, 16). Based on these data, we have produced several anti-CEA scFv-Fc variants with site-specific mutations in the Fc region that show different blood clearance patterns (15). The fastest clearing variant contains two mutations (H310A and H435Q) and exhibits similar blood clearance as that of the minibody. Targeting p185HER2 with this double mutant fragment format did indeed result in almost 3-fold improved tumor uptake as well as reduced kidney activity. Hence, we have shown that size matters in this animal model.
The profile observed for the anti-p185HER2 minibodies regarding their blood clearance is statistically the same to that seen with the anti-CEA minibody, and their stability in the serum also seems very similar. The reason why the anti-p185HER2 minibodies behave so different from the anti-CEA minibody is puzzling as they have equal opportunity to reach the target. The explanation for this difference may be embedded in the tumor models and a reflection of the tumor physiology. Others have targeted p185HER2 using different tumor systems and antibody fragments than those described in this work. In one study, using a 99mTc-labeled 4D5 scFv tetramer, only modest tumor uptake (maximum, 4.3 ± 1.9% ID/g) in nude mice bearing SK-OV-3 tumors (45) was achieved, which is similar to the tumor uptake of our anti-p185HER2 minibodies. The low tumor uptake of the 4D5 tetramer, however, was explained to be possibly due to instability and the orientation and accessibility of the targeted epitope. A more recent study shows excellent tumor uptake with an 111In-DOTA trastuzumab (Fab)2 fragment (maximum, 20.4 ± 6.8% ID/g) in nude mice bearing BT-474 tumors (46). Despite their differences in tumor uptake, the maximum activity in the kidney for both proteins was about 70% ID/g, which is 2- to 3-fold higher than that observed with our minibodies, suggesting that these proteins probably dissociate and clear via the kidneys.
Shifting to the larger scFv-Fc DM optimized the pharmacokinetics and normal tissue distribution in the MCF7/HER2 tumor model. However, the fragments described in this work may behave differently in other tumor models, considering that almost 9% ID/g of the scFv-Fc DM localized in the low expressing MB-MDA 231 tumors at 21 hours. Further work will be required to determine the relationship between signal and antigen density using other cell lines such as SK-BR-3, which has an even higher antigen density than MCF7/HER2 (35). Another difference between MCF-7/HER2 and other tumor models is that these cells have been transfected with p185HER2 to overexpress the receptor, whereas the others inherently overexpress the receptor. Subtle differences between these cells may affect targeting.
To conclude, we have shown that anti-p185HER2 minibodies preferentially localize in the kidneys in mice. Furthermore, we have shown that the kidneys express the antigen in the proximal tubules, which seem to act as a sink for the minibodies. Therefore, these fragments seem not suitable for targeting this antigen. By generating a slightly larger antibody fragment that was also tailored to clear fast, we have shown that increased tumor uptake and reduced kidney uptake can be achieved. These results suggest that the scFv-Fc format may become potentially useful as an imaging agent for HER2-positive tumors.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Chia-wei Cheung, Militza Bococ, Agnes Gardner, Barbara Szpikowska, and Sam Alam for their excellent assay support. We are especially grateful to David Stout, Waldemar Ladno, and Judy Edwards at University of California at Los Angeles for their assistance with the microPET scans. We also thank Nora H. Carter at the Department of Biostatistics (City of Hope National Medical Center) for the statistical analyses and Sofia Loera at the Anatomic Pathology Core Facility (City of Hope Comprehensive Cancer Center) for performing the immunohistochemistry.
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Footnotes |
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9 V.E. Kenanova et al., unpublished data. 
10 V.E. Kenanova et al., unpublished data.
Received 12/22/04. Revised 3/17/05. Accepted 4/19/05.
