Experimental Pretargeting Studies of Cancer with a Humanized anti

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Bioconjugate Chem. 2000, 11, 842−854

Experimental Pretargeting Studies of Cancer with a Humanized anti-CEA × Murine anti-[In-DTPA] Bispecific Antibody Construct and a 99mTc-/188Re-Labeled Peptide H. Karacay,*,† W. J. McBride,† G. L. Griffiths,† R. M. Sharkey,‡ J. Barbet,§ H. J. Hansen,† and D. M. Goldenberg‡ Immunomedics, Inc., Morris Plains, New Jersey, Garden State Cancer Center, Belleville, New Jersey, and IBC Pharmaceuticals, LLC, Morris Plains, New Jersey. Received April 14, 2000; Revised Manuscript Received August 11, 2000

The aim of this study was to localize 99mTc and 188Re radionuclides to tumors, using a bispecific antibody (bsMAb) in a two-step approach where the radionuclides are attached to novel peptides incorporating moieties recognized by one arm of the bsMAb. A chemically cross-linked human/murine bsMAb, hMN14 × 734 (Fab′ × Fab′), anti-carcinoembryonic antigen [CEA] × anti-indium-DTPA was prepared as a prelude to constructing a fully humanized bsMAb for future clinical application. N,N′-o-Phenylenedimaleimide was used to cross-link the Fab′ fragments of the two antibodies at their hinge regions. This construct was shown to be >92% pure and fully reactive with CEA and a divalent (indium)DTPA-peptide. For pretargeting purposes, a peptide, IMP-192 [Ac-Lys(In-DTPA)-Tyr-Lys(In-DTPA)Lys(TscG-Cys-)-NH2 {TscG ) 3-thiosemicarbazonylglyoxyl}], with two indium-DTPAs and a chelate for selectively binding 99mTc or 188Re, was synthesized. IMP-192 was formulated in a “single dose” kit and later radiolabeled with 99mTc (94-99%) at up to 1836 Ci/mmol and with 188Re (97%) at 459-945 Ci/mmol of peptide. [99mTc]IMP-192 was shown to be stable by extensive in vitro and in vivo testing and had no specific uptake in the tumor with minimal renal uptake. The biodistribution of the hMN14 × murine 734 bsMAb was compared alone and in a pretargeting setting to a fully murine antiCEA (F6) × 734 bsMAb that was reported previously [Gautherot, E., Bouhou, J., LeDoussal, J.-M., Manetti, C., Martin, M., Rouvier, E., and Barbet, J. (1997) Therapy for colon carcinoma xenografts with bispecific antibody-targeted, iodine-131-labeled bivalent hapten. Cancer 80 (Suppl.), 2618-2623]. Both bsMAbs maintained their integrity and dual binding specificity in vivo, but the hMN-14 × m734 was cleared more rapidly from the blood. This coincided with an increased uptake of the hMN-14 × m734 bsMAb in the liver and spleen, suggesting an active reticuloendothelial cell recognition mechanism of this mixed species construct in naive mice. Animals bearing GW-39 human colonic cancer xenografts were injected with bsMAb (15 µg) and after allowing 24 or 72 h for the bsMAb constructs to clear from the blood (hMN-14 and murine F6 × 734, respectively), [188Re]IMP-192 (7 µCi) or [99mTc]IMP-192 (10 µCi) was injected at a bsMAb:peptide ratio of 10:1. Tumor uptake of [99mTc] or [188Re]IMP-192 was 12.6 ( 5.2 and 16.9 ( 5.5% ID/g at 3 h postinjection, respectively. Tumor/ nontumor ratios were between 5.6 and 23 to 1 for every major organ, indicating that early imaging with 99mTc will be possible. Radiation absorbed doses showed a 4.8-, 7.2-, and a 12.6 to 1.0 tumor to blood, kidney, and liver ratios when 188Re was used. Although this new bsMAb pretargeting approach requires further optimization, it already shows very promising targeting results for both radioimmunodetection and radioimmunotherapy of colorectal cancer.

INTRODUCTION 1

Radioimmunodetection (RAID) and radioimmunotherapy (RAIT) of cancer rely on the selectivity of radiolabeled antibodies directed against tumor-associated antigens binding to and then subsequently being retained in a tumor while clearing from normal tissues. For imaging, radioactivity uptake in a tumor must exceed that of surrounding tissue within a reasonable period of time and with sufficient signal for detection. For RAIT, the amount of radioactivity delivered must be sufficient such that over a period of time the tumor will be exposed to a radiation dose to effect cell killing. The first dose-limiting * To whom correspondence should be addressed. Phone: (973) 844-7054. Fax: (973) 844-7020. Current address: Garden State Cancer Center, 520 Belleville Ave., Belleville, NJ 07109. † Immunomedics, Inc. ‡ Garden State Cancer Center. § IBC Pharmaceuticals.

toxicity for RAIT, in applications to date, has been hematological. This is due to the high radiosensitivity of red marrow and the relatively high amount of radioactivity circulating in the blood; that is a direct consequence of the extended biological half-life of an intact IgG. Thus, increasing tumor/blood ratios while still delivering an adequate absolute amount of radioactivity to a tumor is an important goal for improving RAIT. 1 Abbreviations: bsMAb, bispecific antibody; CEA, carcinoembryonic antigen; cdr, complementarity-determining region; DMF, dimethylformamide; DTPA, diethylenetriaminepentaacetic acid; HBTU, O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate; HAMA, human anti-murine antibody; HPLC, high-pressure liquid chromatography; HSA, human serum albumin; IACUC, Institutional Animal Care and Use Committee; ID, injected dose; PDM, N,N′-o-phenylenedimaleimide; RAID, radioimmunodetection; RAIT, radioimmunotherapy; RP, reverse phase; SE, size exclusion; TFA, trifluoroacetic acid; TscG, 3-thiosemicarbazonylglyoxylic acid; TscGC, thiosemicarbonylglyoxylcysteinyl group.

10.1021/bc0000379 CCC: $19.00 © 2000 American Chemical Society Published on Web 11/03/2000

Pretargeting with a bsMAb for Cancer Imaging and Therapy

Pretargeting approaches are being investigated as methods proven to be effective for increasing tumor/blood ratios. In a pretargeting method, an antibody conjugate containing a secondary recognition moiety is targeted to tumor, and then allowed to clear from nontarget tissues. Sometime later, a low molecular weight, fast-clearing, radiolabeled ligand recognized by the secondary recognition moiety of the pretargeted antibody conjugate is given. Diverse reagents for antibody-directed pretargeting approaches have been described (Hnatowich et al., 1987; Paganelli et al.,1988; Goodwin et al., 1988; Stickney et al., 1991; Bamias and Epenetos, 1992; Hawkins et al., 1993; Bos et al., 1994). Most have successfully shown improved tumor/nontumor radioactivity ratios as compared to a radiolabeled antibody alone, albeit often at a cost of substantially reduced absolute amounts of radioactivity in the targeted tumor. However, both Axworthy et al. (1994) and Boerman et al. (1999) have reported that the absolute amount of radioactivity delivered to tumors by pretargeting approaches can be comparable to levels achieved with a radiolabeled antibody alone, and could be achieved without sacrificing excellent tumor-to-nontumor ratios. Two pretargeting methodologies have been reported most prominently, those using avidin-biotin and the other bispecific antibody (bsMAb) reagents. An avidinbiotin approach has the advantage of an exceptionally high binding affinity between the pretargeted antibody conjugate (either a biotin or avidin/streptavidin antibody conjugate) and the radiolabeled ligand (either avidin/ streptavidin or biotin) (Rosebrough, 1993; Karacay et al.; Sharkey et al., 1997; Wilbur et al., 1998; Cremonesi et al., 1999). It also has the major disadvantages of the high immunogenicity of the avidins (Chinol et al., 1998), of serum biotinidases that can cleave many biotin conjugates, and the presence of endogenous biotin that can compete for avidin binding sites during pretargeting procedures. Although the alternate bsMAb approach does not engender as high component binding affinities and immunogenicity as the avidin-biotin approach, molecular engineering may be used ultimately to significantly reduce or even eliminate any immunogenicity of the bsMAb construct (Carter and Merchant 1997; Coloma and Morrison, 1997). Also, low molecular weight radiolabeled conjugates (haptens) can be designed that are endogenously unrecognized and are enzymatically resistant. Finally, bsMAb pretargeting results can be improved even more by incorporating radiolabeled bivalent, rather than monovalent, haptens in what has been termed an “affinity enhancement system” (AES) (LeDoussal et al., 1989). In clinical studies of AES, imaging with an 111In-labeled bivalent peptide hapten [N-R(111In/In-DTPA)Tyr-N-(111In/In-DTPA)Lys] given after a pretargeted bsMAb exhibited excellent results in several tumor types (LeDoussal et al., 1990; LeDoussal et al., 1993; Vuillez et al., 1997; Barbet et al., 1998). In these studies, tumors were pretargeted with a murine bsMAb, F6 × 734 (Fab′ × Fab′), anti-carcinoembryonic antigen (CEA) × anti-indium diethylenetriaminepentaacetic acid (DTPA). Although imaging results were impressive, in one study up to 61% of the patients eventually developed a human anti-murine antibody (HAMA) response (Le Doussal et al., 1993). The approach is now being extended to RAIT using the same bsMAb with an 131I-labeled dipeptide (Vuillez et al., 1999). Efforts are currently underway to prepare a fully humanized construct for clinical studies. However, as a prelude to this, a human/murine bsMAb, hMN-14 × 734 (Fab′ × Fab′)(anti-CEA × anti-indium-

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DTPA) has been prepared and tested in animals. Since a humanized antibody might have different clearance and distribution properties in mice than a fully murine construct, comparisons were made to the murine F6 × murine 734 (Fab′ × Fab′) bsMAb that has been used by the French group. Unfortunately, the 734 MAb, raised against indiumDTPA, was found to bind too weakly to yttrium-DTPA to enable any progress toward a yttrium-based bsMAb RAIT agent, and thus preclinical RAIT studies have used radioiodinated (I-131) bivalent haptens (Gautherot et al., 1997, 1998, 2000). Although an 131I-based agent may be a highly effective therapeutic, it is important to investigate higher energy nuclides, such as 90Y and 188Re, since these may be more suitable for tumors >1 cm in diameter. The nuclear properties of 188Re [T1/2 16.9 h; 2.12 and 1.96 MeV beta, 71 and 25% abundant; 155 keV imageable gamma, 15% abundant] would make it an ideal therapeutic isotope when bound to a fast-clearing bivalent hapten in the bsMAb approach. Like 99mTc, 188Re is produced carrier-free (enabling high specific activity labeling of haptens) via a convenient generator system (Knapp et al., 1994). For imaging, 99mTc has multiple advantages over 111In, including low cost, ready availability, ideal nuclear properties (140 keV γ-emission) for γ cameras, reduced radiation exposure to the patient and a shorter half-life (6 h). Thus, in this report, the initial development and preclinical testing of the human/murine hMN-14 × 734 (Fab′ × Fab′) hybrid bsMAb, and the progression from an [111I]DTPA bivalent hapten to an imaging/therapy 99mTc/188Re-based bivalent hapten are described. MATERIALS AND METHODS

