A high-affinity native human antibody neutralizes human cytomegalovirus infection of diverse cell types.

Human cytomegalovirus (HCMV) is the most common infection causing poor outcomes among transplant recipients. Maternal infection and transplacental transmission are major causes of permanent birth defects. Although no active vaccines to prevent HCMV infection have been approved, passive immunization with HCMV-specific immunoglobulin has shown promise in the treatment of both transplant and congenital indications. Antibodies targeting the viral glycoprotein B (gB) surface protein are known to neutralize HCMV infectivity, with high-affinity binding being a desirable trait, both to compete with low-affinity antibodies that promote the transmission of virus across the placenta and to displace nonneutralizing antibodies binding nearby epitopes. Using a miniaturized screening technology to characterize secreted IgG from single human B lymphocytes, 30 antibodies directed against gB were previously cloned. The most potent clone, TRL345, is described here. Its measured affinity was 1 pM for the highly conserved site I of the AD-2 epitope of gB. Strain-independent neutralization was confirmed for 15 primary HCMV clinical isolates. TRL345 prevented HCMV infection of placental fibroblasts, smooth muscle cells, endothelial cells, and epithelial cells, and it inhibited postinfection HCMV spread in epithelial cells. The potential utility for preventing congenital transmission is supported by the blockage of HCMV infection of placental cell types central to virus transmission to the fetus, including differentiating cytotrophoblasts, trophoblast progenitor cells, and placental fibroblasts. Further, TRL345 was effective at controlling an ex vivo infection of human placental anchoring villi. TRL345 has been utilized on a commercial scale and is a candidate for clinical evaluation.

A novel antiangiogenesis therapy using an integrin antagonist or anti-Flk-1 antibody coated 90Y-labeled nanoparticles.

PURPOSE: Integrin alpha(v)beta(3) and vascular endothelial growth factor receptor 2 (Flk-1) have been shown to be involved in tumor-induced angiogenesis. Selective targeting of upregulated alpha(v)beta(3) and Flk-1 on the neovasculature of tumors is a novel antiangiogenesis strategy for treating a wide variety of solid tumors. In the studies described here, we investigated the potential therapeutic efficacy of two three-component treatment regimens using two murine tumor models. METHODS AND MATERIALS: The treatment regimens used nanoparticle (NP) based targeting agents radiolabeled with (90)Y. The small molecule integrin antagonist (IA) 4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)ethoxy]-benzoyl-2-(5)-aminoethylsulfonylamino-beta-alanine, which binds to the integrin alpha(v)beta(3), and a monoclonal antibody against murine Flk-1 (anti-Flk-1 MAb) were used to target the NPs. Murine tumor models K1735-M2 (melanoma) and CT-26 (colon adenocarcinoma) were used to evaluate the treatment efficacy. RESULTS: In K1735-M2 and CT-26 tumors, a single treatment with IA-NP-(90)Y (14.2 microg/g IA, 5 or 6 microCi/g (90)Y) caused a significant tumor growth delay compared to untreated control tumors, as well as tumors treated with IA, IA-NP, and NP-(90)Y, respectively (p < 0.025, Wilcoxon test). In K1735-M2 tumors, a single treatment with anti-Flk-1 MAb-NP-(90)Y (0.36 microg/g anti-Flk-1 MAb, 5 microCi/g (90)Y) also caused a significant tumor growth delay (p < 0.05, Wilcoxon test) compared to untreated tumors, as well as tumors treated with anti-Flk-1 MAb, anti-Flk-1 MAb-NP, and conventional radioimmunotherapy with (90)Y-labeled anti-Flk mAb. Anti-CD31 staining showed a marked decrease in vessel density in tumors treated with anti-Flk-1 MAb-NP-(90)Y, which was associated with a high level of apoptotic death in these tumors, as shown by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. CONCLUSIONS: The present studies provide proof of principle that targeted radiotherapy works using different targeting agents on a nanoparticle, to target both the integrin alpha(v)beta(3) and the vascular endothelial growth factor receptor. These encouraging results demonstrate the potential therapeutic efficacy of the IA-NP-(90)Y and anti-Flk-1 MAb-NP-(90)Y complexes as novel therapeutic agents for the treatment of a variety of tumor types.

Enhancement of the efficacy of an antagonist of an extracellular receptor by attachment to the surface of a biocompatible carrier.

PURPOSE: In order to improve the in vitro and in vivo efficacy of an integrin antagonist (IA) of the extracellular domain of the alphavbeta3 integrin, a receptor upregulated on tumor neovasculature, the IA was attached to the surface of a dextran-coated liposome (DCL). IA-DCLs were characterized in vitro, and the pharmacokinetic and antitumor properties were assessed in vivo. METHODS: The in vitro binding properties were measured with purified integrin, endothelial cells, and melanoma cells. The pharmacokinetic parameters were measured in healthy mice with 14C-labeled IA-DCLs and anti-tumor efficacy was assessed with the M21 human melanoma xenograft mouse model. RESULTS: In vitro, IC50 values for IA-DCLs and IA are similar, and IA-DCLs inhibit cell proliferation relative to controls. IA-DCLs are stable in serum, and the pharmacokinetic half-life in mice is 23 h. In the M21/mouse model, statistically significant inhibition of tumor growth was observed for mice treated with IA-DCLs, whereas controls including saline, DCLs lacking IA, and cyclo(RGDfV) were ineffective. Increased apoptosis and a reduction in vessel counts relative to controls were present in tumors from animals treated with IA-DCLs. CONCLUSIONS: These results demonstrate that IA-DCLs are potent anti-angiogenic therapeutic agents with superior in vivo activity and pharmacology compared to unmodified IA.