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References |
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RI, Fc RII, Fc RIII, and FcRn and design of IgG1 variants with improved binding to the Fc R. J Biol Chem 2001;276:6591–604.This article has been cited by other articles:
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  T. Olafsen, D. Betting, V. E. Kenanova, F. B. Salazar, P. Clarke, J. Said, A. A. Raubitschek, J. M. Timmerman, and A. M. Wu Recombinant Anti-CD20 Antibody Fragments for Small-Animal PET Imaging of B-Cell Lymphomas J. Nucl. Med., September 1, 2009; 50(9): 1500 - 1508. [Abstract] [Full Text] [PDF]
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 J. V. Leyton, T. Olafsen, E. J. Lepin, S. Hahm, K. B. Bauer, R. E. Reiter, and A. M. Wu Humanized Radioiodinated Minibody For Imaging of Prostate Stem Cell Antigen-Expressing Tumors Clin. Cancer Res., November 15, 2008; 14(22): 7488 - 7496. [Abstract] [Full Text] [PDF]
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S. B. Lee, M. Hassan, R. Fisher, O. Chertov, V. Chernomordik, G. Kramer-Marek, A. Gandjbakhche, and J. Capala Affibody Molecules for In vivo Characterization of HER2-Positive Tumors by Near-Infrared Imaging Clin. Cancer Res., June 15, 2008; 14(12): 3840 - 3849. [Abstract] [Full Text] [PDF]
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R. M. Sharkey, H. Karacay, S. Vallabhajosula, W. J. McBride, E. A. Rossi, C.-H. Chang, S. J. Goldsmith, and D. M. Goldenberg Metastatic Human Colonic Carcinoma: Molecular Imaging with Pretargeted SPECT and PET in a Mouse Model Radiology, February 1, 2008; 246(2): 497 - 507. [Abstract] [Full Text] [PDF]
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 S. D. Voss, S. V. Smith, N. DiBartolo, L. J. McIntosh, E. M. Cyr, A. A. Bonab, J. L. J. Dearling, E. A. Carter, A. J. Fischman, S. T. Treves, et al. Positron emission tomography (PET) imaging of neuroblastoma and melanoma with 64Cu-SarAr immunoconjugates PNAS, October 30, 2007; 104(44): 17489 - 17493. [Abstract] [Full Text] [PDF]
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R. M. Sharkey, H. Karacay, W. J. McBride, E. A. Rossi, C.-H. Chang, and D. M. Goldenberg Bispecific Antibody Pretargeting of Radionuclides for Immuno Single-Photon Emission Computed Tomography and Immuno Positron Emission Tomography Molecular Imaging: An Update Clin. Cancer Res., September 15, 2007; 13(18): 5577s - 5585s. [Abstract] [Full Text] [PDF]
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 M. Friedman, E. Nordberg, I. Hoiden-Guthenberg, H. Brismar, G.P. Adams, F.Y. Nilsson, J. Carlsson, and S. Stahl Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor Protein Eng. Des. Sel., April 23, 2007; (2007) gzm011v1. [Abstract] [Full Text] [PDF]
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 A. Orlova, V. Tolmachev, R. Pehrson, M. Lindborg, T. Tran, M. Sandstrom, F. Y. Nilsson, A. Wennborg, L. Abrahmsen, and J. Feldwisch Synthetic Affibody Molecules: A Novel Class of Affinity Ligands for Molecular Imaging of HER2-Expressing Malignant Tumors Cancer Res., March 1, 2007; 67(5): 2178 - 2186. [Abstract] [Full Text] [PDF]
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J. E. Shively 18F Labeling for Immuno-PET: Where Speed and Contrast Meet J. Nucl. Med., February 1, 2007; 48(2): 170 - 172. [Full Text] [PDF]
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 S. Kubetzko, E. Balic, R. Waibel, U. Zangemeister-Wittke, and A. Pluckthun PEGylation and Multimerization of the Anti-p185HER-2 Single Chain Fv Fragment 4D5: EFFECTS ON TUMOR TARGETING J. Biol. Chem., November 17, 2006; 281(46): 35186 - 35201. [Abstract] [Full Text] [PDF]
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 R. M. Sharkey and D. M. Goldenberg Targeted Therapy of Cancer: New Prospects for Antibodies and Immunoconjugates CA Cancer J Clin, July 1, 2006; 56(4): 226 - 243. [Abstract] [Full Text] [PDF]
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A. Orlova, M. Magnusson, T. L.J. Eriksson, M. Nilsson, B. Larsson, I. Hoiden-Guthenberg, C. Widstrom, J. Carlsson, V. Tolmachev, S. Stahl, et al. Tumor Imaging Using a Picomolar Affinity HER2 Binding Affibody Molecule. Cancer Res., April 15, 2006; 66(8): 4339 - 4348. [Abstract] [Full Text] [PDF]
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, hu4D5v8 Mb; 
, hu4D5v8 scFv-Fc DM; 
, T84.66 Mb; 
, T84.66 scFv-Fc DM. Uptake in kidney (B) and liver (C) over time as observed in biodistribution studies. 
, 10H8 mAb; 
, 10H8 Mb; 
, Trastuzumab; 
, hu4D5v8 Mb; 
, T84.66 scFv-Fc DM; 
, T84.66 Mb. The T84.66 minibody data are based on previous publication.