Paul S. Plascjak

A new and convenient method for purification of 86Y using a Sr(II) selective resin and comparison of biodistribution of 86Y and 111In labeled Herceptin

A simple and rapid procedure was developed for purification of cyclotron produced 86Y via the 86Sr(p,n) 86Y reaction. A commercially available Sr(II) selective resin was used to separate 86Y from the cyclotron irradiated Sr(II) target with a recovery of the enriched Sr(II) target while yielding a 75-80% recovery of 86Y suitable for radiolabeling either proteins or peptides. To demonstrate the utility of this methodology, the anti-HER2 monoclonal antibody Herceptin was radiolabeled with the purified 86Y and compared to 111In labeled Herceptin. The biodistribution study demonstrated that 111In-Herceptin, while a suitable surrogate for 90Y in the major organs, did not parallel the uptake of 86Y-Herceptin in the bone, and thus may not accurately predict the level of 90Y accumulation in the bone for clinical RIT applications. This result exemplifies the requirement of employing appropriate matched pair isotopes for imaging and therapy to insure that dosimetry considerations may be addressed accurately.

Effective Treatment of Established Human Breast Tumor Xenografts in Immunodeficient Mice with a Single Dose of the α- Emitting Radioisotope Astatine-211 Conjugated to Anti-HER2/ neu Diabodies

Purpose: Successful radioimmunotherapy strategies depend on selecting radioisotopes with physical properties complementary to the biological properties of the targeting vehicle. Small, engineered antitumor antibody fragments are capable of rapid, highly specific tumor targeting in immunodeficient mouse models. We hypothesized that the C6.5 diabody, a noncovalent anti-HER2 single-chain Fv dimer, would be an ideal radioisotope carrier for the radioimmunotherapy of established tumors using the short-lived α-emitting radioisotope 211At. Experimental Design: Immunodeficient nude mice bearing established HER2/neu–positive MDA-MB-361/DYT2 tumors treated with N-succinimidyl N-(4-[211At]astatophenethyl)succinamate (211At-SAPS)-C6.5 diabody. Additional cohorts of mice were treated with 211At-SAPS T84.66 diabody targeting the carcinoembryonic antigen or 211At-SAPS on a diabody specific for the Müllerian inhibiting substance type II receptor, which is minimally expressed on this tumor cell line. Results: A single i.v. injection of 211At-SAPS C6.5 diabody led to a 30-day delay in tumor growth when a 20 μCi dose was administered and a 57-day delay in tumor growth (60% tumor-free after 1 year) when a 45 μCi dose was used. Treatment of mice bearing the same tumors with 211At-SAPS T84.66 diabody at the same doses led to a delay in tumor growth, but no complete responses, likely due to substantially lower expression of this antigen on the MDA-MB-361/DYT2 tumors. In contrast, a dose of 20 μCi of 211At-SAPS on the anti–Müllerian-inhibiting substance type II receptor diabody did not affect tumor growth rate, demonstrating specificity of the therapeutic effect. Conclusions: These findings indicate that diabody molecules can be effective agents for targeted radioimmunotherapy of solid tumors using powerful, short-lived α-emitting radioisotopes.

Semi-remote production of [64Cu]CuCl2 and preparation of high specific activity [64Cu]Cu-ATSM for PET studies

Summary The PET radionuclide Cu-64 (t1/2 = 12.7 h) can be easily produced utilizing the nuclear reaction 64Ni(p,n)64Cu. A high Cu-64 production rate was obtained using high-purity enriched nickel metal on the internal tangential target of the CS-30 cyclotron. Radio-copper was isolated after column chromatography using a semi-remote apparatus. Use of reagents with little or no carrier copper yields high specific activity Cu-64 pharmaceuticals prepared from the isolated [64Cu]CuCl2. [64Cu]Cu-ATSM was prepared in greater than 1.21±0.40 Ci/mmol (45±15 GBq/mmol) at EOB.

Preparation of 211At-Labeled Humanized Anti-Tac Using 211At Produced in Disposable Internal and External Bismuth Targets

These studies describe the production and purification of 211At as well as the procedure for labeling humanized anti-Tac, the antibody to the alpha-chain of the IL-2 receptor (IL-2R alpha), which has been shown to be a useful target for immunotherapy. The optimized protocol combines the advantages of the two-stage dry distillation procedure with the astatination of trialkylstannyl substances as labeling compounds for proteins. The 211At was produced by bombarding either an external or a recently developed disposable internal bismuth target with alpha-particles from a Cyclotron Corporation CS-30 cyclotron. The 211At was found to contain less than 0.01% 210At. The production rate for the external target was 0.15 mCi +/- 0.056 microA(-1) h(-1) (n = 9) (5.55 MBq mcroA[-1] h[-1]). The production rate for the internal target was 0.44 +/- 0.14 mCi microA(-1) h(-1) (n = 16) (16.28 MBq mcroA[-1] h[-1]).