Antibodies and Peptides. hMN-14 IgG was prepared as described (Sharkey et al., 1995). The F(ab′)2 of hMN14 was derived by pepsin digestion and purified by protein A and ion-exchange chromatography. The antiindium-DTPA antibody 734 F(ab′)2 (Le Doussal et al., 1990) and F6 × 734 (Fab′ × Fab′) bsMAb were supplied by Immunotech (Marseilles, France). A rat anti-idiotypic antibody to hMN-14, termed WI2 (Losman et al., 1994), was used in some of the analytical work with hMN-14containing MAbs to verify lack of discernible impact on the latter’s binding site. The CEA used for immunoreactivity testing was obtained from Scripps (La Jolla, CA). The hMN-14 × 734 bsMAb was prepared by the chemical coupling of the two reduced Fab′ fragments using N,N′-o-phenylenedimaleimide (PDM) by a published method (Glennie et al., 1987). Briefly, MAbs were reduced separately to their Fab′-SH fragments by incubation with 10 mM 2-mercaptoethylamine at 37° C for 1 h. The 734-Fab′-SH was converted to 734-Fab′-maleimide by reaction with 4 mM PDM at room temperature for 1 h. The Fab′-SH (hMN-14) and Fab′-maleimide (734) intermediates was isolated by centrifuged size-exclusion chromatography (Penefsky, 1979; Meares et al., 1984). The hMN-14 × 734-Fab′ × Fab′ conjugate was produced by reaction of a 1:1 mixture of the two fragments overnight at 4° C. It was purified on a SE (size exclusion) HPLC analytical column (Bio-Sil SEC-250, Bio-Rad, Richmond, CA), equipped with an in-line absorbance detector (Waters 486 or Beckman 167), set at 280 nm. The column was eluted at 1 mL/min with 0.2 M sodium phosphate, pH 6.8, containing 0.02% sodium azide. Only the center portion of the peak corresponding to the bsMAb was collected and used in these studies. The level of monospecific 734-F(ab′)2 in hMN-14-F(ab′)2 bsMAb

844 Bioconjugate Chem., Vol. 11, No. 6, 2000

Figure 1. Structures of the peptides IMP-156 and IMP-192, including the 99mTc/188Re chelating thiosemicarbazonyl-glyoxylcysteinyl group of IMP-192.

preparations was determined by mixing radiolabeled bsMAb with CEA, followed by SE-HPLC analysis. The level of monospecific hMN-14-F(ab′)2 in hMN-14- × 734F(ab′)2 bsMAb preparations was determined using a DTPA conjugate of an irrelevant cdr-grafted MAb, hLL2, having the same framework as hMN-14 (Leung et al., 1995). The nonradiolabeled bsMAb and DTPA-hLL2 were mixed and applied to SE-HPLC with in-line UV analysis at 280 nm. In both assays, the percentage of product failing to shift to a higher MW on SE-HPLC was indicative of the levels of monospecific MAbs present in the preparations. Intermediates for the peptide syntheses were obtained from Advanced ChemTech (Louisville, KY). The chelatepeptide bivalent hapten conjugates shown in Figure 1 (termed IMP-156 and IMP-192) were synthesized using standard solid-phase methods. Briefly, for the IMP-192 peptide, the “Fmoc-based” methodology and Rink amide resin were used. Starting with Nalpha-Aloc-Lys(NepsilonFmoc)-OH attached to the resin, Nalpha-Fmoc-Cys(Trt)OH was then added to the side chain of lysine followed by the addition of the 3-thiosemicarbazonylglyoxylic acid (TscG). The latter was prepared by mixing thiosemicarbazone and glyoxylic acid together in a 1:1 ratio in methanol, and evaporating the solvent to generate the desired agent, which was used without further purification. The Aloc group was removed from the lysine using palladium(0)-catalyzed hydrostannolysis (Dangles et al., 1987), the other amino acids were added (Nalpha-FmocTyr(But)-OH, Nalpha-Fmoc-Lys(Nepsilon-Aloc)-OH), the Nterminus was acetylated, and the lysine side-chain protecting groups were removed. The two DTPAs were then added to the lysine side chain using a monoactivated DTPA derivative. This was prepared by dissolving DTPA in methanol containing a 300-M excess of a 1.0-M solution of tetrabutylammonium hydroxide. The reaction mixture was rotary evaporated and the residue was dissolved in 100 mL of DMF. The DMF solution was then concentrated under hi-vacuum on the rotary evaporator. This process was repeated two more times to remove methanol. The residue was then dissolved in DMF and reacted with 1 equiv of O-benzotriazole-N,N,N′,N′-tetramethyluronium-hexafluoro-phosphate (HBTU). The reaction, with 6 equiv of the activated DTPA, proceeded overnight, and was repeated until the resin gave a negative ninhydrin test (Kaiser et al., 1970). The peptide was then cleaved from the resin and purified by reversed-phase HPLC. IMP 156 was synthesized in an analogous manner. Structures were confirmed by electrospray mass spectral analysis (IMP-156, MH+ 1377; IMP-192, MH+ 1590). Radiolabeling. 125I and 111In were obtained from NEN Life Science Products (Boston, MA). The bsMAbs were radioiodinated using the chloramine-T method (Greenwood and Hunter, 1963) and purified using centrifuged

Karacay et al.

size-exclusion columns. A typical 111In labeling of IMP156 is as follows. 111In chloride (1.4 µL, 373 µCi) was mixed with 4.2 µL of 0.5 M sodium acetate, pH 6.1, and IMP-156 (3.3 µL, 4.9 × 10-5 M) in 0.5 M sodium acetate, pH 6.1, was added. After 20 min at room-temperature nonradioactive indium acetate (12.1 µL, 7.5 × 10-5 M in 0.5 M sodium acetate, pH 6.1), was added to bind remaining DTPA moieties. Fresh sodium [99mTc] pertechnetate was purchased from Syncor (Kenilworth, NJ) or Mallinckrodt (Pine Brook, NJ). Sodium [188Re] perrhenate was eluted from an in-house 188W/188Re generator (Oak Ridge National Laboratory, Oak Ridge, TN) with 0.9% sodium chloride solution. Radiolabeled peptides were analyzed on a Waters 4000 HPLC system having a reversed-phase Waters 8 × 100 mm radial Pak cartridge filled with a C-18 Nova-Pak 4 µm stationary phase. The column was eluted at 3 mL/min with a linear gradient of 100% 0.1% trifluoroacetic acid (TFA) in water to 100% 0.1% TFA in 90% acetonitrile/10% water over 10 min. At 10 min, the flow rate was increased to 5 mL/min and remained at 100% 0.1% TFA in 90% acetonitrile/10% water for 5 min before reequilibration to initial conditions. Both a Waters 486 absorbance detector, set at 220 nm, and an in-line radiation detector (Packard Radiomatic Flo-One or Ludlum) were used for detection. For subsequent 99mTc and 188Re radiolabeling, IMP-192 was formulated into lyophilized, single vial, “instant” kits containing excess cold indium. 99mTc kits contained 78 µg of IMP-192, with 100 µg Sn (II) for pertechnetate reduction, ascorbic acid, and a 6-fold excess of cold indium to peptide, for binding to DTPA. These vials were reconstituted with up to 40 mCi of 99mTc pertechnetate in 1.5 mL of saline. After 10 min at room temperature, the leadshielded vials were heated in a boiling water bath for 15 min. For 188Re labeling, kits contained 50 µg of IMP-192, with 660 µg of Sn(II), ascorbic acid, and indium at the same 6:1 indium-to-peptide ratio. These were reconstituted with 17 mCi of freshly eluted 188Re perrhenate in 1.2 mL of saline. For 188Re labeling, vials were heated 1 h in a boiling water bath. In both cases, after cooling, the radiolabeled peptide was diluted in 1.0% human serum albumin (HSA) for animal biodistribution studies. Purity of the di-indium-DTPA peptides after 99mTc and 188Re radiolabeling was evaluated by reversed-phase HPLC (as above), as well as ITLC using saturated sodium chloride as eluent. Unbound isotope eluted with the HPLC void volume, and the radiolabeled peptide eluted at 7.2 min. The ability of the radiolabeled peptides to bind to the bsMAbs was evaluated by SE-HPLC. The elution profiles of the radiolabeled bsMAbs and peptides alone were compared to those obtained after mixing the peptides with 734 F(ab)′2, or with hMN-14 × 734-F(ab)′2 bsMAb, at various ratios. Also, radiolabeled peptides were added to preformed mixtures of CEA and bsMAb, prior to similar SE-HPLC analyses, to investigate the ability of the peptides to bind to CEA-bound bsMAbs. In Vitro Stability and Binding Studies. The in vitro stability of the bsMAb in human serum was tested by incubating the 125I-labeled bsMAb at 37 °C in fresh, sterile-filtered serum taken from a normal donor. Samples were analyzed by SE-HPLC before and after mixing with CEA or WI2. A series of in vitro stability studies were performed on the 99mTc complex of IMP-192 diluted in 0.9% sodium chloride solution, fresh human serum, 1% HSA (prepared diluted in 0.9% saline from 25% HSA; Alpine Biologics, Blauvelt, NY), and 10 mM cysteine. Aliquots were withdrawn and tested by RP-HPLC at


