Siegfried Matzku

A NEW VARIANT OF GLYCOPROTEIN CD44 CONFERS METASTATIC POTENTIAL TO RAT CARCINOMA CELLS

Using a monoclonal antibody (MAb1.1ASML) raised against a surface glycoprotein of the metastasizing rat pancreatic carcinoma cell line BSp73ASML, cDNA clones have been isolated that encode glycoproteins with partial homology to CD44, a presumed adhesion molecule. In one of the clones, pMeta-1, the epitope marks an additional extracellular domain of 162 amino acids inserted into the rat CD44 protein between amino acid positions 223 and 247 (by analogy to human and murine CD44). The new variants are expressed only in the metastasizing cell lines of two rat tumors, the pancreatic carcinoma BSp73 and the mammary adenocarcinoma 13762NF; they are not expressed in the non-metastasizing tumor cell lines nor in most normal rat tissues. Overexpression of pMeta-1 in the nonmetastasizing BSp73AS cells suffices to establish full metastatic behavior.

Immunoreactivity of monoclonal anti-melanoma antibodies in relation to the amount of radioactive iodine substituted to the antibody molecule.

The damage to monoclonal anti-melanoma antibodies caused by iodination was investigated by comparing the results obtained using the chloramine-T method and the 1,3,4,6-tetrachloro-3α,6α-diphenyl-glycoluril (IODOGEN) method at different levels of iodine substitution to the molecule. The level of substitution at which losses in immunoreactivity occurred was evaluated in each monoclonal antibody (MAb) studied. This phenomenon was not dependent on the method of substitution, provided that mild conditions of reaction were used. Lineweaver-Burk plots and — in cases of alterations in binding affinity — Scatchard plots were found to provide an adequate description of the binding behaviour of individual MAbs after labelling. Immunoreactivity was shown to be determined not only by the proportion of bona fide reactive MAb molecules, but also by a substitution-dependent decrease in affinity constants. The practical consequences of altered binding parameters were demonstrated by quantitating specific antibody accumulation in melanoma transplants in vivo.

Tumour targeting with antibody-coupled liposomes: Failure to achieve accumulation in xenografts and spontaneous liver metastases

SummaryThe potential of small unilamellar liposomes coupled to anti-tumour monoclonal antibodies (immunoliposomes) to accumulate in solid tumour tissue was tested in two systems, i.e. a human malignant melanoma xenografted into nude mice and a syngeneic murine lymphoma ESb.Mp exhibiting spontaneous metastasis to the liver. Both monoclonal antibodies tested were partly released from immunoliposomes within a few hours, thus generating a seemingly constant level of circulating antibody. Nevertheless it was possible to follow the biodistribution of intact immunoliposomes by virtue of a radioiodine label incorporated into the lipid moiety. It was found that in both tumor systems, though they differed with respect to the size of lesions and maybe also to the vascular architecture of surrounding tissue, immunoliposome uptake was virtually nil. The blockade of uptake into solid tumour tissue was caused by the limited availability of immunoliposomes due to their moderate stability, but especially by the inability of the particulate carrier to extravasate.

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.

Criteria for selecting monoclonal antibodies with respect to accumulation in melanoma tissue

SummaryImmunohistology provides a necessary but insufficient criterion for selecting monoclonal antibodies (MAbs) capable of tumour targeting in vivo. Additional selection procedures have been evaluated using a panel of anti-melanoma MAbs, including immunoreactivity of (labelled) MAbs, antibody affinity, kinetics of binding and release, apparent antigen density and accumulation in nude mouse transplants. According to these criteria, MAbs M.2.7.6 and M.2.9.4 showed the most favourable properties, i.e. high immunoreactivity and pronounced internalization into melanoma cells. With MAbs M.2.10.15 and KG 6–56, moderate immunoreactivity and a binding pattern characterized by temperature dependence in the absence of internalization was observed. According to the paired label assay, all four MAbs showed specific accumulation into solid melanoma tissue. However, application in the patient still requires evaluation of the side effects of antigen cross-expression on normal human tissues.

Iodination of monoclonal IgG antibodies at a sub-stoichiometric level: immunoreactivity changes related to the site of iodine incorporation.