The Anti-CD25 Monoclonal Antibody 7G7/B6, Armed with the α-Emitter 211At, Provides Effective Radioimmunotherapy for a Murine Model of Leukemia

Radioimmunotherapy of cancer with radiolabeled antibodies has shown promise. alpha-Particles are very attractive for cancer therapy, especially for isolated malignant cells, as is observed in leukemia, because of their high linear energy transfer and short effective path length. We evaluated an anti-CD25 [interleukin-2 receptor alpha (IL-2R alpha)] monoclonal antibody, 7G7/B6, armed with (211)At as a potential radioimmunotherapeutic agent for CD25-expressing leukemias and lymphomas. Therapeutic studies were done in severe combined immunodeficient/nonobese diabetic mice bearing the karpas299 leukemia and in nude mice bearing the SUDHL-1 lymphoma. The results from a pharmacokinetic study showed that the clearance of (211)At-7G7/B6 from the circulation was virtually identical to (125)I-7G7/B6. The biodistributions of (211)At-7G7/B6 and (125)I-7G7/B6 were also similar with the exception of a higher stomach uptake of radioactivity with (211)At-7G7/B6. Therapy using 15 microCi of (211)At-7G7/B6 prolonged survival of the karpas299 leukemia-bearing mice significantly when compared with untreated mice and mice treated with (211)At-11F11, a radiolabeled nonspecific control antibody (P < 0.01). All of the mice in the control and (211)At-11F11 groups died by day 46 whereas >70% of the mice in the (211)At-7G7/B6 group still survived at that time. In summary, (211)At-7G7/B6 could serve as an effective therapeutic agent for patients with CD25-expressing leukemias.

Pretargeted α Emitting Radioimmunotherapy Using 213Bi 1,4,7,10-Tetraazacyclododecane-N,N′,N″,N‴-Tetraacetic Acid-Biotin

Purpose: The use of an α emitter for radioimmunotherapy has potential advantages compared with β emitters. When administered systemically optimal targeting of intact antibodies requires >24 h, therefore limiting the use of short-lived α emitters. This study investigated the biodistribution of bismuth-labeled biotin in A431 tumor-bearing mice pretargeted with antibody B3-streptavidin (B3-SA) and examined the therapeutic efficacy of the α emitter, 213Bi-labeled biotin. Experimental Design: Biotinidase-resistant 7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA)-biotin was radiolabeled with 205,206Bi or 213Bi. Treatment of tumor-bearing mice began by administration of B3-SA (400 μg) to target the tumor sites for 24 h. Then, an agent containing biotin and galactose groups was used to clear the conjugate from the circulation. Four h later, bismuth-radiolabeled DOTA-biotin was given, and biodistribution or therapy was evaluated. Dose escalation treatment from 3.7–74 MBq was performed, and the effects on tumors of different sizes were investigated. Tumor growth, complete blood cell counts, toxicity, and survival were monitored. Results: Radiolabeled biotin cleared rapidly. Rapid tumor uptake resulted in much higher tumor:nontumor targeting ratios than achieved with the directly labeled monoclonal antibody. Dose escalation revealed that 74 MBq caused acute death of mice, whereas 0.37–37 MBq doses inhibited tumor growth and prolonged survival significantly. Evidence of mild hematological toxicity was noted. At therapeutically effective doses renal toxicity was observed. Conclusions:213Bi-DOTA-biotin, directed by the Pretarget method to tumor-targeted B3-SA, showed a therapeutic effect, although the therapeutic index was low. The source of the toxicity was most likely related to the renal toxicity.

Preparation and in vivo evaluation of linkers for 211At labeling of humanized anti-Tac

The syntheses, radiolabeling, antibody conjugation, and in vivo evaluation of new linkers for 211At labeling of humanized anti-Tac (Hu-anti-Tac), an antibody to the alpha-chain of the IL-2 receptor (IL-2Ralpha) shown to be a useful target for radioimmunotherapy are described. Synthesis of the organometallic linker precursors is accomplished by reaction of the corresponding bromo- or iodoaryl esters with bis(tributyltin) in the presence of a palladium catalyst. Subsequent conversion to the corresponding N-succinimidyl ester and labeling with 211At of two new linkers, N-succinimidyl 4-[211At]astato-3-methylbenzoate and N-succinimidyl N-(4-[211At]astatophenethyl)succinamate (SAPS), together with the previously reported N-succinimidyl 4-[211At]astatobenzoate and N-succinimidyl 3-[211At]astato-4-methylbenzoate, are each conjugated to Hu-anti-Tac. The plasma survival times of these conjugates are compared to those of directly iodinated (125I) Hu-anti-Tac. The N-succinimidyl N-(4-[211At]astatophenethyl)succinamate compound (SAPS) emerged from this assay as the most viable candidate for 211At-labeling of Hu-anti-Tac. SAPS, along with the directly analogous radio-iodinated reagent, N-succinimidyl N-(4-[125I]astatophenethyl)succinamate (SIPS), are evaluated in a biodistribution study along with directly iodinated (125I) Hu-anti-Tac. Blood clearance and biological accretion results indicate that SAPS is a viable candidate for further evaluation for radioimmunotherapy of cancer.

