1054
Bioconjugate Chem. 2002, 13, 1054−1070
Pretargeting for Cancer Radioimmunotherapy with Bispecific Antibodies: Role of the Bispecific Antibody’s Valency for the Tumor Target Antigen H. Karacay,† R. M. Sharkey,*,† W. J. McBride,‡ G. L. Griffiths,‡ Z. Qu,‡ K. Chang,§ H. J. Hansen,‡ and D. M. Goldenberg† Center for Molecular Medicine and Immunology, Belleville, New Jersey 07109, Immunomedics, Inc., Morris Plains, New Jersey 07950, and IBC Pharmaceuticals, Inc. Morris Plains, New Jersey 07950. Received February 28, 2002; Revised Manuscript Received June 3, 2002
The use of a divalent effector molecule improves bispecific antibody (bsMAb) pretargeting by enabling the cross-linking of monovalently bound bsMAb on the cell surface, thereby increasing the functional affinity of a bsMAb. In this work, it was determined if a bsMAb with divalency for the primary target antigen would improve bsMAb pretargeting of a divalent hapten. The pretargeting of a 99mTc-labeled divalent DTPA-peptide, IMP-192, using a bsMAb prepared by chemically coupling two Fab′ fragments, one with monovalent specificity to the primary target antigen, carcinoembryonic antigen (CEA), and to indium-loaded DTPA [DTPA(In)], was compared to two other bsMAbs, both with divalency to CEA. One conjugate used the whole anti-CEA IgG, while the other used the anti-CEA F(ab′)2 fragment to make bsMAbs that had divalency to CEA, but with different molecular weights to affect their pharmacokinetic behavior. The rate of bsMAb blood clearance was a function of molecular weight (IgG × Fab′ < F(ab′)2 × Fab′ < Fab′ × Fab′ conjugate). The IgG × Fab′ bsMAb conjugate had the highest uptake and longest retention in the tumor. However, when used for pretargeting, the F(ab′)2 × Fab′ conjugate allowed for superior tumor accretion of the 99mTc-IMP-192 peptide, because its more rapid clearance from the blood enabled early intervention with the radiolabeled peptide when tumor uptake of the bsMAb was at its peak. Excellent peptide targeting was also seen with the Fab′ × Fab′ conjugate, albeit tumor uptake was lower than with the F(ab′)2 × Fab′ conjugate. Because the IgG × Fab′ bsMAb cleared from the blood so slowly, when the peptide was given at the time of its maximum tumor accretion, the peptide was captured predominantly by the bsMAb in the blood. Several strategies were explored to reduce the IgG × Fab′ bsMAb remaining in the blood to take advantage of its 3-4fold higher tumor accretion than the other bsMAb conjugates. A number of agents were tested, including those that could clear the bsMAb from the blood (e.g., galactosylated or nongalactosylated anti-id antibody) and those that could block the anti-DTPA(In) binding arm [e.g., DTPA(In), divalentDTPA(In) peptide, and DTPA coupled to bovine serum albumin (BSA) or IgG]. When clearing agents were given 65 h after the IgG × Fab′ conjugate (time of maximum tumor accretion for this bsMAb), 99m Tc-IMP-192 levels in the blood were significantly reduced, but a majority of the peptide localized in the liver. Increasing the interval between the clearing agent and the time the peptide was given to allow for further processing of the bsMAb-clearing agent complex did not improve targeting. At the dose and level of substitution tested, galacosylated BSA-DTPA(In) was cleared too quickly to be an effective blocking agent, but BSA- and IgG-DTPA(In) conjugates were able to reduce the uptake of the 99mTc-IMP-192 in the blood and liver. Tumor/nontumor ratios compared favorably for the radiolabeled peptide using the IgG × Fab′/blocking agent combination and the F(ab′)2 × Fab′ (no clearing/blocking agent), and peptide uptake 3 h after the blocking agent even exceeded that of the F(ab′)2 × Fab′. However, this higher level of peptide in the tumor was not sustained over 24 h, and actually decreased to levels lower than that seen with the F(ab′)2 × Fab′ by this time. These results demonstrate that divalency of a bsMAb to its primary target antigen can lead to higher tumor accretion by a pretargeted divalent peptide, but that the pharmacokinetic behavior of the bsMAb also needs to be optimized to allow for its clearance from the blood. Otherwise, blocking agents will need to be developed to reduce unwanted peptide uptake in normal tissues.
INTRODUCTION
Pretargeting methods have become an important strategy for conveying targeting specificity to isotopes and drugs for a variety of applications, including tumor * To whom correspondence should be addressed. Phone: (973) 844-7121. Fax: (973) 844-7020. † Center for Molecular Medicine and Immunology. ‡ Immunomedics, Inc.. § IBC Pharmaceuticals, Inc..
imaging and therapy (Chang et al., 2002; Goldenberg, 2002). Unlike direct targeting methods that rely on the association of the effector molecule directly with the antibody, a pretargeting method separates the effector molecule (e.g., isotope or drug) from the primary targeting agent (Goodwin and Meares, 1999). The primary targeting agent in a pretargeting method must have binding specificity for the target and bear a receptor capable of binding to the effector molecule. The vast majority of primary targeting agents used in pretargeting
10.1021/bc0200172 CCC: $22.00 © 2002 American Chemical Society Published on Web 08/31/2002
Pretargeting with Bispecific Antibodies
have been antibodies directed to different tumor antigens. Many effector-binding systems employed by pretargeting methods have used the avidin/streptavidin-biotin recognition system, while others have used bispecific antibodies (bsMAbs)1 and even oligonuclide binding systems (Bos et al., 1994; Cremonesi et al., 1999; Goodwin et al., 1992; Hnatowich et al., 1987; Paganelli et al., 1988; Rusckowski et al., 1997a; Stickney et al., 1991). As initially evaluated by Hnatowich et al. (1987), the avidin-biotin pretargeting method can be configured according to several strategies, but two approaches have prevailed. One approach uses a streptavidin-conjugated antibody as the primary targeting agent, which is then followed by a derivatized biotin as the effector carrier. A third step is used to facilitate the clearance of the primary streptavidin-antibody conjugate from the blood prior to administering the biotin-effector molecule (Axworthy et al., 1994, 2000). The other strategy uses a biotinconjugated antibody as the primary targeting agent (Cremonesi et al., 1999; Paganelli et al., 1988). After allowing sufficient time for the antibody-biotin conjugate to localize the tumor, avidin is given. Because it is highly glycosylated, avidin acts primarily as a clearing agent, binding to the biotin-antibody in the blood and removing it to the liver. Although some avidin could also localize to the tumor-bound biotin-conjugated antibody, streptavidin is also given. Streptavidin is not glycosylated, and therefore it has a longer residence time in the blood, which allows more time for it to localize to the biotinconjugated antibody. Finally, the biotin-effector molecule is given, which then localizes to the biotin-conjugated antibody containing the streptavidin/avidin bridge. Both of these approaches have been used clinically and shown to produce high tumor/background ratios (Breitz et al., 2000; Magnani et al., 2000), and have even been used therapeutically (Breitz et al., 1999, 2000; Knox et al., 2000; Paganelli et al., 2001). Another pretargeting approach relies on the use of bsMAb. The principal behind pretargeting was initially described by Reardon et al. (1985), who suggested that antibodies could be composed of dual binding specificity, one to a particular target and the other capable of binding a chelate. This group subsequently showed that the passive accumulation of an anti-chelate antibody in mice bearing the transplantable KHJJ tumor (Goodwin et al., 1988) could be used to target a radiolabeled chelate. A “chase” step using transferrin-conjugated chelate was introduced prior to administering the radiolabeled chelate as a means of reducing the anti-chelate antibody in the blood. More recently, this same passive targeting model was used to show the feasibility of pretargeting 90Ylabeled chelate for therapy (Lubic et al., 2001). Subsequently, tumor-targeting specificity has been achieved with bispecific antibodies directed against tumor markers and, for the most part for the targeting of radionuclides 1 Abbreviations: AES, affinity enhancement system; BSA, bovine serum albumin; bsMAb, bispecific antibody; c734, chimeric 734 anti-(In)DTPA antibody; CEA, carcinoembryonic antigen; DTPA, diethylenetriaminepentaacetic acid; gal rWI2, galactosylated rWI2; hMN-14, humanized MN-14 anti-CEA antibody; HPLC, high-pressure liquid chromatography; HSA, human serum albumin; IACUC, Institutional Animal Care and Use Committee; ID, injected dose; (In)DTPA, indium-loaded DTPA; RAIT, radioimmunotherapy; RF, reverse phase; rWI2, a rat anti-idiotype monoclonal antibody to MN-14; s.c., subcutaneously; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SE, size-exclusion; SMCC, sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate; T/NT, tumor/ nontumor ratio; TscGc, thiosemicarbazonylglyoxylcysteinyl group.
