Initiation of humoral and cellular immune responses in patients with refractory Hodgkin's disease by treatment with an anti-CD16/CD30 bispecific antibody

Fifteen patients with refractory Hodgkin's disease were treated in a dose-escalation trial with the bispecific monoclonal antibody (bi-mAb) HRS-3/A9, which is directed against the Fcγ receptor III (CD16 antigen) and the Hodgkin's-associated CD30 antigen. Treatment consisted of four cycles of four bi-mAb infusions given over 1 h every 3–4 days at different dose levels ranging from 1 mg/m² to 64 mg/m². Measurable serum levels (above 0.1 μg/ml) of circulating bi-mAb could be detected in patients treated with doses above 4 mg/m², reaching peak levels of 9.5 μg/ml immediately after the end of antibody infusion on the highest dose level. Bi-mAb elimination corresponded to second-order kinetics with a terminal half-life time (t _1/2,β) of 28–32 h. Bi-mAb treatment induced the occurrence of human anti-(mouse Ig) antibodies (HAMA) in 6 out of 13 patients initially testing negative. All 6 patients not only developed anti-isotypic anti-(mouse Ig) but also anti-idiotypic and anti-anti-idiotypic antibodies. While no consistent changes of peripheral blood cell counts, or of any lymphocyte subpopulation including natural killer (NK) cells, has been observed, 4 out of 6 evaluable patients treated with doses of at least 4 mg/m² showed an increase of NK cell activity within 2 weeks after treatment, which lasted for a maximum of 12 weeks. Circulating amounts of soluble CD30 antigen could be detected in the serum of 6 patients. However, like the results and time courses of all the other immunological parameters evaluated, this was not predictive for treatment outcome. Received: 16 September 1999 / Accepted: 6 January 2000     

Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin alpha(2)beta(1)

Evidence was obtained about the mechanism responsible for platelet integrin alpha(2)beta activation by determining effects of various inhibitors on soluble collagen binding, a parameter to assess integrin alpha(2)beta(1) activation, in stimulated platelets. Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin alpha(2)beta(1), but those operating via glycoprotein Ib cannot. Activation of alpha(2)beta(1) induced by low thrombin or collagen-related peptide concentrations was almost completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integrin alpha(2)beta(1) activation, and this was not ADP concentration-dependent. These results suggest that at the low agonist concentrations, the released ADP would be a primary inducer of integrin alpha(2)beta(1) activation, while at the high agonist concentrations, there would be several pathways through which integrin alpha(2)beta(1) activation can be induced. Kinetic analyses revealed that ADP-induced platelets had about the same number of binding sites (B(max)) as thrombin-induced platelets, but their affinity (K(d)) for soluble collagen was 3.7-12.7-fold lower, suggesting that activated integrin alpha(2)beta(1) induced by ADP is different from that induced by thrombin. The data are consistent with an activation mechanism involving released ADP and in which there exists two different states of activated integrin alpha(2)beta(1); these activated forms of integrin alpha(2)beta(1) would have different conformations that determine their ligand affinity.     

Epidermal growth factor negatively regulates chondrogenesis of mesenchymal cells by modulating the protein kinase C-alpha, Erk-1, and p38 MAPK signaling pathways

During limb development, epithelial cells in the apical ectodermal ridge keep the underlying mesenchymal cells in a proliferative state preventing differentiation by secreting signaling molecules such as epidermal growth factor (EGF). We investigated the molecular mechanism of the EGF effect on the regulation of micromass culture-induced chondrogenesis of chick limb bud mesenchymal cells as a model system. We found that expression and tyrosine phosphorylation of the EGF receptor was increased transiently during chondrogenesis. Exogenous EGF inhibited chondrogenic differentiation of mesenchymal cells, and this effect was reversed by the EGF receptor inhibitor AG1478. EGF treatment also inhibited the expression and activation of protein kinase C-alpha, whereas it activated Erk-1 and inhibited p38 mitogen-activated protein kinase, all of which appeared to be involved in the EGF-induced inhibition of chondrogenesis. Stimulation of the EGF receptor blocked precartilage condensation and altered the expression of cell adhesion molecules such as N-cadherin and integrins alpha(5) and beta(1). All these EGF effects were reversible by AG1478. The data indicate that EGF negatively regulate chondrogenesis of chick limb bud mesenchymal cells by inhibiting precartilage condensation and by modulating signaling pathways including those of protein kinase C-alpha, Erk-1, and p38 mitogen-activated protein kinase.     

Mullerian inhibiting substance inhibits breast cancer cell growth through an NFkappa B-mediated pathway

Müllerian inhibiting substance (MIS), a member of the transforming growth factor-beta superfamily, induces regression of the Müllerian duct in male embryos. In this report, we demonstrate MIS type II receptor expression in normal breast tissue and in human breast cancer cell lines, breast fibroadenoma, and ductal adenocarcinomas. MIS inhibited the growth of both estrogen receptor (ER)-positive T47D and ER-negative MDA-MB-231 breast cancer cell lines, suggesting a broader range of target tissues for MIS action. Inhibition of growth was manifested by an increase in the fraction of cells in the G(1) phase of the cell cycle and induction of apoptosis. Treatment of breast cancer cells with MIS activated the NFkappaB pathway and selectively up-regulated the immediate early gene IEX-1S, which, when overexpressed, inhibited breast cancer cell growth. Dominant negative IkappaBalpha expression ablated both MIS-mediated induction of IEX-1S and inhibition of growth, indicating that activation of the NFkappaB signaling pathway was required for these processes. These results identify the NFkappaB-mediated signaling pathway and a target gene for MIS action and suggest a putative role for the MIS ligand and its downstream interactors in the treatment of ER-positive as well as negative breast cancers.     

Structure-activity relationship study of asiatic acid derivatives against beta amyloid (A beta)-induced neurotoxicity

8 Semi-synthetic derivatives of asiatic acid were prepared and their protective effect against A beta-induced neurotoxicity was evaluated. Among them, asiatic acid (2), and 4, 16 showed 97, 92 and 87% of protective effect, respectively.     

Noncovalent thrombin inhibitors incorporating an imidazolylethynyl P1

A series of noncovalent tripeptidic thrombin inhibitors incorporating a unidazolylethynyl moiety at P1 was investigated. A number of compounds of this series were highly potent and selective versus trypsin, and several compounds demonstrated good oral absorption in rats (F=58% for compound 19).     

A Novel Technique for the Effective Production of Short Peptide Analogs from Concatameric Short Peptide Multimers

We designed a basic unit of the modified chicken gonadotropin releasing hormone II (cGnRH-II) peptide containing a trypsin cleavable linker peptide at both ends of the original peptide. We made a synthetic DNA coding for the modified cGnRH-II peptide with asymmetric and complementary cohesive ends of linker nucleotides. A tandemly repeated DNA cassette for the expression of concatameric short peptide multimers was constructed by ligating the basic units. The expressed peptide multimers were purified and subject to amino-terminal sequence analysis, which displayed the amino acid sequences expected from the designed nucleotides of the expression cassette. The monomeric cGnRH-II peptide analogs were generated after trypsin digestion. The present results showed that the technique developed for the production of the concatameric peptide multimers with cleavable linker peptides can be generally applicable to the production of short peptide analogs.