ADPase CD39 Fused to Glycoprotein VI-Fc Boosts Local Antithrombotic Effects at Vascular Lesions.

BACKGROUND: GPVI (Glycoprotein VI) is the essential platelet collagen receptor in atherothrombosis. Dimeric GPVI-Fc (Revacept) binds to GPVI binding sites on plaque collagen. As expected, it did not increase bleeding in clinical studies. GPVI-Fc is a potent inhibitor of atherosclerotic plaque-induced platelet aggregation at high shear flow, but its inhibition at low shear flow is limited. We sought to increase the platelet inhibitory potential by fusing GPVI-Fc to the ectonucleotidase CD39 (fusion protein GPVI-CD39), which inhibits local ADP accumulation at vascular plaques, and thus to create a lesion-directed dual antiplatelet therapy that is expected to lack systemic bleeding risks. METHODS AND RESULTS: GPVI-CD39 effectively stimulated local ADP degradation and, compared with GPVI-Fc alone, led to significantly increased inhibition of ADP-, collagen-, and human plaque-induced platelet aggregation in Multiplate aggregometry and plaque-induced platelet thrombus formation under arterial flow conditions. GPVI-CD39 did not increase bleeding time in an in vitro assay simulating primary hemostasis. In a mouse model of ferric chloride-induced arterial thrombosis, GPVI-CD39 effectively delayed vascular thrombosis but did not increase tail bleeding time in vivo. CONCLUSIONS: GPVI-CD39 is a novel approach to increase local antithrombotic activity at sites of atherosclerotic plaque rupture or injury. It enhances GPVI-Fc-mediated platelet inhibition and presents a potentially effective and safe molecule for the treatment of acute atherothrombotic events, with a favorable risk-benefit ratio.

(211)At-antiCD33 in NMRI nu/nu mice. Biodistribution, in vivo stability and radiotoxicity.

UNLABELLED: The aim of this study is to verify the in vivo stability, to determine the biodistribution and to estimate the unspecific radiotoxicity of an (211)At-labelled CD33-antibody ((211)At-antiCD33) in mice with a view to therapeutic application in treating leukaemia. ANIMALS, METHODS: (211)At was produced via the (209)Bi(a,2n)(211)At reaction and was linked via 3-(211)At-succinimidyl-benzoate to the antiCD33-antibody. The biodistribution and the in vivo stability in serum were determined after i.v.-injection in NMRI nu/nu-mice. For toxicity experiments, mice received either three times 315-650 kBq (211)At-antiCD33 or unlabelled antibody and NaCl-solution respectively. RESULTS: (211)At-antiCD33 showed a characteristic biodistribution complying with the unspecific antibody retention in the reticular endothelial system. The largest proportion of radioactivity remained in blood and blood-rich tissues with a minor accumulation in the thyroid and stomach. After 21 h, >85% of activity in serum still represented intact antibody. Mice showed no difference in unspecific toxicity of (211)At-labelled antibodies over six months compared to those treated with unlabelled antibody and NaCl-solution respectively, with regard to histopathologic lesions, survival time, behaviour and haemograms. CONCLUSION: The radiolabelling method yielded adequate in vivo stability of (211)At-antiCD33. Biodistribution with rapid elimination of free (211)At via kidneys and urine complies with requirements for targeted therapy. Activity doses potentially required for treatment do not elicit radiotoxicity to normal organs in mice. Further development is required to enhance the apparent specific activity and to verify the efficacy in an adequate animal model before phase I clinical studies in leukaemia can be envisaged.

The reciprocal interaction of NK cells with plasmacytoid or myeloid dendritic cells profoundly affects innate resistance functions.