early (1-2 h) postdilution time points and after overnight incubation. The ability of components to retain binding to each other was analyzed using radiomatic size-exclusion HPLC and radiolabeled bsMAb and peptides. 111In or 99mTc/188Re-labeled peptide was injected onto HPLC alone, and immediately after mixing with variable amounts of bsMAb. Incubations were done in the presence of 1% HSA to minimize the possibility of nonspecific binding. When indicated, further incubations of radiolabeled bsMAb, including those radiolabeled by premixing with radiolabeled peptides, were carried out with CEA, WI2 or DTPAhLL2. Preformed complexes of CEA and bsMAb were treated with radiolabeled peptide to demonstrate binding of the radiolabeled peptide to the antigen-bsMAb complex. In all incubations, components were mixed at room temperature and injected onto HPLC immediately, since component binding was very rapid, and did not change with increasing incubation times or temperatures. The precise mole ratio of these mixtures is given in the Results. In Vivo Stability and Animal Targeting Studies. All animal experiments were carried out with IACUCapproved protocols. Studies were conducted in 5-8-weekold, female BALB/c or athymic nude mice (CR-Nu-Nu; Taconic, Germantown, NY). Standard lab chow and water were given ad libitum, and water was supplemented with Lugol’s iodine solution (1 mL/500 mL) whenever radioiodinated agents were to be administered to animals. The GW-39 human colonic tumor cell line (Goldenberg et al., 1966; Goldenberg and Hansen, 1972) was used to demonstrate targeting to this CEA-producing tumor. It was propagated as serial, subcutaneous xenografts in nude mice, as described elsewhere (Sharkey et al., 1997). All test articles were injected intravenously in the lateral tail vein in 0.1-0.2 mL. At the designated time, groups of animals were anesthetized with sodium pentobarbital, bled by cardiac puncture, and then euthanized by cervical dislocation. Tumors and major organs were removed and weighed. The number of animals per grouping for each study is provided in the Results. The gastrointestinal tissues were counted in toto to include their contents. Whenever possible, urine was removed from the bladder at necropsy, weighed, and counted. Tissues, including weighed blood, were placed in 4-mL vials and counted in a γ-counter using appropriate windows for the isotopes. With paired isotopes, windows were set to eliminate or minimize dual-channel crossover, and figures were corrected for crossover counts, when needed, by reference to empirical measurements of radionuclide standards. In addition, in dual isotope studies, tissues were counted immediately and at a later time, for 125I only, after the short-lived 99mTc and 188Re nuclides had decayed. In vivo stability was evaluated in BALB/c mice. 125Ilabeled bsMAbs (5 µCi, 15 µg) were administered and necropsies were performed at 1, 2, 4, and 24 h postinjection. Sera were obtained from these animals at these time points. Retention of binding to CEA, and also to [111In]IMP-156, was examined by SE-HPLC analyses of the serum samples before and after mixing them with CEA or [111In]IMP-156, using energy windows set for 125I (030 keV) or 111In (80-420 keV), respectively. In the pretargeting experiments, animals received tracer amounts of 125I-labeled bsMAbs (5 µCi, 15 µg/animal), to quantitate tissue uptake of the bsMAb. In each pretargeting study, the amount of peptide given was based on a constant ratio of 10:1 (moles of antibody initially injected to the moles of peptide injected). It is important to emphasize


that this ratio was not optimized to provide the best targeting results. The methodology used to calculate radiation doses in mice has been reported (Sharkey et al., 1990). Radiation dose estimates were determined by first integrating the trapezoidal regions (for tumors) or exponential regions (for normal tissues) defined by the time activity data. For the trapezoidal fit of the tumor, the radioactivity at time zero is assumed to be zero, but for most normal tissues, the µCi/g at time zero is estimated from the exponential decay curve. If there is no exponential decay, the microcurie per gram at time zero is estimated by extrapolating the line described by the first data pair to the y-axis (i.e., µCi/g). The resulting integral for each organ is converted to centigray per millicurie with the use of S values appropriate for isotope and organ weight by assuming uniformly distributed activity in small unit density spheres (Siegel and Stabin, 1988). RESULTS

Antibody and Peptide Preparation: Radiolabeling, Immunoreactivity, and In Vitro Stability. On the basis of the starting amount of Fab′, the final yield of the hMN-14 × 734-F(ab′)2 bsMAb after purification was ∼25%. UV analysis at OD280 of SE-HPLC profiles showed the product as a single peak. When this product was mixed with a high molecular weight indium-DTPA conjugate (i.e., [In]DTPA-hLL2-IgG), 92-93% of the UV profile shifted to a higher molecular weight, indicating that less than 8% of the product was possibly monospecific hMN-14-F(ab′)2 or nonimmunoreactive bsMAb. Immunoreactivity testing with CEA and the peptides are described below. The IMP-156 and IMP-192 peptides assayed as single peaks on RP-HPLC and could be stored in bulk for long periods at -20 °C when lyophilized as their TFA salts. When added at a 20-fold molar excess, 111I was quantitatively incorporated in the IMP-156 within 20 min. After radiolabeling was complete, residual DTPA sites on IMP156 were saturated by addition of a 6-fold molar excess of indium to peptide (approximately 3:1 to available DTPA). The IMP-192 peptide, formulated and lyophilized into single vial kits containing indium-saturated DTPA, was reproducibly labeled with sodium 99mTc pertechnetate at up to 1836 Ci/mmol in high yield (94-99%), obviating the need for any postlabeling purification. IMP192, similarly saturated with indium-saturated DTPA, was labeled with 188Re in high yield (96-97%), at a specific activity of 459-945 Ci/mmol of peptide. A series of SE-HPLC studies were performed to show the binding of the hMN-14 × 734 bsMAb to either IMP192 or IMP-156 (Figure 2, upper and lower panels, respectively). The radiolabeled peptides eluted around 14 min (Figure 2A). Since the peptides were diluted in HSA, this was a further indication that they would not associate with this serum protein. Furthermore, when mixed with hMN-14 or CEA alone, the peptide continued to elute at its native-size, indicating no nonspecific binding to these proteins (not shown). After incubating with a molar excess of the hMN-14 × 734 bsMAb (unlabeled), all radioactivity from the two peptides shifted to a higher molecular weight, with the main peak at nearly 9.0 min (Figure 2). It should be noted this elution time is earlier than the antibody alone, which elutes at 10.7 min (refer to Figure 4A). This is because the peptides are divalent and capable of binding to 2 bsMAb molecules. When the peptide was added to the bsMAb that had been premixed with a molar excess of CEA, >90% of the peptide


Karacay et al.

[99m-

Figure 2. Upper panel. SE-HPLC chromatograms of (A) Tc]IMP-192, (B) [99mTc]IMP-192 mixed with hMN-14 × 734 bsMAb at 1:47 peptide: bsMAb mole ratio, and (C) [99mTc]IMP192 added to hMN-14 × 734 bsMAb premixed with CEA, at 1:7 peptide:bsMAb and 1:10 bsMAb:CEA ratio. Lower panel. SEHPLC chromatograms showing binding of hMN-14 × 734 to indium-DTPA and CEA. (A) [111In]In-IMP-156, (B) Panel A mixed with hMN-14 × 734 bsMAb at 1:24 peptide:bsMAb mole ratio, (C) [111In]In-IMP-156 added to hMN-14 × 734 bsMAb premixed with CEA. The bsMAb:CEA and peptide:bsMAb mole ratios were 1:10 and 1:24.

Figure 3. Tissue uptake of [125I]hMN-14 × 734 and [125I]F6 × 734 bsMabs in liver, spleen, kidney, and blood of BALB/c mice, five animals per time point per each bsMAb.

radioactivity migrated to a higher molecular weight, indicating the ability of the 734 arm of the bsMAb to bind to the peptide after first binding to CEA (Figure 2C). The fact that all of the radiolabeled peptides shifted to the higher molecular weight (i.e., to the elution time of a CEA-bsMAb complex rather than the elution time of the bsMAb alone) was further evidence that the bsMAb is essentially free of any cross-linked, immunoreactive 734F(ab′)2 moiety that theoretically might have formed during preparation of the bsMAb. If this product were present, a portion of the radiolabeled peptide would have eluted at either 10 min (antibody alone) or around 8 min (antibody-antibody complexes formed with the divalent peptide). Indeed, when the radiolabeled peptides were added to a molar excess of the 734 F(ab′)2, all of the radioactivity migrated to the higher molecular weight

Figure 4. SE-HPLC chromatograms of (A) [125I]hMN-14 × 734 bsMAb, (B) panel A mixed with CEA at 1:10 molar ratio, (C) serum from BALB/c mice 4 h post [125I]hMN-14 × 734 bsMAb injection, and (D) panel C mixed with excess CEA.

fraction, but as expected, when CEA was added, no further shift was seen in the profile because of 734’s lack of reactivity with CEA (not shown). Similar studies were carried out with the F6 × 734 bsMAb, which showed identical results (not shown). To assess the stability of the bsMAb in serum, aliquots of [125I]hMN-14 × 734-F(ab′)2 were incubated at 37 °C in fresh human serum and then examined by SE-HPLC over 3 days. There was no loss of radioiodine from the antibody, but at 72 h, approximately 4-7% of the radioactivity migrated at a higher molecular weight, which suggested the development of a small amount of aggregate. There was no evidence of any product degradation to Fab′ fragments. It is important to note that the UV profile of the native nonradiolabeled product has not shown any tendency to aggregate or dissociate over a period of 12 months. At each time the radiolabeled bsMAb was tested, a portion was also mixed with a molar excess of either CEA or the anti-idiotypic MAb, WI2. Greater than 90% of the freshly prepared 125I-labeled bsMAb migrated to a higher molecular weight, and by 72 h, there was no change, indicating excellent immunoreactivity retention of the radiolabeled bsMAb. Thus, there was no evidence of instability in vitro. Again, similar studies with the F6 × 734 bsMAb, except that CEA was used to illustrate antigen binding, showed identical results (not shown). In vitro stability studies were also performed with the [99mTc]IMP-192, using a preparation that showed 99% of 99mTc bound at time zero. This product was diluted 10fold in 10 mM cysteine, physiological saline, or human serum (37 °C). Testing at 1, 1.5, and 2.0 h later, respectively, 98-99% of the 99mTc remained bound to IMP-192. Upon retesting at 19-20 h postdilution, the percentages of 99mTc remaining bound to the IMP-192 were 89, 96, and 96%, respectively. In Vivo Stability and Animal Targeting Studies. To assess the stability of the hMN-14 and F6 bsMAbs in vivo, separate groups of BALB/c mice were injected i.v. with 5 µCi (15 µg) of the [125I]bsMAbs. Necropsies were performed at 1, 2, 4, and 24 h after the injection. In addition to quantifying the uptake in the tissues at these times, a portion of the serum was examined by SE-HPLC to assess the molecular integrity of the bsMAb. Signifi-