Thirteen monoclonal antibodies (MAbs) were labeled with 125I to a different degree such as to cover the range from 0.5-20 microCi/micrograms. By SDS polyacrylamide gel electrophoresis, the amount of iodine incorporated into heavy (h) and light (l) chains was determined. Comparing different MAbs, h:1 ratios varied from 0.6-34.6, but virtually no variation was observed with individual MAbs labeled at different levels. Immunoreactivity of labeled MAbs was analyzed with antigen-positive tumor cells according to the Lineweaver Burk method. Immunoreactive fractions were found to decrease with increasing iodine incorporation in 9/12 MAbs, while binding affinities decreased in 5/12 MAbs; only 1 MAb was stable in both respects. Immunoreactivity changes were not linked to preferential h or 1 chain labeling, nor to the isotype. This result indicated incorporation of the first iodine atom to take place at individually distinct residues, with a minimum estimate of two or four sites, depending on whether preferential chain labeling or random incorporation took place. In cases where increasing labeling led to a gradual decrease of binding affinity, a shift in the spectrum of acceptor residues has to be assumed.

Monoclonal antibody uptake in B-cell lymphomas: Experimental studies in nude mouse xenografts

Accumulation of radiolabelled monoclonal antibodies (mAb) in human B-lymphoma xenografts was found to result in two distinct patterns. The basic elements leading to these patterns were elucidated by autoradiographic and immunohistological analysis applied to the nude mouse xenografts BJAB and OCI.LY1. With BJAB, accumulation occurred exclusively in peripheral cell layers of the lymphoma nodule, while central areas were not accessible irrespective of mAb dose. This feature was the consequence of an inefficient transport across intratumoral vessels together with peripheral mAb supply through a subcapsular pseudosinus. With OCI.LY1, intratumoral vessels showed generalized leakiness. Furthermore, interstitial transport was operative to a fair extent, such that in early images multiple sites of mAb extravasation were obvious, which coalesced during the course of prolonged uptake. The pattern of peripheral mAb uptake resulted in a low overall tumour uptake, while multifocal uptake yielded substantial accumulation values.

Radioimmunological Quantitation of Human Group-II Pepsinogens

A solid-phase sandwich radioimmunoassay was developed to quantitate human group-II pepsinogens in plasma. The test detected pepsinogen II in a concentration range of 0.25--64.0 ng/ml using sample volumes of 125 microliter. Purified group I pepsinogens showed no response up to concentrations of 100 microgram/ml. In apparently healthy donors, we observed mean plasma concentrations of 20.3 ng/ml (males) and of 15.5 ng/ml (females).

11C-Butanol for imaging of the blood-flow distribution in tumor-bearing animals

Abstract1-11C-n-Butanol produced semiatomatically using a cyclotron was employed to investigate the whole-body distribution and kinetics of the label of this compound. Following the administration of 11C-butanol into the aorta of two dogs, more than 80% of the activity was cleared from the blood within 1 min. The activity distribution mirrored the cardiac output distribution as determined using 121I microspheres. Within 25 min p.i., a significant release of decay-corrected activity was only observed for the liver, spleen, and kidneys. Muscle and whole-body activity showed constant levels during this period. In 45 tumor transplants from rats, the dynamic behavior of the label was studied. The tissue retention of activity following injection into the a. femoralis was approximately 100% during the 1st 15 s for both tumor and muscle (n=6). The activity release by tumors during the 1st 10 min after intra-aortic injection was 18%±4.5% (n=39; decay corrected). In five different tumor lines (n=10), the initial 11C-butanol uptake was related to that of muscle, and the results were correlated with the tumor-to-muscle retention of 121I-microspheres (r =0.89). In 17 tumors, the correlation between 11C-butanol uptake and the washout rate of 133Xe resulted in a correlation coefficient of 0.96. Tumor-to-muscle uptake ratios could be equally determined using intra-aortic and intravenous injection, as evaluated by intraindividual comparison in 12 rats (y=0.01+0.98x;r=0.98). 11C-Butanol appears to be an appropriate radiotracer for the assessment of blood supply to malignant tumors relative to muscle.

Rat Natural Killer (NK) Cells are not Bone Marrow Dependent

Rats were treated with various doses of 89Strontium. At a dose effecting depletion of the bone marrow (200 muCi), NK activity of spleen, peritoneal and peripheral blood lymphocytes was unaltered or only slightly decreased. At higher doses (400 muCi), a general impairment of the immune system, including T-cell functions, was observed. After application of 700 muCi 89Strontium, rats died within three weeks.