Bifunctional NHS-BAT ester for antibody conjugation and stable technetium-99m labeling: conjugation chemistry, immunoreactivity and kit formulation.

UNLABELLED: Conjugation chemistry and kit formulated binding of the NHS ester of 6-(4'-(4"-carboxyphenoxy)butyl)-2, 10-dimercapto-2,10-dimethyl-4,8-diazaundecane (NHS-BAT ester) to monoclonal antibodies (MAbs) was investigated. The functionalities of the resulting BAT conjugated and 99mTc-labeled MAbs BW 431/26, MAb 425 and bispecific MDX210 (fragment construct) were tested by immunoreactivity and immunoscintigraphy. METHODS: The kinetics and chemistry of the conjugation reaction were monitored by high-performance liquid chromatography, size-exclusion chromatography and positive fast-atom-bombardment mass spectra (FAB-MS). The 99mTc BAT-MAbs were tested with various immunoreactivity assays. The biodistribution of 99mTc-BAT-BW 431/26 in rats was compared with directly labeled BW 431/26. RESULTS: At pH 8.5 and 25 degrees C, the reactivity of the NHS-BAT ester was high with 90% completion after 30 min. The conjugation yield of 19 microM MAb and 228 microM NHS-BAT ester amounted to 30%. Higher NHS-BAT ester concentrations afforded higher BAT-to-MAb ratios. According to FAB-MS, the conjugation competing hydrolysis surprisingly occurred at the NHS ring. Almost quantitative 99mTc labeling was achieved after 5 min at 25 degrees C. Immunoreactivity of the 99mTc-BAT antibodies showed > 90% recovery and proved to be insensitive to BAT-to-MAb ratios of up to 10. The 99mTc-BAT-BW 431/26 showed similar organ distribution but revealed less urinary excretion compared with the directly labeled BW 431/26. Immunoscintigraphy with 99mTc-labeled and BAT-BW 431/26 and BAT-MAb 425 showed the respective biological function in vivo. CONCLUSION: According to straightforward conjugation chemistry, the ease of 99mTc labeling and the application of a simple ultrafiltration technique, the NHS-BAT ester represents a nondestructive, universally applicable biofunctional ligand to introduce stable 99mTc protein binding sites. Kit formulated conjugation/labeling can be performed with little time requirements and laboratory experience.

Granulocyte labelling kit BW 250/183--results of the European multicenter trial.

In 6 European countries a multicenter trial with the technetium-99m labelled monoclonal anti-granulocyte antibody BW 250/183 was conducted in 775 patients. The antibody is used for the immunoscintigraphic visualisation of accumulations of granulocytes. The present study was restricted on the investigation of patients having inflammations, essentially of the peripheral skeleton (mostly of the lower extremities), spinal column, the bowel, or had fever of unknown etiology. The overall sensitivity of the method was 83%, and specificity was of the same order of magnitude (82%). The procedure yielded additional information in 67% of the cases and the treatment strategy was influenced in 35% of the cases to positive effect, even though all conventional methods had previously been exhausted. Human anti mouse-IgG antibodies (HAMA) were detectable only in about 4% of patients investigated for the first time.

Anti-CEA labelling kit BW 431/26. Results of the European multicenter trial.

In seven European countries a multicenter trial with the 99mTc-labelled monoclonal anti-CEA antibody BW 431/26 was conducted in 730 patients. The antibody is used for the immunoscintigraphic visualisation of CEA-expressing tumours. Investigated were in particular colorectal tumours, bladder and breast carcinoma, medullary thyroid carcinoma, adenocarcinoma of the lung and gastric carcinoma. The main area of use is the detection of recurrences and screening for foci in patients with rising serum CEA. The sensitivity amounts to at least 80% in case of primary colorectal tumours (n = 129) and their abdominal or pelvic metastases (n = 33) and to 90% for their recurrences (n = 107). HAMAs were detectable in less than 15% of patients investigated for the first time. In 17% of the patients examined, immunoscintigraphy was the only technique to visualize the lesion whereas all other diagnostic methods had failed. The procedure yielded additional information in 24-51% of cases.

Synthesis, characterization and biodistribution of neutral and lipid-soluble 99mTc-bisaminoethanethiol spiperone derivatives: possible ligands for receptor imaging with SPECT.

