Pretargeting of human mammary carcinoma xenografts with bispecific anti-MUC1/anti-Ga chelate antibodies and immunoscintigraphy with PET.

We recently demonstrated the feasibility of combining enhanced tumor-to-tissue contrast and PET imaging for immunoscintigraphic tumor localization in pancreas and colon carcinoma bearing nude mice. Contrast enhancement was obtained with a multistep targeting technique that consists of the sequential administration of an antitumor/antihapten bispecific antibody (BS-MAb), a blocker to saturate the antihapten binding sites of the BS-MAb that remains in circulation, and a low molecular weight Ga chelate, labeled with the positron emitter 68Ga, which serves as the hapten. To evaluate the efficacy of this pretargeting technique for breast cancer localization, we synthesized a BS-MAb from the F(ab')(2) fragments of the anti-MUC1 MAb 12H12 which reacts with the vast majority of human breast carcinomas, and the F(ab') fragment of an anti-Ga chelate MAb using a bifunctional chemical linker. The BS-MAb was tested for its affinity and its biokinetics in nude mice bearing a human mammary carcinoma. Equilibrium binding of the BS-MAb for mammary carcinoma cells was low (1.2 x 10(7) M(-1)) while the binding capacity of cells was high (8.4 x 10(6) BS-MAbs per cell). Tumor uptake of the 67Ga labeled chelate in pretargeted animals was to 5.8 +/- 0.8% iD/g resulting in a tumor-to-blood ratio of 2.6 at 1h postinjection. This compares with a ratio of 0.65 and 0.85 obtained with 125I-labeled native 12H12 at 24h and 48h postinjection. No difference in the tumor uptake of both the 68Ga and 67Ga labeled chelate was observed. PET imaging of mice, started 1h postinjection of the 68Ga chelate, clearly visualized all tumors.

Gallium-68 chelate imaging of human colon carcinoma xenografts pretargeted with bispecific anti-CD44V6/anti-gallium chelate antibodies.

UNLABELLED: Recently, we demonstrated the feasibility of combining improved tumor-to-tissue contrasts and PET imaging for immunoscintigraphic tumor localization using a multistep targeting technique that consists of the administration of an antitumor/antihapten bispecific monoclonal antibody (BS-MAb), a blocker to saturate the antihapten binding sites of the BS-MAb that are still present in the circulation, and a low molecular weight Ga chelate, labeled with positron emitter 68Ga, serving as the hapten. Due to this technique, the biodistribution of the radiolabeled hapten is governed mainly by the binding characteristics of both the antitumor and the antihapten part of the BS-MAb. For a future clinical implementation of the method, we investigated MAb VFF18, which is reactive with the adhesion molecule CD44V6, a tumor-associated antigen, and up-regulated in colon, squamous cell and pancreas carcinoma, and two anti-Ga chelate MAbs, which are highly selective for only one of the two enantiomers (optical isomers) of the inherently racemic Ga chelate. METHODS: From the VFF18 MAb and the anti-Ga chelate MAbs, two BS-MAbs containing the same antitumor parts, but different antihapten parts, were prepared and tested for multistep targeting in human colon carcinoma-bearing nude mice. RESULTS: Despite identical biodistributions of both BS-MAbs and their very similar affinities for the corresponding Ga chelate enantiomers, tumor uptake of the two enantiomers 1 hr postinjection was significantly different [8.7 +/- 1.9% versus 5.8% +/- 1.6% of the injected dose/g (%i.d./g)], with tumor-to-blood ratios being higher for the BS-MAb showing the lower tumor uptake (7.6 +/- 1.6 versus 4.7 +/- 0.6). From data obtained with each BS-MAb, a similar initial tumor binding of approximately 15.5%i.d./g, but different in vivo half-lives of the corresponding BS-MAb-enantiomer immune complexes, could be estimated. Pretargeting with a mixture of both BS-MAbs followed by the administration of the racemic Ga chelate resulted in the lowest tumor uptake (3.9% +/- 1.5%i.d./g). PET imaging of nude mice with the enantiomeric, as well as with the racemic, 68Ga chelate demonstrated a clear delineation of tumors against blood pool background. CONCLUSION: Multistep immunoscintigraphy with BS-MAbs markedly increases tumor-to-tissue ratios in nude mice and enables PET imaging. Using a BS-MAb containing MAb VFF18, a more sensitive localization of CD44V6-positive tumors in patients should also be obtained.