Bioconjugate Chem., Vol. 13, No. 5, 2002 1055
by means of antibodies to metal chelators. Bispecific antibodies can be conveniently produced by chemically combining Fab′ fragments of one antibody with specificity to a tumor antigen with another antibody specific for the effector molecule. Effector molecules initially were monovalent metal-binding chelates (Goodwin et al., 1988), but then it was shown that a divalent effector molecule greatly enhances the residence time of the effector molecule at the site (Le Doussal et al., 1989). This method was called an affinity enhancement system (AES), since the bivalent effector molecule bridges two bispecific antibodies on the tumor cell surface, thereby increasing their functionally affinity [reviewed by Chang et al. (2002)]. Others have subsequently verified the advantage of a divalent effector molecule over a monovalent form (Goodwin et al., 1992; Boerman et al., 1999). The bsMAb and avidin-biotin pretargeting approaches have their advantages and disadvantages. For example, the avidin-biotin system is subject to potential blockade by endogenous biotin. This is especially problematic when testing in mouse models, since mice have nearly 10 times the amount of biotin in their serum as humans (Rosebrough and Hartley, 1995; Rusckowski et al., 1997b). Using a biotin-deficient diet, the stores of biotin in their bodies can be minimized. Another potential problem with the avidin-biotin strategy is the immunogenic nature of avidin and streptavidin. Antibodies to avidin and streptavidin can develop, which could affect the subsequent use of these agents after an initial exposure. The greatest advantage of the avidin-biotin approach is the strength of the binding between avidin or streptavidin and biotin, such that once biotin is bound to avidin or streptavidin, these complexes are highly stable (Hnatowich et al., 1987). In contrast, the bispecific antibody approach combined with a monovalent effector molecule has a higher dissociation rate. Using a divalent effector molecule greatly enhances the cell binding but, still, antibody dissociation rates are typically several log units higher than that of the avidin-biotin complex (e.g., KD ) 10-15 M for biotin with streptavidin/avidin vs 10-9 M most typically found for antigen-antibody complexes). In this regard, it is important to emphasize that the residence time in the tumor for both bsMAb and avidin/biotin pretargeting approaches is limited by the agent bound to the cell target, i.e., the affinity of an antibody. Since many of the bispecific antibody-pretargeting studies have used bsMAbs with monovalent binding arms (i.e., chemically cross-linked Fab′ × Fab′ or hybrid IgG produced from a quadroma), the bridging of two bsMAbs by a divalent effector molecule was an important binding enhancement for these complexes at the primary target site. However, would a bispecific antibody with divalent binding to the primary target antigen increase tumor uptake of a radiolabeled peptide while maintaining excellent tumor/nontumor ratios typically associated with pretargeting methods? Increasing the amount of the peptide targeting to a tumor would be highly advantageous for use in radioimmunotherapy (RAIT). To test this hypothesis, a pretargeting system involving bispecific antibodies to carcinoembryonic antigen (CEA) and diethylenetriaminepentaacetic acid (DTPA), together with a divalent indium-loaded DTPA [(In)DTPA] peptide capable of binding 99mTc, was used (Karacay et al., 2000). Bispecific antibodies were prepared by chemically conjugating the IgG, F(ab′)2, and Fab′ component of a humanized antibody to CEA to the Fab′ fragment of a chimeric anti-(In)DTPA antibody (c734). A 99mTc-labeled divalent (In)DTPA peptide (IMP-192) was used as the
1056 Bioconjugate Chem., Vol. 13, No. 5, 2002
effector molecule to assess each bsMAb conjugate’s utility for pretargeting. MATERIALS AND METHODS
Antibodies. hMN-14 IgG was prepared as described (Sharkey et al., 1995). The F(ab′)2 of hMN-14 was prepared by pepsin digestion, and purified using protein A and ion-exchange chromatography. A chimeric version of the murine 734 anti-(In)DTPA antibody originally described by Le Doussal et al. (1990) was prepared using the VH and Vλ sequences of murine 734, which were linked to human γ1 and κ constant domain sequences, respectively, using methods that have been described previously (Leung et al., 1994, 1995). The antigen-binding activity of c734 was determined with ELISA using (In)DTPA-BSA-coated wells, and was found to be identical to murine 734. The F(ab′)2 fragment of c734 was prepared by pepsin digestion. The fragment was purified by ammonium sulfate precipitation and then by size exclusion chromatography on a Superdex 200 (Amersham, Pharmacia Bio, Piscataway, NJ) column. Bispecific Antibodies and Other Conjugates. The hMN-14 IgG × c734 Fab′ bsMAb was chemically prepared by cross-linking the Fab′-SH fragment of c734 to maleimide-derivatized hMN-14 IgG. The maleimide group was introduced by modification of the hMN-14 IgG lysine residues with 3×-molar excess of sulfosuccinimidyl 4-(Nmaleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC, Molecular Biosciences, Boulder, CO) at pH 7.2 for 55 min at room temperature. The excess sulfo-SMCC was removed by centrifuged size exclusion chromatography (Penefsky, 1979; Meares et al., 1984) using Sephadex G-50-80 in 50 mM sodium acetate, 0.5 mM EDTA, pH 5.3. c734 Fab′-SH was produced by reduction of the F(ab′)2 fragment in 10 mM mercaptoethylamine, 20 mM HEPES, 150 mM NaCl, 10 mM EDTA, pH 7.2, at 37 °C for 1 h. Centrifuged size exclusion chromatography was used to isolate the Fab′-SH (c734) and IgG-mal (hMN14) intermediates. The IgG × Fab′ conjugate was produced by reaction of a 1:1 mixture of the IgG-mal and Fab′-SH components at pH 5.3 for 1 h at room temperature. Sodium tetrathionate was added to the conjugation mixture to a final concentration of 6 mM. After 5 min at room temperature, excess sodium tetrathionate was removed by centrifuged size exclusion chromatography. The product was isolated following purification by affinity chromatography (Affigel-DTPA column) to isolate antibodies capable of binding DTPA, and then by size exclusion chromatography (Superdex 200, Amersham, Pharmacia Bio, Piscataway, NJ) to separate the anti-CEA × anti-DTPA bsMAb from anti-DTPA Fab′ or possibly F(ab′)2 that might have formed. The hMN-14 F(ab′)2 × c734 Fab′ bsMAb preparation was similar to IgG × Fab′. F(ab′)2 fragment of hMN-14 was treated with 3.5-molar excess of sulfo-SMCC at pH 7.3 for 55 min to modify lysine residue(s) to introduce maleimide groups. Fab′-SH fragment of c734 was prepared as described above. The F(ab′)2-mal and Fab′-SH intermediates were purified on centrifuged size exclusion columns. The F(ab′)2 × Fab′ conjugate was formed by reaction of a 1:1 mixture of the two fragments at room temperature for 1.5 h at pH 5.3. The conjugation mixture was treated with 5 mM sodium tetrathionate, and the excess sodium tetrathionate was removed by centrifuged size exclusion chromatography. The reaction mixture was applied to Affigel-DTPA column to remove impurities containing hMN-14 only. To isolate the pure conjugate,
Karacay et al.