Using parts of the molecular structure of spiperone, two new ligand systems for complexation with [99mTc]technetium were prepared in order to develop potential receptor imaging agents for single photon emission computer tomography (SPECT). The bis-aminoethanethiols (BAT): 1-benzyl-4-(2-mercapto-2-methyl-4-aza-pentyl)-4-(2-mercapto-2-methyl- propylamino)-piperidine (benzylpiperidyl-BAT, BP-BAT) and 1-[3-(4-fluorobenzoyl)-propyl]-4-(2-mercapto-2-methyl-4-aza-pentyl)-4- (2-mercapto-2-methyl-propyl-amino)-piperidine (butyrophenoylpiperidyl-BAT, BUP-BAT) form stable, neutral and lipid soluble complexes with [99mTc]technetium at pH > or = 11 using SnCl2 as reducing agent in nearly quantitative radiochemical yields. Biodistribution of 99mTc-BP-BAT and 99mTc-BUP-BAT in rats showed a moderate clearance from blood and low uptake and retention in the liver, whereas brain uptake was moderate, however with prolonged brain retention. On the other hand, significant accumulations and retentions were observed in heart, kidney and lung with increasing oxygen/blood ratios up to 24 h. Within 24 h p.i. 22 and 29% of the injected dose (i.d.) of 99mTc-BP-BAT and 99mTc-BUP-BAT were eliminated by hepatobiliary excretion whereas 22% i.d. of both 99mTc-BAT complexes were excreted into the urine. Although first biodistribution studies of 99mTc-BP-BAT and 99mTc-BUP-BAT in rats showed relatively low brain uptake, the high uptake in peripheral, receptor rich organs indicates that compounds of this type may be used as a basis for further structural modification to develop agents with optimal properties for cerebral or peripheral receptor imaging with SPECT.

New renal function imaging agents. I. Synthesis and biological characterization of a [99mTc]diaminomercapto(thio)ether (DAMTE).

The present study describes the synthesis of a [99mTc]diaminomercapto(thio)ether (DAMTE-derivative) as a first compound of a new class of 99mTc-complexes which is tubular excreted. 10-Benzoyl-8-keto-7-aza-2-amino-4,10-dithia-decanoic acid (CO2-DAMTE 3) was synthesized by the reaction of succinimidyl-S-benzoyl-thioglycolate and (S)-2-aminoethyl-L-cysteine. The respective technetium complex, 99mTc-CO2-DAMTE was obtained in radiochemical yields of about 70% using stannous chloride as reducing agent. Hydrolysis of the protecting group was performed either prior to the complexation of pertechnetate ("cold kit") or during the labelling reaction ("hot kit"). Organ distribution was determined in Wistar rats. Within 24 h 40% of the activity were excreted into the feces and 43% into the urine, whereas 10% were retained in the kidneys. In contrast, a first human study showed a very fast renal elimination of 99mTc-CO2-DAMTE, a low liver uptake (< 10%) and no retention in the kidneys. The renal clearance of approx. 240 mL/min/1.73 m2 in addition to the protein binding of > 95% suggests an effective tubular excretion of the compound.

[A phosphine-substituted diphosphonic acid (HMPD) for improved scintigraphic detection of bone lesions]. / Eine Phosphin-substituierte Diphosphonsäure (HMPD) zur verbesserten szintigraphischen Darstellung von Skelettläsionen.

Although the radiopharmaceuticals used for bone scintigraphy are of very high quality, the search for an "ideal" agent continues. To optimise the detectability of bone lesions, we analysed 244 different 99mTc-labeled phosphonates in animal experiments. In osteosarcoma-carrying rats 99mTc-labeled 1-Hydroxy-3-methyl-phosphinic-1, 1-propanediphosphonic acid (HMPD) was shown to produce the best lesion/normal bone ratio. 99mTc-MDP was used as reference. The ratio was found to be 1.28 for 99mTc-HMPD. The transferability of our results in animals to the situation in man was studied in 10 patients with bone metastases. There was for 99mTc-HMPD an improvement of the lesion/normal bone ratio by more than 60% but also an additional reduction of the soft tissue contrast by about 40%. 15% of the metastases were detected by scintigraphy using 99mTc-HMPD only and not with 99mTc-MDP. The new agent should make possible a better and earlier discrimination of bone lesions in the scintigram.

[Binding of the monoclonal antibody BW 250/183 to human granulocytes]. / Bindung des monoklonalen Antikörpers BW 250/183 an menschliche Granulozyten.

Lately, the specific binding of appropriate monoclonal antibodies to human granulocytes has been used for the scintigraphic detection of inflammatory foci. Using the antibody BW 250/183 we studied the underlying binding kinetics. As an important requirement for a specific cell binding it has been shown that the labelling procedure does not change the immunoreactivity of the antibody. An affinity constant of 2 x 10(9) l/mol has been calculated from binding studies. Usually, 0.25-1.0 mg of the 99mTc-labelled antibody are applied per patient. In the present study an equilibrium in blood appeared quickly after intravenous application; at steady state about one fourth of the activity was cell-bound. The rest of the activity circulated in the plasma in the form of labelled IgG and was able to react directly with those granulocytes which were already accumulated in the inflamed area. Even a drastic reduction of the applied protein mass did not change this equilibrium. The law of mass action seems not to be directly applicable to this problem. Interferences with components of the plasma can be excluded as explanation for this behaviour. After application of in-vitro labelled granulocytes from which unbound antibodies were removed completely by washing, an identical steady state was observed within 10 min after injection; however, in this situation the intravasal residence time of activity increased distinctly.