cant differences were seen in the distribution properties of the hMN-14 and the murine F6 bsMAb constructs (Figure 3). Overall, there was a higher level of the hMN14 bsMAb in the normal tissues than the murine F6 bsMAb construct at 1 and 4 h. There was nearly a 2-fold higher uptake in the liver and kidneys and almost 4-5fold higher uptake in the spleen, which also displayed a high variability in uptake. There was less pronounced differences in the blood concentrations at these earlier times, but by 24 h, the level of hMN-14 × 734-F(ab′)2 in the blood was 1.5 ( 0.2% ID/g compared to 9.4 ( 1.5% ID/g for the F6 × 734-F(ab′)2. Analysis of the SE-HPLC elution profiles of the serum revealed no significant degradation of either antibody through 4 h (Figure 4, panels A and C, only hMN-14 bsMAb shown). Adding CEA to bsMAb prior to or after it had been injected into animals made little difference in the HPLC profile of the bsMAb-CEA complexes formed (Figure 4, panels B and D). Identical results were obtained with the F6-based bsMAb. Analysis of the molecular profile of the [125I]hMN14 bsMAb at 24 h was not possible, due to insufficient radioactivity remaining in the serum. However, analysis of the profile of the F6 bsMAb revealed that it maintained its molecular integrity (not shown). Thus, the bsMAbs in blood are circulating as intact entities, and are capable of binding to both indium-DTPA complexes and to the target antigen. The differences in distribution properties of these two antibodies, especially the enhanced uptake in the spleen, suggest a recognition of the hMN-14 × 734F(ab′)2 by reticuloendothelial cells, which is different from that of the murine × murine F6 bsMAb construct. This ultimately results in a more rapid blood clearance of the hMN-14 bsMAb. The bispecific binding specificity of the bsMAbs in serum taken from mice was assessed. Twenty microliters of serum was incubated with a prescribed amount of [111In/In]IMP-156 (0.2 µCi, 4.1 × 10-13 mol). The radioactivity profile in the samples was then analyzed by SE-HPLC using an in-line radiation detector with a restricted 111In window. The percentage of [111In/In]IMP-156 that shifted to RT of the bsMAb was 100, 95, and 95 at 1, 2, and 4 h, respectively, for hMN-14 × 734-F(ab′)2. Serum taken at 24 h after the bsMAb injection showed only 10 and 61% of the peptide eluted bound to the hMN-14 × 734-F(ab′)2 and F6 × 734-F(ab′)2, respectively. Retrospective analysis of the amount of bsMAb in the serum, based on the specific activity of the [125I]bsMAb, suggested that this reduction was simply due to an insufficient amount of bsMAb in the serum at 24 h to bind all of the [111In/ In]IMP-156 that was added to the serum in vitro. However, the amount of [111In/In]IMP-156 that comigrated to the molecular size of the bsMAb was in agreement with the amount of bsMAb remaining in blood based on the biodistribution data (Figure 3). For example, at 4 and 24 h, there was 26.4 and 1.5% of the [125I]hMN14 bsMAb in the serum, which corresponded to 7.9 × 10-13 and 4.6 × 10-14 mol in 20 µL of serum. Since 4.1 × 10-13 mol of peptide (containing 8.2 × 10-13 mol of DTPA) was added to each serum sample, the maximum portion of the peptide that could be bound was 96 and 11.2% (4 and 24 h), respectively. The biodistribution of the [99mTc]IMP-192 alone was also examined. In BALB/c mice, it had an extremely rapid blood clearance, with only 0.3% ID/g remaining in circulation at 1 h postinjection (Table 1). The radioactivity was cleared from the body predominantly by urinary excretion, with 169 ( 95% ID/g in the urine within 1 h (or 6.9 ( 6.8% based on the total urine volume collected). Analysis of urine collected from mice injected with

Bioconjugate Chem., Vol. 11, No. 6, 2000 847 Table 1. Biodistribution of [99mTc]IMP-192 Alone in BALB/c Micea percent injected dose per gram of tissue (percent injected dose per organ) 1h

2h

0.27 ( 0.18 0.22 ( 0.16 (0.37 ( 0.21) spleen 0.08 ( 0.01 0.09 ( 0.03 (0.01 ( 0.002) kidney 4.16 ( 0.75 4.05 ( 0.60 (0.60 ( 0.09) lungs 0.50 ( 0.23 0.29 ( 0.08 (0.08 ( 0.02) blood 0.30 ( 0.09 0.21 ( 0.03 (0.41 ( 0.12) stomach 0.39 ( 0.18 0.42 ( 0.18 (0.08 ( 0.04) small int. 1.37 ( 0.75 0.60 ( 0.06 (1.54 ( 0.90) large int. 0.41 ( 0.54 1.53 ( 0.45 (0.29 ( 0.39) muscle 0.10 ( 0.06 0.05 ( 0.00 (ND) (ND) urine 169 ( 95 57 ( 15 (6.9 ( 6.8) (0.011 ( 0.012) liver

4h

24 h

0.09 ( 0.02

0.03 ( 0.01

0.04 ( 0.00 (0.048 ( 0.004) 0.03 ( 0.01 (0.003 ( 0.000) 1.21 ( 0.08 (0.165 ( 0.008) 0.05 ( 0.00 (0.046 ( 0.008) 0.05 ( 0.01 (0.064 ( 0.007) 0.02 ( 0.01 (0.011 ( 0.003) 0.03 ( 0.01 (0.040 ( 0.015) 0.15 ( 0.14 (0.148 ( 0.157) 0.00 ( 0.00

6.30 ( 4.53

0.20 ( 0.02

0.05 ( 0.02 3.21 ( 0.99 0.19 ( 0.04 0.14 ( 0.04 0.27 ( 0.33 0.21 ( 0.09 1.58 ( 0.70

a Values represent the mean with the standard deviation for three animals at each time interval. Values in parenthesis at the 1- and 24-h time intervals indicate the average percentage of [99mTc]IMP-192 in the total tissue mass (total blood volume estimated to be equal to 7.4% of the body weight).

Figure 5. SE-HPLC trace of (A) urine collected from mouse injected with bsMAb and later with [99mTc]IMP-192 and (B) panel A mixed with excess hMN-14 × 734 bsMAb.

[99mTc]IMP-192 showed that it contained the intact peptide, since the radioactivity eluting at 14.18 min on SE-HPLC was shifted to a retention time corresponding to that of bsMAb upon addition of hMN-14 × 734-F(ab′)2 (Figure 5). A minor fraction of the [99mTc]IMP-192 radioactivity was also observed in the small and then the large intestine, suggesting this as another route of excretion. The kidney uptake was about 4% ID/g at 1 h postinjection, but reduced to 1.21 ( 0.08% ID/g by 24 h. The whole-body clearance of the peptide alone, or if given in a pretargeting setting, was tested in tumorbearing nude mice (Figure 6). Groups of five mice were given two different amounts of the [99mTc]IMP-192 peptide, either 1.5 × 10-11 mol (28 µCi) or 1.5 × 10-10 mol (280 µCi). A separate group of animals was first given the 15 µg of the hMN-14 × 734 bsMAb and then 16 h later, the peptide was given. Animals were placed in a dose calibrator at various times after the peptide injection to determine the residual radioactivity remaining. To correct for radioactivity decay, 28 or 280 µCi of [99mTc]IMP-192 were placed in vials and these were read in the dose calibrator before the whole-body readings. The percentage remaining in whole body was calculated from these readings. This study showed >85% of the radioactivity cleared from the animals within 2 to 3 h. This is consistent with the finding in BALB/c mice that the


Figure 6. Whole-body clearance (biological) of [99mTc]IMP-192 (28 µCi, 1.5 × 10-11 mol or 280 µCi, 1.5 × 10-10 mol, (A) alone or (B) given 16 h after hMN-14 × m734 bsMAb was given. Animals were placed in a dose calibrator at 5 and 30 min and then again at 1, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, and 6 h after the peptide alone, or through 3 h for animals given the bsMAb.

majority of the peptide is cleared from the body through urinary excretion rather than fecal elimination. There was no evidence that increasing the amount of peptide affected the clearance rate, and even animals given the bsMAb 16 h earlier cleared the peptide at a similar rate. Pretargeting Studies. To examine the targeting ability of the radiolabeled peptide when used in conjunction with the bsMAb, a study was first performed to assess the temporal relationship between the bsMAb and peptide injections. Nude mice bearing GW-39 human colonic cancer xenografts were first given intravenous injections of 150 pmol (15 µg) of the hMN-14 × 734-F(ab′)2 or the F6 × 734-F(ab′)2 containing a trace amount of each antibody radiolabeled with 125I. At either 4, 16, 24, or 48 h after the hMN-14 bsMAb, 15 pmol of [99mTc]IMP-192 was given. A separate group of animals was given the identical amount of [99mTc]IMP-192 alone. In all groups, the tissue distribution was examined 3 h after the radiolabeled peptide was given. Table 2 shows the results obtained using the hMN-14 bsMAb. An important relationship is the bsMAb and peptide percent uptake in the tumor and blood at each time. When the peptide was injected 4 h after the bsMAb, the concentration of the bsMAb in the blood was too high, and thus a majority of the peptide was bound to the bsMAb in the blood rather than in the tumor. As the bsMAb in the blood cleared, a higher percentage of peptide is found in the tumor, with optimal tumor uptake of the peptide between 16 and 24 h, despite the lower amount of bsMAb in the tumor than at 4 h. Tumor/nontumor ratios continued to improve as the time from the bsMAb injection increased. For example, tumor/blood ratios were 3.6 ( 1.4 when the peptide was given 24 h after the bsMAb, but this increased to 11.3 ( 2.2 when an interval of 48 h was given. However, it is noteworthy that the percent uptake per gram of tumor also decreased, and thus, for therapeutic purposes, the earlier addition of the peptide may

Karacay et al.