[Immunoscintigraphy of breast cancer xenografts. 99m-Tc-labeled anti-mucin monoclonal antibodies BM-7 and 12H12]. / Immunszintigraphie xenotransplantierter Mammakarzinome. 99mTc-markierte monoklonale Anti-Muzinantikörper BM-7 und 12H12.

The present study was undertaken to evaluate the correlation of the favorable in vitro characteristics of the anti-mucin Mabs 12H12 and BM-7 with high tumor accumulation in vivo. They were labeled with 99mTc; their biodistribution in nude mice bearing mammary tumor xenograft AR was examined and immunoscintigraphy was performed after 24 h. 99mTc-labeling of the Mabs 12H12 and BM-7 led to tumor uptakes of 20.7% and 8.8% ID/g, respectively, after 48 h. Tumor-to-muscle ratios were 31 (12H12) and 18 (BM-7). Tumor xenografts were clearly visualized in immunoscintigrams. Combination of Mab 12H12 and 99mTc provides high tumor-to-tissue ratios shortly after administration.

Multistep tumor targeting in nude mice using bispecific antibodies and a gallium chelate suitable for immunoscintigraphy with positron emission tomography.

To improve tumor:tissue ratios in immunoscintigraphy, a three-step targeting method has been developed. The reagents used were (a) a radioactive, low molecular weight chelate prepared from ionic gallium and a phenolic polyaminocarboxylic acid, which can be labeled either with the single-photon emitter 67Ga or with the short-lived positron emitter 68Ga (t1/2 = 68 min); (b) a bispecific monoclonal antibody (bs-mAb) synthesized from the F(ab)2 fragment of the 1.1ASML antibody specific for the glycoprotein CD44v associated with a rat pancreas carcinoma cell line and the F(ab') fragment of an antibody specific for the gallium chelate; and (c) the nonradioactive gallium chelate covalently coupled to transferrin, which served as a high molecular weight blocker to prevent binding of the radioactive gallium chelate to bs-mAbs in the circulation. Targeting experiments in tumor-bearing nude mice with different doses of bs-mAbs, blocker, and 67Ga chelate were adjusted to maximize tumor to tissue contrasts and tumor uptake. Compared with the biodistribution of the 131I-labeled, native 1.1ASML antibody 24 h postinjection, a schedule using 100 pmol bs-mab 24 h later 100 pmol blocker, 15 min later 16 pmol 67Ga chelate, 1 h later examination, increased tumor:blood and tumor: liver ratios by a factor of 5 while keeping the localization of radioactivity in the tumor constant (10.1% injected dose/g). High-contrast images using either 67Ga or 68Ga were obtained within 1 h. The targeting method described enables the use of the short-lived positron emitter 68Ga and thus allows the combination of an improved immunoscintigraphy and positron emission tomography.

Immunoscintigraphy of human mammary carcinoma xenografts using monoclonal antibodies 12H12 and BM-2 labeled with 99mTc and radioiodine.

For immunoscintigraphic localization of human breast cancer two monoclonal antibodies (mabs) 12H12 (immunoglobulin G1) and BM-2 (immunoglobulin G3) were developed. The mabs, directed against two different epitopes on the mucin glycoprotein TAG-12, showed reactivity with 96% of all primary mammary carcinomas. The antibodies were labeled with either 125I or 131I. In addition, 12H12 was directly labeled with 99mTc according to the method of Schwarz and Steinsträsser (A. Schwarz and A. Steinsträsser, J. Nucl. Med., 28:721, 1987). Biodistribution was measured in female nude mice bearing the human mammary carcinoma SF-15. Both radioiodinated mabs showed similar biodistribution with fast tumor uptake (8.5% injected dose/g at 6 h postinjection), which increased to 10-11% injected dose/g at 24 h and subsequently remained constant up to 120 h. 99mTc-Labeling of the mab 12H12 led to an enhanced tumor uptake of 10.5 and 14% injected dose/g at 6 and 24 h postinjection, respectively, and to significantly accelerated blood clearance of radioactivity. Similar results were obtained with a second mammary tumor (AR-1), while an endometrial tumor (EK-3) showed a 3-fold lower accumulation of radioactivity and no difference in uptake of radio-iodinated and 99mTc-labeled 12H12. Scintigraphic imaging of tumor-bearing nude mice with the 99mTc 12H12 at 24 h postinjection clearly demonstrated a diagnostic potential of the new mab for tumor localization and staging.