Effective therapy of murine models of human leukemia and lymphoma with radiolabeled anti-CD30 antibody, HeFi-1.

CD30 is a member of the TNF receptor superfamily. Overexpression of CD30 on some neoplasms versus limited expression on normal tissues makes this receptor a promising target for antibody-based therapy. Radioimmunotherapy of cancer with radiolabeled antibodies has shown promise. In this study, we evaluated the therapeutic efficacy of an anti-CD30 antibody, HeFi-1, armed with 211At in a leukemia (karpas299) model and with 90Y in a lymphoma (SUDHL-1) model. Furthermore, we investigated the combination therapy of 211At-HeFi-1 with unmodified HeFi-1 in the leukemia model. Treatment with unmodified HeFi-1 significantly prolonged the survival of the karpas299-bearing mice compared with the controls (P < 0.001). Treatment with 211At-HeFi-1 showed greater therapeutic efficacy than that with unmodified HeFi-1 as shown by survival of the mice (P < 0.001). Combining these two agents further improved the survival of the mice compared with the groups treated with either 211At-HeFi-1 (P < 0.05) or unmodified HeFi-1 (P < 0.001) alone. In the lymphoma model, the survival of the SUDHL-1-bearing mice was significantly prolonged by the treatment with 90Y-HeFi-1 compared with the controls (P < 0.001). In summary, radiolabeled HeFi-1 is very promising for the treatment of CD30-expressing leukemias and lymphomas, and the combination regimen of 211At-HeFi-1 with unmodified HeFi-1 enhanced the therapeutic efficacy.

Semi-remote production of Br-76 and preparation of high specific activity radiobrominated pharmaceuticals for PET studies

Abstract The PET radionuclide 76Br (t1/2=16.2 h) can be easily produced utilizing the nuclear reaction As(3He,2n)76Br. We use high-purity arsenic targets and isolate radioactive bromide by chromic acid oxidation followed by simple distillation of [76Br] hydrogen bromide using a semi-remote apparatus. Use of reagents with little or no carrier bromine yields high specific activity radiobrominated pharmaceuticals prepared from the distilled [76Br] hydrogen bromide.

Device-dependent activity estimation and decay correction of radionuclide mixtures with application to Tc-94m PET studies.

Multi-instrument activity estimation and decay correction techniques were developed for radionuclide mixtures, motivated by the desire for accurate quantitation of Tc-94m positron emission tomography (PET) studies. Tc-94m and byproduct Tc isotopes were produced by proton irradiation of enriched Mo-94 and natural Mo targets. Mixture activities at the end of bombardment were determined with a calibrated high purity germanium detector. The activity fractions of the greatest mixture impurities relative to 100% for Tc-94m averaged 10.0% (Tc-94g) and 3.3% (Tc-93) for enriched targets and 10.1% (Tc-94g), 11.0% (Tc-95), 255.8% (Tc-96m), and 7.2% (Tc-99m) for natural targets. These radioisotopes have different half-lives (e.g., 52.5 min for Tc-94m, 293 min for Tc-94g), positron branching ratios (e.g., 0.72 for Tc-94m, 0.11 for Tc-94g) and gamma ray emissions for themselves and their short-lived, excited Mo daughters. This complicates estimation of injected activity with a dose calibrator, in vivo activity with PET and blood sample activity with a gamma counter. Decay correction using only the Tc-94m half-life overestimates activity and is inadequate. For this reason analytic formulas for activity estimation and decay correction of radionuclide mixtures were developed. Isotope-dependent sensitivity factors for a PET scanner, dose calibrator, and gamma counter were determined using theoretical sensitivity models and fits of experimental decay curves to sums of exponentials with fixed decay rates. For up to 8 h after the end of bombardment with activity from enriched and natural Mo targets, decay-corrected activities were within 3% of the mean for three PET studies of a uniform cylinder, within 3% of the mean for six dose calibrator decay studies, and within 6% of the mean for four gamma counter decay studies. Activity estimation and decay correction for Tc-94m mixtures enable routine use of Tc-94m in quantitative PET, as illustrated by application to a canine Tc-94m sestamibi study.