be preferred. Tumor uptake of the [99mTc]IMP-192 given alone was only 0.10 ( 0.02% ID/g, indicating that there is selective uptake of the peptide due to the bsMAb in the tumor. Another noteworthy item was the observation that in the animals given the radiolabeled peptide 4 h after the bsMAb, there was a very high percentage of 125I radioactivity in the stomach. Since the uptake of [99mTc]IMP-192 in the stomach was so much lower (i.e., 1.8%), the 125I radioactivity in stomach likely represents a catabolized product. This is very likely since as was seen in Figure 4, there was considerable early accretion of the [125I]hMN-14 bsMAb in the liver and the spleen. Although no significant amount of nonantibody-associated 125I radioactivity was found in the serum at these early times, there is a clear indication that a rapid degradation of the radiolabeled hMN-14 bsMAb is occurring with uptake and excretion of the radioactivity through the GI tract. On the basis of the previous biodistribution of the 125Ilabeled bsMAb alone (Figure 2), there did not appear to be any evidence that the bsMAb’s distribution or blood clearance was altered by the addition of the peptide. This was confirmed in a separate study where the bsMAb was given alone or the peptide was given 16 h after the bsMAb. Tumor and blood uptake of [125I]bsMAb alone was 3.7 ( 1.5 and 1.9 ( 0.3, respectively, and 2.5 ( 0.3 and 0.8 ( 0.08% ID/g at 19 and 27 h, respectively. In the animals given the [125I]bsMAb followed 16 h later with the peptide, tumor and blood uptake was 3.4 ( 1.1 and 1.7 ( 0.2% ID/g, respectively, at 19 h, and by 27 h, [125I]bsMAb uptake was 3.6 ( 0.9 and 0.8 ( 0.10% ID/g for tumor and blood, respectively. By virtue of having the highest tumor uptake and reasonable tumor/nontumor ratios for radiolabeled peptide, 24 h was selected for further evaluation with the hMN-14 bsMAb. Because of the slower blood clearance of the F6 antibody, an interval of 72 h was selected for the following pretargeting studies. A time course study was designed to assess the distribution of the 99mTc- and 188Re-labeled IMP-192 over a 24- and 72-h period, respectively. These results are shown in Tables 3-6. By allowing time to let the individual bsMAb clear from the blood, it was evident that even within just 30 min after the [99mTc]IMP-192 injection, there was a higher uptake of the peptide in the tumor than in any of the normal tissues (Tables 3 and 4). Tumor uptake of the [125I]hMN-14 bsMAb ranged between 5 and 6% ID/g through 3 h after the [99mTc]IMP192 injection and was reduced to 3.3 ( 0.7% ID/g by 24 h (Table 3). During this same time period, the average tumor uptake of the [99mTc]IMP-192 was between 11 and 14% ID/g, reducing to 8.7 ( 3.3% ID/g at 24 h. Tumor/ blood ratios for the hMN-14 bsMAb pretargeting system were 2.7 ( 0.7 within 30 min of the [99mTc]IMP-192 injection and peaked at 16.1 ( 6.4 at 24 h after injection. The tumor/blood ratio in animals given only the [99mTc]IMP-192 was 0.29 ( 0.14 at 3 h (Table 2) compared to 7.3 ( 2.3 in the pretargeting system at this same time (Table 3). Even tumor/kidney ratios were >1.0 within 30 min in each bsMAb pretargeting system. In contrast to when the [99mTc]IMP-192 was given alone, higher concentrations of the peptide were seen in the blood and the other normal tissues in the pretargeting system (e.g., liver, spleen, lungs). This is expected since there was still residual bsMAb remaining in the blood and tissues even at these later times following the bsMAb injection. Thus, the bsMAb-bound peptide would have a delayed clearance in comparison to the peptide alone.



Table 2. Biodistribution of [99mTc]IMP-192 in Nude Mice Bearing GW-39 Tumorsa percent injected dose per gram of tissue (values in parentheses are the tumor/nontumor ratios for the radiolabeled peptide) peptide alone tissue tumor liver spleen kidney lungs blood stomach small int. large int.

4 h clearance

16 h clearance

24 h clearance

99mTc

125I

99mTc

125I

99mTc

0.10 ( 0.02 0.23 ( 0.01 (0.43 ( 0.09) 0.07 ( 0.01 (1.45 ( 0.27) 1.87 ( 0.14 (0.05 ( 0.01) 0.14 ( 0.04 (0.72 ( 0.13) 0.44 ( 0.35 (0.29 ( 0.14) 0.56 ( 0.16 (0.19 ( 0.08) 1.29 ( 0.38 (0.08 ( 0.03) 0.65 ( 0.15 (0.15 ( 0.03)

11.2 ( 3.2 5.0 ( 0.6

5.5 ( 1.1 8.0 ( 0.9 (0.7 ( 0.1) 12.6 ( 4.4 (0.5 ( 0.1) 12.4 ( 2.8 (0.5 ( 0.1) 9.7 ( 2.8 (0.6 ( 0.1) 28.8 ( 7.7 (0.2 ( 0.03) 1.8 ( 0.4 (3.1 ( 0.7) 8.9 ( 1.0 (0.6 ( 0.1) 1.2 ( 0.7 (5.6 ( 2.9)

5.1 ( 1.1 0.7 ( 0.07

8.8 ( 3.6 2.5 ( 0.2 (3.5 ( 1.3) 2.6 ( 0.5 (3.4 ( 1.1) 5.3 ( 1.0 (1.6 ( 0.4) 3.5 ( 0.3 (2.6 ( 1.0) 11.9 ( 0.9 (0.7 ( 0.3) 0.8 ( 0.2 (11.2 ( 3.3) 3.2 ( 1.0 (2.7 ( 0.3) 1.7 ( 0.6 (6.7 ( 5.7)

12.0 ( 3.7 7.5 ( 1.9 7.7 ( 2.0 15.0 ( 3.8 48.0 ( 5.3 3.3 ( 0.7 1.7 ( 0.6

1.1 ( 0.6 1.0 ( 0.1 1.2 ( 0.1 2.3 ( 0.2 6.5 ( 0.8 0.5 ( 0.02 0.5 ( 0.07

125I

48 h clearance

99mTc

4.8 ( 2.3 0.6 ( 0.2 0.6 ( 0.2 0.6 ( 0.2 2.3 ( 1.1 0.9 ( 0.2 8.7 ( 1.4 0.4 ( 0.1 0.9 ( 0.06

125I

14.3 ( 6.9 1.3 ( 0.2 (11.5 ( 5.5) 0.8 ( 0.3 (21.3 ( 14) 4.5 ( 1.2 (3.4 ( 1.9) 3.1 ( 1.7 (7.1 ( 6.3) 3.3 ( 0.5 (4.2 ( 1.8) 1.5 ( 1.5 (14.3 ( 9.5) 2.7 ( 0.5 (5.0 ( 1.9) 0.3 ( 0.1 (59.0 ( 50.7)

99mTc

2.0 ( 0.9 0.3 ( 0.1

4.8 ( 1.1 0.3 ( 0.0 (18.1 ( 3.6) 0.3 ( 0.1 (15.8 ( 4.3) 3.3 ( 0.7 (1.5 ( 0.2) 0.5 ( 0.2 (11.5 ( 3.3) 0.4 ( 0.2 (11.3 ( 2.2) 0.2 ( 0.2 (32.0 ( 18) 0.3 ( 0.1 (20.0 ( 5.5) 1.3 ( 0.2 (3.6 ( 0.6)

0.3 ( 0.1 0.4 ( 0.1 0.3 ( 0.2 0.1 ( 0.01 0.3 ( 0.2 0.1 ( 0.01 0.2 ( 0.1

a Animals were either given the [99mTc]IMP-192 alone (1.5 × 10-11 mol) or the animals first received [125I]hMN-14 × 734-bsMAb (1.5 × 10-10 mol), and then after 4, 16, 24, and 48 h, they received the radiolabeled peptide. All values were obtained 3 h after the [99mTc]IMP192 (1.5 × 10-11 mol), injection and are presented as the mean ( standard deviation with five animals per interval. The average GW-39 weights for each respective group in order shown were 0.113 ( 0.043, 0.102 ( 0.008, 0.120 ( 0.029, 0.404 ( 0.277, and 0.512 ( 0.352 g.

Table 3. Biodistribution of [99mTc]IMP-192 in Mice Pretargeted with [125I]hMN-14 × m734a percent injected dose per gram of tissue (values in parentheses are the tumor/nontumor ratios for the radiolabeled peptide) 30 min

1h

125I

99mTc

tumor liver

4.9 ( 1.1 0.6 ( 0.1

spleen

0.8 ( 0.3

kidney

0.5 ( 0.1

11.4 ( 4.8 1.4 ( 0.3 (7.9 ( 1.7) 1.2 ( 0.4 (9.4 ( 1.0) 9.9 ( 6.1 (1.2 ( 0.2) 4.2 ( 3.4 (3.7 ( 1.7) 4.3 ( 1.2 (2.7 ( 0.7) 3.6 ( 4.8 (17.9 ( 20.9) 1.4 ( 0.3 (8.2 (3.3) 0.5 ( 0.3 (29.3 ( 12.3)

tissue

lungs

0.9 ( 0.3

blood

0.9 ( 0.3

stomach

5.0 ( 1.7

small int.

0.4 ( 0.1

large int.

0.7 ( 0.4

125I

6.0 ( 2.3 0.5 ( 0.2 0.7 ( 0.3 0.5 ( 0.1 0.8 ( 0.2 1.2 ( 0.4 6.1 ( 3.3 0.4 ( 0.08 0.5 ( 0.09

3h

24 h

99mTc

125I

99mTc

14.3 ( 3.6 0.9 ( 0.2 (15.7 ( 5.4) 0.8 ( 0.2 (19.5 ( 8.6) 4.6 ( 0.7 (3.1 ( 0.6) 3.6 ( 1.9 (5.5 ( 3.6) 3.5 ( 0.9 (4.2 ( 1.3) 1.0 ( 1.2 (25.0 ( 14.3) 1.4 ( 0.2 (10.3 ( 1.6) 0.3 ( 0.05 (56.3 ( 13.0)

5.5 ( 1.1 0.5 ( 0.1

12.6 ( 5.2 0.6 ( 0.1 (20.7 ( 7.6) 0.5 ( 0.1 (22.9 ( 7.5) 2.4 ( 0.5 (5.2 ( 1.5) 1.0 ( 0.3 (13.5 ( 7.1) 1.7 ( 0.4 (7.3 ( 2.3) 0.5 ( 0.2 (30.7 ( 14.5) 1.0 ( 0.5 (14.1 ( 7.6) 0.9 ( 0.3 (14.6 ( 6.7)

0.7 ( 0.2 0.5 ( 0.1 0.8 ( 0.3 1.1 ( 0.3 5.7 ( 3.3 0.5 ( 0.2 0.8 ( 0.4

125I

3.3 ( 0.7 0.1 ( 0.02 0.2 ( 0.03 0.1 ( 0.02 0.3 ( 0.01 0.2 ( 0.07 0.8 ( 0.3 0.09 ( 0.02 0.1 ( 0.03

99mTc

8.7 ( 3.3 0.4 ( 0.1 (22.3 ( 7.4) 0.4 ( 0.2 (23.8 ( 11.7) 1.2 ( 0.3 (7.3 ( 1.9) 0.3 ( 0.1 (30.8 ( 14.4) 0.6 ( 0.2 (16.1 ( 6.4) 0.3 ( 0.2 (42.4 ( 33.3) 0.3 ( 0.07 (35.7 ( 11.7) 0.5 ( 0.2 (17.3 ( 8.1)

a Nude mice bearing GW-39 human colonic cancer xenografts were given [125I]hMN-14 × m734 (Fab′ × Fab′, 5 µCi, 1.5 × 10-10 mol). Twenty-four hours later, [99mTc]IMP-192 (10 µCi, 1.6 × 10-11 mol) was injected. All times given in the tables are the means and standard deviations at the hours indicated after the [99mTc]IMP-192 injection (five animals per time point). The average GW-39 weights for each respective group in order shown were 0.382 ( 0.106, 0.373 ( 0.062, 0.265 ( 0.094, and 0.358 ( 0.110 g.