Preclinical and clinical testing of radiolabelled monoclonal antibodies for immunoscintigraphy.

Immunoscintigraphy is a new method for in vivo diagnosis of diseases using monoclonal antibodies. Emphasis is placed on diagnosis of malignant tumors although the range of application includes a number of non-malignant diseases. To date, no European country has issued clear guidelines on the testing and registration of those monoclonal antibodies labelled with a radionuclide and used for diagnostic. This involves the risk of overregulation which would considerably reduce the applicability of the method. This holds particularly true since the complications initially anticipated with the use of such compounds did not occur. The conduct of immunoscintigraphy has evolved during the last few years. For reasons of applicability, but also and mainly for reasons of radiation hygiene, I-131 and finally also In-111 were abandoned as labelling nuclides and replaced by Tc-99m. The protein amount involved was reduced. Some false estimations, which were due to particularities or artifacts of the iodinated antibodies used at the beginning, had to be corrected: the representation of the liver in a scintigram is part of the physiological distribution of antibodies; and fragments of antibodies normally do not present the anticipated kinetic advantages. The clinical results obtained with colon carcinomas show that not only recurrencies and metastases, but also primary tumors can be detected with equally high sensitivity. In contrast, radioimmunotherapy does not yet seem as successful, at least against solid tumors.

[Age dependence of kidney function]. / Altersabhängigkeit der Nierenfunktion.

Although kidney function is immature in infants and decreases with increasing age in adults, when assessing the kidney clearance, it is not common to make an adjustment for age. On the other hand, correction for the standard body surface is generally accepted. Previous studies have yielded contradictory results, probably due to the small numbers of patients studied. To obtain the statistically significant relationship between age and kidney clearance, we compiled more than 1000 studies. These studies were divided into three groups: (1) children under 2 years of age (n = 71); (2) children and adolescents from 2 to 15 years (n = 64 male/58 female); (3) adults (n = 474 male/424 female; age: 16-80 years). Total clearance determinations were only considered if there were no differences between the two kidneys and if there was no obstruction of micturition or other pathological findings. Surprisingly, the statistical analysis showed no differences in clearance values between sexes. The maximal clearance values were found in the groups with children about 8 years old and the greatest scatter of values was seen in children under 2 years of age (a discrepancy between maturation age and calendar age). The complete set of data was based on the presumption that continuous function consisting of three trunks describes the course of normal kidney clearance in relation to patient age. If one determines an arbitrary reference value (e.g., 25 years), correction factors can easily be derived from the function described in order to compare individual clearance with a normal value.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioimmunoimaging for diagnosis of bone marrow involvement in breast cancer and malignant lymphoma.

Granulopoietic bone-marrow was scintigraphically imaged in 15 patients with carcinoma of the breast and known skeletal metastases, 10 patients with malignant lymphomas, and 15 controls without suspected malignant disease, with a technetium-99m labelled murine monoclonal IgG1 antibody directed against nonspecific cross-reacting antigen (NCA-95) and carcinoembryonic antigen. Immunoscintigraphy revealed more lesions than did bone scanning in both patient groups. This method offers a sensitive, cost-effective, and easy-to-perform whole body technique for evaluating metastatic spread.

Effect of DTPA to antibody ratio on chemical, immunological and biological properties of the 111In-labelled F(ab')2 fragment of the monoclonal antibody 431/31.

When used for immunoscintigraphic detection of malignant tumours, monoclonal antibodies are frequently labelled with indium-111. This study investigated whether antibody properties are affected by DTPA, the chelating agent with which the antibody has to be bound before labelling with indium-111. Chromatography, cell binding assays and animal studies indicated marked changes in the chemical, immunological and biological properties of the F(ab')2 antibody fragment 431/31 at DTPA:F(ab')2 ratios greater than 1. It is concluded that, in order to avoid lasting effects on the normal biokinetic behaviour of this antibody, the DTPA:F(ab')2 ratio for the antibody should not exceed 1.

Quantitative considerations supporting the irrelevance of circulating serum CEA for the immunoscintigraphic visualization of CEA expressing carcinomas.

