Fusion protein mediated prodrug activation (FMPA) in vivo

A two component system, consisting of a fusion protein and an appropriate prodrug, suited to perform selective tumor therapy in vivo, is presented. The fusion protein, owing to its humanized carcinoembryonic antigen (CEA)-specific variable region, specifically binds to CEA-expressing tumors and has an enzymatic activity comparable to human β-glucuronidase. The prodrug is a nontoxic glucuronide-spacer-derivative of doxorubicin decomposing to doxorubicin by enzymatic deglucuronidation. In vivo studies in nude mice bearing human CEA-expressing tumor xenografts revealed that 7 d after injection of 20 mg/kg fusion protein, a high specificity ratio (>100:1) was obtained between tumor and plasma. Injection of 250 mg/kg of prodrug at d 7 resulted in tumor therapeutic effects superior to conventional chemotherapy without any detectable toxicity. These superior therapeutic effects that were observed using established human tumor xenografts can be explained by the approx 10-fold higher drug concentrations found in tumors of mice treated with fusion protein and prodrug than in those treated with the maximal tolerable dose of drug alone.     

Biological properties and physical map of the genome of a new papovavirus, HD virus.

The superhelical DNA of the HD papovavirus is heterogeneous and consists of two discrete size classes with molecular weights of 3.45 X 10(6) and 3.25 X 10(6). Both size classes of DNA are encapsidated into HD virion particles. Their relative intracellular amounts differ, depending on the cell system. Vero-76 carrier cultures in which HD virus was detected contain both size classes of DNA, with the larger molecules prevailing by a factor of 10. Five clonal lines derived from Vero-76 cell cultures contain exclusively the larger DNA. On the other hand, after cocultivation of Vero-76 with CV-1 cells for several passages, minicircular DNA is accumulated such that both size classes are synthesized in equal amounts. Any of the originally viral DNA-producing cell lines may, upon subcultivation, cease yielding virus. The RITA cell line of Cercopithecus aethiops origin is the only cell line among numerous ones tested which upon infection permits the establishment of a one-step growth cycle. However, between 6 and 8 days after infection, viral DNA synthesis is discontinued, and a persistent viral infection cannot be established. Physical maps of the genomes were constructed, and it could be shown that the smaller, minicircular DNA had originated from the larger DNA as the result of a deletion. The sequences missing in the minicircular DNA are confined to the relative map position 0.15 to 0.21.     

Simultaneous high-performance liquid chromatographic determination of a glucuronyl prodrug of doxorubicin, doxorubicin and its metabolites in human lung tissue

A rapid and sensitive method was developed for the simultaneous determination of the new doxorubicin glucuronide prodrug HMR 1826, the parent drug doxorubicin and its metabolites in human lung tissue samples. Homogenization of frozen tissue samples with the micro-dismembrator was followed by a silver nitrate precipitation step. By removing the exceeding silver ions with sodium chloride further purification steps could be omitted. Compounds were separated by isocratic high-performance liquid chromatography on a LiChrospher 100 RP18 column and a mobile phase consisting of citric acid buffer–acetonitrile–methanol–tetrahydrofuran within 30 min and quantified with fluorescence detection. The method showed good recoveries for all compounds (86–99%) and a linear calibration range of 20 ng/g–80 μg/g for doxorubicin and 1–600 μg/g for HMR 1826.     

Synthesis and cytotoxic activity of a glucuronylated prodrug of nornitrogen mustard

A new glucuronylated prodrug of nornitrogen mustard, incorporating the same spacer group as the doxorubicin prodrug HMR 1826, has been prepared. Upon exposure to E. coli beta-glucuronidase, fast hydrolysis occurs but a lower cytotoxicity against LoVo cancer cells is observed compared to the nornitrogen mustard alone. This is explained by cyclization of the intermediate carbamic acid to the inactive chloroethyl oxazolidinone.     

Clonal analysis of expression of tumor-associated transplantation antigens and of metastatic capacity

Summary ESb, a spontaneous high metastatic variant of the chemically induced T lymphoma Eb, was found previously to express a tumor-associated transplantation antigen (TATA) that was different from that of the parental line. Syngeneic tumor-specific cytolytic T lymphocytes (CTL) were able to recognize the different TATAs of Eb and ESb in vitro and could therefore be used for routine typing. The object of this study was to investigate tumor antigen expression on a clonal level and to compare the in vitro data with the in vivo behavior of the same cell lines.Our CTL typing analysis of cloned tumor lines revealed that the two populations, Eb and ESb, are distinct and relatively homogeneous with regard to their TATA expression. Furthermore, all ESb clones formed rosettes with antibody-coated erythrocytes, while none of the parental type Eb clones showed this characteristic. The sensitivity to tumor-specific CTL lysis varied with time of tumor cell culture in vitro in a clone-dependent manner.Variability was also noted in vivo in tumor growth and metastatic spread. Of over 50 ESb clones tested, the majority were highly metastatic while a minority were significantly lower in metastatic capacity. High and low metastatic ESb clones could not be distinguished by their expression of TATAs and of Fc receptors. There was also a considerable individual variability in the hosts, although they were genetically identical. This variability was most probably due to differences in the immune status of the animals.     