Interestingly, the percent injected dose per gram of the [99mTc]IMP-192 in tumors was similar in animals pretargeted with either the F6 bsMAb or the hMN-14 bsMAb, despite the nearly 2-fold higher amount of the F6 bsMAb in the tumor (Table 4). This is most likely explained by the fact that there was also nearly 2-fold more F6 bsMAb than hMN-14 bsMAb in the blood, which would have reduced the amount of [99mTc]IMP-192 available for tumor binding. As stated earlier, these studies were not conducted under conditions to optimize each targeting system fully. The pretargeting studies were repeated using [188Re]IMP-192 in place of [99mTc]IMP-192, at the same 10:1 bsMAb to IMP-192 ratio (Tables 5 and 6). Because of the longer half-life of 188Re, biodistributions were performed at 3, 24, 48, and 72 h following [188Re]IMP-192 injection. Again, uptake of the [188Re]IMP-192 was higher in the tumor than all the normal tissues as early as 3 h after injection, due to both the rapid clearance of small molecular weight [188Re]IMP-192 and the ready recognition of the radiolabeled peptide by the tumor-localized bsMAb. In contrast to tumor uptake of the [188Re]IMP192 alone (7 µCi, 1.5 × 10-11 mol of peptide), which at 3

h was 0.36 ( 0.15 (Table 5), [188Re]IMP-192 tumor uptake was 16.9 ( 5.5% ID/g when used with the hMN-14 bsMAb pretargeting system. Overall, no meaningful differences were seen between the 99mTc- and the 188Relabeled peptide in either bsMAb pretargeting system at 3 and 24 h, times that were common for the two studies. Dosimetry. To predict the therapeutic potential of [188Re]IMP-192 in animals pretargeted with hMN-14 734 bsMAb, the biodistribution studies were used to calculate radiation-absorbed doses to the tissues and tumor (Table 7). Even under these conditions which were not fully optimized, the tumor is predicted to receive a radiation dose nearly 12-fold higher than the liver, 7 times higher than the kidneys, and 5-fold higher than the blood. Radiation doses to the gastrointestinal tract, most notably the stomach, were higher than the other organs. DISCUSSION

Pretargeting is a very attractive modality for the delivery of radionuclides to tumors for both RAID and RAIT, although its complexity requires multiple problems to be solved before it can be applied optimally in a clinical


Karacay et al.

Table 4. Tissue Uptake of [99mTc]IMP-192 in Mice Pretargeted with [125I]F6 × m734 bsMAba percent injected dose per gram of tissue (values in parentheses are the tumor/nontumor ratios for the radiolabeled peptide) 30 min

1h

125I

99mTc

tumor liver

11.1 ( 3.2 0.4 ( 0.1

15.6 ( 3.9 0.4 ( 0.03

spleen

0.4 ( 0.1

kidney

0.7 ( 0.3

lungs

0.8 ( 0.3

blood

1.6 ( 0.5

stomach

3.8 ( 1.6

11.4 ( 3.9 1.7 ( 0.4 (7.0 ( 3.2) 1.9 ( 1.3 (7.5 ( 4.9) 20.8 ( 19.9 (1.1 ( 1.0) 3.1 ( 1.2 (4.4 ( 2.8) 8.0 ( 3.2 (1.7 ( 0.9) 0.8 ( 0.6

small int.

0.3 ( 0.1

0.3 ( 0.04

large int.

0.3 ( 0.2

1.4 ( 0.5 (10.6 ( 4.5) 1.5 ( 0.4 (10.1 ( 4.6)

tissue

3h

125I

0.4 ( 0.001 0.6 ( 0.1 1.0 ( 0.1 1.8 ( 0.3 2.2 ( 0.7

0.2 ( 0.04

99mTc

125I

11.1 ( 1.4 1.7 ( 0.5 (6.8 ( 1.4) 1.2 ( 0.1 (9.5 ( 0.2) 7.1 ( 1.0 (1.5 ( 0.03) 3.4 ( 0.9 (3.3 ( 0.5) 7.6 ( 1.6 (1.5 ( 0.1) 1.2 ( 0.3 (9.2 ( 1.0) 2.1 ( 0.3 (5.4 ( 0.1) 0.8 ( 0.3 (14.4 ( 2.9)

11.3 ( 1.5 0.4 ( 0.1 0.4 ( 0.1 0.4 ( 0.1 0.6 ( 0.2 1.2 ( 0.2 2.3 ( 0.8 0.2 ( 0.04 0.2 ( 0.09

24 h 99mTc

125I

99mTc

14.3 ( 6.7 0.9 ( 0.1 (16.3 ( 5.9) 0.8 ( 0.2 (18.4 ( 8.4) 4.1 ( 0.5 (3.4 ( 1.3) 1.1 ( 0.2 (12.6 ( 4.5) 3.4 ( 0.2 (4.2 ( 1.8) 0.8 ( 0.6 (23.2 ( 12.6) 1.4 ( 0.5 (10.6 ( 4.5) 1.5 ( 0.4 (10.2 ( 4.6)

8.1 ( 1.0 0.3 ( 0.01

10.6 ( 1.4 0.6 ( 0.1 (17.4 ( 3.1) 0.5 ( 0.1 (21.9 ( 4.5) 2.3 ( 0.3 (4.6 ( 0.6) 0.5 ( 0.1 (20.7 ( 3.6) 1.3 ( 0.2 (8.1 ( 1.5) 0.4 ( 0.2 (31.2 ( 10.1) 0.4 ( 0.09 (31.1 ( 5.8) 0.6 ( 0.2 (19.0 ( 7.0)

0.3 ( 0.04 0.3 ( 0.04 0.4 ( 0.06 0.8 ( 0.08 1.1 ( 0.2 0.2 ( 0.02 0.2 ( 0.04

a Nude mice bearing GW-39 human colonic cancer xenografts were given [125I]F6 × m734 (Fab′ × Fab′, 5 µCi, 1.5 × 10-10 mol). Three days later, [99mTc]IMP-192 (10 µCi, 1.6 × 10-11 mol) was injected. All times given in the tables are the means and standard deviations at the hours indicated after the [99mTc]IMP-192 injection (five animals per time point). The average GW-39 weights for each respective group in order shown were 0.902 ( 0.874, 0.342 ( 0.005, 0.588 ( 0.221, and 0.666 ( 0.215 g.

Table 5. Tissue Uptake of [188Re]IMP-192 in Mice Pretargeted with [125I]hMN-14 × m734a percent injected dose per gram of tissue (values in parentheses are the tumor/nontumor ratios for the radiolabeled peptide) peptide alone

3h

24 h

48 h

188Re

125I

188Re

125I

188Re

tumor liver

0.36 ( 0.15 0.36 ( 0.11

6.9 (1.8 0.4 ( 0.06

4.5 ( 0.6 0.2 ( 0.04

spleen

0.24 ( 0.07

1.4 ( 1.5

kidney

1.20 ( 0.24

0.5 ( 0.06

lungs

0.36 ( 0.13

0.8 ( 0.1

blood

0.75 ( 0.45

1.0 ( 0.1

stomach

3.62 ( 1.79

4.5 ( 1.4

small int.

1.38 ( 0.80

0.3 ( 0.08

large int.

1.04 ( 0.36

0.3 ( 0.2

16.9 ( 5.5 0.9 ( 0.2 (17.4 ( 3.8) 1.2 ( 1.0 (20.3 ( 11.3) 2.4 ( 0.3 (6.8 ( 1.8) 1.4 ( 0.3 (11.7 ( 2.8) 2.9 ( 0.9 (5.9 ( 2.1) 7.1 ( 1.5 (2.4 ( 0.8) 2.3 ( 0.3 7.2 ( 1.9 0.3 ( 0.2 (94.5 ( 104.7)

11.1 ( 1.6 0.4 ( 0.03 (30.4 ( 6.1) 0.4 ( 0.1 (27.6 ( 6.5) 0.6 ( 0.1 (17.9 ( 5.3) 0.4 ( 0.05 (29.3 ( 7.5) 0.8 ( 0.1 (14.7 ( 4.0) 0.3 ( 0.1 (43.2 ( 34.0) 0.3 ( 0.1 (49.1 ( 26.4) 0.4 ( 0.1 (28.5 ( 17.9)

tissue

0.3 ( 0.05 0.3 ( 0.04 0.4 ( 0.06 0.5 ( 0.07 1.1 ( 0.3 0.1 ( 0.01 0.1 ( 0.03

125I

2.5 ( 0.5 0.1 ( 0.01 0.1 ( 0.01 0.1 ( 0.02 0.1 ( 0.03 0.2 ( 0.03 0.2 ( 0.04 0.06 ( 0.01 0.05 ( 0.00

72 h 188Re

125I

188Re

4.9 ( 1.3 0.2 ( 0.01 (23.9 ( 5.2) 0.3 ( 0.1 (18.8 ( 6.4) 0.3 ( 0.04 (17.3 ( 4.2) 0.1 ( 0.05 (36.5 ( 12.5) 0.3 ( 0.1 (16.1 ( 3.1) 0.08 ( 0.03 (67.0 ( 27.8) 0.07 ( 0.02 (76.4 ( 22.5) 0.08 ( 0.06 (73.8 ( 47.5)

1.7 ( 0.6 ND

3.1 ( 1.4 ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

a Nude mice bearing GW-39 human colonic cancer xenografts were given [125I]hMN-14 × m734 Fab′ × Fab′ (5 µCi, 1.5 × 10-10 mol). Twenty-four hours later, [188Re]IMP-192 (7 µCi, 1.6 × 10-11 mol of peptide) was injected. All times given in the tables are the means and standard deviations at the hours indicated after the [188Re]IMP-192 injection (five animals per time point). The data for the [188Re]IMP192 alone is 3 h after its injection. The average GW-39 weights for each respective group in order shown were 0.098 ( 0.042, 0.160 ( 0.065, 0.136 ( 0.036, and 0.266 ( 0.168. ND, insufficient radioactivity in normal tissue to reliably calculate percent uptake.

