Preclinical studies of monoclonal antibodies for intravesical radioimmunotherapy of human bladder cancer

Eighty percent of bladder cancers present as superficial disease. Many are multifocal, and apparently successful treatment is frequently followed by recurrence. The use of monoclonal antibodies (MAbs) to target radiotherapy to these tumors offers great potential, especially since they can be administered directly into the bladder (intravesically) bypassing many of the side effects encountered to date with systemic MAb-based therapy. Implantation of human bladder cancer cell lines in the bladder wall of nude rats results in tumor formation, providing an excellent model to test this. Tumor size can be monitored by X-ray analysis after administration of urograffin. Comparative studies of two murine MAbs, BLCA-8, IgG3, and C1-137, IgG1, against malignant human bladder cancer cells have been performed. Radio-immunoconjugates produced with¹²⁵Iodine (¹²⁵I) have been used for biodistribution studies following administration directly into rat bladder. Radioiodinated intact MAbs or Fabs administered intravesically into nontumor bearing rats did not leak into the systemic circulation and were stable in urine for up to 100 h. Biodistribution studies carried out following intravesical administration of radio-immunoconjugates to tumor-bearing nude rats indicate better tumor uptake of C1-137 than BLCA-8. Further studies to test two-step intravesical administration of biotinylated MAb followed by radioiodinated streptavidin are in progress. Our studies indicate that the C1-137 MAb may have considerable potential for intravesical radioimmunotherapy of patients with superficial bladder tumors.     

Drosophila rab GDI mutants disrupt development but have normal rab membrane extraction

Summary: Rab GTPases are essential for vesicular transport. Rab GDP dissociation inhibitor (GDI) binds to GDP‐bound rabs, removes rabs from acceptor membranes and delivers rabs to donor membranes. We isolated lethal GDI mutations in Drosophila and analyzed their developmental phenotypes. To learn how these mutations affect GDI structure, the crystal structure of Drosophila GDI was determined by molecular replacement to a resolution of 3.0 Å. Two hypomorphic, missense mutations are located in domain II of GDI at highly conserved positions, but not in previously identified sequence conserved regions. The mutant GDIs were tested for ability to extract rabs from membranes and showed wild‐type levels of rab membrane extraction. The two missense alleles showed intragenic complementation, indicating that domain II of GDI may have two separable functions. This study indicates that GDI function is essential for development of a complex, multicellular organism and that puparium formation and pole cell formation are especially dependent on GDI function. genesis 31:17–29, 2001. © 2001 Wiley‐Liss, Inc.     

Fetal losses and pathologic findings of clones derived from serum-starved versus serum-fed bovine ovarian granulosa cells

The objective was to evaluate development of cloned embryos constructed with serum-starved versus fed ovarian granulosa cells. Fusion of somatic cells with cytoplasts (50.7 and 50.7%; SEM=5.5), development of cloned embryos to 8-16-cell (59.4 and 62.6%; SEM=6.4), and compact morula/blastocyst-stages (24.9 and 26.5%; SEM=8.5) was similar for serum-starved and fed groups. More heifers were confirmed pregnant with clones derived from serum-starved cells (9/13, 67.2% versus 11/25, 44%). However, embryonic loss between 29-50 days was greater for clones constructed with serum-starved (88.9%) versus fed (36.4%) cells. Development of clones derived from serum-fed cells through placentation and differentiation was not predictive of competency to term. Fetal deaths occurred in the majority of late term pregnancies as a result of complications from hydrallantois. Only one fetus derived from serum-fed ovarian granulosa cells developed to term (278 days). At birth and for approximately 9 consecutive months thereafter, routine veterinary examinations confirmed expected growth patterns. In summary, the use of serum fed granulosa cells for nuclear transfer was coincident with a high incidence of third trimester losses associated with hydrallantois and fetal oversize.     

Potent and selective inhibitors of Helicobacter pylori glutamate racemase (MurI): Pyridodiazepine amines

An SAR study of an HTS screening hit generated a series of pyridodiazepine amines as potent inhibitors of Helicobacter pylori glutamate racemase (MurI) showing highly selective anti-H. pylori activity, marked improved solubility, and reduced plasma protein binding. X-ray co-crystal E–I structures were obtained. These uncompetitive inhibitors bind at the MurI dimer interface.