the c734-containing portion from the Affigel-DTPA column was purified by size-exclusion chromatography (Superdex 200). The hMN-14 Fab′ × c734 Fab′ bsMAb was prepared by the chemical coupling of the two reduced Fab′-SH fragments using N,N′-o-phenylenedimaleimide (PDM) by a standard literature method (Glennie et al., 1987). Briefly, MAbs were separately reduced to their Fab′-SH fragments by incubation with 10 mM 2-mercaptoethylamine at 37 °C for 1 h. The c734-Fab′-SH was converted to c734-Fab′-maleimide by reaction with 4 mM PDM at room temperature for 1 h. Centrifuged size exclusion chromatography was used to isolate the Fab′-SH (hMN14) and Fab′-maleimide (c734) intermediates. Crosslinking of hMN-14-Fab′-SH to c734-Fab′-maleimide proceeded for 95 min at room temperature at a 1:1 molar ratio. The bsMAb was isolated after purification by DTPA affinity chromatography and by size exclusion chromatography (Superdex-200). The purity and specificity of the bsMAbs were analyzed by SDS-PAGE (reducing and nonreducing conditions), as well as by SE-HPLC. SE-HPLC was used to examine the chromatographic properties of the bsMAbs alone and when mixed with an excess of CEA and after mixing with 111 In-labeled IMP-156, another divalent DTPA peptide (Karacay et al., 2000), or both CEA and the radiolabeled peptide. In both assays, the percentage of product failing to shift to a higher MW on SE-HPLC (shown by in-line monitoring of either UV or radioactivity) indicated the level of monospecific MAbs present in the preparations. DTPA-conjugated bovine serum albumin (BSA) and DTPA-IgG, used as blocking/clearing agents, were prepared according to published procedures (Hnatowich et al., 1982). BSA and diethylenetriaminepentaacetic acid anhydride were obtained from Sigma (St. Louis, MO). The IgG was humanized LL2 anti-CD22 (Leung et al., 1995). MALDI-mass spectral analysis determined there were four DTPA conjugated to the BSA, and eight DTPA per IgG. The BSA- and IgG-DTPA conjugates were preloaded with indium on the day of their use by adding 20-times mole excess of indium to a prescribed amount of each conjugate. Rat WI2 (rWI2) is an anti-idiotype antibody that is reactive with the hMN-14 IgG (Losman et al., 1994). It, along with a galactosylated conjugate of this antibody, has been used as a clearing agent to remove MN-14 IgG or MN-14 IgG-streptavidin conjugates from the blood (Karacay et al., 1997; Sharkey et al., 1997). BSA-DTPA and rWI2 were conjugated with galactose (43:1 and 49:1 mole substitution, respectively) using the methodology reported by others (Lee et al., 1976; Mattes, 1987). The ability of rWI2 and gal-rWI2 and BSA- and IgG-DTPA(In) conjugates to bind to hMN-14 × 734 bsMAb was tested by SE-HPLC. A molar excess of each agent was mixed with 125I-labeled bsMAb and after a brief incubation period the mixture was loaded on a Bio-Sil SE 250 size-exclusion HPLC column (BioRad, Richmond, CA). In each instance, the resulting product was shifted to a higher molecular weight than that of the 125I-bsMAb alone. Radiolabeling. IMP-192, a divalent DTPA peptide containing a thiosemicarbonylglyoxyl-cysteinyl group (TscGc) for radiolabeling with 99mTc, was prepared as previously reported by Karacay et al. (2000). For 99mTcradiolabeling, IMP-192 was formulated in individually lyophilized vials that contained 50 µg of the peptide with Sn(II) for pertechnetate reduction and a 6-fold excess of nonradioactive indium to peptide for binding to DTPA. These vials were reconstituted with up to 77 mCi of
Pretargeting with Bispecific Antibodies
Bioconjugate Chem., Vol. 13, No. 5, 2002 1057
99m
Tc-pertechnetate (Mallinckrodt, Pine Brook, NJ) in 1.5 mL of saline. After 10 min at room temperature, the vials were heated in a water bath for 15 min. Quality assurance checks of the 99mTc-labeled IMP-192 included instant thin-layer chromatography (ITLC), which showed >95% of the radioactivity bound to the peptide, as well as an incubation with a mole excess of the bsMAb, which always showed >95% of the radioactivity shifted to a higher molecular weight peak by size-exclusion, highpressure liquid chromatography (SE-HPLC). The bsMAb was radiolabeled with Na125I (PerkinElmer, Boston, MA) by the chloramine-T method (Greenwood and Hunter, 1963) to a specific activity of 10-14 mCi/mg. Quality assurance of the radiolabeled bsMAb included ITLC, SE-HPLC, and immunoreactive fraction. All radiolabeled bsMAb preparations had to have e 5% unbound radioiodine by ITLC, g85% of the product migrating at the expected SE-HPLC elution volume for the bsMAb (i.e., no appreciable aggregation or other products), and an immunoreactive fraction when mixed with CEA >85%. The immunoreactive fraction of the bsMAb was tested against CEA by mixing 0.5 µCi of the radiolabeled bsMAb with 5 × the moles of CEA (Scripps Laboratories, San Diego, CA) for 5 min at room temperature. The dual specificity of the bsMAbs for CEA and DTPA was also spot-checked by incubating the 125IbsMAb with both CEA and another divalent DTPApeptide, IMP-156, radiolabeled with 111In (Karacay et al., 2000). In Vitro Stability and Binding Studies. The in vitro stability of the bsMAbs in human serum was tested by incubating the 125I-labeled bsMAb at 37 °C in fresh, sterile-filtered serum taken from a healthy donor. Samples were analyzed by SE-HPLC before and after mixing with CEA. The ability of the bsMAbs to retain binding ability to the peptide and CEA was analyzed using size-exclusion HPLC and radiolabeled bsMAbs and peptides. 