Monoclonal antibodies against 5'-nucleotidase from a human pancreatic tumor cell line: their characterization and inhibitory capacity on tumor cell adhesion to fibronectin substratum.

Four mouse monoclonal antibodies (PTN63, PTN108, PTN124, PTN514) against the ecto-5'-nucleotidase purified from a human pancreatic adenocarcinoma cell line (PaTu II) have been raised and characterized. All four monoclonal antibodies recognize the protein moiety of the glycosylated ecto-5'-nucleotidase. In competition assays it was demonstrated that three of the antibodies (PTN63, PTN108, PTN514) recognize different epitopes within the protein moiety. Furthermore, PTN108, PTN124, and PTN514 reduced the 5'-nucleotidase AMPase activity in contrast to PTN63 having no inhibitory effect. The antibodies show no cross-reactivity with ecto-5'-nucleotidases from rat liver, bull seminal plasma, chicken gizzard and human peripheral blood cells. When assayed by indirect immunofluorescence the antibodies react with the plasma membrane of human pancreatic tumor cells with varying staining intensity. Immunocytochemistry on paraffin sections of normal human pancreas revealed a prominent staining of the pancreatic duct cells. No staining of the acinar and islet cells could be detected. Thus, 5'-nucleotidase is a marker enzyme for pancreatic duct cells and can be used to determine the origin of pancreatic tumor cells. PTN63 reduced the attachment to fibronectin substratum of a human pancreatic adenocarcinoma tumor cell line possessing a high amount of plasma membrane bound ecto-5'-nucleotidase, but had no effect on a cell line lacking the membrane bound AMPase. In contrast, PTN108 and PTN514, which inhibit the AMPase activity, exhibited no influence on the adhesion of human pancreatic tumor cells to fibronectin substratum.     

Immunohistochemical and immunochemical characterization of a new endothelial cell-specific antigen

A new monoclonal antibody (moab BW 200) of IgG3 kappa-isotype was generated which recognizes an epitope located on an antigen molecule restricted to human neoplastic and non-neoplastic endothelial cells. The molecular weight of the antigen was determined using immunoprecipitation analysis followed by SDS-PAGE. Despite its similar molecular weight to FVIII-RAG, the antigen detected by moab BW 200 was shown to be different from FVIII-RAG.     

Attachment of rhodosaminylanthracyclinone-type anthracyclines to the hinge region of monoclonal antibodies

We have found that a maleimidobenzoyl spacer attached to OH-4' of the rhodosamine moiety of rhodosaminylanthracyclinone-type anthracyclines is most suitable for the attachment of these drugs to carriers, providing important advantages: The spacer is selectively and most readily introduced into the rhodosamine moiety of the drugs, is stable enough for proper handling of the derivatives, and can easily be attached to thiol groups of carrier systems such as reduced monoclonal antibodies. The anthracyclines can be liberated from the conjugates by mere hydrolysis, requiring neither hydrolytic enzymes nor acidic pH. Liberation of the drugs can, moreover, be affected by the presence of the appropriate substituents Z on the phenylene ring of the spacer, thus allowing slowed or enhanced liberation of the cytostatically active drug. The corresponding p-maleimidobenzoyl derivatives of beta-rhodomycin I, N,N-dimethyldaunorubicin, and rodorubicin have been attached to thiol groups of the hinge region of reduced monoclonal antibody BW 494/32, directed against a pancreatic cancer associated glycoprotein antigen, resulting in MoAb BW 494/32 conjugates, carrying 4.8-6.8 mol of cytotoxic residues/mol of MoAb. Rodorubicin was similarly attached to MoAb BW 575/931/2, directed against a small cell lung cancer associated antigen and to MoAb BW 431/26, recognizing an epitope detectable on carcinoembryonic antigen. The results provide evidence that the newly developed method of coupling of anthracyclines to the hinge region of monoclonal antibodies may be of broader use.     

Metastatic tumor cell variants with increased resistance to infection by Semliki Forest virus

Semliki Forest virus (SFV) is an interesting virus for cell interaction studies because it binds directly to the cells' major histocompatibility antigens. We used this reagent to study the expression and functional properties of H-2 molecules on murine tumor lines that are closely related but differ greatly in metastatic capacity. Tumor cell variants with high metastatic capacity showed an increased resistance to virus infection, an effect that was selective for SFV. Although the high metastatic tumor lines did not express less H-2 antigens than the low metastatic ones, they bound much less of the SFV viral glycoproteins.     

A monoclonal antibody with binding and inhibiting activity towards human pancreatic carcinoma cells

Summary The murine monoclonal antibody (MAb) BW 494 was characterized in relation to its tissue specificity, the epitope recognized, in vitro and in vivo radiolocalization and its potential to mediate antibody dependent cellular cytotoxicity (ADCC) and complement mediated cytolysis (CMC). The MAb defined carbohydrate epitope located on a >200 k daltons glycoprotein was mainly expressed on the majority of well differentiated adenocarcinomas of the pancreas. Furthermore, the epitope is accessible to MAb BW 494 in vivo, allowing an enrichment of radioactive antibody at the tumor site in nude mice. Additionally, MAb BW 494 is able to use human peripheral blood lymphocytes as effector cells for ADCC reactions against appropriate tumor target cells in vitro. In contrast, the antibody does not mediate human or rabbit CMC.