A reciprocal activating interaction between NK cells and dendritic cells (DC) has been suggested to play a role in the functional regulation of these cells in immunity, but it has been studied only using in vitro generated bone marrow- or monocyte-derived DC. We report that human peripheral blood plasmacytoid DC (pDC) and myeloid DC are necessary to induce NK cell function depending on the type of microbial stimulus. pDC and myeloid DC are required for strongly increased NK cytolytic activity and CD69 expression, in response to inactivated influenza virus or CpG-containing oligonucleotides and poly(I:C), respectively. Secreted type I IFN is required and sufficient for the augmentation of NK cell cytolytic activity in the coculture with pDC or myeloid DC, whereas CD69 expression is dependent on both type I IFN and TNF. In addition, in response to poly(I:C), myeloid DC induce NK cells to produce IFN-gamma through a mechanism dependent on both IL-12 secretion and cell contact between NK cells and myeloid DC, but independent of type I IFN. IL-2-activated NK cells have little to no cytolytic activity for immature myeloid DC and pDC, but are able to induce maturation of these cells. Moreover, IL-2-activated NK cells induce, in the presence of a suboptimal concentration of CpG-containing oligonucleotides, a strong IFN-alpha and TNF production. These data suggest that the reciprocal functional interaction between NK cells and either pDC or myeloid DC may play an important physiological role in the regulation of both innate resistance and adaptive immunity to infections.

Reduced phagocytosis of colloidal carriers using soluble CD47.

PURPOSE: This study was designed to illustrate the feasibility of using soluble CD47 protein to antagonize phagocytosis of colloidal drug carriers by macrophages. METHODS: Expression of CD47-streptavidin (CD47-SA) fusion protein was achieved in B21CodonPlus host cells following IPTG induction. Murine macrophage cell line J774A.1, expressing high levels of SIRPalpha, was selected as the biologic model system for phagocytosis. FITC-labeled perfluorocarbon (PFC) emulsions were used as the colloidal carriers to trigger phagocytosis. Microscopy (inverted light and UV-fluorescence) and flow cytometry were used to qualitatively and quantitatively determine the degree of phagocytosis, respectively. RESULTS: The bacterially expressed, purified CD47-SA had neither cytotoxic nor cytostatic effects when incubated with J774A.1 cells up to a concentration of 400 nM for 24 h. Phagocytosis of FITC-labeled PFC emulsions was significantly diminished when macrophages were pretreated with 100 nM CD47-SA for 1 h. CONCLUSIONS: We demonstrated that soluble CD47-SA antagonized phagocytosis of colloidal carriers to a significant degree by interaction with macrophage SIRPalpha.

Cytotoxic potency of CD22-ricin A depends on intracellular routing rather than on the number of internalized molecules.

Cytotoxicity of immunotoxins (ITs) varies considerably depending on factors like the capability of the target antigen to internalize IT molecules, intracellular processing and routing of the IT. We studied factors that may influence cytotoxicity of CD22-ricin A IT to several B cell lines. The antigen density varied from 5.9 x 10(3) to 6.0 x 10(4) molecules/cell. The ID50, determined by protein synthesis inhibition, varied from 2.1 x 10(-12) to 3.8 x 10(-11) M IT in absence and from 2.8 x 10(-14) M to 5.2 x 10(-12) M IT in presence of the cytotoxicity enhancer NH4Cl (6 mM). In absence as well as in presence of NH4Cl no correlation could be found between antigen density and ID50. No relation was observed either with the rate of cytotoxicity. Even in cell lines with a low antigen density, such as KM3, protein synthesis was quickly inhibited. In order to investigate whether the cytotoxicity was dependent on the number of internalized molecules the kinetics of internalization and exocytosis of degraded 125I-labelled CD22 molecules were studied. After 24 h the number of internalized CD22 molecules was highest in Ramos (154,500), followed by Daudi (110,300) and KM3 (69,900). However, despite the higher internalization rate of Daudi the rate of cytotoxicity of 10(-8) M IT was comparable with KM3. NH4Cl did not influence the number of internalized molecules but postponed degradation of CD22. In conclusion, CD22-ricin A is a very potent and fast acting IT even for elimination of target cells that express low numbers of antigen. These results may have implication for treatment of different B cell malignancies with CD22-ricin A.