setting. Previously, we examined a streptavidin-antibody conjugate/radiolabeled biotin pretargeting approach analogous to the one described by Axworthy et al. (1994). These investigators were able to deliver the same amount of radioactivity to a tumor using radiolabeled biotin as was possible with a radiolabeled antibody conjugate. We also found that excellent tumor/nontumor ratios could be achieved with reasonable absolute tumor uptake using such a system (Sharkey et al., 1997). While animal studies continue to evaluate the therapeutic benefit of the antibody-streptavidin/radiolabeled biotin pretargeting approach in comparison to directly radiolabeled antibody, we continue to have serious concerns with the system, particularly because of the immunogenicity of streptavidin (Chinol et al., 1998). Knowing of the possibilities for future engineering (Carter and Merchant

1997; Coloma and Morrison, 1997) of a humanized agent with minimal, if any, immunogenicity, efforts were undertaken to investigate a bispecific antibody pretargeting approach useful for RAIT. For these initial studies, we decided to evaluate our humanized antibody to CEA, hMN-14, since this is presently being used in clinical trials. This antibody was paired with the murine antibody specific for [In]DTPA, termed 734, that was used previously by the French investigators with their murine anti-CEA antibody, F6 (Chetanneau et al., 1990). Using established technology (Glennie et al., 1987), a Fab′ fragment of each of these MAbs was coupled site-specifically in their hinge-regions via a thioether linkage to produce a bispecific F(ab′)2. Rigorous SE-HPLC analyses were performed to show bsMAb purity to be greater than 90%, with excellent



Table 6. Tissue Uptake of [188Re]IMP-192 in Mice Pretargeted with [125I]F6 × m734a percent injected dose per gram of tissue (values in parentheses are the tumor/nontumor ratios for the radiolabeled peptide) 3h tissue tumor liver

125I

14.7 ( 3.1 0.3 ( 0.1

spleen

0.3 ( 0.05

kidney

0.4 ( 0.05

lungs

0.4 ( 0.1

blood

1.2 ( 0.2

stomach

1.5 ( 0.4

small int.

0.2 ( 0.04

large int.

0.1 ( 0.01

24 h 188Re

19.3 ( 4.2 1.3 ( 0.3 (14.9 ( 4.0) 1.0 ( 0.3 (20.7 ( 8.1) 3.3 ( 0.4 (5.9 ( 0.9) 1.5 ( 0.4 (13.6 ( 5.2) 5.1 ( 1.2 (3.9 ( 1.3) 5.9 ( 1.2 (3.3 ( 0.5) 2.0 ( 0.6 (10.0 ( 2.3) 1.2 ( 0.5 (20.5 ( 13.7)

125I

13.9 ( 4.3 0.2 ( 0.1 0.2 ( 0.1 0.2 ( 0.1 0.3 ( 0.2 0.6 ( 0.3 0.8 ( 0.3 0.1 ( 0.05 0.1 ( 0.03

48 h 188Re

125I

20.3 ( 6.8 0.5 ( 0.2 (40.5 ( 9.1) 0.4 ( 0.1 (57.5 ( 11.3) 0.7 ( 0.2 (29.9 ( 8.6) 0.4 ( 0.3 (63.8 ( 24.4) 1.3 ( 0.8 (16.9 ( 5.1) 0.3 ( 0.1 (84.3 ( 29.5) 0.3 ( 0.1 (82.8 ( 31.8) 0.4 ( 0.2 (40.0 ( 14.9)

6.5 ( 2.0 0.1 ( 0.02 0.1 ( 0.02 0.1 ( 0.03 0.1 ( 0.03 0.3 ( 0.1 0.4 ( 0.2 0.07 ( 0.01 0.06 ( 0.01

72 h 188Re

8.5 ( 2.8 0.3 ( 0.1 (27.3 ( 10.1) 0.2 ( 0.1 (51.9 ( 18.8) 0.4 ( 0.1 (24.1 ( 8.7) 0.2 ( 0.1 (60.9 ( 34.4) 0.6 ( 0.2 (15.4 ( 6.1) 0.1 ( 0.03 (79.8 ( 23.9) 0.1 ( 0.03 (77.0 ( 24.6) 0.1 ( 0.05 (64.1 ( 23.5)

125I

6.2 ( 1.9 0.1 ( 0.04 0.1 ( 0.02 0.1 ( 0.03 0.1 ( 0.01 0.3 ( 0.1 0.3 ( 0.1 0.07 ( 0.02 0.05 ( 0.02

188Re

6.5 ( 2.6 0.3 ( 0.1 (20.0 ( 5.7) 0.2 ( 0.1 (44.0 ( 49) 0.3 ( 0.1 (21.5 ( 6.0) 0.2 ( 0.1 (39.7 ( 18.9) 0.5 ( 0.1 (13.4 ( 6.2) 0.05 ( 0.03 (165.3 ( 123.3) 0.09 ( 0.01 (77.2 ( 31.8) 0.10 ( 0.04 (65.2 ( 23.9)

a Nude mice bearing GW-39 human colonic cancer xenografts were given [125I]F6 × m734 Fab′ × Fab′ (5 µCi, 1.5 × 10-10 mol). Three days later, [188Re]IMP-192 (7 µCi, 1.6 × 10-11 mol of peptide) was injected. All times given in the tables are the means and standard deviations at the hours indicated after the [188Re]IMP-192 injection (five animals per time point). The average GW-39 weights for each respective group in order shown were 0.091 ( 0.036, 0.133 ( 0.048, 0.199 ( 0.036, and 0.096 ( 0.028 g.

Table 7. Mean Cumulative Radiation Absorbed Dose Delivered to Tissues Based on Biodistribution of [188Re]IMP-192 in Mice Pretargeted with hMN-14 × 734 bsMAb tissue

dose (cGy/mCi)

tumor liver spleen kidney lungs blood stomach small int. large int.

1954 155 132 269 150 407 621 383 87

retention of binding both to CEA and to [111In/In]DTPA. All of radiolabeled bivalent hapten, [111In/In]IMP-156, could be shifted to a higher molecular weight fraction when combined with bsMAb and CEA, indicating that all of the binding 734 arm was associated with a hMN14 arm. About 92-93% of the bsMAb migrated to a higher molecular weight by SE-HPLC when combined with [In]DTPA-LL2-IgG, indicating that 7-8% hMN-14 × hMN-14 (Fab′ × Fab′) was likely present. Since it would be preferable to have excess monospecific anti-CEA MAb present rather than excess monospecific anti-DTPA MAb, the 734 Fab′ was selected to be activated with the dimaleimide. In vitro, the hMN-14 × 734 bsMAb was comparable to the previously described all-murine F6 × 734 bsMAb, in that both molecules maintained molecular integrity, as well as capacity for binding to CEA or indium-DTPA. The murine F6 anti-CEA × 734 construct was used primarily as a positive control to show that substituting the hMN-14 in bsMAb would not affect the quality of the bsMAb. In vitro studies certainly confirmed this, but in vivo, an unexpected rapid blood clearance of the hMN14 × 734-F(ab′)2 (humanized × murine) compared to the F6 × 734-(Fab′)2 (murine × murine) was found. Thioether-linked F(ab′)2 is not susceptible to reduction and, indeed, was previously shown to have an extended blood circulation time compared to a natural F(ab)2 fragment (Siddiqui et al., 1995), so the linkage chemistry could not explain the rapid clearance seen with the humanizedmurine hybrid bsMAb. SE-HPLC was used to assess catabolic products in the serum (e.g., Fab′ fragments or even smaller molecular weight byproducts). Given the amount of radioactivity administered to the animals, the

radiolabeled hMN-14 × 734 bsMAb could only be evaluated over 4 h. During this time, there was no evidence of catabolic byproducts to suggest an instability of the bsMAb. Previous studies with hMN-14 F(ab′)2 in mice have shown a similar rapid blood clearance (Leung et al., 1999) in comparison with the corresponding murine F(ab′)2, and we have also noted differences in the clearance properties of murine IgG1 and murine IgG2a and of human IgG1 in nude mice (Sharkey et al., 1991). This observation was ultimately traced to varying amounts of murine IgG2a in different strains of mice (Reddy et al., 1998), but it is uncertain whether a similar mechanism can explain the differences in the clearance of bsMAb fragments, since these molecules lack the Fc-portion of the immunoglobulin molecule that is most frequently associated with its recognition by immune cells. However, an initially high uptake was noted in both the spleen and liver for the hMN-14 bsMAb in comparison to the all murine F6 bsMAb, suggesting that there is a recognition mechanism involved in this enhanced clearance. The elevated uptake of radioiodine in the stomach was a further indication that the hMN-14 bsMAb was likely being catabolized in these organs. The humanized MN14 × murine 734 bsMAb F(ab′)2 is presently being prepared for clinical use, where it will be possible to reassess its distribution and clearance. However, since clinical studies comparing the murine MN-14 and humanized MN-14 IgG revealed very similar distribution and clearance properties (Sharkey et al., 1995), it is more likely that this preclinical observation is unique to the mouse model. Thus, it might be better to use murine bsMAbs in murine models, as this would maintain the species similarities that will ultimately occur when a fully humanized bsMAb is used clinically. Despite the observed differences in the clearance rate of the two bsMAbs, the subsequent targeting of both the [99mTc]- and [188Re]IMP-192 peptides to either conjugate was excellent. Even though a higher tumor uptake was observed with the radioiodinated F6-based bsMAb compared to the hMN-14 bsMAb, initial tumor uptake of the radiolabeled peptide in both pretargeting systems was very similar. This was clearly due to differences in the amount of bsMAb remaining in the blood at the time the peptide was given. This illustrates the importance of fully optimizing the relationship between the amount of bsMAb remaining in the blood at the time the peptide is