99mTclabeled peptide was diluted in 1.0% HSA and injected onto HPLC alone, and immediately after mixing with variable amounts of bsMAb. To demonstrate binding of the radiolabeled peptide to the antigen-bsMAb complex, bsMAb was mixed with CEA before addition of the radiolabeled peptide. Animal Model. All animal studies were carried out with IACUC approved protocols. Female nude mice (Taconic, Germantown, NY) approximately 6-8 weeks old were inoculated subcutaneously (s.c.) with a 10% suspension of the serially transplanted GW-39 human colonic cancer cell line (Goldenberg et al., 1966; Goldenberg and Hansen, 1972). After allowing 2-3 weeks for tumor growth, the animals were given an intravenous injection of the bsMAb conjugate that contained a trace amount of 125I-bsMAb so that its distribution properties could be evaluated. In all studies, a constant amount, 1.5 × 10-10 mol, of the bsMAb was administered (15, 22.5, and 30 µg of the Fab′ × Fab′, F(ab′)2 × Fab′, and IgG × Fab′ conjugates, respectively). After waiting a defined period of time based on the conjugate used, the 99mTcIMP-192 peptide was given at a constant bsMAb:peptide mole ratio of 10:1 based on the moles of bsMAb administered. Animals were then scheduled for necropsy at 3 and/or 24 h after the peptide was given. A number of studies were performed with the IgG × Fab′ bsMAb conjugate using a variety of clearing or blocking agents at a prescribed time after the bsMAb’s injection. The timing of the administration of these agents after the bsMAb, the dose administered, and the time allotted before the 99mTc-IMP-192 peptide was given are provided for each study under Results. The dosage of these agents
Figure 1. Size-exclusion HPLC chromatograms of the hMN14 × c734 bsMAbs (IgG × Fab′ bsMAb, left panel; F(ab′)2 × Fab′ bsMAb, middle panel; Fab′ × Fab′ bsMAb, right panel) showing the radioactivity traces for 111In-IMP-156 alone (A), when mixed with the bsMAb (B), and mixed with the bsMAb after addition of CEA (C). The bsMAb was added in a 20-fold mole excess to the 111In-IMP-156 (row B). A 5-fold mole excess of CEA was added to the bsMAb followed by the addition of the peptide (20:1, bsMAb/peptide ratio).
was based on a prescribed molar ratio in relation to the average amount of bsMAb in the blood as determined from samples taken by retroorbital bleeding of a minimum of three anesthetized animals, using the specific activity of the 125I-bsMAb to estimate its concentration. At the scheduled time of necropsy, groups of animals (n ) 4-5) were anesthetized, approximately 0.2 mL of blood was removed by cardiac puncture, and then they were euthanized prior to dissection. Organs were removed, weighed, and placed in glass vials for counting 125 I- and 99mTc-activity. Windows were set to exclude 125I from the 99mTc-window, and samples were first counted for their 99mTc content and then after allowing 2 days for decay, recounted for 125I. For each counting session, standards were placed along with the tissues to calculate the total injected radioactivity. The amount of radioactivity in each tissue was first divided by the weight of the tissue and then by the total radioactivity injected to derive the percent injected dose per gram of tissue (% ID/g). Tumor to nontumor tissue ratios (T/NT) were derived by dividing the percent injected dose per gram of the tumor by the percentage in the respective tissue. A ratio of the % ID/g of 99mTc-IMP-192 peptide in each tissue compared to the % ID/g of 125I-bsMAb (peptide/ bsMAb ratio) was also calculated in a similar manner. Statistical analysis of these data was performed using an ANOVA with a two-tailed F-test. RESULTS
In Vitro and in Vivo Properties of ChemicallyStabilized bsMAb Constructs. BsMAb composed of hMN-14 IgG, F(ab′)2, or Fab′ chemically cross-linked to the Fab′ fragment of c734 were prepared, and according to SE-HPLC and SDS-PAGE, each conjugate contained the anti-CEA and anti-DTPA antibody at a 1:1 mole ratio. Figure 1 shows the SE-HPLC chromatograms of the bsMAb preparations when mixed in a molar excess with 111 In-IMP-156 divalent-DTPA peptide alone or with CEA and then the peptide. All of the peptide shifted to a higher molecular weight consistent primarily with the formation of complexes composed of 2 mol of bsMAb/peptide, and with a smaller fraction of bsMAb with 1 mol of peptide (Figure 1, row B). When added to the bsMAb after CEA addition, the complexes uniformly migrated as a higher molecular weight (Figure 1, row C). This shows that all of the antibody in each bsMAb preparation that is capable of binding the DTPA-peptide is also able to bind to CEA.
1058 Bioconjugate Chem., Vol. 13, No. 5, 2002 Table 1. Biodistribution of
125I-Labeled
Karacay et al. hMN-14 × c734 bsMAb Constructs Alonea % ID/g
4h tumor liver kidney blood tumor wt (g)
2.71 ( 1.33 2.81 ( 0.42 2.93 ( 0.45 10.2 ( 1.2 0.18 ( 0.112
tumor liver kidney blood tumor wt (g)
2.61 ( 0.60 2.20 ( 0.35 2.86 ( 0.47 7.00 ( 0.94 0.31 ( 0.288
tumor liver kidney blood tumor wt (g)
2.86 ( 0.53 1.43 ( 0.50 3.98 ( 0.59 3.31 ( 0.44 0.24 ( 0.115
day 1
day 2
IgG × Fab′ 7.42 ( 2.04 12.8 ( 2.7 1.31 ( 0.15 1.01 ( 0.09 1.62 ( 0.23 1.42 ( 0.29 5.67 ( 0.74 3.89 ( 0.44 0.20 ( 0.128 0.32 ( 0.029 F(ab′)2 × Fab 2.59 ( 0.81 2.52 ( 0.71 0.40 ( 0.04 0.20 ( 0.02 0.44 ( 0.03 0.31 ( 0.08 0.74 ( 0.10 0.19 ( 0.03 0.31 ( 0.097 0.44 ( 0.304 Fab′ × Fab′ 1.46 ( 0.33 0.58 ( 0.19 0.15 ( 0.01 0.07 ( 0.01 0.28 ( 0.08 0.12 ( 0.03 0.12 ( 0.01 0.04 ( 0.01 0.42 ( 0.08 0.30 ( 0.103
day 3
day 7
11.8 ( 1.6 0.80 ( 0.06 0.95 ( 0.12 3.01 ( 0.17 0.34 ( 0.054
5.92 ( 1.50 0.23 ( 0.07 0.32 ( 0.11 0.77 ( 0.32 0.80 ( 0.318
1.37 ( 0.24 0.12 ( 0.02 0.18 ( 0.03 0.06 ( 0.01 0.44 ( 0.146
ND
0.37 ( 0.11 0.06 ( 0.01 0.12 ( 0.03 0.02 ( 0.01 0.47 ( 0.128
ND
a Nude mice bearing GW-39 human colon cancer xenografts were injected i.v., with 1.5 × 10-10 mol of each bsMAb that contained a tracer amount (10 µCi) of the 125I-bsMAb. Animals were necropsied at the times indicated, and the percentage of 125I-bsMAb in the tissues shown was determined. Values represent the means ( SD (n ) 5 per group).
