A novel, blocking, Fc-silent anti-CD40 monoclonal antibody prolongs nonhuman primate renal allograft survival in the absence of B cell depletion.

CD40-CD154 pathway blockade prolongs renal allograft survival in nonhuman primates (NHPs). However, antibodies targeting CD154 were associated with an increased incidence of thromboembolic complications. Antibodies targeting CD40 prolong renal allograft survival in NHPs without thromboembolic events but with accompanying B cell depletion, raising the question of the relative contribution of B cell depletion to the efficacy of anti-CD40 blockade. Here, we investigated whether fully silencing Fc effector functions of an anti-CD40 antibody can still promote graft survival. The parent anti-CD40 monoclonal antibody HCD122 prolonged allograft survival in MHC-mismatched cynomolgus monkey renal allograft transplantation (52, 22, and 24 days) with accompanying B cell depletion. Fc-silencing yielded CFZ533, an antibody incapable of B cell depletion but still able to potently inhibit CD40 pathway activation. CFZ533 prolonged allograft survival and function up to a defined protocol endpoint of 98-100 days (100, 100, 100, 98, and 76 days) in the absence of B cell depletion and preservation of good histological graft morphology. CFZ533 was well-tolerated, with no evidence of thromboembolic events or CD40 pathway activation and suppressed a gene signature associated with acute rejection. Thus, use of the Fc-silent anti-CD40 antibody CFZ533 appears to be an attractive approach for preventing solid organ transplant rejection.

C-terminal interaction is essential for surface trafficking but not for heteromeric assembly of GABA(b) receptors.

Assembly of fully functional GABA(B) receptors requires heteromerization of the GABA(B(1)) and GABA(B(2)) subunits. It is thought that GABA(B(1)) and GABA(B(2)) undergo coiled-coil dimerization in their cytoplasmic C termini and that assembly is necessary to overcome GABA(B(1)) retention in the endoplasmatic reticulum (ER). We investigated the mechanism underlying GABA(B(1)) trafficking to the cell surface. We identified a signal, RSRR, proximal to the coiled-coil domain of GABA(B(1)) that when deleted or mutagenized allows for surface delivery in the absence of GABA(B(2)). A similar motif, RXR, was recently shown to function as an ER retention/retrieval (ERR/R) signal in K(ATP) channels, demonstrating that G-protein-coupled receptors (GPCRs) and ion channels use common mechanisms to control surface trafficking. A C-terminal fragment of GABA(B(2)) is able to mask the RSRR signal and to direct the GABA(B(1)) monomer to the cell surface, where it is functionally inert. This indicates that in the heteromer, GABA(B(2)) participates in coupling to the G-protein. Mutagenesis of the C-terminal coiled-coil domains in GABA(B(1)) and GABA(B(2)) supports the possibility that their interaction is involved in shielding the ERR/R signal. However, assembly of heteromeric GABA(B) receptors is possible in the absence of the C-terminal domains, indicating that coiled-coil interaction is not necessary for function. Rather than guaranteeing heterodimerization, as previously assumed, the coiled-coil structure appears to be important for export of the receptor complex from the secretory apparatus.

Ontogenic expression of anterior pituitary GABA(B) receptor subunits.

gamma-Aminobutyric acid (GABA) is involved in the neuroendocrine control of hypophyseal secretion, acting both in the central nervous system and directly at the pituitary. We have characterized the properties of anterior pituitary GABA(B) receptors. In this work the ontogeny of rat anterior pituitary GABA(B) receptors and the pattern of subunit expression in rats of both sexes were determined. Western blot analysis showed a temporal decrease in GABA(B) subunits GABA(B(1a)) and GABA(B(1b)) expression in female anterior pituitary membranes from day 4 to adulthood, with GABA(B(1a)) being significantly more abundant than GABA(B(1b)) at early stages of development; the GABA(B(2)) subunit was barely detectable. In the male, GABA(B(1a)) followed a similar pattern and appeared to be significantly less abundant than in 4- and 12-day-old females; GABA(B(1b)) and GABA(B(2)) expression in the male was barely detectable. Scatchard plot analysis showed a temporal decrease in binding sites in female anterior pituitary membranes, in agreement with the western blot results. The number of binding sites was significantly higher in female than in male 4-day-old membranes. Dissociation constant values were similar for both sexes at all ages studied. This study reports for the first time the ontogeny of anterior pituitary GABA(B) receptors, showing a particular developmental pattern of subunit expression and a clear sexual dimorphism.

Conformations of synthetic beta peptides in solid state and in aqueous solution: relation to toxicity in PC12 cells.

The secondary structures of peptides beta 25-35 (the active toxic fragment) and beta 35-25 (reverse sequence and non-toxic fragment), as well as of the amidated beta (25-35)-NH2 peptide were investigated in aqueous solution and in the solid state by means of Fourier-transformed infrared spectroscopy and circular dichroism spectroscopy. The conformations of the beta 25-35 and beta 35-25 in solid state were identical and contained mostly beta-sheet structures. In solid state the amidated beta (25-35)-NH2 peptide also contained mostly beta-sheet structures. Freshly prepared aqueous solutions of the beta 25-32 (0.5 - 3.8 mM) contained a mixture of beta-sheet and random coil structures. Within 30-60 min incubation at 37 degrees C in water or in phosphate-buffered saline solution (PBS), beta 25-35 was almost fully converted to a beta-sheet structure. Decreasing the temperature from 37 degrees C to 20 degrees C decreased the rate of conversion from random coil to beta-sheet structures, 1-2 h being required for complete conversion. In contrast beta 35-25 in water or in PBS buffer had mostly a random coil structure and remained so for 6 days. The amidated beta(25-35)-NH2 peptide in water (2.7 mM) was also mostly random coil. However, when this peptide (2-2.7 mM) was dissolved in PBS (pH 7.4) or in 140 mM NaCl, a gel was formed and its conformation was mostly beta-sheet. Decreasing the concentration of beta (25-35)-NH2 peptide in 140 mM NaCl aqueous solution from 2 mM to 1 mM or below favored the conversion from beta-sheet structures to random coil structures. The beta 25-35 was toxic to PC12 cells while beta 35-25 was not. The amidated peptide beta (25-35)-NH2 was at least 500-fold less toxic than beta 25-35. Structural differences between these beta peptides in aqueous solutions may explain the difference in their respective toxicities.