Jochen Schuhmacher

Evaluation of Positron Emission Tomography Imaging Using [68Ga]-DOTA-D Phe1-Tyr3-Octreotide in Comparison to [111In]-DTPAOC SPECT. First Results in Patients with Neuroendocrine Tumors

PURPOSE [111In]-DTPAOC (Octreoscan(R)) has been shown to be very useful in the detection of somatostatin receptor (SSTR) positive tumors and their metastases using either conventional scintigraphy or single photon emission computed tomography (SPECT). The main drawback of this method is the limited spatial resolution and a somewhat low receptor affinity of the radiopeptide. Due to the increased spatial resolution and the ability of quantification, an agent for positron emission tomography (PET) imaging of SSTR is desirable. This communication shows our initial experience using [68Ga]-DOTA-D-Phe(1)-Tyr(3)-Octreotide (DOTATOC) in comparison to [111In]-DTPAOC-SPECT in patients with neuroendocrine tumors. PROCEDURES Four patients, two male and two female (46-55 years old) have been examined by [111In]-DTPAOC scintigraphy and within one month by [68Ga]-DOTATOC-PET. All of them suffered from neuroendocrine tumors and/or their metastases. DOTATOC has been labeled using the positron-emitting generator-nuclide 68Ga (t(1/2) 68 minutes). In two patients with previously known localization of tumor, dynamic PET scans after intravenous bolus-injection of 181+/-17 MBq [68Ga]-DOTATOC until 120 minutes post-injection were acquired. In all patients, the static PET-scans have been acquired after 45 or 60 minutes post-injection (SUV1) and 140 minutes post-injection (SUV2). RESULTS Similar to [111In]-DTPAOC, [68Ga]-DOTATOC showed the highest uptake in the spleen, followed by the kidneys and the liver. A clear delineation of the pituitary gland could only be achieved by PET. The highest SUVs were found at a plateau between 45 and 90 minutes with a maximum 60 minutes post-injection. Due to the fast tracer accumulation in the tumor and the rapid clearance of the compound, resulting in high tumor to background ratios even 40 minutes after injection, the short half life of 68Ga is reasonable. In two patients more findings have been revealed by [68Ga]-DOTATOC-PET as compared to the [111In]-DTPAOC-SPECT. In comparison to the [111In]-DTPAOC-SPECT [68Ga]-DOTATOC-PET seems to be superior especially concerning small findings with low tracer uptake. Both [111In]-DTPAOC-SPECT and [68Ga]-DOTATOC-PET were less sensitive in the detection of liver metastases of neuroendocrine tumors compared to computerized tomography CT because they showed a lower uptake than the surrounding liver tissue. CONCLUSIONS According to our initial experiences in a limited number of patients, [68Ga]-DOTATOC is a promising PET tracer for imaging neuroendocrine tumors and their metastases. In comparison to the [111In]-DTPAOC-scan it seems to be superior especially in detecting small tumors or tumors bearing only a low density of SSTRs. It offers excellent imaging properties and very high tumor to background ratios. Further evaluation of [68Ga]-DOTATOC in a larger number of patients is certainly justified.

Treatment of Human B Cell Lymphoma Xenografts with a CD3 × CD19 Diabody and T Cells

The use of anti-CD3 × antitumor bispecific Abs is an attractive and highly specific approach in cancer therapy. Recombinant Ab technology now provides powerful tools to enhance the potency of such immunotherapeutic constructs. We designed a heterodimeric diabody specific for human CD19 on B cells and CD3ε chain of the TCR complex. After production in Escherichia coli and purification, we analyzed its affinity, stability, and pharmacokinetics, and tested its capacity to stimulate T cell proliferation and mediate in vitro lysis of CD19+ tumor cells. The effect of the diabody on tumor growth was investigated in an in vivo model using immunodeficient mice bearing a human B cell lymphoma. The CD3 × CD19 diabody specifically interacted with both CD3- and CD19-positive cells, was able to stimulate T cell proliferation in the presence of tumor cells, and induced the lysis of CD19+ cells in the presence of activated human PBL. The lytic potential of the diabody was enhanced in the presence of an anti-CD28 mAb. In vivo experiments indicated a higher stability and longer blood retention of diabodies compared with single chain Fv fragments. Treatment of immunodeficient mice bearing B lymphoma xenografts with the diabody and preactivated human PBL efficiently inhibited tumor growth. The survival time was further prolonged by including the anti-CD28 mAb. The CD3 × CD19 diabody is a powerful tool that should facilitate the immunotherapy of minimal residual disease in patients with B cell leukemias and malignant lymphomas.

Stable expression of soluble therapeutic peptides in eukaryotic cells by multimerisation: application to the HIV-1 fusion inhibitory peptide C46.