Bispecific antibody conjugates placed in sterile-filtered mouse serum for 3 days were examined for stability by size-exclusion HPLC. After 3 days, 93%, 86%, and 76% of the IgG, F(ab′)2, and Fab′ bsMAb conjugates shifted to a higher molecular weight fraction when mixed with CEA. The distribution and clearance properties of the bsMAbs in tumor-bearing nude mice followed a predictable behavior based on the molecular size of the conjugates; e.g., the larger the construct, the slower it cleared from the blood (Table 1). Tumor uptake was highest for the IgG × Fab′ construct, but it took 2 days before it reached its maximum accretion level of 12.8 ( 2.7% ID/g. The maximum accretion in the tumor of the F(ab′)2 × Fab′ and the Fab′ × Fab′ conjugates was observed at the earliest sampling time (4 h after the bsMAb injection). The F(ab′)2 × Fab′ construct maintained this level for a period of 2 days, whereas the Fab′ × Fab′ conjugate steadily declined over the next 3 days, sustaining a loss of about 50% each day. The IgG × Fab′ conjugate stayed at its highest level for at least 2 days (days 2 and 3 after bsMAb injection), but by day 7 had decreased to 50% of its maximum level. The Fab′ × Fab′ fragment had somewhat higher renal uptake at 4 h than the other bsMAb constructs, but none of the constructs had sustained renal retention. This is most likely explained by the fact that all of these conjugates are a molecular size too large to be cleared directly by renal excretion, and because these were all radioiodinated by conventional means, any iodinated catabolic product would be rapidly filtered though the kidneys rather than retained in any major organ (e.g., liver). Pretargeting 99mTc-IMP-192 with Each of the bsMAb Conjugates. Our earlier experience (Karacay et al., 2000) indicated that optimal pretargeting occurred when sufficient time was given for the bsMAb conjugate to clear from the blood prior to giving the radiolabeled peptide. Based on the data in Figure 1, a clearance time of 6 days, 49 h, and 19 h was selected for the initial testing of the IgG ×, F(ab′)2 ×, and Fab′ × Fab′ conjugates, respectively. The protein dosage of the bsMAb was adjusted so that each conjugate was administered in equal mole amounts [1.5 × 10-10 mol; the molecular weights were assumed to be 200 000 for the IgG × Fab′, 150 000 for the F(ab′)2 × Fab′, and 100 000 for the Fab′
× Fab′]. After allowing the allotted time for bsMAb clearance, 99mTc-IMP-192 was administered (1.5 × 10-11 mol; i.e., a bsMAb/peptide ratio of 10:1). Animals were then necropsied at 3 and 24 h after the peptide injection. As shown in Table 2, the 125I-IgG × Fab′ bsMAb had the highest concentration in the tumor at 21.2 ( 9.7% ID/g 3 h after the peptide was injected, compared to the F(ab′)2 × Fab′ conjugate, which was only 3.88 ( 0.66% ID/g, and the Fab′ × Fab′ at 1.45 ( 0.35% ID/g. At the time intervals selected, blood concentrations of the F(ab′)2 × Fab′ and Fab′ × Fab′ conjugates were similar, but the blood concentration of the IgG × Fab′ was nearly 10-fold higher. Thus, despite having a higher concentration of bsMAb in the tumor, the peptide uptake was higher for the F(ab′)2 × Fab′ and Fab′ × Fab′ conjugates than the IgG × Fab′. These results suggest that the peptide was more likely to bind to the IgG × Fab′ bsMAb in the blood, which could have reduced its availability for binding to the tumor. The ratio of the amount of peptide to the amount of bsMAb localized in the tumor (peptide/bsMAb ratio) was determined to illustrate the peptide’s ability to localize with the bsMAb (see Table 2). For example, the peptide/bsMAb ratio was only 0.34 ( 0.15 for the IgG × Fab′ conjugate, which was 7 times lower than the peptide/bsMAb ratio for the F(ab′)2 × Fab conjugate (2.38 ( 0.56) and 11 times lower than that observed for the Fab′ × Fab′ conjugate (3.79 ( 0.16). The IgG × Fab′ conjugate only had 1 mol of peptide for every 37 mol of bsMAb, whereas the F(ab′)2 × Fab′ and Fab′ × Fab′ conjugate in the tumor had 1 mol of peptide for every 3-4 mol of bsMAb. Thus, the mole substitution level for the F(ab′)2 × Fab′ and Fab′ × Fab′ conjugates was close to the theoretically ideal ratio for an affinity enhancement model, i.e., 1 mol of peptide for every 2 mol of bsMAb. Although there were significant differences in the peptide/bsMAb ratios in the blood among the three groups, unlike the ratios seen in the tumor, the magnitude separating them was no greater than a factor of 2.0. It was estimated, using the radiotracers to determine the number of bsMAb for each mole of peptide in the blood at 3 h, that there was 4.4, 6.3, and 2.6 mol of the IgG ×, F(ab′)2 ×, and Fab′ × Fab′ conjugates, respectively. The comparison of the peptide/bsMAb in the blood for these three conjugates may provide some insight into the
Pretargeting with Bispecific Antibodies
Bioconjugate Chem., Vol. 13, No. 5, 2002 1059
Table 2. Pretargeting Using Various Forms of hMN-14 × c734 bsMAb Conjugates and 3 hb % bsMAb/g tumor liver kidney blood tumor wt (g)
21.2 ( 9.7 0.93 ( 0.29 0.89 ( 0.32 2.71 ( 0.59
tumor liver kidney blood tumor wt (g)
3.88 ( 0.66 0.23 ( 0.03 0.23 ( 0.04 0.33 ( 0.05
tumor liver kidney blood tumor wt (g)
1.45 ( 0.35 0.19 ( 0.03 0.31 ( 0.04 0.29 ( 0.03
% peptide/g
99mTc-IMP-192
Peptidea
24 hb T/NT pep
pep/bsMAb
% bsMAb/g
IgG × Fab′ (6 Day Interval) 6.04 ( 0.86 0.34 ( 0.15 26.0 ( 10.4 8.15 ( 2.31 0.81 ( 0.34 8.97 ( 0.98 0.74 ( 0.09 3.57 ( 0.98 1.84 ( 0.71 ND 0.82 ( 0.19 6.24 ( 1.96 1.07 ( 0.45 2.28 ( 0.32 2.51 ( 0.34 0.38 ( 0.112 F(ab′)2 × Fab′ (49 h Interval) 9.00 ( 1.40 2.38 ( 0.56 2.12 ( 0.58 0.34 ( 0.05 27.3 ( 7.3 1.50 ( 0.11 0.11 ( 0.01 1.90 ( 0.28 4.88 ( 1.24 ND 0.16 ( 0.02 0.52 ( 0.10 18.1 ( 5.3 1.59 ( 0.21 0.12 ( 0.03 0.15 ( 0.023 Fab′ × Fab′ (19 h Interval) 5.47 ( 1.23 3.79 ( 0.16 1.58 ( 0.24 0.36 ( 0.04 15.1 ( 2.7 2.26 ( 0.71 0.10 ( 0.01 2.40 ( 0.31 2.30 ( 0.52 ND 0.17 ( 0.03 1.11 ( 0.11 4.90 ( 0.99 3.60 ( 1.22 0.08 ( 0.01 0.11 ( 0.028
% peptide/g
T/NT pep
pep/bsMAb
10.0 ( 2.5 3.85 ( 0.66 2.68 ( 0.85 2.59 ( 0.46 4.07 ( 1.57 3.19 ( 0.50 3.21 ( 0.93 0.32 ( 0.108
0.46 ( 0.26 4.61 ( 1.60 ND 1.68 ( 0.96
