Phase I study and preliminary pharmacology of the novel innate immune modulator rBBX-01 in gynecologic cancers.

PURPOSE: A recombinant protein product, rBBX-01, is the first innate immunostimulator derived from a protozoan (Eimeria protozoan) and has shown potent preclinical in vivo and in vitro activities. This phase I trial was done to determine the safety and basic pharmacology of rBBX-01. EXPERIMENTAL DESIGN: Eligible patients had recurrent incurable gynecologic malignancies. The study was divided into three components: a starting low-dose phase (0.85, 2.0, and 4.0 microg/m2), an intrapatient dose acceleration phase (4.0-1,024.0 microg/m2), and a high-dose phase (1,000 and 2,000 microg/m2). All treatment doses were administered daily for 5 days. Patients were allowed a second cycle of treatment if there was evidence of response. RESULTS: Sixteen patients received a total of 20 cycles of rBBX-01. All patients tolerated the drug well, exhibiting no local or systemic, acute or delayed, adverse reactions. Plasma levels of rBBX-01 were detectable in all patients over the entire dose range, although changes in the pharmacodynamic marker (interleukin-12) exhibited patient-to-patient variability. Of 14 patients with ovarian, primary peritoneal, or endometrial cancer with elevated CA125 biomarkers at the start of treatment, 4 responded with decreased levels of CA125. One patient showed decreasing CA125 levels for 10 months and received no additional chemotherapy for 11 months. Those patients exhibiting reductions in CA125 also exhibited increased levels of plasma interleukin-12 during the week of therapy. CONCLUSION: The immunostimulator rBBX-01 was safe in multidose regimens in heavily pretreated women. Of the 14 patients with elevated CA125 levels, an approximately 30% response rate was detected. rBBX-01 should receive additional testing in the clinical setting.

Liposomal polyethyleneglycol and polyethyleneglycol-peptide combinations for active targeting to liver in vivo.

This report describes the development and evaluation of a range of polyethyleneglycol and polyethyleneglycol-peptide liposome formulations that effectively target liver in vivo. A 19-amino-acid sequence from the N-terminal region of the circumsporozoite protein of Plasmodium berghei was attached to the distal end of di22:1-aminopropane-polyethyleneglycol(3400), and incorporated into liposomes containing di22:1-phosphatidylcholine and di22:1-phosphatidylethanolamine-polyethyleneglycol(5000). By systematically varying the mole fractions of both the lipid-polyethyleneglycol and the lipid-polyethyleneglycol-peptide conjugates, and screening for serum-induced aggregation in vitro, a serum-stable range of formulations was established. These stable formulations were tested for binding to Hepa 1-6 liver cells in culture, and from these results three formulations were prepared for intravenous administration in mice. All three formulations exhibited effective liposome targeting to the liver, with approximately 80% of the total injected dose recovered in the liver within 15 min. Uptake by liver cells was more than 600-fold higher than uptake by those in the heart, and more than 200-fold higher than uptake by lung or kidney cells. Effective targeting to liver in vivo was successful after repeated (up to three) administrations to the host at 14-day intervals. All formulations prepared for in vivo administration were stable in the presence of serum, as measured by complete retention of entrapped calcein dye. The formulation with the lowest mole fractions of peptide and polyethyleneglycol was the most cost-effective in terms of encapsulation efficiency and minimal use of peptide and polymer compounds. The in vitro biophysical screening, followed by cell culture testing, reduced the number of animals required to develop an effective set of targeted liposome formulations for in vivo application.

Cloning and preliminary characterization of the dihydroorotase from Toxoplasma gondii.

A full-length dihydroorotase (DHOase) sequence was cloned from a Toxoplasma gondii tachyzoite cDNA library. The sequence had a calculated molecular mass of 44.2 kDa and a pI of 5.72, and was most similar to type IIa DHOases. A recombinant protein was expressed and purified with a yield of approximately 20 mg L(-1) of cell culture. Polyclonal antibodies raised against purified recombinant protein reacted with a band of the expected molecular mass in tachyzoite extracts. Specific activities of 18.3 micromol/min/mg in the biosynthetic direction and 18.4 micromol/min/mg in the degradative direction, with K(m, carbamyl aspartate) = 323 microM and K(m, dihydroorotate) = 64.3 microM, were measured for purified recombinant protein. Size exclusion chromatography/laser light scattering showed a single, monodisperse peak with a molecular mass of 45.6 kDa, suggesting that the native protein is a monomer.

Plasmodium falciparum TryThrA antigen synthetic peptides block in vitro merozoite invasion to erythrocytes.

Tryptophan-threonine-rich antigen (TryThrA) is a Plasmodium falciparum homologue of Plasmodium yoelii-infected erythrocyte membrane pypAg-1 antigen. pypAg-1 binds to the surface of uninfected mouse erythrocytes and has been used successfully in vaccine studies. The two antigens are characterized by an unusual tryptophan-rich domain, suggesting similar biological properties. Using synthetic peptides spanning the TryThrA sequence and human erythrocyte we have done binding assays to identify possible TryThrA functional regions. We describe four peptides outside the tryptophan-rich domain having high activity binding to normal human erythrocytes. The peptides termed HABPs (high activity binding peptides) are 30884 ((61)LKEKKKKVLEFFENLVLNKKY(80)) located at the N-terminal and 30901 ((401)RKSLEQQFGDNMDKMNKLKKY(420)), 30902 ((421)KKILKFFPLFNYKSDLESIM(440)) and 30913 ((641)DLESTAEQKAEKKGGKAKAKY(660)) located at the C-terminal. Studies with polyclonal goat antiserum against synthetic peptides chosen to represent the whole length of the protein showed that TryThrA has fluorescence pattern similar to PypAg-1 of P. yoelii. All HABPs inhibited merozoite in vitro invasion, suggesting that TryThrA protein may be participating in merozoite-erythrocyte interaction during invasion.

Protein sorting in Plasmodium falciparum-infected red blood cells permeabilized with the pore-forming protein streptolysin O.

Plasmodium falciparum is an intracellular parasite of human red blood cells (RBCs). Like many other intracellular parasites, P. falciparum resides and develops within a parasitophorous vacuole which is bound by a membrane that separates the host cell cytoplasm from the parasite surface. Some parasite proteins are secreted into the vacuolar space and others are secreted, by an as yet poorly defined pathway, into the RBC cytosol. The transport of proteins from the parasite has been followed mainly using morphological methods. In search of an experimental system that would allow (i) dissection of the individual steps involved in transport from the parasite surface into the RBC cytosol, and (ii) an assessment of the molecular requirements for the process at the erythrocytic side of the vacuolar membrane, we permeabilized infected RBCs with the pore-forming protein streptolysin O using conditions which left the vacuole intact. The distribution of two parasite proteins which served as markers for the vacuolar space and the RBC cytosol respectively was analysed morphologically and biochemically. In permeabilized RBCs the two marker proteins were sorted to the same compartments as in intact RBCs. The protein which was destined for the RBC cytosol traversed the vacuolar space before it was translocated across the vacuolar membrane. Protein transport could be arrested in the vacuole by removing the RBC cytosol. Translocation across the vacuolar membrane required ATP and a protein source at the erythrocytic face of the membrane, but it was independent of the intracellular ionic milieu of the RBC.