No abstract

The involvement of platelets and the c-mpl receptor in the regulation of thrombopoietin (TPO) plasma concentrations and tissue mRNA levels was investigated in both normal mice and mice defective in c-mpl (c-mpl- /-). Although c-mpl-/- mice have fewer platelets and higher plasma TPO activity than normal mice, there was no increase in TPO mRNA levels as measured by an S1 nuclease protection assay. After the intravenous injection of 125I-TPO, specific uptake of radioactivity by the spleen and blood cells was present in the normal mice, but absent in the c-mpl- /- mice. Platelet-rich plasma (PRP) from normal mice was able to bind and internalize 125I-TPO, whereas PRP from c-mpl-/- mice lacked this ability. Analysis of 125I-TPO binding to normal PRP indicated that binding was specific and saturable, with an approximate affinity of 560 pmol/L and 220 receptors per platelet. PRP from normal mice was also able to degrade 125I-TPO into lower molecular weight fragments. After the intravenous injections, c-mpl-/- mice cleared a dose of 125I-TPO at a much slower rate than did normal mice. Injection of washed platelets from normal mice into c-mpl-/- mice resulted in a dramatic, but transient, decrease in plasma TPO levels. These data provide evidence that platelets regulate plasma TPO levels via binding to the c-mpl receptor on circulating platelets.

Bispecific antibodies and antibody fragments in various formats have been explored as a means to recruit cytolytic T cells to kill tumor cells. Encouraging clinical data have been reported with molecules such as the anti-CD19/CD3 bispecific T cell engager (BiTE) blinatumomab. However, the clinical use of many reported T cell-recruiting bispecific modalities is limited by liabilities including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing challenges. We describe a B cell-targeting anti-CD20/CD3 T cell-dependent bispecific antibody (CD20-TDB), which is a full-length, humanized immunoglobulin G1 molecule with near-native antibody architecture constructed using "knobs-into-holes" technology. CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo. In cynomolgus monkeys, CD20-TDB potently depletes B cells in peripheral blood and lymphoid tissues at a single dose of 1 mg/kg while demonstrating pharmacokinetic properties similar to those of conventional monoclonal antibodies. CD20-TDB also exhibits activity in vitro and in vivo in the presence of competing CD20-targeting antibodies. These data provide rationale for the clinical testing of CD20-TDB for the treatment of CD20-expressing B cell malignancies.

Clinical results from the latest strategies for T-cell activation in cancer have fired interest in combination immunotherapies that can fully engage T-cell immunity. In this study, we describe a trastuzumab-based bispecific antibody, HER2-TDB, which targets HER2 and conditionally activates T cells. HER2-TDB specifically killed HER2-expressing cancer cells at low picomolar concentrations. Because of its unique mechanism of action, which is independent of HER2 signaling or chemotherapeutic sensitivity, HER2-TDB eliminated cells refractory to currently approved HER2 therapies. HER2-TDB exhibited potent antitumor activity in four preclinical model systems, including MMTV-huHER2 and huCD3 transgenic mice. PD-L1 expression in tumors limited HER2-TDB activity, but this resistance could be reversed by anti-PD-L1 treatment. Thus, combining HER2-TDB with anti-PD-L1 yielded a combination immunotherapy that enhanced tumor growth inhibition, increasing the rates and durability of therapeutic response. Cancer Res; 74(19); 5561-71. Ó2014 AACR.

A cDNA clone (designated as GP2‐7) encoding a novel 5‐hydroxytryptamine (5‐HT) receptor was isolated from a guinea pig hippocampal library. The receptor shares amino acid homology within the hydrophobic domains with other cloned 5‐HT receptor subtypes (34–48%). The sequence of GP2‐7 is homologous to that described for a novel receptor previously cloned from a rat brain cDNA library and provisionally designated as 5‐HT7. mRNA for GP2‐7 was detected in cortical and limbic brain regions. Transiently expressed GP2‐7 showed high‐affinity binding to [3H]5‐HT (pKi = 9.0) with the following rank order of affinities: 5‐carboxyamidotryptamine (5‐CT) > 5‐HT = 5‐methoxytryptamine (5‐MeOT) > methiothepin > 8‐hydroxy‐2‐(dipropylamino)tetralin (8‐OH‐DPAT) > spiperone ≫ sumatriptan. Adenylyl cyclase activity in CHO‐K1 cells transiently transfected with GP2‐7 was stimulated by several analogues of 5‐HT with the following order of potency: 5‐CT > 5‐HT = 5‐MeOT > dipropyl‐5‐CT > 8‐OH‐DPAT. Methiothepin and spiperone were potent antagonists. Preliminary analysis suggests that GP2‐7 closely resembles a receptor in the guinea pig hippocampus that exhibits a high affinity toward 5‐CT.