6.39 ( 1.86 0.21 ( 0.03 30.3 ( 9.5 0.74 ( 0.12 8.75 ( 2.65 0.15 ( 0.04 46.2 ( 19.3 0.25 ( 0.183
3.24 ( 1.19 1.94 ( 0.12 ND 1.21 ( 0.19
3.75 ( 0.52 0.18 ( 0.02 21.6 ( 3.6 0.64 ( 0.10 5.94 ( 1.12 0.34 ( 0.02 10.9 ( 1.5 0.19 ( 0.058
2.39 ( 0.10 1.75 ( 0.07 ND 4.22 ( 0.51
a Data include the percent injected dose per gram for 125I-bsMAb and 99mTc-peptide, the tumor/nontumor ratio for 99mTc-peptide (T/NT pep), and the ratio of the % bsMAb and % peptide in the tissues (pep/bsMAb). 1.5 × 10-10 mol of bsMAb and 1.5 × 10-11 mol of 99mTcIMP-192 were given for each conjugate. Values represent mean ( SD; n ) 5 for each observation. The interval is the amount of time between the bsMAb and peptide injections. b Time after 99mTc-IMP-192 given.
relative efficiency of each conjugate for binding the peptide in vivo. For example, in the case of the F(ab′)2 × Fab′ and the Fab′ × Fab conjugates, the concentration of bsMAb in the blood, as measured by the 125I-bsMAb, was not significantly different (p ) 0.105), yet the amount of peptide found in the blood at 3 h was significantly higher with the Fab′ (p ) 0.023), and the difference between the peptide/bsMAb ratios for these two groups was significantly different at a level of p ) 0.007. This assessment suggests that the Fab′ × Fab′ bsMAb conjugate was able to bind more avidly to the peptide than the F(ab′)2 × Fab′ conjugate. This is not unexpected since the chemical coupling process of the hMN-14 Fab′ and the c734 Fab′ more effectively orients the twp molecules by binding each molecule at its hinge region. Because the Fab′ is coupled to lysine groups on the IgG and F(ab′)2, it can be randomly positioned on the molecule, which could affect its ability to bind to the peptide. Although the IgG × Fab′ conjugate had higher peptide/bsMAb ratio in the blood compared to the F(ab′)2 × Fab′, it is more likely that this occurred because the IgG × Fab′ conjugate had nearly 10 times the amount of bsMAb in the blood at the time the peptide was given, rather than an indication of it being more capable of avidly binding the peptide than the F(ab′)2 × Fab′. Thus, this assessment can only apply to the F(ab′)2 × Fab′ and Fab′ × Fab′ conjugates, since the blood concentration at the time of the injection was the same. It is also important to point out that Karacay et al. (2000) had previously shown 99mTc-IMP-192 was rapidly cleared from the blood, and since there is no detectable binding of the peptide to other serum proteins, such as albumin, as determined by SEHPLC of 99mTc-IMP-192 incubated in plasma (not shown), it is reasonable to assume that any peptide remaining in the blood is most likely bound to the bsMAb. There was also a substantial amount of 99mTc-IMP-192 in the liver with the IgG × Fab′ conjugate. This could have been a consequence of peptide binding directly to the bsMAb in the liver, but it is also possible that a portion of the liver uptake was a consequence of either mono- or dimeric complexes of the bsMAb formed with the peptide in the blood, which were then transported to the liver. Renal uptake of the 99mTc)IMP-192 alone (i.e., without pretargeting) was previously shown to be about
2.0% ID/g (Karacay et al., 2000). In a pretargeting setting, renal uptake is not much higher unless there is a substantial level of bsMAb in the blood. This suggests that a substantial portion of the bsMAb is not itself sequestered in the kidneys, leading to peptide localization. Twenty-four hours after the peptide injection, the tumor uptake of the 99mTc-IMP-192 increased from 6.0 ( 0.9 to 10.0 ( 2.4% ID/g for the IgG × Fab′ conjugate, while it decreased for the other conjugates. Some of this increase could have been due to the subsequent tumor localization of the IgG × Fab′-99mTc-IMP-192 complexes that were formed in the blood. Indeed, the bsMAb99m Tc-IMP-192 complexes in the blood at 3 h after the peptide injection were more highly laden with peptide, having 1 mol of peptide for every 4.3 mol of bsMAb, than the bsMAb in the tumor, which had only 1 mol of peptide for every 37 ( 22 mol of bsMAb. As a consequence of higher tumor accretion of the peptide with lower amounts in the blood, the 99mTc-IMP192 tumor/blood ratios for the F(ab′)2 × Fab′ and Fab′ × Fab′ bsMAb were higher than the IgG × Fab′ conjugate (Table 2). Although there was some improvement in the IgG × Fab′ conjugate by 24 h, the tumor/blood ratios never exceeded that seen with the other 2 conjugates. Thus, despite having the highest level of bsMAb uptake in the tumor, the higher concentration of IgG × Fab′ in the blood at the time of the peptide injection inhibited tumor accretion. Since the peptide was given to animals when the amount of the F(ab′)2 × Fab′ and Fab′ × Fab′ conjugate in the blood were the same, the higher tumor uptake of the F(ab′)2 × Fab′ resulted in this conjugate having the highest tumor/nontumor ratios for the peptide under these targeting conditions. Examination of Clearing and Blocking Agents. Although the targeting results described above favored the F(ab′)2 × Fab′ conjugate, the fact that the IgG × Fab′ conjugate had nearly 5 times the concentration of bsMAb in the tumor as the F(ab′)2 × Fab′ conjugate meant that it might be possible to enhance peptide accretion in the tumor further if the blood concentration of the IgG × Fab′ could be reduced to a lower level at the time the peptide was given. A number of strategies were examined to determine a means of maintaining a high amount of IgG
a hMN-14 IgG × c734 Fab′ (1.5 × 10-10 mol; 10 µCi) was given intravenously. Forty-eight hours later, the clearing/blocking agent was given, and after 3 h, the 99mTc-IMP-192 (1.5 × 10-11 mol) was given. Three hours later, the animals were necropsied, and the 125I-bsMAb and 99mTc-IMP-192 radioactivity levels were determined. b gal-rWI2 was given at a 5:1 mole ratio, and In-DTPA was given at a 1:1 mole ratio based on the amount of bsMAb in blood at the time of their administration. The group that was not given a clearing/blocking agent received the 99mTc-IMP-192 51 h after the bsMAb and necropsied 3 h later. Values represent the means ( SD with 5 animals per group.
