Bispecific antibodies targeting tumor-associated antigens and neutralizing complement regulators increase the efficacy of antibody-based immunotherapy in mice.

The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4-25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluated in a human-SCID model of Burkitt lymphoma. The distribution profile of bsAbs mimics the data obtained by studying the pharmacokinetics of anti-CD20 antibodies, showing a peak in the tumor mass 3-4 days after injection. The treatment with bsAbs completely prevented the development of human/SCID lymphoma. The tumor growth was blocked by the activation of the C cascade and by the recruitment of macrophages, polymorphonuclear and natural killer cells. This strategy can easily be applied to the other anti-tumor C-fixing antibodies currently used in the clinic or tested in preclinical studies using the same vector with the appropriate modifications.

Innate immunity, through late complement components activation, contributes to the development of early vascular inflammation and morphologic alterations in experimental diabetes.

OBJECTIVE: To verify if innate immunity, and namely the assembly of terminal complement complex (TCC) could be involved in the development of early diabetic vascular damage. METHODS AND RESULTS: At first in 2 groups of diabetic or non-diabetic Wistar rats the occurrence of basal or stimulated stable adherence to the endothelial layer and extravasation of circulating fluorescently-labelled leukocytes was assessed by using an in vivo videomicroscopy technique. In a second part of the study, the development of vascular damage in short term diabetes was studied in the genetically C6 deficient rats of the PVG strain, and compared with those observed in the wild-type C6 sufficient animals. Here, the analysis of mesentery vascular expression of mRNA for vascular cell adhesion molecule (VCAM)-1, transforming growth factor-ß (TGF-ß), connective tissue growth factor (CTGF), and platelet-derived growth factor (PDGF), the evaluation of intravascular protein levels of VCAM-1, TGF-ß, CTGF, proliferative cell nuclear antigen (PCNA), as well as the assessment of structural changes and Complement components deposition at the mesentery arterial vascular wall were also performed. CONCLUSIONS: Leukocyte trafficking, mesentery arteries hypertrophy, extracellular matrix deposition, local vascular gene and protein expression of VCAM-1, TGF-ß, CTGF and PCNA, as well as PGDF gene expression were all increased by short term diabetes, but all significantly reduced in the C6 deficient diabetic animals, thus suggesting an active role for TCC in the development of vascular inflammation in the early phases of experimental diabetes.

Mannose-binding lectin is produced by vaginal epithelial cells and its level in the vaginal fluid is influenced by progesterone.

Mannose-binding lectin (MBL) is a recognition molecule of the complement (C) system and binds to carbohydrate ligands present on a wide range of pathogenic bacteria, viruses, fungi, and parasites. MBL has been detected in the cervico-vaginal cavity where it can provide a first-line defence against infectious agents colonizing the lower tract of the reproductive system. Analysis of the cervico-vaginal lavage (CVL) obtained from 11 normal cycling women at different phases of the menstrual cycle revealed increased levels of MBL in the secretive phase. Part of this MBL derives from the circulation as indicated by the presence of transferrin in CVL tested as a marker of vascular and tissue permeability. The local synthesis of MBL is suggested by the finding that its level is substantially higher than that of transferrin in the secretive phase. The contribution of endometrium is negligible since the MBL level did not change before and after hysterectomy. RT-PCR and in situ RT-PCR analysis showed that the vaginal tissue, and in particular the basal layer of the epithelium, is a source of MBL which binds to the basal membrane and to cells of the outer layers of the epithelium. In conclusion, we have shown that MBL detected in CVL derives both from plasma as result of transudation and from local synthesis and its level is progesterone dependent increasing in the secretive phase of the menstrual cycle.

Fluvastatin treatment inhibits leucocyte adhesion and extravasation in models of complement-mediated acute inflammation.

Complement activation plays a relevant role in the development of tissue damage under inflammatory conditions, and clinical and experimental observations emphasize its contribution to inflammatory vasculitides. Statins have recently been shown to reduce cardiovascular morbidity independently of plasma cholesterol lowering and in vitro studies support a direct anti-inflammatory action of these drugs. The aim of this study was to verify the in vivo effect of fluvastatin on complement-mediated acute peritoneal inflammation. The effect of oral treatment with fluvastatin was investigated in normo-cholesterolaemic rats that received intraperitoneal injection of either yeast-activated rat serum (Y-act RS) or lipopolysaccharide to induce peritoneal inflammation monitored by the number of PMN recruited in peritoneal fluid washes. In addition, vascular adherence and extravasation of leucocytes were evaluated by direct videomicroscopy examination on mesentery postcapillary venules topically exposed to Y-act RS. The number of PMN in the peritoneal washes of rats treated with fluvastatin was 38% lower than that of untreated animals (P < 0.05) 12 h after LPS injection, and was even lower (56%) in rats treated with Y-act RS already 8 h after injection (P < 0.02). Firm adhesion to endothelium and extravasation of leucocytes evaluated under direct videomicroscopy observation were significantly inhibited in fluvastatin treated rats (77% and 72%, respectively; P < 0.01), 120 min after treatment with Y-act RS. Our results demonstrate that fluvastatin inhibits in vivo complement-dependent acute peritoneal inflammation and suggest a role for statins in preventing the inflammatory flares usually associated with complement activation in chronic diseases, such as SLE or rheumatoid arthritis.