Starting from the phenomenon that the amount of circulating CEA in patients' sera did not significantly influence immunoscintigraphic visualization of CEA expressing tumors, we built up an in vitro model to explain this phenomenon. Blocking experiments in this model system showed that the CEA specific MAbs BW 431/26 and BW 431/31 could not be inhibited in their binding to cell associated CEA, if they were preincubated with a 20 molar excess of serum CEA. In contrast, the CEA-NCA cross reactive MAbs could be inhibited in their binding to tumor associated CEA under identical conditions. These data combined with western blotting analysis of patients' sera and affinity constant determinations argue that conformational changes in serum CEA cause a decreased affinity of the CEA specific MAbs to serum CEA allowing a preferential binding to tumor associated CEA.

Investigations on the effect of metamizol on ureteral motility.

The aim of this comparative study was to obtain quantitative information on the effect that metamizol has on ureteral motility. Renal Tc-99m-MDP excretion and ureteral urine transport against gravity were examined in five patients, once without (control) and, several days later, with metamizol treatment. In each of these ten scanning sessions, renograms of both kidneys were recorded. They were corrected for background activity and compared with respect to the following parameters: the curve amplitude at 30 min p.i. normalized to the respective maximum amplitude; the 6-30 min and 40 s-6 min integrals of a corrected curve. Under metamizol medication, the flow of radioactivity to the bladder was thus found to be significantly reduced (p less than 0.001). The comparison of the Tc-99m-MDP retention curves obtained with and without metamizol treatment did not reveal a statistically significant drug effect on renal Tc-99m-MDP excretion.

Biophysical investigations of the dose-effect relationship in chromosome aberrations of human lymphocytes caused by thorotrast deposits. I. Physical aspects.

Thorotrast is a X-ray contrast medium which has been used in the years 1930-1950. It is hardly ever eliminated from the body and is stored in the organs of the reticulohistiocytic system (RHS). Since Thorotrast contains all radionuclides of the thorium decay chain, the effect of internal irradiation by these radionuclides can be investigated in the so called "Thorotrast patients". It is the purpose of this study to determine the radiation dose absorbed by the lymphocytes of the peripheral blood of these persons, and to correlate it with their chromosome aberration rate. Part I of the paper describes the physical aspects of dose evaluation. The chromosome aberration rates of Thorotrast patients were subjected to a repeated statistical test. They show a logarithmic normal distribution at a mean value of about seven dicentric chromosomes per 100 cells. The corresponding dose has to be determined. The method for evaluating size and distribution of Thorotrast conglomerates in the examined tissue is demonstrated, and the function and resolution power of the applied "texture analysing system" (TAS) is described. Size-distributions of Thorotrast conglomerates of lymph nodes from the neck, the lung and from the lower aorta (bifurcatio) are given. In the lymphatic system more than 90% of the absorbed dose in tissue is due to alpha-particles. The computation method of calculating the self-absorption of alpha-particles in the conglomerates and of calculating the annual doses in the lymph nodes is described. The values lie between 135 and 1200 rad/year. The contribution to irradiation from outside the lymphatic system can be neglected. The method of dose determination is discussed and compared with microdosimetric methods. With these methods, the dose of a lymphocyte caused by one hit of an alpha-particle lies between 80 and 920 rad. An energy dissipation does not take place and the total energy emitted acts on the lymphocytes thus inducing chromosome aberrations.

Biophysical investigations of the dose-effect relationship in chromosome aberrations of human lymphocytes caused by thorotrast deposits. II. Biological and medical aspects.

The biological effect of alpha-radiation, which has been examined in this investigation, is the occurrence of chromosome aberrations in lymphocytes from the peripheral blood of Thorotrast patients. To set up a dose-effect relationship, it is necessary to estimate the absorbed dose of a single lymphocyte found in a blood sample. Then it would be possible to use chromosome aberrations of lymphocytes from peripheral blood as an indicator for estimating the effective radiation dose. The physical aspects of this investigation have been described in part I of this paper. In part II, biomedical considerations are described and discussed. The irradiation of the lymphocytes takes place in the whole lymphatic system, but the lymph node could have been used as a model for the lymphatic system. Based on the evaluation of size and distribution of Thorotrast conglomerates, the average annual dose in different structures of the lymph node has been determined. The Thorotrast content in lymph nodes depends on the region of the body from where the lymph nodes originate. It has been tried to compile the distribution of the lymph nodes in the different regions of the body. Annual doses have been calculated in these different regions in order to find a representative value for the whole body. The values describing the kinetics of the lymphocytes in the body have been used to convert the annual dose in lymph nodes into the dose of a lymphocyte found in a sample of peripheral blood. It is pointed out that the calculated dose of the lymphocytes, being 250 rad, is an estimation of an upper value. The lymphocytes are "in vivo" irradiated by the Thorotrast, but most of the calibrations used for biological dosimetry are referred to as "in vitro" experiments. The discrepancy between "in vivo" and "in vitro" results are discussed in the findings of this study and in other results.