pep/bsMAb
0.14 ( 0.03 2.19 ( 0.18 ND 1.18 ( 0.15 2.1 ( 0.39 5.8 ( 0.63 0.36 ( 0.05 3.2 ( 0.63 0.64 ( 0.13 11.3 ( 2.9 0.19 ( 0.05 0.53 ( 0.09 16.5 ( 7.9 2.7 ( 0.35 3.1 ( 0.59 9.5 ( 1.40 0.19 ( 0.03 2.20 ( 0.19 ND 1.12 ( 0.20 2.6 ( 0.53 6.9 ( 1.18 0.39 ( 0.14 3.7 ( 0.31 0.71 ( 0.11 11.1 ( 1.5 0.23 ( 0.02 0.80 ( 0.22 14.1 ( 3.3 3.1 ( 0.33 3.5 ( 0.50 10.1 ( 1.0 0.09 ( 0.03 1.54 ( 0.19 ND 0.49 ( 0.08 1.5 ( 0.43 14.3 ( 2.1 0.10 ( 0.03 1.2 ( 0.18 1.24 ( 0.32 0.9 ( 0.28 1.84 ( 0.87 0.37 ( 0.12 16.0 ( 3.5 9.3 ( 1.01 1.4 ( 0.35 1.8 ( 0.50 tumor liver kidney blood tumor wt (g)
T/NT peptide % ID/g peptide % ID/g bsMAb
% ID/g peptide
T/NT peptide
pep/bsMAb
% ID/g bsMAb
% ID/g peptide
T/NT peptide
pep/bsMAb
% ID/g bsMAb
no clearing/blocking agentb In-DTPA (Blocking)b gal-rat WI2 (clearing)b
Table 3. Assessment of Pretargeting Using the hMN-14 IgG × c734 Fab′ bsMAb with or without Clearing/Blocking Agents Prior to the Administration of
× Fab′ bsMAb in the tumor while removing or blocking it in the blood. Clearing Agents. We previously used galactosylated and nongalactosylated rat WI2 anti-MN-14 idiotype antibody as a clearing agent for an avidin-biotin pretargeting procedure (Karacay et al., 1997; Sharkey et al., 1997). Thus, these clearing agents were examined in combination with the hMN-14 IgG × c734 Fab′ in an attempt to improve pretargeting with the 99mTc-IMP-192 peptide. In the first studies, galactosylated rWI2 (galrWI2) was given 48 h after the bsMAb at a 5:1 mole ratio based on the concentration of bsMAb in the blood immediately prior to its administration. After waiting 3 h to allow for bsMAb clearance, 99mTc-IMP-192 was given, and then 3 h later, animals were necropsied to determine the distribution of the bsMAb and peptide. As shown in Table 3, gal-rWI2 was effective in clearing the bsMAb from the blood, reducing it from 9.5 ( 1.4% ID/g seen in the group that did not receive any clearing agent to 1.8 ( 0.50% ID/g in the group given gal-rWI2 clearing agent. With a lower amount of bsMAb in the blood, animals given the gal-rWI2 had only 0.9 ( 0.28% ID/g of the peptide in the blood, whereas the group that was not given the clearing agent had 11.3 ( 2.9% ID/g of the injected peptide in the blood. Despite the significant reduction of peptide binding in the blood, tumor uptake of the peptide for the animals given the gal-rWI2 was not any higher than that of the control animals. Liver accretion of the peptide, however, increased significantly in the animals given the gal-rWI2 (14.3 ( 2.1 vs 5.8 ( 0.6% ID/g for the control; p < 0.001), suggesting that bsMAb had been transported to the liver by the clearing agent, but had not been sufficiently processed to prevent its binding to the peptide, which in turn reduced the availability of the peptide for tumor localization. Therefore, despite improving tumor/blood ratios as compared to the control group (1.84 ( 0.87 vs 0.19 ( 0.05), the tumor/nontumor ratios and amount of peptide targeted to the tumor were not higher than those seen with the F(ab′)2 × Fab′ or Fab′ × Fab′ bsMAbs. It was also noted that the peptide/bsMAb ratio in the blood for the control group was 1.18 ( 0.15 (representing 1 peptide for every 8.4 mol of bsMAb). In contrast, the animals given the gal-rWI2 had a peptide/bsMAb ratio of 0.49 ( 0.08 (1 mol of peptide for every 20 mol of bsMAb in the blood). This substantially lower peptide/bsMAb ratio could be a result of less favorable kinetics for peptide binding to the lower amount of bsMAb in the blood of the gal-rWI2 group, or steric hindrance created by the binding of the gal-rWI2 to the bsMAb. In the next study, galactosylated and nongalactosylated rWI2 were independently evaluated as clearing agents, and additional modifications were made in an attempt to reduce the amount of bsMAb that would be present at the time the 99mTc-IMP-192 was given. In this regard, the IgG × Fab′ bsMAb was allowed to clear for 65 h (rather than 48 h) before giving the clearing agents, and the radiolabeled peptide was not given until either 5 or 24 h after the clearing agents (rather than 3 h). Increasing the interval between the clearing agent and the peptide injection was intended to allow more time for the bsMAb-anti-id antibody complexes diverted to the liver to be processed, and therefore inaccessible at the time the peptide is given. Immune complexes cleared by the gal-rWI2 and nongalactosylated rWI2 would also be expected to localize to different cell types in the liver, which could also influence the processing of the bsMAb and peptide. The same amount of bsMAb and peptide were given as before (1.5 × 10-10 and 1.5 × 10-11 mol,
Karacay et al. 99mTc-IMP-192a
1060 Bioconjugate Chem., Vol. 13, No. 5, 2002
Pretargeting with Bispecific Antibodies
Bioconjugate Chem., Vol. 13, No. 5, 2002 1061
Table 4. Evaluation of Galactosylated and Nongalactosylated Rat Anti-MN-14 Idiotype Antibody, rWI2, as a Clearing Agent for Improving Pretargeting Using hMN-14 IgG × c734 Fab′ bsMAba rWI2, 5 h clearance tumor liver kidney blood tumor wt (g)
% bsMAb/g
% peptide/g
T/NT pep
13.7 ( 4.1 5.20 ( 1.77 1.26 ( 0.33 2.31 ( 0.71
3.12 ( 0.74 17.9 ( 4.2 0.19 ( 0.09 2.88 ( 0.52 1.11 ( 0.31 1.41 ( 1.05 3.07 ( 2.02 0.44 ( 0.396
% bsMAb/g
% peptide/g
rWI2, 24 h clearance pep/bsMAb
% bsMAb/g
% peptide/g
0.25 ( 0.09 3.59 ( 0.72 ND 0.65 ( 0.59
7.25 ( 2.14 0.98 ( 0.57 0.28 ( 0.12 0.61 ( 0.30
7.16 ( 1.68 6.97 ( 3.83 1.66 ( 1.70 2.19 ( 0.43 3.41 ( 1.25 1.50 ( 1.09 9.25 ( 9.92 0.49 ( 0.185