A bifunctional HBED-derivative for labeling of antibodies with 67Ga, 111In and 59Fe. Comparative biodistribution with 111In-DPTA and 131I-labeled antibodies in mice bearing antibody internalizing and non-internalizing tumors.

To investigate whether bifunctional ligands containing chelating structures other than EDTA and DTPA and metallic radiotracers other than 111In will reduce the non-specific radioactivity uptake in the liver during immunoscintigraphy, we synthetized an isothiocyanato-substituted phenolic polyaminocarboxylic acid (HBED-CI) for labeling of MAbs with 67Ga, 111In and 59Fe. Biodistribution of HBED-CI-labeled MAbs was compared to that of 131I and 111In-DTPA labeled MAbs in nude mice bearing tumors, which differ with regard to intracellular internalization and catabolism of the corresponding MAb-antigen complex. In the liver a continuous radioactivity excretion for 67Ga-HBED-CI-labeled MAbs was observed with kinetics that parallel 131I clearance after administration of 131I-MAbs, while 111In-HBED-CI-labeling led to a constant 111In liver level quite similar to that of 111In-DTPA-MAbs. In tumors, 67Ga-HBED-CI-MAb uptake again paralleled that of 131I-MAbs, showing continuous accumulation in tumor tissues when internalization of the MAb-antigen complex was not involved. A much lower uptake, which peaked between 24 and 48 h, was found in the case of MAb-antigen internalization. 111In of 111In-HBED-CI- and 111In-DTPA-labeled MAbs continuously accumulated in both types of tumors. Compared with 111In-DTPA-MAbs, an improvement in tumor-to-liver ratios, due to the reduced liver radioactivity associated with 67Ga-HBED-CI-labeled MAbs, could only be obtained with non-internalizing tumors. The time course of radioactivity distribution in the liver and in MAb-internalizing tumors after administration of 67Ga-HBED-CI-, 111In-HBED-CI- and 111In-DTPA-labeled MAbs further indicates a dominating influence of the metallic radiotracer rather than the ligand on retention or excretion of radioactivity in MAb-catabolizing tissues.

Uptake of indium-111 in the liver of mice following administration of indium-111-DTPA-labeled monoclonal antibodies: influence of labeling parameters, physiologic parameters, and antibody dose.

Liver uptake of indium-111 (111In) in mice was investigated following administration of 111In-DTPA murine monoclonal antibodies (111In-DTPA-MAbs) labeled by the cyclic anhydride method. Biodistribution of HPLC-purified 111In-DTPA-MAb preparations was checked with a low (0.2 micrograms) and a high (8.0 micrograms) MAb dose. Using Bio Gel P-30 for desalting the MAb-conjugates, 111In uptake in the liver amounted to 8%-9% of the injected dose (ID) and was independent from the MAb dose, the DTPA-to-MAb molar ratio, tumor growth and biologic variability (different MAbs and different strains of mice). Using Sephadex G-25 for desalting, 0.2 micrograms doses from 7 out of 26 preparations showed increased liver accumulation of 111In in non-tumor mice ranging from 15%-25% of ID. Corresponding high doses led to a "normal" value of 8%-9%. Increased liver uptake of the low dose could not be reduced by coadministration of the unconjugated MAb, but was normal after reinjection of "in vivo filtered" material. An inverse intracellular distribution of 111In activity between sediment and supernatant of liver homogenates, following the administration of the low and the high MAb dose, indicated an artifact of the labeling procedure rather than an inherent biological property of labeled MAbs.