given. The best radiolabeled peptide ratios correlated with the highest bsMAb tumor uptakes and the lowest bsMAb blood levels. It is also important to note that there was no evidence that the injection of the radiolabeled peptide affected the blood clearance rate or the organ distribution of the bsMAb. Given the divalent nature of the peptide, it is possible that complexes of 2 bsMAb molecules could form in the blood, and they might be cleared differently or even cause the bound peptide or antibody to distribute differently in the organs. It is possible that at the concentrations of the bsMAb and peptide used in these studies and the times that were examined failed to detect altered kinetics and distribution. This issue should be reexamined once an optimized therapeutic system is developed. Indeed, as shown by Gautherot et al. (1998, 2000), careful optimization is required to achieve superior therapeutic results. The IMP-156 is not suitable for therapeutic applications with Y-90, because the 734 MAb was unable to bind tightly to the yttrium-DTPA complex. Additionally, [90Y]DTPA species, wherein one carboxyl group is unavailable due to amide bond formation, are well-known to have poor in vivo stability (Harrison et al., 1991). Although a species containing mixed DOTA and DTPA chelates may be prepared, the similarities of labeling chemistries between yttrium and indium might make selective indium labeling of DTPA and yttrium labeling of DOTA problematic. Another option would be to prepare a new anti-yttrium-DOTA MAb so that a new bsMAb could be used, but this too would restrict the applicability of such a bsMAb to this single system. Instead, we are presently exploring a single peptide-binding MAb that could be used with a peptide that itself could be modified to accommodate multiple binding possibilities. The approach taken here was to work with the 734 antibody, but to construct a new peptide with another binding specificity. Since the chemistries involved in technetium/ rhenium radiolabeling are quite distinct from those used in indium chelation, this presented an opportunity to explore the selective radiolabeling of a Tc/Re chelatepeptide that also contained indium-di-DTPA. This peptide, IMP-192, was assembled with a thiosemicarbonylglyoxylcysteinyl group (TscGC), which is a thiol-containing ligand for Tc/Re. It was then formulated with excess nonradioactive indium to saturate DTPA chelate sites and lyophilized into single-vial kits containing stannous ion, for later reconstitution with either 99mTc or 188Re. 99mTc-formulated kits were 99mTc labeled at 1836 Ci/mmol/ mmol of peptide (94-99% incorporation), while kits formulated for 188Re were labeled at 459-945 Ci/mmol (96-97%). Successful labeling of both Tc and Re IMP192 kit formulations indicated that the presence of free and DTPA-bound indium did not interfere with 99mTc or 188Re labeling of the TscGC chelate. Further studies are planned to determine if higher specific activities can be achieved for therapeutic applications. Biodistribution studies of [99mTc]IMP-192 and [188Re]IMP-192 bivalent haptens alone in normal BALB/c mice showed rapid blood clearance. As with the IMP-156 peptide, the presence of the two DTPAs on the IMP-192 peptide probably aids in rapid renal excretion, and it was gratifying to find that attachment of the TscGC moiety did not materially change the behavior of the IMP-192 di-DTPA peptide. In addition, the in vitro and in vivo studies clearly demonstrate stability of these complexes, with unchanged radiolabeled IMP-192 excreted in urine at early time points postinjection. Unlike radiolabeled biotin (Rusckowski et al., 1996; Sharkey et al., 1997), there was no evidence of selective tumor accretion (i.e.,

Karacay et al.

tumor/nontumor ratios > 1.0) of the radiolabeled IMP192 peptide when administered alone to mice. Using this pretargeting system, tumor-to-liver, -kidney, -lungs, and -blood ratios were between 3- and 22.9:1 at 1 and 3 h after the [99mTc]IMP-192 peptide was given, indicating that early imaging will be possible. In comparison, a [99mTc]Fab′ anti-CEA, prepared by direct labeling of hinge-region thiol groups, and tested in the same preclinical tumor model, gave tumor-to-liver, -kidney, -lungs, and -blood ratios of 1.6-, 0.06-, 1.2-, and 0.9to-1 at 2 h postinjection (Hansen et al., 1990). With the hMN-14 × 734-F(ab′)2 bsMAb pretargeting method, even the kidney shows a positive tumor-to-nontumor ratio at only 30 min postinjection of [99mTc]IMP-192. The pretargeted [188Re]IMP-192 peptide gave tumor-to-nontumor ratios between 14.7- and 63.8-to-1 for the major tissues by 24 h postinjection of the 188Re-labeled peptide, and ratios were decidedly positive for each tissue even by 3 h postinjection of the [188Re]IMP-192. For comparison, a 188 Re direct-labeled IgG in a similar tumor xenograft model (LS-174T) showed tumor-to-nontumor ratios at 24 h postinjection of 1.8- to 6.1:1, and none of these ratios was positive at 2 h postinjection (Griffiths et al., 1991). Of particular note with these 99mTc- and 188Re-labeled peptides is the absence of renal retention of the radiometal when compared to radiometal-labeled MAb fragments developed for RAID and RAIT (Behr et al., 1995). The excellent tumor/nontumor ratios and very reasonable tumor uptakes already suggest that this bsMAbpeptide combination has promising RAID and RAIT applications. RAIT prospects would be improved further if an increase in absolute tumor uptake of radioactivity at these same tumor-to-tissue ratios could be achieved. In the present studies, only small doses of the bsMAb (i.e., 15 µg) were given to each animal, but for RAIT, higher bsMAb protein doses will be used to increase the number of peptide-binding sites localized to the tumor. The use of other bsMAb constructs could also increase the amount of bsMAb localized in the tumor. For instance, use of bsMAb comprising a divalent anti-CEA combined with either a monovalent or even a divalent anti-hapten antibody is of interest. Changing bsMAb valency would increase the size of the bsMAb and likely slow its blood clearance, and then more time would be needed before radiolabeled peptide administration to allow for adequate blood clearance of the bsMAb. Others (Axworthy et al., 1994; Schuhmacher et al., 1995) have used clearing agents to reduce the blood level of nonlocalized pretargeting agents after maximum tumor uptake has been achieved, although clearance mechanisms involving blockade of secondary binding sites can adversely affect subsequent targeting of the low molecular weight RAID/RAIT agent. In this regard, we have an anti-idiotypic MAb (WI2) to the MN-14 anti-CEA arm, which was successfully used for clearance in previous work with MAb-streptavidin studies (Sharkey et al., 1997) and has since been humanized (Losman et al., 1999). We are presently evaluating alternate bsMAb targeting with hMN-14 IgG × 734-Fab′, and hMN-14 F(ab′)2 × 734-Fab′ conjugates, to assess valency and molecular size issues. Ultimately, bsMAbs will be prepared through molecular engineering (Coloma and Morrison, 1997; Merchant and Carter, 1997), at sizes indicated best for either RAID or RAIT application from the current studies. The molecular engineering approach will also provide an opportunity to enhance the productivity and availability of bsMAb constructs, while simultaneously minimizing or eliminating any immunogenicity. In summary, these studies provide highly promising


preclinical targeting data with human-murine and murine-murine bsMAb (Fab′)2. Furthermore, a novel peptide containing metal-selective chelates for indium and for 99mTc and 188Re radiolabeling allows parallel development for both RAID and RAIT applications. The pretargeting results presented here suggest that early time-point imaging using [99mTc]IMP-192, with the high tumor-totissue ratios versus major internal organs achieved, could provide a new standard for RAID. The data are also very encouraging for therapy with [188Re]IMP-192. ACKNOWLEDGMENT

We thank Immunotech S. A., Marseilles, France, for providing supplies of the F6 and 734 antibodies. We also thank Ms. H. Spiegelman, L. Reed, and L. Osorio for their assistance with the animal studies and Mr. T. Jackson for radiolabeling. Supported in part by USPHS Grants CA 81760 and CA 37895 from the NIH. LITERATURE CITED (1) Axworthy, D. B., Fritzberg, A. R., Hylarides, M. D., Mallett, R. W., Theodore, L. J., Gustavson, L. M., Su, F.-M., Beaumier, P. L., and Reno, J. M. (1994) Preclinical evaluation of an antitumor monoclonal antibody/streptavidin conjugate for pretargeted 90Y radioimmunotherapy in a mouse xenograft model. J. Immunother. 16, 158. (2) Bamias, A., and Epenetos, A. A. (1992) Two-step strategies for the diagnosis and treatment of cancer with bioconjugates. Antibody, Immunoconjugates, Radiopharm. 5, 385-395. (3) Barbet, J., Peltier, P., Bardet, S., Vuillez, J. P., and Chatal, J. F. (1998) Radioimmunodetection of medullary thyroid carcinoma using indium-111 bivalent hapten and anti-CEA × anti-DTPA-indium bispecific antibody. J. Nucl. Med. 39, 1172-1178. (4) Behr, T. M., Sharkey, R. M., Juweid, M. E., Blumenthal, R. D., Dunn, R. M., Griffiths, G. L., Bair, H.-J, Wolf, F. G., Becker, W. S., and Goldenberg, D. M. (1995) Reduction in the renal uptake of radiolabeled monoclonal antibody fragments by cationic amino acids and their derivatives. Cancer Res. 55, 3825-3834. (5) Boerman, O. C., Kranenborg, M. H. G. C., Oosterwijk, E., Griffiths, G. L., McBride, W. J., Oyen, W. J. G., de Weijert, M., Oosterwijk-Wakka, J., Hansen, H. J., and Corstens, F. H. M. (1999) Pretargeting of renal cell carcinoma: improved tumor targeting with a bivalent chelate. Cancer Res. 59, 4400-4405. (6) Bos, E. S., Kuijpers, W. H. A., Meesters-Winters, M., Pham, D. T., deHaan, A. S., van Doormalen, A. M., Kasperson, F. M., van Boeckel, C. A. A., and Gougeon-Bertrand, F. (1994) In vitro evaluation of DNA-DNA hybridization as a two-step approach in radioimmunotherapy of cancer. Cancer Res. 54, 3479-3486. (7) Carter, P., and Merchant, A. M. (1997) Engineering antibodies for imaging and therapy. Curr. Opin. Biotechnol. 8, 449454. (8) Chetanneau, A., Baum, R. P., Lehur, P. A., Liehn, J. C., Perkins, A. C., Bares, R., Bourguet, P., Herry, J. Y., Saccavini, J. C., and Chatal, J. F. (1990) Multi-centre immunoscinitgraphic study using indium-111-labeled CEA-specific and/or 19-9 monoclonal antibody F(ab′)2 fragments. Eur. J. Nucl. Med. 17, 223-229. (9) Chinol, M., Casalini, P., Maggiolo, M., Canevari, S., Omodeo, E. S., Caliceti, P., Veronese, F. M., Cremonesi, M., Chiolerio, F., Nardone, E., Siccardi, A. G., and Paganelli, G. (1998) Biochemical modifications of avidin improve pharmacokinetics and biodistribution, and reduce immunogenicity. Br. J. Cancer 78, 189-197. (10) Coloma, M. J., and Morrison, S. L. (1997) Design and production of novel tetravalent bispecific antibodies. Nat. Biotechnol. 15, 159-163. (11) Cremonesi, M., Ferrari, M., Chinol, M. Stabin, M. G., Grana, C., Prisco, G., Robertson, C., Tosi, G., and Paganelli, G. (1999) Three-step radioimmunotherapy with yttrium-90

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BC0000379

Experimental Pretargeting Studies of Cancer with a Humanized anti

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