pep/bsMAb
% bsMAb/g
% peptide/g
0.27 ( 0.09 2.38 ( 0.40 ND 0.57 ( 0.39
7.64 ( 1.54 2.66 ( 0.47 0.37 ( 0.15 0.55 ( 0.39
pep/bsMAb
% bsMAb/g
% peptide/g
0.31 ( 0.06 4.87 ( 0.86 ND 2.11 ( 0.63
14.8 ( 6.5 1.67 ( 0.68 1.35 ( 0.46 4.71 ( 1.82
3.45 ( 1.3 10.4 ( 5.3 0.45 ( 0.32 3.41 ( 0.65 1.07 ( 0.55 12.4 ( 7.2 0.40 ( 0.33 0.61 ( 0.550
gal-rWI2, 5 h clearance tumor liver kidney blood tumor wt (g)
10.9 ( 2.77 9.72 ( 1.78 0.62 ( 0.11 0.88 ( 0.46
T/NT pep
2.84 ( 0.66 23.0 ( 4.9 0.13 ( 0.06 1.19 ( 0.14 2.42 ( 0.57 0.47 ( 0.29 10.4 ( 11.2 0.44 ( 0.077
tumor liver kidney blood tumor wt (g)
% peptide/g
T/NT pep
11.7 ( 2.7 2.63 ( 0.73 2.43 ( 0.18 7.21 ( 1.68
3.60 ( 1.20 13.1 ( 4.9 0.29 ( 0.18 5.49 ( 0.89 0.57 ( 0.04 15.5 ( 6.0 0.18 ( 0.04 0.86 ( 0.281
pep/bsMAb 1.08 ( 0.47 7.90 ( 4.55 ND 2.87 ( 2.86
gal-rWI2, 24 h
no clearing agent, 70 h (n ) 4) % bsMAb/g
T/NT pep
T/NT pep
7.27 ( 1.49 17.3 ( 2.4 0.43 ( 0.14 1.91 ( 0.58 3.95 ( 0.67 1.00 ( 1.13 19.0 ( 18.8 0.82 ( 0.293
pep/bsMAb 0.97 ( 0.23 6.66 ( 1.47 ND 1.48 ( 0.80
no clearing agent, 90 h T/NT pep
pep/bsMAb 0.27 ( 0.14 5.97 ( 0.97 ND 2.60 ( 1.2
a Nude mice bearing GW-39 tumors were given an intravenous injection of hMN-14 IgG × c734 Fab′ bsMAb (1.5 × 10-10 mol) that contained a trace amount of 125I-bsMAb. Sixty-five hours later, the clearing agent was given intravenously, and then after waiting either 5 or 24 h, 99mTc-IMP-192 (1.5 × 10-11 mol) was administered. Animals (n ) 5, except where indicated) were necropsied 3 h later, and the amount of 125I-bsMAb and 99mTc-IMP-192 was determined. Control animals not given any clearing agent received the 99mTc-IMP-192 at either 70 or 90 h after the bsMAb injection.
respectively), and animals were all necropsied 3 h after the peptide injection. The peptide was also given to animals that did not receive a clearing agent at either 70 or 90 h after the bsMAb injection so the time of necropsy would coincide closely with the animals given the clearing agents for comparison. Table 4 shows the % ID/g for the bsMAb and peptide, tumor/nontumor ratios for the peptide, and the peptide/bsMAb ratios obtained in these groups. Delaying the 99mTc-IMP-192 injection until 70 and 90 h after the bsMAb was given reduced the blood concentration of the bsMAb to 7.21 ( 1.68 and 4.71 ( 1.82% ID/g, respectively, compared to 9.5 ( 1.40% ID/g seen in the previous study. However, the 99mTc-IMP-192 continued to show higher concentrations of the peptide in the blood and liver than the tumor. Both the rWI2 and galrWI2 effectively reduced the blood concentration of the IgG × Fab′ bsMAb, with the gal-rWI2 showing a greater ability to reduce the bsMAb level in the blood more quickly than the rWI2. For example, the bsMAb in blood was 2.31 ( 0.71% ID/g for the rWI2 group, which was about 4-fold lower than the group that was given no clearing agent (7.11 ( 1.68 at 70 h), but the blood concentration of the bsMAb in animals gal-WI2 was reduced to 0.88 ( 0.46% ID/g. When the period between the time the clearing agent and the peptide was extended to 24 h, then the level of bsMAb in the blood of galactosylated and nongalactosylated rWI2 was the same (0.55 ( 0.39 and 0.61 ( 0.30% ID/g, respectively), which was about 8-fold lower than if no clearing agent was given (4.71 ( 1.82% ID/g). It was also interesting to note that in the groups receiving the clearing agents, the ratio of peptide/bsMAb in the blood for the 5 h clearance group was 0.57 to 0.65, but with a 24 h clearance interval, this ratio increased to 1.48 to 2.87. This indicates that the bsMAb in the blood at the earlier time was not as receptive to binding the peptide as the bsMAb in the blood after 24 h, supporting the possibility of steric hindrance.
Tumor uptake of the bsMAb in the treated groups with a 5 h clearance time and control group (70 h) was the same, but despite having lower blood concentrations of the bsMAb, the animals given the clearing agents had the same amount of 99mTc-IMP-192 in the tumor as the control animals. In each of the treated groups, there was considerable peptide uptake in the liver with highly unfavorable tumor/liver ratios. Increasing the interval between the clearing agent and the time the peptide was given to 24 h reduced the bsMAb concentration in the blood to levels that were comparable to those seen with the F(ab′)2 × Fab′ conjugate (0.61 ( 0.30, 0.55 ( 0.39, and 0.33 ( 0.15% ID/g for rWI2 and gal rWI2, and F(ab′)2 × Fab′, respectively). Under these conditions, tumor uptake of the 99mTc-IMP-192 was 7.16 ( 1.68 and 7.27 ( 1.49% ID/g for the rWI2 and gal-rWI2, respectively, which was not significantly different (p ) 0.156) to the peptide uptake seen with the F(ab′)2 × Fab′ (9.0 ( 1.4% ID/g). Although tumor/blood ratios in animals given the clearing agents with a 24 h interval before administering the peptide were highly variable, the individual animal data revealed that when the blood concentration of the bsMAb was reduced to about 0.2-0.4% ID/g, tumor/blood ratios were between 13 and 26, thus being comparable to that achieved with the F(ab′)2 × Fab′ conjugate. However, there continued to be considerable liver accretion of the peptide, most noticeably with the gal-WI2 clearing agent. In conclusion, tumor/liver ratios for the 99m Tc-IMP-192 using IgG × Fab′ bsMAb with either clearing agent and a 24 h interval were much lower than that seen with the F(ab′)2 × Fab′ bsMAb (i.e.,