Labeling of monoclonal antibodies with a 67Ga-phenolic aminocarboxylic acid chelate. Part I. Chemistry and labeling technique.

As a chelating agent for labeling antibodies (Abs) with metallic radionuclides, a propionic acid substituted ethylenediamine N,N'-di-[(o-hydroxyphenyl) acetic acid] (P-EDDHA), which tightly complexes 67Ga, was synthesized. The 67Ga-P-EDDHA chelate was coupled in aqueous solution to IgG at a molar ratio of 1:1 via carbodiimide. The average coupling yield was 15%. A specific activity of 4 mCi/mg IgG could be obtained with commercially supplied 67Ga. In vitro stability was evaluated in human serum at 37 degrees C and showed a half-life of about 120 h for the release of 67Ga from the labeled Ab during the initial phase of incubation. This in vitro halflife is similar to that measured for 111In-DTPA labeled Abs. Because of the high stability of the 67Ga-P-EDDHA chelate, the in vivo formation of radioactive labeled transferrin by transchelation, as described for 111In-DTPA labeled Abs, should, however, be reduced by this labeling technique.

The pharmacokinetics of thyrotropin-releasing hormone (TRH) and deamido-TRH in the rat.

The pharmacokinetics of TRH and its metabolite deamido-TRH (TRH-OH) were evaluated and compared. After a 60- to 90-min infusion of the appropriate peptide into rats, the MCR, half-life of disappearance (t 1/2), and volume of distribution were calculated. The MCR of TRH averaged 4 ml/min, whereas that of TRH-OH was 6.5 ml/min. The serum concentrations of both TRH and TRH-OH decreased in a biexponential manner after stopping the corresponding peptide infusions. The t 1/2 values were 2.4 and 3.9 min for the initial phase of disappearance and 14.1 and 20.6 min for the later phase of disappearance for TRH and TRH-OH, respectively. The volume of distribution of TRH-OH was twice that of TRH. The fractional conversion rate of TRH to TRH-OH, based on serum TRH-OH concentrations measured during TRH infusions, was 0.7-1.4%. Less than 1 ng TRH-OH was excreted in the urine during the last 30 min of TRH infusion. The rapid clearance of TRH-OH and low fractional conversion of TRH to TRH-OH, as measured in serum, suggest that little TRH secretion will be reflected in serum TRH-OH concentrations.

Antineoplastic activity of ASTA Z 7557 (INN mafosfamide) in transplanted and autochthonous experimental rodent tumors.

The antineoplastic activities of ASTA Z 7557 and cyclophosphamide (CPA) were compared in advanced transplanted AKR lymphoma by determining the optimal dose using single dose and twofold applications. Autochthonous DMBA-induced leukemias and MNU-induced mammary carcinomas were treated with fractionated doses over 3 and 5 weeks, respectively. In the respective optimal dosages ASTA Z 7557 exhibited an antitumor effect comparable to that of CPA in all three models. The results obtained by treatment of the autochthonous models indicate that Z 7557 seems to have advantages over CPA in the treatment of malignancies with impaired bone marrow function as for instance acute leukemias and in fractionated dose schedules.

A Ga-68-labeled tetrabromophthalein (Ga-68 BP-IDA) for positron imaging of hepatobiliary function: concise communication.

We describe the chemical synthesis of an iminodiacetic-acid-substituted tetrabromo-o-cresolphthalein (BP-IDA), which complexes Ga-68 tightly. The liver uptake, bile excretion, and urinary excretion of the complex were examined in rats. Maximum liver uptake reached 60%, and 1-hr cumulative bile excretion was 75% of injected dose. Urinary excretion in rats with ligated common bile duct remained below 1%. Competitive action of exogenous bilirubin on hepatobiliary excretion of the Ga complex was less pronounced than that of bromosulfophthalein. The absolute activity determination of the positron emitter Ga-68, the high accumulation in the liver, the low urinary excretion, and the weak competition from exogenous bilirubin are promising features of this radiopharmaceutical for the quantitative study of hepatobiliary function.