A major drawback of therapeutic peptides is their short half‐life, which results in the need for multiple applications and high synthesis costs. To overcome this, we established a eukaryotic expression system that allows the stable expression of small therapeutic peptides by multimerisation. By inserting the sequence encoding the therapeutic peptide between a signal peptide and the multimerising domain of the α‐chain from the human C4bp plasma protein, therapeutic peptides as small as 5 kDa are secreted as multimers from transfected cells; this allows easy purification. As proof of principle, we show that the T20‐derived HIV‐1 fusion inhibitory peptide C46 in its multimeric form: i) was efficiently secreted, ii) was more stable than the current antiviral drug T20 in vitro and in vivo, and iii) inihibited HIV‐1 entry with similar efficiency in vitro. Besides the gain in stability, multimerisation also leads to increased valency and allows the combination of several therapeutic functions. Furthermore, by expressing the multimers from cells, post‐translational modifications could easily be introduced.

Exceptional increase in somatostatin receptor expression in pancreatic neuroendocrine tumour, visualised with 68Ga-DOTATOC PET

After complete resection, histology confirmed a neuroendocrine (NE) tumour of the pancreas. According to the new WHO classification (2000), it was classified as borderline between 1a (NE tumour with high differentiation) and 1b (NE carcinoma with high differentiation). Ki67-proliferation activity was only slightly increased (2%). Gene-Chip analysis (HG-U133A, Affymetrix Inc, Santa Clara, CA, USA) revealed an increase in the gene expression for the somatostatin receptor subtype 2 by a factor of 33.7 (subtype 1: 1.2; subtype 3: 0.9; subtype 4: 7.1; subtype 5: 2.1). The preparation and application of 68Ga-DOTATOC for PET imaging of somatostatin receptors was initially described in meningiomas [1] and neuroendocrine tumours [2, 3]. In the presented patient, extremely high tumoural uptake of 68Ga-DOTATOC was detected. The mean SUV in the pancreatic tumour was 166.4 (maximum 199.2). This was much higher even than the uptake in the normal spleen, which is the organ with the highest physiological DOTATOC uptake (mean 26.2, maximum 33.1). Mean SUV in normal tissues was as follows: kidneys, 10.7 (maximum 13.8); liver, 6.2 (maximum 7.4); pancreas, 3.1 (maximum 4.9); muscle, 0.9 (maximum 1.9); and lung, 0.7 (maximum 1.1). References

Development of a bispecific monoclonal antibody against a gallium-67 chelate and the human melanoma-associated antigen p97 for potential use in pretargeted immunoscintigraphy

A bispecific monoclonal antibody (bsmAb) has been developed against the human melanoma-associated antigen p97 and an octahedral gallium chelate (Ga-HBED) using the hybrid hybridoma technology. As tetradomas were expected to produce a maximum of ten different molecular species of immunoglobulins, the bispecific antibody was purified from this mixture by consecutive protein A affinity and cation-exchange chromatographic techniques. Although it was established by sodium dodecyl sulphate/polyacrylamide gel electrophoresis that the heavy (H) and light (L) chains of the two parental immunoglobulins were mismatched in the bispecific antibody, results from cell enzyme-linked immunosorbent assay indicated significant dual specific binding to both the melanoma cells and67Ga-HBED. Other in vitro techniques further confirmed that the bsmAb Bi 5-56-II-17 still retained about 30%–40% simultaneous binding capacity to both the antigens, as would have been expected in a bsmAb that has ideally matched H and L chains. Preliminary in vivo experiments using nude mice bearing the human melanoma xenografts showed that the bsmAb Bi 5-56-II-17 was able to target the radioactive gallium chelate to the tumours twice as efficiently compared to the monospecific, bivalent gallium chelate antibody.

Contribution of paramagnetic trace elements to the spin-lattice relaxation time in the liver.

The relaxation times of water protons in rat liver tissue were measured with a NMR spectrometer at 20 MHz. The paramagnetic trace elements Cu, Fe, and Mn were determined by neutron activation analysis. No shortening of T1 could be observed when liver Cu or Fe concentration was increased in the microgram range. T1 was strongly correlated with the liver Mn concentration of untreated animals and animals whose liver Mn concentration was artificially increased or decreased by intravenous injection of manganous acetate or a metal chelating agent with high affinity for hepatobiliary excretion. Deviations from this Mn-T1 correlation were found in the initial phase of liver cirrhosis induced by thioacetamide (elongated T1, normal Mn concentration) and after stimulation of liver growth by phenobarbital (normal T1, decreased Mn concentration). An increased or decreased enhancement factor for Mn may have contributed to the observed deviations during phenobarbital and thioacetamide treatment.

Labeling of monoclonal antibodies with a 67Ga-phenolic aminocarboxylic acid chelate

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 tighly 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° 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.

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.

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

SummaryThe 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.

Enhancement of incorporation of 131Iododeoxyuridine into tumors after application of Clostridium oncolyticum s. butyricum (M 55)

The application of spores of the bacterium Clostridium oncolyticum s. butyricum (M 55) results in an increased incorporation of 131Iododeoxyuridine in tumors (amelanotic melanoma Fortner III of the golden hamster and Brown-Pearce carcinoma of the rabbit). This leads to an improved scintigraphic demonstration of the tumors.